G2Cdb::Gene report

Gene id
G00002149
Gene symbol
SNCA (HGNC)
Species
Homo sapiens
Description
synuclein, alpha (non A4 component of amyloid precursor)
Orthologue
G00000900 (Mus musculus)

Databases (7)

Gene
ENSG00000145335 (Ensembl human gene)
6622 (Entrez Gene)
194 (G2Cdb plasticity & disease)
SNCA (GeneCards)
Literature
163890 (OMIM)
Marker Symbol
HGNC:11138 (HGNC)
Protein Sequence
P37840 (UniProt)

Synonyms (3)

  • NACP
  • PD1
  • alpha-synuclein

Literature (548)

Pubmed - other

  • Alpha-synuclein locus duplication as a cause of familial Parkinson's disease.

    Chartier-Harlin MC, Kachergus J, Roumier C, Mouroux V, Douay X, Lincoln S, Levecque C, Larvor L, Andrieux J, Hulihan M, Waucquier N, Defebvre L, Amouyel P, Farrer M and Destée A

    Unité INSERM 508, 1 rue du Pr Calmette, BP 245, 59019 Lille Cedex, France.

    Genomic triplication of the alpha-synuclein gene (SNCA) has been reported to cause hereditary early-onset parkinsonism with dementia. These findings prompted us to screen for multiplication of the SNCA locus in nine families in whom parkinsonism segregates as an autosomal dominant trait. One kindred was identified with SNCA duplication by semiquantitative PCR and confirmed by fluorescent in-situ hybridisation analysis in peripheral leucocytes. By contrast with SNCA triplication families, the clinical phenotype of SNCA duplication closely resembles idiopathic Parkinson's disease, which has a late age-of-onset, progresses slowly, and in which neither cognitive decline nor dementia are prominent. These findings suggest a direct relation between SNCA gene dosage and disease progression.

    Lancet (London, England) 

  • Causal relation between alpha-synuclein gene duplication and familial Parkinson's disease.

    Ibáñez P, Bonnet AM, Débarges B, Lohmann E, Tison F, Pollak P, Agid Y, Dürr A and Brice A

    INSERM U289, Neurologie et Thérapeutique Expérimentale, Hôpital de la Pitié-Salpêtrière, AP-HP, Paris, France.

    The alpha-synuclein gene (SNCA) has been implicated in autosomal dominant forms of Parkinson's disease. We screened 119 individuals from families with this rare form of the disease for SNCA duplications by semiquantitative multiplex PCR. Two patients had duplications, which were confirmed by analysis of intragenic and flanking microsatellite markers. The phenotype in both patients was indistinguishable from idiopathic Parkinson's disease and no atypical features were present, by contrast with reports of families with triplication of the same gene. These results indicate that SNCA is more frequently associated with familial Parkinson's disease than previously thought, and that there is a clear dosage effect according to the number of supernumerary copies of this gene.

    Lancet (London, England) 

  • Genome-wide association study confirms SNPs in SNCA and the MAPT region as common risk factors for Parkinson disease.

    Edwards TL, Scott WK, Almonte C, Burt A, Powell EH, Beecham GW, Wang L, Züchner S, Konidari I, Wang G, Singer C, Nahab F, Scott B, Stajich JM, Pericak-Vance M, Haines J, Vance JM and Martin ER

    John P. Hussman Institute for Human Genomics, University of Miami, FL 33136, USA.

    Parkinson disease (PD) is a chronic neurodegenerative disorder with a cumulative prevalence of greater than one per thousand. To date three independent genome-wide association studies (GWAS) have investigated the genetic susceptibility to PD. These studies implicated several genes as PD risk loci with strong, but not genome-wide significant, associations. In this study, we combined data from two previously published GWAS of Caucasian subjects with our GWAS of 604 cases and 619 controls for a joint analysis with a combined sample size of 1752 cases and 1745 controls. SNPs in SNCA (rs2736990, p-value = 6.7 x 10(-8); genome-wide adjusted p = 0.0109, odds ratio (OR) = 1.29 [95% CI: 1.17-1.42] G vs. A allele, population attributable risk percent (PAR%) = 12%) and the MAPT region (rs11012, p-value = 5.6 x 10(-8); genome-wide adjusted p = 0.0079, OR = 0.70 [95% CI: 0.62-0.79] T vs. C allele, PAR%= 8%) were genome-wide significant. No other SNPs were genome-wide significant in this analysis. This study confirms that SNCA and the MAPT region are major genes whose common variants are influencing risk of PD.

    Funded by: NIA NIH HHS: U24 AG021886; NINDS NIH HHS: NS39764, P50 NS039764, P50 NS039764-10S40003

    Annals of human genetics 2010;74;2;97-109

  • Differential phospholipid binding of alpha-synuclein variants implicated in Parkinson's disease revealed by solution NMR spectroscopy.

    Bodner CR, Maltsev AS, Dobson CM and Bax A

    Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892-0520, USA.

    Three familial variants of the presynaptic protein alpha-synuclein (alphaS), A30P, E46K, and A53T, correlate with rare inherited Parkinson's disease (PD), while wild-type alphaS is implicated in sporadic PD. The classic manifestation of both familiar and sporadic PD is the formation of fibrillar structures of alphaS which accumulate as the main component in intraneuronal Lewy bodies. At presynaptic termini, the partitioning of alphaS between disordered cytosolic and membrane-bound states likely mediates its proposed role in regulation of reserve pools of synaptic vesicles. Previously, we reported on multiple distinct phospholipid binding modes of alphaS with slow binding kinetics. Here, we report the phospholipid binding properties of the disease variants, viewed by solution NMR in a residue-specific manner. Our results agree qualitatively with previous biophysical studies citing overall decreased lipid affinity for the A30P mutation, comparable affinity for A53T, and an increased level of binding of E46K, relative to wild-type alphaS. Additionally, our NMR results describe the distribution of lipid-bound states for alphaS: the population of the SL1 binding mode (residues 3-25 bound as a helix) is augmented by each of the disease variants, relative to wild-type alphaS. We propose that the SL1 binding mode, which anchors the N-terminus of alphaS in the lipoprotein complex while the hydrophobic NAC region remains dynamically disordered, is prone to intermolecular interactions which progress toward disease-associated oligomers and fibrils. The elevation of the SL1 binding mode, unchecked by a proportionate population of binding modes incorporating the full N-terminal domain, may well account for the increased toxicity of the A30P, E46K, and A53T disease variants of alphaS.

    Funded by: Intramural NIH HHS; Wellcome Trust

    Biochemistry 2010;49;5;862-71

  • Characterization of inhibitor-bound alpha-synuclein dimer: role of alpha-synuclein N-terminal region in dimerization and inhibitor binding.

    Yamaguchi Y, Masuda M, Sasakawa H, Nonaka T, Hanashima S, Hisanaga S, Kato K and Hasegawa M

    Department of Structural Biology and Biomolecular Engineering, Graduate School of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabe-dori, Mizuho-ku, Nagoya 467-8603, Japan. yyoshiki@riken.jp

    alpha-Synuclein is a major component of filamentous inclusions that are histological hallmarks of Parkinson's disease and other alpha-synucleinopathies. Previous analyses have revealed that several polyphenols inhibit alpha-synuclein assembly with low micromolar IC(50) values, and that SDS-stable, noncytotoxic soluble alpha-synuclein oligomers are formed in their presence. Structural elucidation of inhibitor-bound alpha-synuclein oligomers is obviously required for the better understanding of the inhibitory mechanism. In order to characterize inhibitor-bound alpha-synucleins in detail, we have prepared alpha-synuclein dimers in the presence of polyphenol inhibitors, exifone, gossypetin, and dopamine, and purified the products. Peptide mapping and mass spectrometric analysis revealed that exifone-treated alpha-synuclein monomer and dimer were oxidized at all four methionine residues of alpha-synuclein. Immunoblot analysis and redox-cycling staining of endoproteinase Asp-N-digested products showed that the N-terminal region (1-60) is involved in the dimerization and exifone binding of alpha-synuclein. Ultra-high-field NMR analysis of inhibitor-bound alpha-synuclein dimers showed that the signals derived from the N-terminal region of alpha-synuclein exhibited line broadening, confirming that the N-terminal region is involved in inhibitor-induced dimerization. The C-terminal portion still predominantly exhibited the random-coil character observed in monomeric alpha-synuclein. We propose that the N-terminal region of alpha-synuclein plays a key role in the formation of alpha-synuclein assemblies.

    Journal of molecular biology 2010;395;3;445-56

  • Towards multiparametric fluorescent imaging of amyloid formation: studies of a YFP model of alpha-synuclein aggregation.

    van Ham TJ, Esposito A, Kumita JR, Hsu ST, Kaminski Schierle GS, Kaminski CF, Dobson CM, Nollen EA and Bertoncini CW

    Department of Genetics, University of Groningen, Groningen, The Netherlands.

    Misfolding and aggregation of proteins are characteristics of a range of increasingly prevalent neurodegenerative disorders including Alzheimer's and Parkinson's diseases. In Parkinson's disease and several closely related syndromes, the protein alpha-synuclein (AS) aggregates and forms amyloid-like deposits in specific regions of the brain. Fluorescence microscopy using fluorescent proteins, for instance the yellow fluorescent protein (YFP), is the method of choice to image molecular events such as protein aggregation in living organisms. The presence of a bulky fluorescent protein tag, however, may potentially affect significantly the properties of the protein of interest; for AS in particular, its relative small size and, as an intrinsically unfolded protein, its lack of defined secondary structure could challenge the usefulness of fluorescent-protein-based derivatives. Here, we subject a YFP fusion of AS to exhaustive studies in vitro designed to determine its potential as a means of probing amyloid formation in vivo. By employing a combination of biophysical and biochemical studies, we demonstrate that the conjugation of YFP does not significantly perturb the structure of AS in solution and find that the AS-YFP protein forms amyloid deposits in vitro that are essentially identical with those observed for wild-type AS, except that they are fluorescent. Of the several fluorescent properties of the YFP chimera that were assayed, we find that fluorescence anisotropy is a particularly useful parameter to follow the aggregation of AS-YFP, because of energy migration Förster resonance energy transfer (emFRET or homoFRET) between closely positioned YFP moieties occurring as a result of the high density of the fluorophore within the amyloid species. Fluorescence anisotropy imaging microscopy further demonstrates the ability of homoFRET to distinguish between soluble, pre-fibrillar aggregates and amyloid fibrils of AS-YFP. Our results validate the use of fluorescent protein chimeras of AS as representative models for studying protein aggregation and offer new opportunities for the investigation of amyloid aggregation in vivo using YFP-tagged proteins.

    Journal of molecular biology 2010;395;3;627-42

  • Residue Glu83 plays a major role in negatively regulating alpha-synuclein amyloid formation.

    Waxman EA, Emmer KL and Giasson BI

    Department of Pharmacology, University of Pennsylvania School of Medicine, 3620 Hamilton Walk, 125 John Morgan Building, Philadelphia, PA 19104-6084, USA.

    Alpha-synuclein (alpha-syn) amyloid filaments are the major ultrastructural component of pathological inclusions that define several neurodegenerative disorders, including Parkinson disease and other disorders that are collectively termed synucleinopathies. Since the aggregation of alpha-syn is associated with the etiology of these diseases, defining the molecular elements that influence this process may have important therapeutics implication. The deletions of major portions of the hydrophobic region of alpha-syn (Delta74-79 and Delta71-82) impair the ability to form amyloid. However, mutating residue E83 to an A restored the ability of these proteins to form amyloid. Additionally supporting an inhibitory role of residue E83 on amyloid formation, mutating this residue to an A enhanced amyloid formation in the presence of small molecule inhibitors, such as dopamine and EGCG. Our data, therefore, suggest that the presence and placement of the highly charged E83 residue plays a significant inhibitory role in alpha-syn amyloid formation and these findings provide important insights in the planning of therapeutic agents that may be capable of preventing alpha-syn amyloid formation.

    Funded by: NIA NIH HHS: AG09215, P01 AG009215, P01 AG009215-160009, P01 AG009215-170009, P01 AG009215-180009, P01 AG009215-190009, P01 AG009215-200009, T32 AG000255-10, T32 AG000255-11A1, T32 AG00255; NINDS NIH HHS: NS053488, P50 NS053488, P50 NS053488-01A20004, P50 NS053488-020004, P50 NS053488-02S10004, P50 NS053488-030004, P50 NS053488-03S10004

    Biochemical and biophysical research communications 2010;391;3;1415-20

  • Dopamine facilitates alpha-synuclein oligomerization in human neuroblastoma SH-SY5Y cells.

    Yamakawa K, Izumi Y, Takeuchi H, Yamamoto N, Kume T, Akaike A, Takahashi R, Shimohama S and Sawada H

    Clinical Research Center, Utano National Hospital, National Hospital Organization, Kyoto 616-8255, Japan.

    Parkinson's disease is characterized by selective loss of dopaminergic neurons in the substantia nigra and by the appearance of Lewy bodies. Fibrillar alpha-synuclein is the main component of Lewy bodies. Previous studies have suggested that dopamine promotes alpha-synuclein oligomerization and that partially aggregated or oligomeric alpha-synuclein could be cytotoxic. To confirm this hypothesis using cell cultures, we performed size exclusion chromatography as a pretreatment method prior to Western blotting to more clearly detect a small amount of alpha-synuclein oligomers in wild-type alpha-synuclein-overexpressing SH-SY5Y cells. Using this method, we confirmed that stable overexpression of alpha-synuclein in SH-SY5Y cells indeed increased the amounts of alpha-synuclein oligomers in these cells and exposure of the cells to dopamine for 6h facilitated alpha-synuclein oligomerization. These dopamine-induced alpha-synuclein oligomers continued to exist for the following 24h. However, the dopamine-treated cells did not undergo cell death or apoptosis in spite of the presence of increased oligomeric alpha-synuclein. Our data may contribute to the understanding of the mechanisms underlying alpha-synuclein oligomer formation and its suspected cytotoxicity toward dopaminergic neurons.

    Biochemical and biophysical research communications 2010;391;1;129-34

  • Forebrain overexpression of alpha-synuclein leads to early postnatal hippocampal neuron loss and synaptic disruption.

    Lim Y, Kehm VM, Li C, Trojanowski JQ and Lee VM

    Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA.

    Transgenic (Tg) mouse models of Parkinson's disease (PD) generated to date have primarily been designed to overexpress human alpha-synuclein (alpha-syn) to recapitulate PD-like motor impairments as well as PD-like nigrostriatal degeneration and alpha-syn pathology. However, cognitive impairments and cortical alpha-syn pathology are also common in PD patients. To model these features of PD, we created forebrain-specific conditional Tg mice that overexpress human wild type (WT) or A53T mutant alpha-syn. Here we show that both WT and A53T mutant alpha-syn lead to massive degeneration of postmitotic neurons in the hippocampal dentate gyrus (DG) during postnatal development, with hippocampal synapse loss as evidenced by reduced levels of pre- and postsynaptic markers. However, when mutant and WT alpha-syn expression was repressed until the Tg mice were mature postnatally and then induced for several months, no hippocampal neuron loss was observed. These data imply that developing neurons are more vulnerable to degenerate than mature neurons as a consequence of forebrain WT and mutant alpha-syn overexpression.

    Funded by: NIA NIH HHS: AG 09215, P01 AG009215, P01 AG009215-130006; NINDS NIH HHS: NS 053488, P50 NS053488, P50 NS053488-03S10003

    Experimental neurology 2010;221;1;86-97

  • Interaction with synphilin-1 promotes inclusion formation of alpha-synuclein: mechanistic insights and pathological implication.

    Xie YY, Zhou CJ, Zhou ZR, Hong J, Che MX, Fu QS, Song AX, Lin DH and Hu HY

    State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 320 Yueyang Road, Shanghai, China.

    alpha-Synuclein (alpha-Syn) is the major component of Lewy bodies (LBs) deposited in the brains of patients with Parkinson's disease. Synphilin-1 (Sph1) is a novel alpha-Syn-interacting protein also present in the LBs. However, the roles of alpha-Syn-Sph1 interaction in LB formation and in the related pathogenesis are still unclear. We have studied the interaction between alpha-Syn and Sph1 by biochemical and structural approaches and found that the central coiled-coil domain of Sph1 specifically interacts with the N-terminal stretch of alpha-Syn. When overexpressed in HEK 293T cells, Sph1 forms inclusions together with alpha-Syn, but the Sph1-positive inclusions cannot recruit the N-terminally truncated alpha-Syn. The central portion of Sph1 can also recruit alpha-Syn and induce inclusion formation through its coiled-coil domain. These observations demonstrate that the alpha-Syn-Sph1 interaction significantly promotes the formation of cytoplasmic alpha-Syn inclusions, which may have implications for LB formation in neural cells. We have also elucidated solution structure of the coiled-coil domain of Sph1 and its interaction with the N-terminal peptide of alpha-Syn. The specific interaction between alpha-Syn and Sph1 provides mechanistic insights into the inclusion-body formation in cells and pathological implication in Parkinson's disease.

    FASEB journal : official publication of the Federation of American Societies for Experimental Biology 2010;24;1;196-205

  • Lrrk2 interaction with alpha-synuclein in diffuse Lewy body disease.

    Qing H, Zhang Y, Deng Y, McGeer EG and McGeer PL

    Kinsmen Laboratory of Neurological Research, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z3.

    Mutations of the leucine-rich repeat kinase 2 (LRRK2) gene are the leading cause of genetically inherited Parkinson's disease (PD) and its more severe variant diffuse Lewy body disease (DLB). Pathological mutations in Lrrk2 are autosomal dominant, suggesting a gain of function. Mutations in alpha-synuclein also produce autosomal dominant disease. Here we report an interaction between Lrrk2 and alpha-synuclein in a series of diffuse Lewy body (DLB) cases and in an oxidative stress cell based assay. All five cases of DLB, but none of five controls, showed co-immunoprecipitation of Lrrk2 and alpha-synuclein in soluble brain extracts. Colocalization was also found in pathological deposits in DLB postmortem brains by double immunostaining. In HEK cells transfected simultaneously with plasmids expressing Lrrk2 and alpha-synuclein, co-immunoprecipitation of Lrrk2 and alpha-synuclein was detected when they were exposed to oxidative stress by H(2)O(2). Taken together, these results suggest the possibility that in PD and related synucleinopathies, oxidative stress upregulates alpha-syn and Lrrk2 expression, paving the way for pathological interactions. New therapeutic approaches to PD and the synucleinopathies may result from limiting the interaction between Lrrk2 and alpha-synuclein.

    Biochemical and biophysical research communications 2009;390;4;1229-34

  • Tryptophan fluorescence reveals structural features of alpha-synuclein oligomers.

    van Rooijen BD, van Leijenhorst-Groener KA, Claessens MM and Subramaniam V

    Biophysical Engineering Group, MESA+ Institute for Nanotechnology, University of Twente, P.O. Box 217, 7500 AE Enschede, The Netherlands.

    Oligomeric alpha-synuclein (alphaS) is considered to be the potential toxic species responsible for the onset and progression of Parkinson's disease, possibly through the disruption of lipid membranes. Although there is evidence that oligomers contain considerable amounts of secondary structure, more detailed data on the structural characteristics and how these mediate oligomer-lipid binding are critically lacking. This report is, to our knowledge, the first study that aimed to address the structure of oligomeric alphaS on a more detailed level. We have used tryptophan (Trp) fluorescence spectroscopy to gain insight into the structural features of oligomeric alphaS and the structural basis for oligomer-lipid interactions. Several single Trp mutants of alphaS were used to gain site-specific information about the microenvironments of monomeric alphaS, oligomeric alphaS and lipid-bound oligomeric alphaS. Acrylamide quenching and spectral analyses indicate that the Trp residues are considerably more solvent protected in the oligomeric form compared with the monomeric protein. In the oligomers, the negatively charged C-terminus was the most solvent exposed part of the protein. Upon lipid binding, a blue shift in fluorescence was observed for alphaS mutants where the Trp is located within the N-terminal region. These results suggest that, as in the case of monomeric alphaS, the N-terminus is critical in determining oligomer-lipid binding.

    Journal of molecular biology 2009;394;5;826-33

  • Alpha-synuclein polymorphisms are associated with Parkinson's disease in a Saskatchewan population.

    Rajput A, Vilariño-Güell C, Rajput ML, Ross OA, Soto-Ortolaza AI, Lincoln SJ, Cobb SA, Heckman MG, Farrer MJ and Rajput A

    Division of Neurology, University of Saskatchewan and Saskatoon Health Region, Saskatoon, Saskatchewan, Canada.

    Alpha-synuclein gene (SNCA) mutations cause familial Parkinsonism but the role of SNCA variability in idiopathic Parkinson's disease (PD) remains incompletely defined. We report a study of SNCA genetic variation in 452 idiopathic PD cases and 245 controls. SNCA copy number mutations were not associated with early-onset disease in this population. The minor allele "G" at rs356165 was associated with increased odds of PD (P = 0.013) and genetic variation in D4S3481 (Rep1) was associated with age of disease onset (P = 0.007). There was a trend toward association between variation at rs2583988 and rapid PD progression.

    Funded by: NINDS NIH HHS: P50 NS40256

    Movement disorders : official journal of the Movement Disorder Society 2009;24;16;2411-4

  • Genetic association studies of methamphetamine use disorders: A systematic review and synthesis.

    Bousman CA, Glatt SJ, Everall IP and Tsuang MT

    Department of Psychiatry, Center for Behavioral Genomics, University of California San Diego, La Jolla, 92037, USA.

    Efforts to understand the biological processes that increase susceptibility to methamphetamine (METH) use disorders (i.e., abuse, dependence, and psychosis) have uncovered several putative genotypic variants. However, to date a synthesis of this information has not been conducted. Thus, systematic searches of the current literature were undertaken for genetic-association studies of METH use disorders. Each gene's chromosomal location, function, and examined polymorphic markers were extracted. Frequencies, odds ratios and 95% confidence intervals for risk alleles, as well as sample size and power, were calculated. We uncovered 38 studies examining 39 genes, of which 18 were found to have a significant genotypic, allelic, and/or haplotypic association with METH use disorders. Three genes (COMT, DRD4, and GABRA1) were associated with METH abuse, nine (ARRB2, BDNF, CYP2D6, GLYT1, GSTM1, GSTP1, PDYN, PICK1, and SLC22A3) with METH dependence, two (AKT1 and GABRG2) with METH abuse/dependence, and four (DTNBP1, OPRM1, SNCA, and SOD2) with METH psychosis. Limitations related to phenotypic classification, statistical power, and potential publication bias in the current literature were noted. Similar to other behavioral, psychiatric, and substance use disorders, the genetic epidemiology of METH use disorders is complex and likely polygenic. National and international collaborative efforts are needed to increase the availability of large population-based samples and improve upon the power to detect genetic associations of small magnitude. Further, replication of the findings reviewed here along with further development of more rigorous methodologies and reporting protocols will aid in delineating the complex genetic epidemiology of METH use disorders.

    American journal of medical genetics. Part B, Neuropsychiatric genetics : the official publication of the International Society of Psychiatric Genetics 2009;150B;8;1025-49

  • Chaperone proteostasis in Parkinson's disease: stabilization of the Hsp70/alpha-synuclein complex by Hip.

    Roodveldt C, Bertoncini CW, Andersson A, van der Goot AT, Hsu ST, Fernández-Montesinos R, de Jong J, van Ham TJ, Nollen EA, Pozo D, Christodoulou J and Dobson CM

    Department of Chemistry, University of Cambridge, Cambridge, UK. cintia.roodveldt@cabimer.es

    The ATP-dependent protein chaperone heat-shock protein 70 (Hsp70) displays broad anti-aggregation functions and has a critical function in preventing protein misfolding pathologies. According to in vitro and in vivo models of Parkinson's disease (PD), loss of Hsp70 activity is associated with neurodegeneration and the formation of amyloid deposits of alpha-synuclein (alphaSyn), which constitute the intraneuronal inclusions in PD patients known as Lewy bodies. Here, we show that Hsp70 depletion can be a direct result of the presence of aggregation-prone polypeptides. We show a nucleotide-dependent interaction between Hsp70 and alphaSyn, which leads to the aggregation of Hsp70, in the presence of ADP along with alphaSyn. Such a co-aggregation phenomenon can be prevented in vitro by the co-chaperone Hip (ST13), and the hypothesis that it might do so also in vivo is supported by studies of a Caenorhabditis elegans model of alphaSyn aggregation. Our findings indicate that a decreased expression of Hip could facilitate depletion of Hsp70 by amyloidogenic polypeptides, impairing chaperone proteostasis and stimulating neurodegeneration.

    Funded by: Biotechnology and Biological Sciences Research Council: 9015651/1; Wellcome Trust

    The EMBO journal 2009;28;23;3758-70

  • Genome-wide association study identifies common variants at four loci as genetic risk factors for Parkinson's disease.

    Satake W, Nakabayashi Y, Mizuta I, Hirota Y, Ito C, Kubo M, Kawaguchi T, Tsunoda T, Watanabe M, Takeda A, Tomiyama H, Nakashima K, Hasegawa K, Obata F, Yoshikawa T, Kawakami H, Sakoda S, Yamamoto M, Hattori N, Murata M, Nakamura Y and Toda T

    Division of Neurology/Molecular Brain Science, Kobe University Graduate School of Medicine, Kobe, Japan.

    To identify susceptibility variants for Parkinson's disease (PD), we performed a genome-wide association study (GWAS) and two replication studies in a total of 2,011 cases and 18,381 controls from Japan. We identified a new susceptibility locus on 1q32 (P = 1.52 x 10(-12)) and designated this as PARK16, and we also identified BST1 on 4p15 as a second new risk locus (P = 3.94 x 10(-9)). We also detected strong associations at SNCA on 4q22 (P = 7.35 x 10(-17)) and LRRK2 on 12q12 (P = 2.72 x 10(-8)), both of which are implicated in autosomal dominant forms of parkinsonism. By comparing results of a GWAS performed on individuals of European ancestry, we identified PARK16, SNCA and LRRK2 as shared risk loci for PD and BST1 and MAPT as loci showing population differences. Our results identify two new PD susceptibility loci, show involvement of autosomal dominant parkinsonism loci in typical PD and suggest that population differences contribute to genetic heterogeneity in PD.

    Nature genetics 2009;41;12;1303-7

  • Genome-wide association study reveals genetic risk underlying Parkinson's disease.

    Simón-Sánchez J, Schulte C, Bras JM, Sharma M, Gibbs JR, Berg D, Paisan-Ruiz C, Lichtner P, Scholz SW, Hernandez DG, Krüger R, Federoff M, Klein C, Goate A, Perlmutter J, Bonin M, Nalls MA, Illig T, Gieger C, Houlden H, Steffens M, Okun MS, Racette BA, Cookson MR, Foote KD, Fernandez HH, Traynor BJ, Schreiber S, Arepalli S, Zonozi R, Gwinn K, van der Brug M, Lopez G, Chanock SJ, Schatzkin A, Park Y, Hollenbeck A, Gao J, Huang X, Wood NW, Lorenz D, Deuschl G, Chen H, Riess O, Hardy JA, Singleton AB and Gasser T

    Laboratory of Neurogenetics, National Institute on Aging, National Institutes of Health, Bethesda, Maryland, USA.

    We performed a genome-wide association study (GWAS) in 1,713 individuals of European ancestry with Parkinson's disease (PD) and 3,978 controls. After replication in 3,361 cases and 4,573 controls, we observed two strong association signals, one in the gene encoding alpha-synuclein (SNCA; rs2736990, OR = 1.23, P = 2.24 x 10(-16)) and another at the MAPT locus (rs393152, OR = 0.77, P = 1.95 x 10(-16)). We exchanged data with colleagues performing a GWAS in Japanese PD cases. Association to PD at SNCA was replicated in the Japanese GWAS, confirming this as a major risk locus across populations. We replicated the effect of a new locus detected in the Japanese cohort (PARK16, rs823128, OR = 0.66, P = 7.29 x 10(-8)) and provide supporting evidence that common variation around LRRK2 modulates risk for PD (rs1491923, OR = 1.14, P = 1.55 x 10(-5)). These data demonstrate an unequivocal role for common genetic variants in the etiology of typical PD and suggest population-specific genetic heterogeneity in this disease.

    Funded by: Intramural NIH HHS: Z01 AG000949-03; Medical Research Council: G0400000, G0701075, G0901254, G108/638; NCATS NIH HHS: UL1 TR000448; NCRR NIH HHS: UL1 RR024992; NIA NIH HHS: Z01 AG000949; NIEHS NIH HHS: Z01 ES101986; NINDS NIH HHS: R01 NS041509, R01 NS041509-09, R01 NS050425, R01 NS050425-05, R01 NS058714, R01 NS058714-03; Parkinson's UK: G-0907, G-0909, J-0804

    Nature genetics 2009;41;12;1308-12

  • Exogenous alpha-synuclein fibrils seed the formation of Lewy body-like intracellular inclusions in cultured cells.

    Luk KC, Song C, O'Brien P, Stieber A, Branch JR, Brunden KR, Trojanowski JQ and Lee VM

    Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Center for Neurodegenerative Disease Research, Institute on Aging, Philadelphia, PA 19104-4283, USA.

    Cytoplasmic inclusions containing alpha-synuclein (alpha-Syn) fibrils, referred to as Lewy bodies (LBs), are the signature neuropathological hallmarks of Parkinson's disease (PD). Although alpha-Syn fibrils can be generated from recombinant alpha-Syn protein in vitro, the production of fibrillar alpha-Syn inclusions similar to authentic LBs in cultured cells has not been achieved. We show here that intracellular alpha-Syn aggregation can be triggered by the introduction of exogenously produced recombinant alpha-Syn fibrils into cultured cells engineered to overexpress alpha-Syn. Unlike unassembled alpha-Syn, these alpha-Syn fibrils "seeded" recruitment of endogenous soluble alpha-Syn protein and their conversion into insoluble, hyperphosphorylated, and ubiquitinated pathological species. Thus, this cell model recapitulates key features of LBs in human PD brains. Also, these findings support the concept that intracellular alpha-Syn aggregation is normally limited by the number of active nucleation sites present in the cytoplasm and that small quantities of alpha-Syn fibrils can alter this balance by acting as seeds for aggregation.

    Funded by: NIA NIH HHS: AG09215, P01 AG009215; NINDS NIH HHS: NS053488, P50 NS053488

    Proceedings of the National Academy of Sciences of the United States of America 2009;106;47;20051-6

  • Molecular insights into the interaction between alpha-synuclein and docosahexaenoic acid.

    De Franceschi G, Frare E, Bubacco L, Mammi S, Fontana A and de Laureto PP

    CRIBI Biotechnology Centre, University of Padova, Viale G. Colombo 3, 35121 Padova, Italy.

    alpha-Synuclein (alpha-syn) is a 140-residue protein of unknown function, involved in several neurodegenerative disorders, such as Parkinson's disease. Recently, the possible interaction between alpha-syn and polyunsaturated fatty acids has attracted a strong interest. Indeed, lipids are able to trigger the multimerization of the protein in vitro and in cultured cells. Docosahexaenoic acid (DHA) is one of the main fatty acids (FAs) in cerebral gray matter and is dynamically released following phospholipid hydrolysis. Moreover, it has been found in high levels in brain areas containing alpha-syn inclusions in patients affected by Parkinson's disease. Debated and unsolved questions regard the nature of the molecular interaction between alpha-syn and DHA and the effect exerted by the protein on the aggregated state of the FA. Here, we show that alpha-syn is able to strongly interact with DHA and that a mutual effect on the structure of the protein and on the physical state of the lipid derives from this interaction. alpha-Syn acquires an alpha-helical conformation in a simple two-state transition. The binding of the protein to the FA leads to a reduction of the size of the spontaneously formed aggregated species of DHA as well as of the critical aggregate concentration of the lipid. Specifically, biophysical methods and electron microscopy observations indicated that the FA forms oil droplets in the presence of alpha-syn. Limited proteolysis experiments showed that, when the protein is bound to the FA oil droplets, it is initially cleaved in the 89-102 region, suggesting that this chain segment is sufficiently flexible or unfolded to be protease-sensitive. Subsequent proteolytic events produce fragments corresponding to the first 70-80 residues that remain structured and show high affinity for the lipid. The fact that a region of the polypeptide chain remains accessible to proteases, when interacting with the lipid, suggests that this region could be involved in other interactions, justifying the ambivalent propensity of alpha-syn towards folding or aggregation in the presence of FAs.

    Journal of molecular biology 2009;394;1;94-107

  • The molecular chaperone Hsp90 modulates intermediate steps of amyloid assembly of the Parkinson-related protein alpha-synuclein.

    Falsone SF, Kungl AJ, Rek A, Cappai R and Zangger K

    Institute of Chemistry, University of Graz, Heinrichstrasse 28, A-8010 Graz, Austria. fabio.falsone@uni-graz.at

    Alpha-synuclein is an intrinsically unstructured protein that binds to membranes, forms fibrils, and is involved in neurodegeneration. We used a reconstituted in vitro system to show that the molecular chaperone Hsp90 influenced alpha-synuclein vesicle binding and amyloid fibril formation, two processes that are tightly coupled to alpha-synuclein folding. Binding of Hsp90 to monomeric alpha-synuclein occurred in the low micromolar range, involving regions of alpha-synuclein that are critical for vesicle binding and amyloidogenesis. As a consequence, both processes were affected. In the absence of ATP, the accumulation of non-amyloid alpha-synuclein oligomers prevailed over fibril formation, whereas ATP favored fibril growth. This suggests that Hsp90 modulates the assembly of alpha-synuclein in an ATP-dependent manner. We propose that Hsp90 affects these folding processes by restricting conformational fluctuations of alpha-synuclein.

    The Journal of biological chemistry 2009;284;45;31190-9

  • A Swedish family with de novo alpha-synuclein A53T mutation: evidence for early cortical dysfunction.

    Puschmann A, Ross OA, Vilariño-Güell C, Lincoln SJ, Kachergus JM, Cobb SA, Lindquist SG, Nielsen JE, Wszolek ZK, Farrer M, Widner H, van Westen D, Hägerström D, Markopoulou K, Chase BA, Nilsson K, Reimer J and Nilsson C

    Department of Neurology, Lund University Hospital, Sweden; Department of Clinical Science, Section of Geriatric Psychiatry, Lund University, Sweden. andreas.puschmann@med.lu.se

    A de novo alpha-synuclein A53T (p.Ala53 Th; c.209G > A) mutation has been identified in a Swedish family with autosomal dominant Parkinson's disease (PD). Two affected individuals had early-onset (before 31 and 40 years), severe levodopa-responsive PD with prominent dysphasia, dysarthria, and cognitive decline. Longitudinal clinical follow-up, EEG, SPECT and CSF biomarker examinations suggested an underlying encephalopathy with cortical involvement. The mutated allele (c.209A) was present within a haplotype different from that shared among mutation carriers in the Italian (Contursi) and the Greek-American Family H kindreds. One unaffected family member carried the mutation haplotype without the c.209A mutation, strongly suggesting its de novo occurrence within this family. Furthermore, a novel mutation c.488G > A (p.Arg163His; R163H) in the presenilin-2 (PSEN2) gene was detected, but was not associated with disease state.

    Funded by: NIA NIH HHS: P01 AG017216, P01 AG017216-100005, P01AG017216, R01 AG015866-09, R01AG015866; NINDS NIH HHS: P50 NS40256, R15 NS043162, R15 NS043162-01A2

    Parkinsonism & related disorders 2009;15;9;627-32

  • Clearance and phosphorylation of alpha-synuclein are inhibited in methionine sulfoxide reductase a null yeast cells.

    Oien DB, Shinogle HE, Moore DS and Moskovitz J

    Department of Pharmacology and Toxicology, School of Pharmacy, University of Kansas, Lawrence, KS 66045, USA.

    Aggregated alpha-synuclein and the point mutations Ala30Pro and Ala53Thr of alpha-synuclein are associated with Parkinson's disease. The physiological roles of alpha-synuclein and methionine oxidation of the alpha-synuclein protein structure and function are not fully understood. Methionine sulfoxide reductase A (MsrA) reduces methionine sulfoxide residues and functions as an antioxidant. To monitor the effect of methionine oxidation to alpha-synuclein on basic cellular processes, alpha-synucleins were expressed in msrA null mutant and wild-type yeast cells. Protein degradation was inhibited in the alpha-synuclein-expressing msrA null mutant cells compared to alpha-synuclein-expressing wild-type cells. Increased inhibition of degradation and elevated accumulations of fibrillated proteins were observed in SynA30P-expressing msrA null mutant cells. Additionally, methionine oxidation inhibited alpha-synuclein phosphorylation in yeast cells and in vitro by casein kinase 2. Thus, a compromised MsrA function combined with alpha-synuclein overexpression may promote processes leading to synucleinopathies.

    Funded by: NIA NIH HHS: AG027363, R03 AG027363

    Journal of molecular neuroscience : MN 2009;39;3;323-32

  • Tyrosine and serine phosphorylation of alpha-synuclein have opposing effects on neurotoxicity and soluble oligomer formation.

    Chen L, Periquet M, Wang X, Negro A, McLean PJ, Hyman BT and Feany MB

    Department of Pathology, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115, USA.

    Mutations in the neuronal protein alpha-synuclein cause familial Parkinson disease. Phosphorylation of alpha-synuclein at serine 129 is prominent in Parkinson disease and influences alpha-synuclein neurotoxicity. Here we report that alpha-synuclein is also phosphorylated at tyrosine 125 in transgenic Drosophila expressing wild-type human alpha-synuclein and that this tyrosine phosphorylation protects from alpha-synuclein neurotoxicity in a Drosophila model of Parkinson disease. Western blot analysis of fly brain homogenates showed that levels of soluble oligomeric species of alpha-synuclein were increased by phosphorylation at serine 129 and decreased by tyrosine 125 phosphorylation. Tyrosine 125 phosphorylation diminished during the normal aging process in both humans and flies. Notably, cortical tissue from patients with the Parkinson disease-related synucleinopathy dementia with Lewy bodies showed less phosphorylation at tyrosine 125. Our findings suggest that alpha-synuclein neurotoxicity in Parkinson disease and related synucleinopathies may result from an imbalance between the detrimental, oligomer-promoting effect of serine 129 phosphorylation and a neuroprotective action of tyrosine 125 phosphorylation that inhibits toxic oligomer formation.

    Funded by: NINDS NIH HHS: P50-NS38372, R01-NS41536

    The Journal of clinical investigation 2009;119;11;3257-65

  • Genetic regulation of alpha-synuclein mRNA expression in various human brain tissues.

    Linnertz C, Saucier L, Ge D, Cronin KD, Burke JR, Browndyke JN, Hulette CM, Welsh-Bohmer KA and Chiba-Falek O

    Institute for Genome Sciences & Policy, Duke University Medical Center, Durham, North Carolina, United States of America.

    Genetic variability across the SNCA locus has been repeatedly associated with susceptibility to sporadic Parkinson's disease (PD). Accumulated evidence emphasizes the importance of SNCA dosage and expression levels in PD pathogenesis. However whether genetic variability in the SNCA gene modulates the risk to develop sporadic PD via regulation of SNCA expression remained elusive. We studied the effect of PD risk-associated variants at SNCA 5' and 3'regions on SNCA-mRNA levels in vivo in 228 human brain samples from three structures differentially vulnerable to PD pathology (substantia-nigra, temporal- and frontal-cortex) obtained from 144 neurologically normal cadavers. The extensively characterized PD-associated promoter polymorphism, Rep1, had an effect on SNCA-mRNA levels. Homozygous genotype of the 'protective', Rep1-259 bp allele, was associated with lower levels of SNCA-mRNA relative to individuals that carried at least one copy of the PD-risk associated alleles, amounting to an average decrease of approximately 40% and >50% in temporal-cortex and substantia-nigra, respectively. Furthermore, SNPs tagging the SNCA 3'-untranslated-region also showed effects on SNCA-mRNA levels in both the temporal-cortex and the substantia-nigra, although, in contrast to Rep1, the 'decreased-risk' alleles were correlated with increased SNCA-mRNA levels. Similar to Rep1 findings, no difference in SNCA-mRNA level was seen with different SNCA 3'SNP alleles in the frontal-cortex, indicating there is brain-region specificity of the genetic regulation of SNCA expression. We provide evidence for functional consequences of PD-associated SNCA gene variants in disease relevant brain tissues, suggesting that genetic regulation of SNCA expression plays an important role in the development of the disease.

    Funded by: NIA NIH HHS: P30 AG028377

    PloS one 2009;4;10;e7480

  • Characterization of hydrophobic residue requirements for alpha-synuclein fibrillization.

    Waxman EA, Mazzulli JR and Giasson BI

    Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA.

    alpha-Synuclein is the major component of pathological inclusions characteristic of diseases like Parkinson's disease, dementia with Lewy bodies, and multiple-system atrophy. A role for alpha-synuclein in neurodegenerative diseases is further supported by point mutations and duplication and triplication of the alpha-synuclein gene (SNCA) that are causative of these disorders. The middle hydrophobic region of the alpha-synuclein protein, also termed the "non-Abeta component of Alzheimer's disease amyloid plaque (NAC)" domain, is required for alpha-synuclein to polymerize into amyloid filaments, which are the major components of alpha-synuclein pathological inclusions. In this study, we assessed the importance of specific stretches of hydrophobic residues in driving the intrinsic ability of alpha-synuclein to polymerize. Several small deletions, even one with as few as two amino acid residues (A76 and V77), dramatically impaired the ability of alpha-synuclein to polymerize into mature amyloidogenic fibrils, and instead, it preferentially formed oligomers. However, this inhibition of filament assembly was clearly dependent on the spatial context, since similar and larger hydrophobic deletions in other parts of the NAC domain reduced only the rate of fibril formation, without abrogating filament assembly. Further, mutation of residue E83 to an A rescued the ability of mutant Delta76-77 alpha-synuclein to polymerize. These findings support the notion that while both the location and hydrophobicity of protein segments are important elements that affect the propensity to form amyloid fibrils, the intrinsic ability of a polypeptide to fold structurally into amyloid is also critical.

    Funded by: NIA NIH HHS: AG09215, P01 AG009215, P01 AG009215-190009, P01 AG009215-200009, T32 AG000255-11A1, T32 AG000255-12, T32AG00255; NINDS NIH HHS: NS053488, P50 NS053488, P50 NS053488-020004, P50 NS053488-030004

    Biochemistry 2009;48;40;9427-36

  • Alpha-synuclein knockdown attenuates MPP+ induced mitochondrial dysfunction of SH-SY5Y cells.

    Wu F, Poon WS, Lu G, Wang A, Meng H, Feng L, Li Z and Liu S

    Department of Anatomy, Shandong University School of Medicine, 44 Wen-hua Xi Road, Jinan, Shandong Province 250012, PR China.

    Alpha-synuclein is one of the main constituents of Lewy bodies and plays an important role in the pathology of Parkinson's disease. Mutation or overexpression of alpha-synuclein causes Parkinson's disease, and downregulation of alpha-synuclein resists MPP(+)-induced cell death, but the mechanism remains elusive. In this study, we attempted to explore the effect of alpha-synuclein knockdown on mitochondrial function in MPP(+)-treated SH-SY5Y cells. We reconstructed the short hairpin RNA expression vector, pGenesil-2, specially targeting alpha-synuclein mRNA, and it was stably transfected into SH-SY5Y cells. Cell viability, nuclear morphology, and mitochondrial membrane potential were then detected, and the expression of alpha-synuclein, cytochrome c, Bcl-2 and Bax were analyzed by Western blotting. The results showed that after exposure to 500 microM MPP(+) for 24 h, about 41.0+/-1.5% control cells showed low mitochondrial membrane potential. However, the percentage was 13.6+/-1.2% in MPP(+) treated alpha-synuclein knockdown cells. MPP(+) induced cytochrome c release significantly, which was about 3.1-fold compared with that of control. However, in alpha-synuclein knockdown cells, the release of cytochrome c was blocked, which was about 1.4-fold compared with that of control. The Bcl-2/Bax ratio of SH-SY5Y cells reduced to 35.5+/-3.8% after MPP(+) treatment, and this ratio was 85.2+/-3.0% in MPP(+) treated alpha-synuclein knockdown cells. These data suggest that knockdown of alpha- synuclein might be an effective means in rescuing MPP(+)-induced mitochondrial dysfunction of SH-SY5Y cells.

    Brain research 2009;1292;173-9

  • Transglutaminase-mediated intramolecular cross-linking of membrane-bound alpha-synuclein promotes amyloid formation in Lewy bodies.

    Nemes Z, Petrovski G, Aerts M, Sergeant K, Devreese B and Fésüs L

    Department of Psychiatry, Signaling and Apoptosis Research Group, Hungarian Academy of Sciences, Research Center for Molecular Medicine, University of Debrecen, Debrecen H-4012, Hungary. znemes@dote.hu

    The alpha-synuclein immunopositive and chaotrope-insoluble material from human brains with Lewy body pathology was analyzed by mass spectrometry. From the proteinase K-cleavable peripheral fraction of Lewy bodies, which was densely cross-linked by gamma-glutamyl-epsilon-lysine bonds between HspB1 and ubiquitin in a pattern similar to neurofibrillary tangles (Nemes, Z., Devreese, B., Steinert, P. M., Van Beeumen, J., and Fésüs, L. (2004) FASEB J. 18, 1135-1137), 53 proteins were identified. In the core of Lewy bodies only alpha-synuclein was found, and it contained a low amount of intramolecular cross-links between Gln-99 and Lys-58. In vitro cross-linking of alpha-synuclein by transglutaminases 1-3 and 5 produced a heterogeneous population of variably cross-linked alpha-synucleins in solution, which inhibited the aggregation of the protein into amyloid. However, in the presence of phosphatidylserine-rich membranes and micromolar calcium concentrations, the cross-linking by transglutaminases 1, 2, and 5 showed specificity toward the utilization of Gln-99 and Lys-58. As shown by thioflavin T fluorescence monitoring, the formation of this cross-link accelerated the aggregation of native alpha-synuclein. Chemical cross-linking of residues 58-99 triggered amyloid formation, whereas such bonding of residues 99 to 10 was inhibitory. Our findings reveal the pivotal role of membrane attachment and transglutaminase-mediated intermolecular cross-linking for the propagative misfolding and aggregation of alpha-synuclein.

    The Journal of biological chemistry 2009;284;40;27252-64

  • The effect of amino acid substitution in the imperfect repeat sequences of alpha-synuclein on fibrillation.

    Harada R, Kobayashi N, Kim J, Nakamura C, Han SW, Ikebukuro K and Sode K

    Department of Biotechnology, Graduate School of Engineering, Tokyo University of Agriculture and Technology, Japan.

    Human alpha-synuclein is the causative protein of several neurodegenerative diseases, such as Parkinson's disease (PD) and dementia with Lewy Bodies (DLB). The N-terminal half of alpha-synuclein contains seven imperfect repeat sequences. One of the PD/DLB-causing point mutations, E46K, has been reported in the imperfect repeat sequences of alpha-synuclein, and is prone to form amyloid fibrils. The presence of seven imperfect repeats in alpha-synuclein raises the question of whether or not mutations corresponding to E46K in the other imperfect KTKE(Q)GV repeats have similar effects on aggregation and fibrillation, as well as their propensities to form alpha-helices. To investigate the effect of E(Q)/K mutations in each imperfect repeat sequence, we substituted the amino acid corresponding to E46K in each of the seven repeated sequences with a Lys residue. The mutations in the imperfect KTKE(Q)GV repeat sequences of the N-terminal region were prone to decrease the lag time of fibril formation. In addition, AFM imaging suggested that the Q24K mutant formed twisted fibrils, while the other mutants formed spherical aggregates and short fibrils. These observations indicate that the effect of the mutations on the kinetics of fibril formation and morphology of fibrils varies according to their location.

    Biochimica et biophysica acta 2009;1792;10;998-1003

  • Alpha-synuclein overexpression and aggregation exacerbates impairment of mitochondrial functions by augmenting oxidative stress in human neuroblastoma cells.

    Parihar MS, Parihar A, Fujita M, Hashimoto M and Ghafourifar P

    School of Studies in Biotechnology & Zoology, Vikram University, Ujjain, MP, India.

    Overexpression of alpha-synuclein and oxidative stress has been implicated in the neuronal cell death in Parkinson's disease. Alpha-synuclein associates with mitochondria and excessive accumulation of alpha-synuclein causes impairment of mitochondrial functions. However, the mechanism of mitochondrial impairment caused by alpha-synuclein is not fully understood. We recently reported that alpha-synuclein associates with mitochondria and that overexpression of alpha-synuclein causes nitration of mitochondrial proteins and release of cytochrome c from the mitochondria [Parihar M.S., Parihar A., Fujita M., Hashimoto M., Ghafourifar P. Mitochondrial association of alpha-synuclein causes oxidative stress. Cell Mol Life Sci. 2008a;65:1272-1284]. The present study shows that overexpression of alpha-synuclein A53T or A30P mutants or wild-type in human neuroblastoma cells augmented aggregation of alpha-synuclein. Immunoblotting and immuno-gold electron transmission microscopy show localization of alpha-synuclein aggregates within the mitochondria of overexpressing cells. Overexpressing cells show increased mitochondrial reactive oxygen species, increased protein tyrosine nitration, decreased mitochondrial transmembrane potential, and hampered cellular respiration. These findings suggest an important role for mitochondria in cellular responses to alpha-synuclein.

    Funded by: NIA NIH HHS: AG023264-02

    The international journal of biochemistry & cell biology 2009;41;10;2015-24

  • Mutant alpha-synuclein overexpression mediates early proinflammatory activity.

    Su X, Federoff HJ and Maguire-Zeiss KA

    Center for Aging and Developmental Biology, Aab Institute for Biomedical Research, University of Rochester School of Medicine and Dentistry, Rochester, NY, USA.

    Microglia provide immune surveillance for the brain through both the removal of cellular debris and protection against infection by microorganisms and "foreign" molecules. Upon activation, microglia display an altered morphology and increased expression of proinflammatory molecules. Increased numbers of activated microglia have been identified in a number of neurodegenerative diseases including Parkinson's disease (PD). What remains to be determined is whether activated microglia result from ongoing cell death or are involved in disease initiation and progression. To address this question we utilized a transgenic mouse model that expresses a mutated form of a key protein involved in Parkinson's disease, alpha-synuclein. Herein, we report an increase in activated microglia and proinflammatory molecules in 1-month-old transgenic mice well before cell death occurs in this model. Frank microglial activation is resolved by 6 months of age while a subset of proinflammatory molecules remain elevated for 12 months. Both tyrosine hydroxylase mRNA expression and alpha-synuclein protein are decreased in the striatum of older animals evidence of dystrophic neuritic projections. To determine whether mutated alpha-synuclein could directly activate microglia primary microglia-enriched cell cultures were treated with exogenous mutated alpha-synuclein. The data reveal an increase in activated microglia and proinflammatory molecules due to direct interaction with mutated alpha-synuclein. Together, these data demonstrate that mutated alpha-synuclein mediates a proinflammatory response in microglia and this activity may participate in PD pathogenesis.

    Funded by: NIEHS NIH HHS: R01 ES014470-01A1, R01 ES014470-02, R01 ES014470-03, R01ES014470

    Neurotoxicity research 2009;16;3;238-54

  • Genetic variants of the alpha-synuclein gene SNCA are associated with multiple system atrophy.

    Al-Chalabi A, Dürr A, Wood NW, Parkinson MH, Camuzat A, Hulot JS, Morrison KE, Renton A, Sussmuth SD, Landwehrmeyer BG, Ludolph A, Agid Y, Brice A, Leigh PN, Bensimon G and NNIPPS Genetic Study Group

    MRC Centre for Neurodegeneration Research, King's College London, Department of Clinical Neuroscience, Institute of Psychiatry, and NIHR Biomedical Research Centre, London, United Kingdom. ammar@iop.kcl.ac.uk

    Background: Multiple system atrophy (MSA) is a progressive neurodegenerative disorder characterized by parkinsonism, cerebellar ataxia and autonomic dysfunction. Pathogenic mechanisms remain obscure but the neuropathological hallmark is the presence of alpha-synuclein-immunoreactive glial cytoplasmic inclusions. Genetic variants of the alpha-synuclein gene, SNCA, are thus strong candidates for genetic association with MSA. One follow-up to a genome-wide association of Parkinson's disease has identified association of a SNP in SNCA with MSA.

    We evaluated 32 SNPs in the SNCA gene in a European population of 239 cases and 617 controls recruited as part of the Neuroprotection and Natural History in Parkinson Plus Syndromes (NNIPPS) study. We used 161 independently collected samples for replication. Two SNCA SNPs showed association with MSA: rs3822086 (P = 0.0044), and rs3775444 (P = 0.012), although only the first survived correction for multiple testing. In the MSA-C subgroup the association strengthened despite more than halving the number of cases: rs3822086 P = 0.0024, OR 2.153, (95% CI 1.3-3.6); rs3775444 P = 0.0017, OR 4.386 (95% CI 1.6-11.7). A 7-SNP haplotype incorporating three SNPs either side of rs3822086 strengthened the association with MSA-C further (best haplotype, P = 8.7 x 10(-4)). The association with rs3822086 was replicated in the independent samples (P = 0.035).

    We report a genetic association between MSA and alpha-synuclein which has replicated in independent samples. The strongest association is with the cerebellar subtype of MSA.

    ClinicalTrials.gov NCT00211224.

    Funded by: Medical Research Council: G0400017; Parkinson's UK: J-0804

    PloS one 2009;4;9;e7114

  • A single nucleotide polymorphism in the 3'UTR of the SNCA gene encoding alpha-synuclein is a new potential susceptibility locus for Parkinson disease.

    Sotiriou S, Gibney G, Baxevanis AD and Nussbaum RL

    Department of Medicine, University of California San Francisco, San Francisco, CA 94143, USA.

    In Parkinson disease, the second most common neurodegenerative disorder in humans, increased alpha-synuclein (SNCA) levels are pathogenic, as evidenced by gene copy number mutations and increased alpha-synuclein levels detected in some familial and sporadic PD cases, respectively. Gene expression can be regulated at the post-transcriptional level by elements in the 3' untranslated region (3'UTR) of mRNAs. The goal of this study was to determine whether the 3'UTR of human SNCA can affect gene expression. Comparative sequence analysis revealed very high conservation across the entire 3'UTR of human SNCA over millions of years, suggesting the presence of multiple functionally important domains. EST and RT-PCR analyses showed that four different polyadenylation events occur in the 3'UTR of human SNCA. Finally, using luciferase assays, we examined the effect of the minor allele of five naturally occurring single nucleotide polymorphisms (SNPs) in the 3'UTR of SNCA on gene expression. The minor allele of SNP rs17016074 increased luciferase expression by 32% in a transient transfection assay in SHSY5Y neuroblastoma cells. Understanding the role of the 3'UTR of human SNCA and identifying functionally important naturally occurring SNPs using reporter assays can complement disease association studies in humans, uncovering potential susceptibility or protective polymorphisms in Parkinson disease. Our findings demonstrate that the 3'UTR of human SNCA, as a whole, and rs17016074, in particular, are loci of potential clinical importance for Parkinson disease.

    Funded by: NIA NIH HHS: R21 AG033941-01

    Neuroscience letters 2009;461;2;196-201

  • Controlling aggregation propensity in A53T mutant of alpha-synuclein causing Parkinson's disease.

    Kumar S, Sarkar A and Sundar D

    Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology Delhi, Hauz Khas, New Delhi 110016, India.

    Understanding alpha-synuclein in terms of fibrillization, aggregation, solubility and stability is fundamental in Parkinson's disease (PD). The three familial mutations, namely, A30P, E46K and A53T cause PD because the hydrophobic regions in alpha-synuclein acquire beta-sheet configuration, and have a propensity to fibrillize and form amyloids that cause cytotoxicity and neurodegeneration. On simulating the native form and mutants (A30P, E46K and A53T) of alpha-synuclein in water solvent, clear deviations are observed in comparison to the all-helical 1XQ8 PDB structure. We have identified two crucial residues, (40)Val and (74)Val, which play key roles in beta-sheet aggregation in the hydrophobic regions 36-41 and 68-78, respectively, leading to fibrillization and amyloidosis in familial (A53T) PD. We have also identified V40D_V74D, a double mutant of A53T (the most amyloidogenic mutant). The simultaneous introduction of these two mutations in A53T nearly ends its aggregation propensity, increases its solubility and positively enhances its thermodynamic stability.

    Biochemical and biophysical research communications 2009;387;2;305-9

  • 19F NMR studies of alpha-synuclein conformation and fibrillation.

    Li C, Lutz EA, Slade KM, Ruf RA, Wang GF and Pielak GJ

    Department of Chemistry, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA.

    Fibrils of the intrinsically disordered protein alpha-synuclein are hallmarks of Parkinson's disease. The fluorescent dye thioflavin T is often used to characterize fibrillation, but this assay may not provide quantitative information about structure and mechanism. To gain such information, we incorporated the 19F-labeled amino acid, 3-fluorotyrosine, into recombinant human alpha-synuclein at its endogenous tyrosine residues. Tyrosine 39 is in the positively charged N-terminal region of this 140-residue protein. The other three tyrosines, 125, 133, and 136, are near the C-terminus. 19F nuclear magnetic resonance spectroscopy was used to study several properties of labeled alpha-synuclein, including its conformation, conformational changes induced by urea, spermine, and sodium dodecyl sulfate (SDS), its interaction with SDS micelles, and the kinetics of fibril formation. The results show that the tyrosines are in disordered regions but that there is some structure near position 39 that is disrupted by urea. SDS binding alters the conformation near position 39, but the C-terminal tyrosines are disordered under all conditions. The NMR data also indicate that SDS-micelle-bound alpha-synuclein and the free protein exchange on the 10 ms time scale. Studies of fibrillation show the utility of 19F-labeled NMR. The data indicate that fibrillation is not accompanied by the formation of large quantities of low molecular weight intermediates. Although dye binding and 19F NMR data show that 1 mM SDS and 1 mM spermine accelerate aggregation compared to buffer alone, only the NMR data indicate that the species formed in SDS are smaller than those formed in buffer or buffer plus spermine. We conclude that 19F NMR spectroscopy is useful for obtaining residue-level, quantitative information about the structure, binding, and aggregation of alpha-synuclein.

    Funded by: NIH HHS: DP1 OD000783, DP1 OD000783-01, DP1 OD000783-02, DP1 OD000783-03, DP1 OD000783-04, DP1OD783

    Biochemistry 2009;48;36;8578-84

  • Expanding the clinical phenotype of SNCA duplication carriers.

    Nishioka K, Ross OA, Ishii K, Kachergus JM, Ishiwata K, Kitagawa M, Kono S, Obi T, Mizoguchi K, Inoue Y, Imai H, Takanashi M, Mizuno Y, Farrer MJ and Hattori N

    Department of Neurology, Juntendo University School of Medicine, Bunkyo-ku, Tokyo, Japan.

    SNCA duplication is a recognized cause of familial Parkinson's disease (PD). We aimed to explore the genetic and clinical variability in the disease manifestation. Molecular characterization was performed using real-time PCR, SNP arrays, and haplotype analysis. We further studied those patients who were found to harbor SNCA duplication with olfactory function tests, polysomnography, and PET. We identified four new families and one sporadic patient with SNCA duplication. Eleven symptomatic patients from these four families presented with parkinsonism, of which three subsequently developed dementia. The lifetime estimate of overall penetrance was 43.8%. FDG-PET study of symptomatic patients showed hypometabolism in the occipital lobe, whereas asymptomatic carriers of SNCA duplication demonstrated normal glucose metabolism. Symptomatic patients showed abnormal olfactory function and polysomnography and asymptomatic carriers showed normal results. The clinical features of SNCA duplication include parkinsonism with or without dementia. Asymptomatic carriers displayed normal test results with the eldest individual aged 79 years; thus, even a carrier of SNCA duplication may escape the development of PD. This difference in age-associated penetrance may be due to the genetic background or environmental exposures. Further studies of SNCA duplication carriers will help identify disease-modifiers and may open novel avenues for future treatment.

    Movement disorders : official journal of the Movement Disorder Society 2009;24;12;1811-9

  • Lrrk2 phosphorylates alpha synuclein at serine 129: Parkinson disease implications.

    Qing H, Wong W, McGeer EG and McGeer PL

    Kinsmen Laboratory of Neurological Research, University of British Columbia, 2255 Wesbrook Mall, Vancouver, BC, Canada.

    Mutations in the alpha synuclein gene (SNCA) are the most potent cause of autosomal dominant Parkinson disease (PD) while mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most common cause. We hypothesized that a direct interaction may exist between their protein products. Here we show that full-length Lrrk2 or fragments containing its kinase domain have a significant capacity to phosphorylate recombinant alpha synuclein (Asyn) at serine 129. Such phosphorylated Asyn is the major component of pathological deposits in PD. We further show that the G2019S mutation in Lrrk2, which is the most common genetic determinant of PD, has a significantly greater capacity than wild-type Lrrk2 to phosphorylate Asyn. This suggests that the G2019S mutant protein may cause PD by generating pathological levels of phosphorylated Asyn. Controlling Lrrk2 Asyn phosphokinase activity may be an approach to disease modifying therapy for PD and other synucleinopathies.

    Biochemical and biophysical research communications 2009;387;1;149-52

  • Expansion of the Parkinson disease-associated SNCA-Rep1 allele upregulates human alpha-synuclein in transgenic mouse brain.

    Cronin KD, Ge D, Manninger P, Linnertz C, Rossoshek A, Orrison BM, Bernard DJ, El-Agnaf OM, Schlossmacher MG, Nussbaum RL and Chiba-Falek O

    Center for Human Genome Variation, Institute for Genome Sciences and Policy, Duke University, Durham, NC 27708, USA.

    Alpha-synuclein (SNCA) gene has been implicated in the development of rare forms of familial Parkinson disease (PD). Recently, it was shown that an increase in SNCA copy numbers leads to elevated levels of wild-type SNCA-mRNA and protein and is sufficient to cause early-onset, familial PD. A critical question concerning the molecular pathogenesis of PD is what contributory role, if any, is played by the SNCA gene in sporadic PD. The expansion of SNCA-Rep1, an upstream, polymorphic microsatellite of the SNCA gene, is associated with elevated risk for sporadic PD. However, whether SNCA-Rep1 is the causal variant and the underlying mechanism with which its effect is mediated by remained elusive. We report here the effects of three distinct SNCA-Rep1 variants in the brains of 72 mice transgenic for the entire human SNCA locus. Human SNCA-mRNA and protein levels were increased 1.7- and 1.25-fold, respectively, in homozygotes for the expanded, PD risk-conferring allele compared with homozygotes for the shorter, protective allele. When adjusting for the total SNCA-protein concentration (endogenous mouse and transgenic human) expressed in each brain, the expanded risk allele contributed 2.6-fold more to the SNCA steady-state than the shorter allele. Furthermore, targeted deletion of Rep1 resulted in the lowest human SNCA-mRNA and protein concentrations in murine brain. In contrast, the Rep1 effect was not observed in blood lysates from the same mice. These results demonstrate that Rep1 regulates human SNCA expression by enhancing its transcription in the adult nervous system and suggest that homozygosity for the expanded Rep1 allele may mimic locus multiplication, thereby elevating PD risk.

    Funded by: NINDS NIH HHS: 5P50-NS038375

    Human molecular genetics 2009;18;17;3274-85

  • Absence of alpha-synuclein pathology in postencephalitic parkinsonism.

    Jellinger KA

    Institute of Clinical Neurobiology, 1070 Vienna, Austria. kurt.jellinger@univie.ac.at

    Postencephalitic parkinsonism (PEP), a chronic complication of encephalitis lethargica, is a tauopathy characterized by multisystem neuronal loss and gliosis with widespread neurofibrillary lesions composed of both 3- and 4-repeat (3R and 4R) tau isoforms. Previous immunohistochemical studies in a small number of PEP cases demonstrated absence of Lewy bodies as well as the lack of other alpha-synuclein pathology, classifying PEP as a "pure" tauopathy. Neuropathologic examination of 10 brains with clinico-pathologically verified PEP confirmed widespread neurodegeneration in subcortical and brainstem areas associated with multifocal neurofibrillary pathology comprising both 3R and 4R tau. Very rare beta-amyloid deposits were observed in two elderly patients, while Lewy bodies and neurites or any other alpha-synuclein deposits were completely absent. The causes and molecular background of total absence of alpha-synuclein pathology in PEP, in contrast to most other tauopathies, remain as unknown as the pathogenesis of PEP.

    Acta neuropathologica 2009;118;3;371-9

  • Alpha-Synuclein contributes to GSK-3beta-catalyzed Tau phosphorylation in Parkinson's disease models.

    Duka T, Duka V, Joyce JN and Sidhu A

    Department of Biochemistry, Molecular and Cellular Biology, Georgetown University, Washington, District of Columbia, USA.

    We have shown in the parkinsonism-inducing neurotoxin MPP(+)/MPTP model that alpha-Synuclein (alpha-Syn), a presynaptic protein causal in Parkinson's disease (PD), contributes to hyperphosphorylation of Tau (p-Tau), a protein normally linked to tauopathies, such as Alzheimer's disease (AD). Here, we investigated the kinase involved and show that the Tau-specific kinase, glycogen synthase kinase 3beta (GSK-3beta), is robustly activated in various MPP(+)/MPTP models of Parkinsonism (SH-SY5Y cotransfected cells, mesencephalic neurons, transgenic mice overexpressing alpha-Syn, and postmortem striatum of PD patients). The activation of GSK-3beta was absolutely dependent on the presence of alpha-Syn, as indexed by the absence of p-GSK-3beta in cells lacking alpha-Syn and in alpha-Syn KO mice. MPP(+) treatment induced translocation and accumulation of p-GSK-3beta in nuclei of SH-SY5Y cells and mesencephalic neurons. Through coimmunoprecipitation (co-IP), we found that alpha-Syn, pSer396/404-Tau, and p-GSK-3beta exist as a heterotrimeric complex in SH-SY5Y cells. GSK-3beta inhibitors (lithium and TDZD-8) protected against MPP(+)-induced events in SH-SY5Y cells, preventing cell death and p-GSK-3beta formation, by reversing increases in alpha-Syn accumulation and p-Tau formation. These data unveil a previously unappreciated role of alpha-Syn in the induction of p-GSK-3beta, and demonstrate the importance of this kinase in the genesis and maintenance of neurodegenerative changes associated with PD.

    Funded by: NIA NIH HHS: R01 AG028108-01A2, R01AG028108; NINDS NIH HHS: R01 NS045326-01A1, R01NS45326

    FASEB journal : official publication of the Federation of American Societies for Experimental Biology 2009;23;9;2820-30

  • Functional dissection of the alpha-synuclein promoter: transcriptional regulation by ZSCAN21 and ZNF219.

    Clough RL, Dermentzaki G and Stefanis L

    Division of Basic Neuroscience, Biomedical Research Foundation of the Academy of Athens, Athens 11527, Greece. lclough@bioacademy.gr

    Alpha-synuclein (SNCA) is an abundant neuronal protein involved in synaptic neurotransmission. SNCA expression levels have been strongly implicated in Parkinson's disease pathogenesis. We have previously demonstrated that in the PC12 cell line elements in intron 1 may mediate SNCA transcriptional regulation in response to neurotrophins. We have now identified transcription factor (TF) binding sites in intron 1 and the 5'-promoter of SNCA. A binding site for the TF zinc finger and SCAN domain containing (ZSCAN)21 in the 5'-region of intron 1 is required for intron 1 transcriptional activity. Small interfering RNA against ZSCAN21 inhibits activation in the luciferase assay and diminishes SNCA protein levels in naïve and neurotrophin-treated PC12 cells and in primary cultured cortical neurons, demonstrating that ZSCAN21 is a novel transcriptional regulator of SNCA in neuronal cells. The 5'-promoter of SNCA has a complex architecture, including multiple binding sites for the TF zinc finger protein (ZNF)219, which functions as both an activator and a repressor. Targeting ZSCAN21 or other TFs controlling SNCA transcriptional activity may provide novel therapeutic avenues not only for Parkinson's disease but also for other synucleopathies.

    Journal of neurochemistry 2009;110;5;1479-90

  • Striatal transplantation for multiple system atrophy--are grafts affected by alpha-synucleinopathy?

    Stefanova N, Hainzer M, Stemberger S, Couillard-Després S, Aigner L, Poewe W and Wenning GK

    Department of Clinical Neurobiology, Innsbruck Medical University, Innsbruck, Austria. nadia.stefanova@i-med.ac.at

    Multiple system atrophy (MSA), a fatal neurodegenerative disorder, is the second most common cause of parkinsonism and frequently associated with autonomic failure. Previous work from our laboratory has shown that striatal grafts survive and exert functional effects in toxin-induced rodent models of MSA-P, the parkinson variant characterized by levodopa resistance due to loss of striatal medium-sized spiny neurons. It is unknown whether oligodendroglial alpha-synuclein signature lesions affect graft survival in MSA. Recent reports on neurotransplantation in Parkinson's disease patients suggest a possible host-to-graft disease propagation of alpha-synuclein pathology which may be relevant to transplantation in MSA as well. We here demonstrate that embryonic E14 striatal allografts show reduced p-zone volume and dopaminergic graft re-innervation accompanied by increased gliosis in a transgenic MSA mouse model featuring alpha-synuclein oligodendrogliopathy. Oligodendrocytes expressing host-specific alpha-synuclein migrate into the graft tissue after 3 months of survival. Our data suggest that the presence of MSA-like alpha-synuclein oligodendrogliopathy and related to it pro-inflammatory microenvironment may compromise the connectivity and neurorestorative outcome of striatal grafts.

    Funded by: Austrian Science Fund FWF: P 17905-B05, P 19989-B05, W 1206-B18

    Experimental neurology 2009;219;1;368-71

  • Structural reorganization of alpha-synuclein at low pH observed by NMR and REMD simulations.

    Wu KP, Weinstock DS, Narayanan C, Levy RM and Baum J

    Department of Chemistry and Chemical Biology, Rutgers University, Piscataway, NJ 08854, USA.

    alpha-Synuclein is an intrinsically disordered protein that appears in aggregated forms in the brains of patients with Parkinson's disease. The conversion from monomer to aggregate is complex, and aggregation rates are sensitive to changes in amino acid sequence and environmental conditions. It has previously been observed that alpha-synuclein aggregates faster at low pH than at neutral pH. Here, we combine NMR spectroscopy and molecular simulations to characterize alpha-synuclein conformational ensembles at both neutral and low pH in order to understand how the altered charge distribution at low pH changes the structural properties of these ensembles and leads to an increase in aggregation rate. The N-terminus, which has a small positive charge at neutral pH due to a balance of positively and negatively charged amino acid residues, is very positively charged at low pH. Conversely, the acidic C-terminus is highly negatively charged at neutral pH and becomes essentially neutral and hydrophobic at low pH. Our NMR experiments and replica exchange molecular dynamics simulations indicate that there is a significant structural reorganization within the low-pH ensemble relative to that at neutral pH in terms of long-range contacts, hydrodynamic radius, and the amount of heterogeneity within the conformational ensembles. At neutral pH, there is a very heterogeneous ensemble with transient contacts between the N-terminus and the non-amyloid beta component (NAC); however, at low pH, there is a more homogeneous ensemble that exhibits strong contacts between the NAC and the C-terminus. At both pH values, transient contacts between the N- and C-termini are observed, the NAC region shows similar exposure to solvent, and the entire protein shows similar propensities to secondary structure. Based on the comparison of the neutral- and low-pH conformational ensembles, we propose that exposure of the NAC region to solvent and the secondary-structure propensity are not factors that account for differences in propensity to aggregate in this context. Instead, the comparison of the neutral- and low-pH ensembles suggests that the change in long-range interactions between the low- and neutral-pH ensembles, the compaction of the C-terminal region at low pH, and the uneven distribution of charges across the sequence are key to faster aggregation.

    Funded by: NIDDK NIH HHS: 3T90DK070135, 5R90DK071502; NIGMS NIH HHS: GM 30580, GM 45302, R01 GM030580, R01 GM030580-27, R01 GM045302-16

    Journal of molecular biology 2009;391;4;784-96

  • A cellular model to monitor proteasome dysfunction by alpha-synuclein.

    Nonaka T and Hasegawa M

    Department of Molecular Neurobiology, Tokyo Institute of Psychiatry, Tokyo Metropolitan Organization for Medical Research, 2-1-8 Kamikitazawa, Setagaya-ku, Tokyo 156-8585. nonakat@prit.go.jp

    Impairment of the ubiquitin-proteasome degradation system has recently been suggested to be related to the onset of neurodegenerative disorders such as Alzheimer's disease and Parkinson's disease. In this study, we investigated whether intracellular alpha-synuclein affects proteasome activity in SH-SY5Y cells. To monitor intracellular proteasome activity, we used a reporter consisting of a short peptide degron fused to the carboxyl-terminus of green fluorescent protein (GFP-CL1), which is known to be degraded by proteasome. The level of intact GFP-CL1 was dramatically increased by coexpression of GFP-CL1 and alpha-synuclein, as judged by confocal microscopic and immunoblot analyses. Expression of two pathogenic mutants of alpha-synuclein, A30P and A53T, and phosphomimetic S129D mutant increased the intensities of GFP more effectively than did wild-type alpha-synuclein. GFP fluorescence in cells transfected with Delta73-83 mutant or beta-synuclein, which does not assemble into filaments in vitro, was not changed as compared with that in cells expressing GFP-CL1 alone. Thus, the ability of alpha-synuclein to inhibit proteasome activity is related to its propensity to assemble into filaments. Furthermore, we observed that some compounds inhibiting alpha-synuclein filament formation in vitro prevented the alpha-synuclein-mediated proteasome dysfunction in cells transfected with both GFP-CL1 and alpha-synuclein. The cellular model expressing both GFP-CL1 and alpha-synuclein may be a useful tool to screen compounds protecting neurons from alpha-synuclein-mediated proteasome dysfunction.

    Biochemistry 2009;48;33;8014-22

  • Aggregation-defective alpha-synuclein mutants inhibit the fibrillation of Parkinson's disease-linked alpha-synuclein variants.

    Koo HJ, Choi MY and Im H

    Department of Molecular Biology, Sejong University, 98 Gunja-dong, Kwangjin-gu, Seoul 143-747, Republic of Korea.

    Alpha-synuclein comprises the fibrillar core of Lewy bodies, which is one of the histologically defining lesions of Parkinson's disease. Previously, we screened for alpha-synuclein substitution mutants that do not form fibrils. For preventative or therapeutic uses, it is essential to suppress the oligomerization/fibrillation of the wild-type and PD-linked alpha-synuclein proteins. Here we have examined the effects of fibrillation-retarded alpha-synuclein mutants on fibril formation by wild-type and PD-linked alpha-synuclein molecules. Six self-aggregation-defective alpha-synuclein mutants completely inhibit the fibrillation of both wild-type and Parkinson's disease-linked alpha-synuclein variants. These results suggest future applications for gene therapy: the transplantation of a fibrillation-blocking mutant alpha-synuclein gene into individuals who carry an early-onset PD-associated alpha-synuclein allele. Short synthetic peptides derived from these mutant sequences may also serve as a lead compound for the development of therapeutics for Parkinson's disease.

    Biochemical and biophysical research communications 2009;386;1;165-9

  • Inclusion formation and neuronal cell death through neuron-to-neuron transmission of alpha-synuclein.

    Desplats P, Lee HJ, Bae EJ, Patrick C, Rockenstein E, Crews L, Spencer B, Masliah E and Lee SJ

    Department of Neurosciences and Pathology, School of Medicine, University of California at San Diego, La Jolla, CA 92093, USA.

    Neuronal accumulation of alpha-synuclein and Lewy body formation are characteristic to many neurodegenerative diseases, including Parkinson's disease (PD). This Lewy pathology appears to spread throughout the brain as the disease progresses. Furthermore, recent studies showed the occurrence of Lewy pathology in neurons grafted into the brains of PD patients, suggesting the spread of pathology from the host tissues to the grafts. The mechanism underlying this propagation is unknown. Here, we show that alpha-synuclein is transmitted via endocytosis to neighboring neurons and neuronal precursor cells, forming Lewy-like inclusions. Moreover, alpha-synuclein was transmitted from the affected neurons to engrafted neuronal precursor cells in a transgenic model of PD-like pathology. Failure of the protein quality control systems, especially lysosomes, promoted the accumulation of transmitted alpha-synuclein and inclusion formation. Cells exposed to neuron-derived alpha-synuclein showed signs of apoptosis, such as nuclear fragmentation and caspase 3 activation, both in vitro and in vivo. These findings demonstrate the cell-to-cell transmission of alpha-synuclein aggregates and provide critical insights into the mechanism of pathological progression in PD and other proteinopathies.

    Funded by: NIA NIH HHS: AG02074, AG10435, AG18440, AG5131

    Proceedings of the National Academy of Sciences of the United States of America 2009;106;31;13010-5

  • Rescue of alpha-synuclein cytotoxicity by insulin-like growth factors.

    Kao SY

    Eaton Peabody Laboratory, Massachusetts Eye and Ear Infirmary, 243 Charles Street, Boston, MA 02114, USA. shyan-yuan_kao@meei.harvard.edu

    While mutation of alpha-synuclein is a cause of autosomal-dominant Parkinson's disease (PD), it is still elusive as to how alpha-synuclein is involved in the pathogenesis of PD. Here, we show that dopamine-dependent accumulation of alpha-synuclein in cultured cells results in apoptosis. Furthermore, activation of insulin-like growth factor 1 (IGF-1) pathway can rescue alpha-synuclein toxicity and suppress alpha-synuclein aggregation through the activation of PI3K/Akt pathways. These results suggest the therapeutic potential of IGF-1 pathway in Parkinson disease.

    Biochemical and biophysical research communications 2009;385;3;434-8

  • Binding of Cu(II) to human alpha-synucleins: comparison of wild type and the point mutations associated with the familial Parkinson's disease.

    Hong L and Simon JD

    Department of Chemistry, Duke University, Durham, NC 27708, USA.

    The Cu(II)-alpha-synuclein interaction has been invoked as an important process in the pathogenesis of Parkinson's disease. Herein, we report binding constants and stoichiometry under near-physiological conditions for the binding of Cu(II) to human alpha-synuclein. Specifically, we compare the binding of Cu(II) to wild-type (WT) protein and two separate single mutation proteins that are associated with familial Parkinson's diseases: A30P and A53T. Cu(II) binds to all three alpha-synuclein proteins with a 1:1 stoichiometry. The Cu(II) binding constants, however, vary among the proteins studied. Cu(II) binding to WT and A53T at 37 degrees C is similar with a pH-dependent binding constant (K) of approximately 2.4 x 10(9) and approximately 4.8 x 10(9) M(-1) at pH 7.2 and 7.4, respectively. Cu(II) binding to A30P, however, exhibits two binding constants. The major binding site of A30P, characteristic of >90% of the bound Cu(II), has binding constants of 1.6 x 10(9) and 3.6 x 10(9) M(-1) at pH 7.2 and 7.4, respectively, slightly lower ( approximately 70%) than that characteristic of WT or A53T at the corresponding pH. The second less populated binding exhibited by A30P has a large binding constant, approximately 10(10) M(-1). Our size exclusion analysis ruled out the contribution of protofibrils to the strong Cu(II) binding. Previous studies indicated that A30P had a larger proportion of intermediate species (e.g., small oligomeric species, such as dimers and trimers) relative to WT and A53T. Thus, we propose that the high affinity site is attributed to the binding of Cu(II) to those small oligomeric species.

    The journal of physical chemistry. B 2009;113;28;9551-61

  • Contribution of endogenous G-protein-coupled receptor kinases to Ser129 phosphorylation of alpha-synuclein in HEK293 cells.

    Sakamoto M, Arawaka S, Hara S, Sato H, Cui C, Machiya Y, Koyama S, Wada M, Kawanami T, Kurita K and Kato T

    Department of Neurology, Hematology, Metabolism, Endocrinology, and Diabetology, Faculty of Medicine, Yamagata University, 2-2-2 Iida-nishi, Yamagata 990-9585, Japan.

    The majority of alpha-synuclein (alphaS) deposited in Lewy bodies, the pathological hallmark of Parkinson's disease (PD), is phosphorylated at serine 129 (Ser129). Ser129 phosphorylation of alphaS has been demonstrated to enhance the alphaS toxicity to dopaminergic neurons in a Drosophila model of PD. Phosphorylation of alphaS at Ser129 seems to play a crucial role in the pathogenesis of PD. Here, we assessed the contribution of ubiquitously expressing members of the G-protein-coupled receptor kinase family (GRK2, GRK3, GRK5, and GRK6) to Ser129 phosphorylation of alphaS in HEK293 cells. To selectively reduce the endogenous expression of each member of the GRK family in cells, we used small interfering RNAs. Knockdown of GRK3 or GRK6 significantly decreased Ser129 phosphorylation of alphaS; however, knockdown of GRK2 or GRK5 did not decrease alphaS phosphorylation. The results indicate that endogenous GRK3 and GRK6, but not GRK2 or GRK5, contribute to Ser129 phosphorylation of alphaS in HEK293 cells.

    Biochemical and biophysical research communications 2009;384;3;378-82

  • Alpha-synuclein, alcohol use disorders, and Parkinson disease: a case-control study.

    Brighina L, Schneider NK, Lesnick TG, de Andrade M, Cunningham JM, Mrazek D, Rocca WA and Maraganore DM

    Department of Neuroscience and Biomedical Technologies, University of Milano-Bicocca, Monza, Italy.

    Collaborative pooled analyses demonstrated that allele length variability of the dinucleotide repeat sequence within the alpha-synuclein gene promoter (SNCA REP1) is associated with Parkinson disease (PD) worldwide. Other studies demonstrated that variability in the SNCA promoter is also associated with alcohol use disorders, but not consistently. Yet other studies demonstrated that alcohol use disorders are inversely associated with PD, but not consistently. The aim of this study was to clarify the patterns of association between REP1 genotype, alcohol use disorders, and PD. Cases were recruited from the Department of Neurology of the Mayo Clinic in Rochester, MN. The controls included unaffected siblings and unrelated controls. We assessed alcohol use via a structured telephone interview and screened for alcohol use disorders using the CAGE questionnaire. REP1 genotyping was performed using an ABI 3730XL platform. Odds ratios (ORs) and 95% confidence intervals (CIs) were determined using conditional logistic regression models. We recruited 893 case-control pairs. There was an increasing risk of PD with increasing SNCA REP1 allele length (OR 1.18 for each REP1 genotype score unit, 95% CI 1.02-1.35; p=0.02). There was a decreasing risk of PD with increasing CAGE score (p=0.01). The association of REP1 score with PD remained significant after adjusting for CAGE score, and the association of CAGE score with PD remained significant after adjusting for REP1 score. There were no pairwise interactions. Our findings suggest that SNCA REP1 genotype and alcohol use disorders are independently associated with PD.

    Funded by: NIEHS NIH HHS: ES10751, R01 ES010751-09; NINDS NIH HHS: NS33978

    Parkinsonism & related disorders 2009;15;6;430-4

  • Leucine-rich repeat kinase 2 expression leads to aggresome formation that is not associated with alpha-synuclein inclusions.

    Waxman EA, Covy JP, Bukh I, Li X, Dawson TM and Giasson BI

    Department of Pharmacology, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6084, USA.

    Mutations in leucine-rich repeat kinase-2 (LRRK2) are the most common known cause of Parkinson disease, but how this protein results in the pathobiology of Parkinson disease is unknown. Moreover, there is variability in pathology among cases, and alpha-synuclein (alpha-syn) neuronal inclusions are often present, but whether LRRK2 is present in these pathological inclusions is controversial. This study characterizes novel LRRK2 antibodies, some of which preferentially recognize an aggregated form of LRRK2, as observed in cell culture models. Large perinuclear aggregates containing LRRK2 were promoted by proteasome inhibition and prevented by microtubule polymerization inhibition. Furthermore, they were vimentin- and gamma-tubulin- but not lamp1-immunoreactive, suggesting that these structures fit the definition of aggresomes. Inhibition of heat shock protein 90 led to the degradation of only the soluble/cytosolic pool of LRRK2, suggesting that the aggresomes formed independent of the stability provided by the heat shock protein 90. Although these novel anti-LRRK2 antibodies identified aggregates in model cell systems, they did not immunostain pathological inclusions in human brains. Furthermore, coexpression of LRRK2 and alpha-syn did not recruit alpha-syn into aggresomes in cultured cells, even in the presence of proteasome inhibition. Thus, although LRRK2 is a model system for aggresome formation, LRRK2 is not present in alpha-syn pathological inclusions.

    Funded by: NIA NIH HHS: AG09215, P01 AG009215, P01 AG009215-180009, P01 AG009215-190009, P01 AG009215-200009, T32 AG000255-10, T32 AG000255-12, T32 AG00255; NINDS NIH HHS: NS053488, NS38377, P50 NS038377-10, P50 NS053488, P50 NS053488-020004, P50 NS053488-030004

    Journal of neuropathology and experimental neurology 2009;68;7;785-96

  • Molecular mechanisms of alpha-synuclein neurodegeneration.

    Waxman EA and Giasson BI

    Department of Pharmacology, University of Pennsylvania School of Medicine, 125 John Morgan Building, Philadelphia, PA 19104-6084, USA.

    alpha-Synuclein is an abundant highly charged protein that is normally predominantly localized around synaptic vesicles in presynaptic terminals. Although the function of this protein is still ill-defined, genetic studies have demonstrated that point mutations or genetic alteration (duplications or triplications) that increase the number of copies of the alpha-synuclein (SCNA) gene can cause Parkinson's disease or the related disorder dementia with Lewy bodies. alpha-Synuclein can aberrantly polymerize into fibrils with typical amyloid properties, and these fibrils are the major component of many types of pathological inclusions, including Lewy bodies, which are associated with neurodegenerative diseases, such as Parkinson's disease. Although there is substantial evidence supporting the toxic nature of alpha-synuclein inclusions, other modes of toxicity such as oligomers have been proposed. In this review, some of the evidence for the different mechanisms of alpha-synuclein toxicity is presented and discussed.

    Funded by: NIA NIH HHS: AG09215, P01 AG009215, P01 AG009215-18, P01 AG009215-190009, P01 AG009215-200009, T32 AG000255-10, T32 AG00255; NINDS NIH HHS: NS053488, P50 NS053488, P50 NS053488-01A20004, P50 NS053488-020004

    Biochimica et biophysica acta 2009;1792;7;616-24

  • Relative contribution of simple mutations vs. copy number variations in five Parkinson disease genes in the Belgian population.

    Nuytemans K, Meeus B, Crosiers D, Brouwers N, Goossens D, Engelborghs S, Pals P, Pickut B, Van den Broeck M, Corsmit E, Cras P, De Deyn PP, Del-Favero J, Van Broeckhoven C and Theuns J

    Neurodegenerative Brain Diseases Group, Department of Molecular Genetics, VIB, Antwerp, Belgium.

    The relative contribution of simple mutations and copy number variations (CNVs) in SNCA, PARK2, PINK1, PARK7, and LRRK2 to the genetic etiology of Parkinson disease (PD) is still unclear because most studies did not completely analyze each gene. In a large group of Belgian PD patients (N = 310) and control individuals (N = 270), we determined the mutation frequency of both simple mutations and CNVs in these five PD genes, using direct sequencing, multiplex amplicon quantification (MAQ), and real-time PCR assays. Overall, we identified 14 novel heterozygous variants, of which 11 were absent in control individuals. We observed eight PARK2 (multiple) exon multiplications in PD patients and one exon deletion in a control individual. Furthermore, we identified one SNCA whole-gene duplication. The PARK2 and LRRK2 mutation frequencies in Belgian PD patients were similar to those reported in other studies. However, at this stage the true pathogenic nature of some heterozygous mutations in recessive genes remains elusive. Furthermore, though mutations is SNCA, PINK1, and PARK7 are rare, our identification of a SNCA duplication confirmed that screening of these genes remains meaningful.

    Human mutation 2009;30;7;1054-61

  • Alpha-synuclein and familial Parkinson's disease.

    Pankratz N, Nichols WC, Elsaesser VE, Pauciulo MW, Marek DK, Halter CA, Wojcieszek J, Rudolph A, Pfeiffer RF, Foroud T and Parkinson Study Group - PROGENI Investigators

    Department of Medical and Molecular Genetics, Indiana University Medical Center, Indianapolis, Indiana 46202, USA. npankrat@iupui.edu

    Whole gene duplications and triplications of alpha-synuclein (SNCA) can cause Parkinson's disease (PD), and variation in the promoter region (Rep1) and 3' region of SNCA has been reported to increase disease susceptibility. Within our cohort, one affected individual from each of 92 multiplex PD families showing the greatest evidence of linkage to the region around SNCA was screened for dosage alterations and sequence changes; no dosage or non-synonymous sequence changes were found. In addition, 737 individuals (from 450 multiplex PD families) that met strict diagnostic criteria for PD and did not harbor a known causative mutation, as well as 359 neurologically normal controls, were genotyped for the Rep1 polymorphism and four SNPs in the 3' region of SNCA. The four SNPs were in high LD (r(2) > 0.95) and were analyzed as a haplotype. The effects of the Rep1 genotype and the 3' haplotype were evaluated using regression models employing only one individual per family. Cases had a 3% higher frequency of the Rep1 263 bp allele compared with controls (OR = 1.54; empirical P-value = 0.02). There was an inverse linear relationship between the number of 263 bp alleles and age of onset (empirical P-value = 0.0004). The 3' haplotype was also associated with disease (OR = 1.29; empirical P-value = 0.01), but not age of onset (P = 0.40). These data suggest that dosage and sequence changes are a rare cause of PD, but variation in the promoter and 3' region of SNCA convey an increased risk for PD.

    Funded by: NCRR NIH HHS: M01 RR000750, MO1 RR-00750; NIA NIH HHS: U24 AG021886; NINDS NIH HHS: R01 NS037167, R01 NS37167

    Movement disorders : official journal of the Movement Disorder Society 2009;24;8;1125-31

  • Alpha-synuclein multiplications with parkinsonism, dementia or progressive myoclonus?

    Puschmann A, Wszolek ZK, Farrer M, Gustafson L, Widner H and Nilsson C

    Department of Neurology, Lund University Hospital, Sweden. andreas.puschmann@med.lu.se

    Duplications and triplications of the alpha-synuclein (SNCA) gene have been reported in Parkinson's disease patients belonging to the Southern Swedish "Lister family". Further genealogical research has now shown that these individuals are descended from a large kindred characterized by Herman Lundborg in 1901-1913. In the expanded pedigree, a total of 25 individuals had Parkinson's disease with an autosomal dominant pattern of inheritance. Hereditary dementia, and, historically, dementia praecox have been described in other family members. Furthermore, an autosomal recessively inherited pediatric disease with nocturnal tonic-clonic fits, subsequent progressive myoclonus, startle reactions, tremor and muscle rigidity was described by Lundborg in the same pedigree. The entity was later designated Unverricht-Lundborg disease (ULD) or progressive myoclonus epilepsy type 1 (EPM1). However, Lundborg's clinical description of this disease, based on 17 patients within this kindred, differs from the modern definition of EPM1, which relies on patients with a mutation in the cystatin B (CSTB) gene. We hypothesize that the former pediatric disease, as well as the parkinsonism and dementia phenotypes, are associated with duplications, triplications and possibly higher-order multiplications of the alpha-synuclein (SNCA) gene. This hypothesis is supported by the distribution of afflicted family members within the pedigree and by recently obtained genealogical information.

    Funded by: NINDS NIH HHS: P50 NS40256

    Parkinsonism & related disorders 2009;15;5;390-2

  • Anti-aggregation and fibril-destabilizing effects of sex hormones on alpha-synuclein fibrils in vitro.

    Hirohata M, Ono K, Morinaga A, Ikeda T and Yamada M

    Department of Neurology and Neurobiology of Aging, Kanazawa University Graduate School of Medical Science, Kanazawa 920-8640, Japan.

    The alpha-synuclein aggregation in the brain is the hallmark of Lewy body diseases, including Parkinson's disease and dementia with Lewy bodies, and multiple system atrophy. Some epidemiological studies have revealed that estrogen therapy reduces the risk of Parkinson's disease in females. We examined the effects of estriol, estradiol, estrone, androstenedione, and testosterone on the formation and destabilization of alpha-synuclein fibrils at pH 7.5 and 37 degrees C in vitro, using fluorescence spectroscopy with thioflavin S and electron microscopy. These sex hormones, especially estriol, significantly exert anti-aggregation and fibril-destabilizing effects; and hence, could be valuable preventive and therapeutic agents for alpha-synucleinopathies.

    Experimental neurology 2009;217;2;434-9

  • Inducible over-expression of wild type alpha-synuclein in human neuronal cells leads to caspase-dependent non-apoptotic death.

    Vekrellis K, Xilouri M, Emmanouilidou E and Stefanis L

    Division of Basic Neurosciences, Biomedical Research Foundation of the Academy of Athens, Soranou Efesiou, Athens, Greece. vekrellis@bioacademy.gr

    Alpha-synuclein (ASYN) is central in Parkinson's disease pathogenesis. Converging pieces of evidence suggest that the levels of ASYN expression play a critical role in both familial and sporadic Parkinson's disease. To elucidate the mechanism underlying wild type (WT) ASYN-mediated neurotoxicity, we have generated a novel Tet-Off SHSY-5Y cell line, conditionally expressing WT ASYN. Induction of human WT ASYN in retinoic acid-differentiated SHSY-5Y cells leads to accumulation of soluble ASYN oligomers, in the absence of inclusions, and to gradual cellular degeneration. Morphologically, the death observed is non-apoptotic. Caspases other than caspase 3, including caspase 9, are activated and caspase inhibition diminishes death by acting at a point upstream of cytochrome c release. Application of Scyllo-inositol, an oligomer-stabilizing compound, prevents neuronal death in this model. These findings are consistent with a model in which oligomeric ASYN triggers the initial activation of the apoptotic pathway, which is however blocked downstream of the mitochondrial checkpoint, thus leading to a death combining in a unique fashion both apoptotic and non-apoptotic features. This novel inducible cell model system may prove valuable in the deciphering of WT ASYN-induced pathogenic effects and in the assessment and screening of potential therapeutic strategies.

    Funded by: NINDS NIH HHS: R21 NS055693

    Journal of neurochemistry 2009;109;5;1348-62

  • E46K Parkinson's-linked mutation enhances C-terminal-to-N-terminal contacts in alpha-synuclein.

    Rospigliosi CC, McClendon S, Schmid AW, Ramlall TF, Barré P, Lashuel HA and Eliezer D

    Department of Biochemistry and Program in Structural Biology, Weill Cornell Medical College, New York, NY 10065, USA.

    Parkinson's disease (PD) is associated with the deposition of fibrillar aggregates of the protein alpha-synuclein (alphaS) in neurons. Intramolecular contacts between the acidic C-terminal tail of alphaS and its N-terminal region have been proposed to regulate alphaS aggregation, and two originally described PD mutations, A30P and A53T, reportedly reduce such contacts. We find that the most recently discovered PD-linked alphaS mutation E46K, which also accelerates the aggregation of the protein, does not interfere with C-terminal-to-N-terminal contacts and instead enhances such contacts. Furthermore, we do not observe a substantial reduction in such contacts in the two previously characterized mutants. Our results suggest that C-terminal-to-N-terminal contacts in alphaS are not strongly protective against aggregation, and that the dominant mechanism by which PD-linked mutations facilitate alphaS aggregation may be altering the physicochemical properties of the protein such as net charge (E46K) and secondary structure propensity (A30P and A53T).

    Funded by: NIA NIH HHS: AG019391, AG025440, R01 AG019391, R01 AG019391-09, R01 AG025440, R01 AG025440-04, R37 AG019391; NIGMS NIH HHS: P41 GM066354, P41 GM66354; NINDS NIH HHS: NS058137

    Journal of molecular biology 2009;388;5;1022-32

  • Formation of dopamine-mediated alpha-synuclein-soluble oligomers requires methionine oxidation.

    Leong SL, Pham CL, Galatis D, Fodero-Tavoletti MT, Perez K, Hill AF, Masters CL, Ali FE, Barnham KJ and Cappai R

    Department of Pathology, The University of Melbourne, Parkville, VIC, Australia.

    alpha-Synuclein is the major component of the intracellular Lewy body inclusions present in Parkinson disease (PD) neurons. PD involves the loss of dopaminergic neurons in the substantia nigra and the subsequent depletion of dopamine (DA) in the striatum. DA can inhibit alpha-synuclein fibrillization in vitro and promote alpha-synuclein aggregation into soluble oligomers. We have studied the mechanism by which DA mediates alpha-synuclein aggregation into soluble oligomers. Reacting alpha-synuclein with DA increased the mass of alpha-synuclein by 64 Da. NMR showed that all four methionine residues were oxidized by DA, consistent with the addition of 64 Da. Substituting all four methionines to alanine significantly reduced the formation of DA-mediated soluble oligomers. The (125)YEMPS(129) motif in alpha-synuclein can modulate DA inhibition of alpha-synuclein fibrillization. However, alpha-synuclein ending before the (125)YEMPS(129) motif (residues 1-124) could still form soluble oligomers. The addition of exogenous synthetic YEMPS peptide inhibited the formation of soluble oligomers and resulted in the YEMPS peptide being oxidized. Therefore, the (125)YEMPS(129) acts as an antioxidant rather than interacting directly with DA. Our study defines methionine oxidation as the dominant mechanism by which DA generates soluble alpha-synuclein oligomers and highlights the potential role for oxidative stress in modulating alpha-synuclein aggregation.

    Free radical biology & medicine 2009;46;10;1328-37

  • At low concentrations, 3,4-dihydroxyphenylacetic acid (DOPAC) binds non-covalently to alpha-synuclein and prevents its fibrillation.

    Zhou W, Gallagher A, Hong DP, Long C, Fink AL and Uversky VN

    Department of Chemistry and Biochemistry, University of California at Santa Cruz, Santa Cruz, CA 95064, USA.

    Several studies have shown that catecholamines can inhibit the fibrillation of alpha-synuclein (alpha-Syn), a small presynaptic protein whose aggregation is believed to be a critical step in the etiology of Parkinson's disease and several other neurodegenerative disorders. However, the mechanism of this inhibition is uncertain. We show here that substoichiometric concentrations of 3,4-dihydroxyphenylacetic acid (DOPAC), a normal product of the metabolism of dopamine, can inhibit the fibrillation of alpha-Syn, due to non-covalent binding of DOPAC to alpha-Syn monomer. Intriguingly, the presence of alpha-Syn accelerates the spontaneous oxidation of DOPAC, and the oxidized form of DOPAC (the quinone) is responsible for the fibrillation inhibition. In addition, the presence of DOPAC leads to the oxidation of the methionine residues of alpha-Syn, probably due to the H(2)O(2) production as a by-product of DOPAC oxidation. The lack of fibrillation results from the formation of stable oligomers, which are very similar to those observed transiently at early stages of the alpha-Syn fibrillation. A possible explanation for this phenomenon is that DOPAC stabilizes the normally transient oligomers and prevents them from subsequent fibril formation. The analysis of the alpha-Syn Y39W variant suggests that DOPAC binds non-covalently to the same N-terminal region of alpha-Syn as lipid vesicles, probably in the vicinity of residue 39. In contrast to the compounds with 1,2-dihydroxyphenyl groups (DOPAC and catechol), their 1,4-dihydroxyphenyl isomers (hydroquinone and homogentisic acid) are able to modify alpha-Syn covalently, probably due to the less steric hindrance in the Michael addition.

    Funded by: NIGMS NIH HHS: GM 071714-01A2, R01 GM071714-03; NINDS NIH HHS: R01 NS 39985, R01 NS039985-03; NLM NIH HHS: R01 LM 007688-01A1, R01 LM007688-01A1S1

    Journal of molecular biology 2009;388;3;597-610

  • SNCA variants are associated with increased risk for multiple system atrophy.

    Scholz SW, Houlden H, Schulte C, Sharma M, Li A, Berg D, Melchers A, Paudel R, Gibbs JR, Simon-Sanchez J, Paisan-Ruiz C, Bras J, Ding J, Chen H, Traynor BJ, Arepalli S, Zonozi RR, Revesz T, Holton J, Wood N, Lees A, Oertel W, Wüllner U, Goldwurm S, Pellecchia MT, Illig T, Riess O, Fernandez HH, Rodriguez RL, Okun MS, Poewe W, Wenning GK, Hardy JA, Singleton AB, Del Sorbo F, Schneider S, Bhatia KP and Gasser T

    Laboratory of Neurogenetics, National Institute on Aging, National Institutes of Health, 35 Convent Drive, Bethesda, MD 20892, USA. scholzs@mail.nih.gov

    To test whether the synucleinopathies Parkinson's disease and multiple system atrophy (MSA) share a common genetic etiology, we performed a candidate single nucleotide polymorphism (SNP) association study of the 384 most associated SNPs in a genome-wide association study of Parkinson's disease in 413 MSA cases and 3,974 control subjects. The 10 most significant SNPs were then replicated in additional 108 MSA cases and 537 controls. SNPs at the SNCA locus were significantly associated with risk for increased risk for the development of MSA (combined p = 5.5 x 10(-12); odds ratio 6.2) [corrected].

    Funded by: Medical Research Council: G108/638; NIA NIH HHS: Z01 AG000957-06, Z01-AG 000957-06; NIEHS NIH HHS: Z01-ES 101986; Telethon: GTB07001

    Annals of neurology 2009;65;5;610-4

  • Do polymorphisms in the familial Parkinsonism genes contribute to risk for sporadic Parkinson's disease?

    Sutherland GT, Halliday GM, Silburn PA, Mastaglia FL, Rowe DB, Boyle RS, O'Sullivan JD, Ly T, Wilton SD and Mellick GD

    Eskitis Institute for Cell and Molecular Therapies, School of Biomolecular and Physical Sciences, Griffith University, Brisbane, Queensland, Australia.

    Recent whole genome association studies provided little evidence that polymorphisms at the familial Parkinsonism loci influence the risk for Parkinson's disease (PD). However, these studies are not designed to detect the types of subtle effects that common variants may impose. Here, we use an alternative targeted candidate gene approach to examine common variation in 11 genes related to familial Parkinsonism. PD cases (n = 331) and unaffected control subjects (n = 296) were recruited from three specialist movement disorder clinics in Brisbane, Australia and the Australian Electoral Roll. Common genetic variables (76 SNPs and 1 STR) were assessed in all subjects and haplotype, genotype, and allele associations explored. Modest associations (uncorrected P < 0.05) were observed for common variants around SNCA, UCHL1, MAPT, and LRRK2 although none were of sufficient magnitude to survive strict statistical corrections for multiple comparisons. No associations were seen for PRKN, PINK1, GBA, ATP13A2, HTRA2, NR4A2, and DJ1. Our findings suggest that common genetic variables of selected PD-related loci contribute modestly to PD risk in Australians.

    Movement disorders : official journal of the Movement Disorder Society 2009;24;6;833-8

  • Detecting morphologically distinct oligomeric forms of alpha-synuclein.

    Emadi S, Kasturirangan S, Wang MS, Schulz P and Sierks MR

    Department of Chemical Engineering, Arizona State University, Tempe, Arizona 85287-6006, USA.

    Neuropathologic and genetics studies as well as transgenic animal models have provided strong evidence linking misfolding and aggregation of alpha-synuclein to the progression of Parkinson disease (PD) and other related disorders. A growing body of evidence implicates various oligomeric forms of alpha-synuclein as the toxic species responsible for neurodegeneration and neuronal cell death. Although numerous different oligomeric forms of alpha-synuclein have been identified in vitro, it is not known which forms are involved in PD or how, when, and where different forms contribute to the progression of PD. Reagents that can interact with specific aggregate forms of alpha-synuclein would be very useful not only as tools to study how different aggregate forms affect cell function, but also as potential diagnostic and therapeutic agents for PD. Here we show that a single chain antibody fragment (syn-10H scFv) isolated from a phage display antibody library binds to a larger, later stage oligomeric form of alpha-synuclein than a previously reported oligomeric specific scFv isolated in our laboratory. The scFv described here inhibits aggregation of alpha-synuclein in vitro, blocks extracellular alpha-synuclein-induced toxicity in both undifferentiated and differentiated human neuroblastoma cell lines (SH-SY5Y), and specifically recognizes naturally occurring aggregates in PD but not in healthy human brain tissue.

    The Journal of biological chemistry 2009;284;17;11048-58

  • Synphilin-1A inhibits seven in absentia homolog (SIAH) and modulates alpha-synuclein monoubiquitylation and inclusion formation.

    Szargel R, Rott R, Eyal A, Haskin J, Shani V, Balan L, Wolosker H and Engelender S

    Department of Pharmacology, The B. Rappaport Faculty of Medicine and Institute of Medical Research, Technion-Israel Institute of Technology, Haifa 31096, Israel.

    Parkinson disease (PD) is characterized by the presence of ubiquitylated inclusions and the death of dopaminergic neurons. Seven in absentia homolog (SIAH) is a ubiquitin-ligase that ubiquitylates alpha-synuclein and synphilin-1 and is present in Lewy bodies of PD patients. Understanding the mechanisms that regulate the ubiquitylation of PD-related proteins might shed light on the events involved in the formation of Lewy bodies and death of neurons. We show in this study that the recently described synphilin-1 isoform, synphilin-1A, interacts in vitro and in vivo with the ubiquitin-protein isopeptide ligase SIAH and regulates its activity toward alpha-synuclein and synphilin-1. SIAH promotes limited ubiquitylation of synphilin-1A that does not lead to its degradation by the proteasome. SIAH also increases the formation of synphilin-1A inclusions in the presence of proteasome inhibitors, supporting the participation of ubiquitylated synphilin-1A in the formation of Lewy body-like inclusions. Synphilin-1A/SIAH inclusions recruit PD-related proteins, such as alpha-synuclein, synphilin-1, Parkin, PINK1, and UCH-L1. We found that synphilin-1A robustly increases the steady-state levels of SIAH by decreasing its auto-ubiquitylation and degradation. In addition, synphilin-1A blocks the ubiquitylation and degradation of the SIAH substrates synphilin-1 and deleted in colon cancer protein. Furthermore, synphilin-1A strongly decreases the monoubiquitylation of alpha-synuclein by SIAH and the formation of alpha-synuclein inclusions, supporting a role for monoubiquitylation in alpha-synuclein inclusion formation. Our results suggest a novel function for synphilin-1A as a regulator of SIAH activity and formation of Lewy body-like inclusions.

    The Journal of biological chemistry 2009;284;17;11706-16

  • Familial Parkinsonism and early onset Parkinson's disease in a Brazilian movement disorders clinic: phenotypic characterization and frequency of SNCA, PRKN, PINK1, and LRRK2 mutations.

    Camargos ST, Dornas LO, Momeni P, Lees A, Hardy J, Singleton A and Cardoso F

    Movement Disorders Group, Neurology Service, Department of Internal Medicine, Federal University of Minas Gerais, Minas Gerais, Brazil.

    The aim of the study was to evaluate the frequency and to perform phenotypic and genotypic characterization of familial Parkinsonism and early onset Parkinson's disease (EOPD) in a Brazilian movement disorder unit. We performed a standardized clinical assessment of patients followed by sequencing of PRKN, PINK1 in EOPD cases and SNCA, LRRK2 in familial Parkinsonism individuals. During the period of study (January through December, 2006), we examined 575 consecutive patients of whom 226 (39.3%) met the diagnosis of Parkinsonism and idiopathic Parkinson's disease (IPD) was diagnosed in 202 of the latter. Of the IPD cases, 45 (22.3%) had EOPD. The age at onset in the EOPD cases (n = 45) was 34.8 +/- 5.4 years (mean +/- standard deviation). The age at onset in the familial late-onset PD patients (n = 8) was 52.3 +/- 12.2 years. In the early onset cases, we identified five known mutations in PRKN, two single heterozygous and three compound heterozygous (P153R, T240M, 255Adel, W54R, V3I); in addition, we identified one novel mutation in PINK1 (homozygous deletion of exon 7). In the familial cases (late onset), 1 patient had a novel LRRK2 variant, Q923H, but no SNCA mutations were identified. We have demonstrated that EOPD accounts for a high frequency of IPD cases in our tertiary referral center. PRKN was the most commonly mutated gene, but we also identified a novel mutation in PINK1 and a novel variant in LRRK2.

    Funded by: Intramural NIH HHS: Z01 AG000957-05; Medical Research Council: G0701075; NIA NIH HHS: Z01 AG000957; Parkinson's UK: G-0907

    Movement disorders : official journal of the Movement Disorder Society 2009;24;5;662-6

  • Regional differences in the severity of Lewy body pathology across the olfactory cortex.

    Silveira-Moriyama L, Holton JL, Kingsbury A, Ayling H, Petrie A, Sterlacci W, Poewe W, Maier H, Lees AJ and Revesz T

    Reta Lila Weston Institute for Neurological Studies, UCL Institute of Neurology, London, UK.

    We studied alpha-synuclein pathology in the rhinencephalon of ten cases of Parkinson's disease (PD) and twelve neurologically normal controls, of which seven had incidental Lewy bodies in the substantia nigra at autopsy and five had no pathological evidence of neurological disease. In all PD and incidental Lewy bodies cases, alpha-synuclein pathology was found in all five subregions of the primary olfactory cortex that were sampled, and amongst them the pathology was significantly more severe in the temporal division of the piriform cortex than in the frontal division of the piriform cortex, olfactory tubercle or anterior portions of the entorhinal cortex. The orbitofrontal cortex, which is an area of projection from the primary olfactory cortex, was affected in some cases but overall the alpha-synuclein pathology was less severe in this area than in the primary olfactory cortex. Because different areas of the rhinencephalon are likely to play different roles in olfaction and our data indicate a differential involvement by alpha-synuclein deposition of structures implicated in smell, future prospective studies investigating the pathophysiological basis of hyposmia in PD should consider to examine the areas of primary olfactory cortex separately.

    Neuroscience letters 2009;453;2;77-80

  • [Analysis of the alpha-synuclein gene dosage variation associated with autosomal dominant form of ParkinsonTs disease].

    Semenova EV, Shadrina MI, Slominskiĭ PA, Illarioshkin SN, Bagyeva GKh, Karabanov AV, Ivanova-Smolenskaia IA and Limborskaia SA

    Fifty-two patients that had ParkinsonTs disease with autosomal dominant type of inheritance were analyzed for the presence of duplications and triplications in exons 4--6 of alpha-synuclein gene using real-time PCR with Taq-Man probes. No mutations involving the examined exons dosage were revealed in alpha-synuclein gene. Thus, mutations modifying copy number of alpha-synuclein gene do not significantly affect the pathogenesis of the autosomal dominant form of ParkinsonTs disease in patients from Russia.

    Genetika 2009;45;4;573-6

  • Increased frequency of alpha-synuclein in the substantia nigra in human immunodeficiency virus infection.

    Khanlou N, Moore DJ, Chana G, Cherner M, Lazzaretto D, Dawes S, Grant I, Masliah E, Everall IP and HNRC Group

    Department of Pathology and Laboratory Medicine, Division of Neuropathology, David Geffen School of Medicine, Ronald Reagan UCLA Medical Center, Los Angeles, California, USA.

    The frequency of neurodegenerative markers among long surviving human immunodeficiency virus (HIV)-infected individuals is unknown, therefore, the present study investigated the frequency of alpha-synuclein, beta-amyloid, and HIV-associated brain pathology in the brains of older HIV-infected individuals. We examined the substantia nigra of 73 clinically well-characterized HIV-infected individuals aged 50 to 76 years from the National NeuroAIDS Tissue Consortium. We also examined the frontal and temporal cortical regions of a subset of 36 individuals. Neuritic alpha-synuclein expression was found in 16% (12/73) of the substantia nigra of the HIV+cases and none of the older control cases (0/18). beta-Amyloid deposits were prevalent and found in nearly all of the HIV+cases (35/36). Despite these increases of degenerative pathology, HIV-associated brain pathology was present in only 10% of cases. Among older HIV+adults, HIV-associated brain pathology does not appear elevated; however, the frequency of both alpha-synuclein and beta-amyloid is higher than that found in older healthy persons. The increased prevalence of alpha-synuclein and beta-amyloid in the brains of older HIV-infected individuals may predict an increased risk of developing neurodegenerative disease.

    Funded by: NIMH NIH HHS: MH 62512, MH62512, R24 MH059745, R24 MH59745, U01 MH083506

    Journal of neurovirology 2009;15;2;131-8

  • Lewy body/alpha-synucleinopathy in schizophrenia and depression: a preliminary neuropathological study.

    Jellinger KA

    Institute of Clinical Neurobiology, Kenyongasse 18, 1070, Vienna, Austria. kurt.jellinger@univie.ac.at

    The role of alpha-synuclein (alphaSyn) in schizophrenia is unknown, whereas in a recent animal model of depression, alpha- and gamma-synuclein have been related to its pathophysiology. Previous biochemical studies in Brodmann area 9 showed significant reduction of alphaSyn in both chronic schizophrenia and bipolar disorder. Here, prevalence and cerebral distribution of alphaSyn were examined in 80 autopsy cases of elderly subjects (41 chronic schizophrenia, 12 late live depression/LLD and bipolar disorder/BD, and 27 age-matched controls without neuropsychiatric disorders). Using immunohistochemistry, alphaSyn-positive lesions (Lewy bodies and neurites) were assessed semiquantitatively. Among 41 chronic schizophrenics, all except one showing low neuritic Braak stages (mean 1.46), three brains (7.3%) revealed only few alphaSyn-positive inclusions restricted to medullary nuclei. Among 12 LLD and BD patients with mean Braak stage 2.25, alphaSyn-positive pathology was seen in two cases (16.7%) with clinical LLD, but none in BD. Among 27 controls, showing mean neuritic Braak stage 2.6, seven brains (26%) with higher mean age showed alphaSyn-positive lesions, either isolated in substantia nigra and nucleus basalis of Meynert (n = 2 each), in medullary nuclei, locus ceruleus and substantia nigra (n = 2), with additional involvement of nucleus basalis (n = 1). This first preliminary study in non-demented psychiatric disorders indicates that alphaSyn/Lewy pathology in chronic schizophrenia is significantly less frequent than in clinically healthy elderly people (P < 0.01), showing 10-30% of so-called incidental Lewy body disease. Among chronic affective disorders, according to our small cohort, the incidence of Lewy-pathology in LLD appears to be comparable to a healthy elderly population, whereas its occurence in BD is to be elucidated.

    Acta neuropathologica 2009;117;4;423-7

  • Membrane-associated farnesylated UCH-L1 promotes alpha-synuclein neurotoxicity and is a therapeutic target for Parkinson's disease.

    Liu Z, Meray RK, Grammatopoulos TN, Fredenburg RA, Cookson MR, Liu Y, Logan T and Lansbury PT

    Center for Neurologic Diseases, Brigham and Women's Hospital and Department of Neurology, Harvard Medical School, 65 Landsdowne Street, Cambridge, MA 02139, USA.

    Ubiquitin C-terminal hydrolase-L1 (UCH-L1) is linked to Parkinson's disease (PD) and memory and is selectively expressed in neurons at high levels. Its expression pattern suggests a function distinct from that of its widely expressed homolog UCH-L3. We report here that, in contrast to UCH-L3, UCH-L1 exists in a membrane-associated form (UCH-L1(M)) in addition to the commonly studied soluble form. C-terminal farnesylation promotes the association of UCH-L1 with cellular membranes, including the endoplasmic reticulum. The amount of UCH-L1(M) in transfected cells is shown to correlate with the intracellular level of alpha-synuclein, a protein whose accumulation is associated with neurotoxicity and the development of PD. Reduction of UCH-L1(M) in cell culture models of alpha-synuclein toxicity by treatment with a farnesyltransferase inhibitor (FTI-277) reduces alpha-synuclein levels and increases cell viability. Proteasome function is not affected by UCH-L1(M), suggesting that it may negatively regulate the lysosomal degradation of alpha-synuclein. Therefore, inhibition of UCH-L1 farnesylation may be a therapeutic strategy for slowing the progression of PD and related synucleinopathies.

    Funded by: Intramural NIH HHS; NIA NIH HHS: Z01 AG000953

    Proceedings of the National Academy of Sciences of the United States of America 2009;106;12;4635-40

  • Conversion of wild-type alpha-synuclein into mutant-type fibrils and its propagation in the presence of A30P mutant.

    Yonetani M, Nonaka T, Masuda M, Inukai Y, Oikawa T, Hisanaga S and Hasegawa M

    Department of Molecular Neurobiology, Tokyo Institute of Psychiatry, 2-1-8 Kamikitazawa, Setagaya-ku, 156-8585 Tokyo, Japan.

    Fibrillization or conformational change of alpha-synuclein is central in the pathogenesis of alpha-synucleinopathies, such as Parkinson disease. We found that the A30P mutant accelerates nucleation-dependent fibrillization of wild type (WT) alpha-synuclein. Electron microscopy observation and ultracentrifugation experiments revealed that shedding of fragments occurs from A30P fibrils and that these fragments accelerate fibrillization by serving as seeds. Immunochemical analysis using epitope-specific antibodies and biochemical analyses of protease-resistant cores demonstrated that A30P fibrils have a distinct conformation. Interestingly, WT fibrils formed with A30P seeds exhibited the same character as A30P fibrils, as did A30P fibrils formed with WT seeds, indicating that the A30P mutation affects the conformation and fibrillization of both WT and A30P. These effects of A30P mutation may explain the apparent conflict between the association of A30P with Parkinson disease and the slow fibrillization of A30P itself and therefore provide new insight into the molecular mechanisms of alpha-synucleinopathies.

    The Journal of biological chemistry 2009;284;12;7940-50

  • Dynamic changes in presynaptic and axonal transport proteins combined with striatal neuroinflammation precede dopaminergic neuronal loss in a rat model of AAV alpha-synucleinopathy.

    Chung CY, Koprich JB, Siddiqi H and Isacson O

    Neuroregeneration Laboratories, Harvard Medical School, McLean Hospital, Belmont, Massachusetts 02478, USA. cychung@mclean.harvard.edu

    Little is known about key pathological events preceding overt neuronal degeneration in Parkinson's disease (PD) and alpha-synucleinopathy. Recombinant adeno-associated virus 2-mediated delivery of mutant (A53T) human alpha-synuclein into the substantia nigra (SN) under a neuron-specific synapsin promoter resulted in protracted neurodegeneration with significant dopaminergic (DA) neuron loss by 17 weeks. As early as 4 weeks, there was an increase in a dopamine metabolite, DOPAC and histologically, DA axons in the striatum were dystrophic with degenerative bulbs. Before neuronal loss, significant changes were identified in levels of proteins relevant to synaptic transmission and axonal transport in the striatum and the SN. For example, striatal levels of rabphilin 3A and syntaxin were reduced. Levels of anterograde transport motor proteins (KIF1A, KIF1B, KIF2A, and KIF3A) were decreased in the striatum, whereas retrograde motor proteins (dynein, dynamitin, and dynactin1) were increased. In contrast to reduced levels in the striatum, KIF1A and KIF2A levels were elevated in the SN. There were dramatic changes in cytoskeletal protein levels, with actin levels increased and alpha-/gamma-tubulin levels reduced. In addition to these alterations, a neuroinflammatory response was observed at 8 weeks in the striatum, but not in the SN, demonstrated by increased levels of Iba-1, activated microglia and increased levels of proinflammatory cytokines, including IL-1beta, IFN-gamma and TNF-alpha. These results demonstrate that changes in proteins relevant to synaptic transmission and axonal transport coupled with neuroinflammation, precede alpha-synuclein-mediated neuronal death. These findings can provide ideas for antecedent biomarkers and presymptomatic interventions in PD.

    Funded by: NINDS NIH HHS: NS39793, P50 NS039793-08, P50 NS039793-09

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2009;29;11;3365-73

  • Alterations in corticostriatal synaptic plasticity in mice overexpressing human alpha-synuclein.

    Watson JB, Hatami A, David H, Masliah E, Roberts K, Evans CE and Levine MS

    Department of Psychiatry and Biobehavioral Sciences, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095, USA. jwatson@mednet.ucla.edu

    Most forms of Parkinson's disease (PD) are sporadic in nature, but some have genetic causes as first described for the alpha-synuclein gene. The alpha-synuclein protein also accumulates as insoluble aggregates in Lewy bodies in sporadic PD as well as in most inherited forms of PD. The focus of the present study is the modulation of synaptic plasticity in the corticostriatal pathway of transgenic (Tg) mice that overexpress the human alpha-synuclein protein throughout the brain (ASOTg). Paired-pulse facilitation was detected in vitro by activation of corticostriatal afferents in ASOTg mice, consistent with a presynaptic effect of elevated human alpha-synuclein. However basal synaptic transmission was unchanged in ASOTg, suggesting that human alpha-synuclein could impact paired-pulse facilitation via a presynaptic mechanism not directly related to the probability of neurotransmitter release. Mice lacking alpha-synuclein or those expressing normal and A53T human alpha-synuclein in tyrosine hydroxylase-containing neurons showed, instead, paired-pulse depression. High-frequency stimulation induced a presynaptic form of long-term depression solely in ASOTg striatum. A presynaptic, N-methyl-d-aspartate receptor-independent form of chemical long-term potentiation induced by forskolin (FSK) was enhanced in ASOTg striatum, while FSK-induced cAMP levels were reduced in ASOTg synaptoneurosome fractions. Overall the results suggest that elevated human alpha-synuclein alters presynaptic plasticity in the corticostriatal pathway, possibly reflecting a reduction in glutamate at corticostriatal synapses by modulation of adenylyl cyclase signaling pathways. ASOTg mice may recapitulate an early stage in PD during which overexpressed alpha-synuclein dampens corticostriatal synaptic transmission and reduces movement.

    Funded by: NIEHS NIH HHS: NIH U54 ES012078, U54 ES012078-03, U54 ES012078-04, U54 ES012078-05; NINDS NIH HHS: NIH NS 38367, P50 NS038367-06A20002, P50 NS038367-06A29005, P50 NS038367-070002, P50 NS038367-079005, P50 NS038367-080002, P50 NS038367-089005

    Neuroscience 2009;159;2;501-13

  • Curvature dynamics of alpha-synuclein familial Parkinson disease mutants: molecular simulations of the micelle- and bilayer-bound forms.

    Perlmutter JD, Braun AR and Sachs JN

    Department of Biomedical Engineering, University of Minnesota, Minneapolis, Minnesota 55455, USA.

    Alpha-synuclein remains a protein of interest due to its propensity to form fibrillar aggregates in neurodegenerative disease and its putative function in synaptic vesicle regulation. Herein, we present a series of atomistic molecular dynamics simulations of wild-type alpha-synuclein and three Parkinson disease familial mutants (A30P, A53T, and E46K) in two distinct environments. First, in order to match recent NMR experiments, we have simulated each protein bound to an SDS detergent micelle. Second, in order to connect more closely to the true biological environment, we have simulated the proteins bound to a 1,2-dioleoyl-sn-glycero-3-phosphoserine lipid bilayer. In the micelle-bound case, we find that the wild type and all of the variants of alpha-synuclein flatten the underlying micelle, decreasing its surface area. A30P is known to lessen alpha-synuclein/membrane affinity and, consistent with experiment, destabilizes the simulated secondary structure. In the case of A53T, our simulations reveal a range of stabilizing hydrogen bonds that form with the threonine. In both environments, the E46K mutation, which is known to increase bilayer affinity, leads to an additional hydrogen bond between the protein and either the detergent or lipid. Simulations indicate that alphaS and its variants are less dynamic in the bilayer than in the micelle. Furthermore, the simulations of the mutants suggest how changes in the structure and dynamics of alpha-synuclein may affect its biological role.

    The Journal of biological chemistry 2009;284;11;7177-89

  • Phosphorylation does not prompt, nor prevent, the formation of alpha-synuclein toxic species in a rat model of Parkinson's disease.

    Azeredo da Silveira S, Schneider BL, Cifuentes-Diaz C, Sage D, Abbas-Terki T, Iwatsubo T, Unser M and Aebischer P

    Brain Mind Institute, Ecole Polytechnique Fédérale de Lausanne, CH-1015 Lausanne, Switzerland.

    Phosphorylation is involved in numerous neurodegenerative diseases. In particular, alpha-synuclein is extensively phosphorylated in aggregates in patients suffering from synucleinopathies. However, the share of this modification in the events that lead to the conversion of alpha-synuclein to aggregated toxic species needed to be clarified. The rat model that we developed through rAAV2/6-mediated expression of alpha-synuclein demonstrates a correlation between neurodegeneration and formation of small filamentous alpha-synuclein aggregates. A mutation preventing phosphorylation (S129A) significantly increases alpha-synuclein toxicity and leads to enhanced formation of beta-sheet-rich, proteinase K-resistant aggregates, increased affinity for intracellular membranes, a disarrayed network of neurofilaments and enhanced alpha-synuclein nuclear localization. The expression of a mutation mimicking phosphorylation (S129D) does not lead to dopaminergic cell loss. Nevertheless, fewer but larger aggregates are formed, and signals of apoptosis are also activated in rats expressing the phosphorylation-mimicking form of alpha-synuclein. These observations strongly suggest that phosphorylation does not play an active role in the accumulation of cytotoxic pre-inclusion aggregates. Unexpectedly, the study also demonstrates that constitutive expression of phosphorylation-mimicking forms of alpha-synuclein does not protect from neurodegeneration. The role of phosphorylation at Serine 129 in the early phase of Parkinson's disease is examined, which brings new perspective to therapeutic approaches focusing on the modulation of kinases/phosphatases activity to control alpha-synuclein toxicity.

    Human molecular genetics 2009;18;5;872-87

  • Alpha-synuclein aggregation variable temperature and variable pH kinetic data: a re-analysis using the Finke-Watzky 2-step model of nucleation and autocatalytic growth.

    Morris AM and Finke RG

    Department of Chemistry, Colorado State University, Fort Collins, CO 80523, USA.

    The aggregation of proteins is believed to be intimately connected to many neurodegenerative disorders. We recently reported an "Ockham's razor"/minimalistic approach to analyze the kinetic data of protein aggregation using the Finke-Watzky (F-W) 2-step model of nucleation (A-->B, rate constant k(1)) and autocatalytic growth (A+B-->2B, rate constant k(2)). With that kinetic model we have analyzed 41 representative protein aggregation data sets in two recent publications, including amyloid beta, alpha-synuclein, polyglutamine, and prion proteins (Morris, A. M., et al. (2008) Biochemistry 47, 2413-2427; Watzky, M. A., et al. (2008) Biochemistry 47, 10790-10800). Herein we use the F-W model to reanalyze protein aggregation kinetic data obtained under the experimental conditions of variable temperature or pH 2.0 to 8.5. We provide the average nucleation (k(1)) and growth (k(2)) rate constants and correlations with variable temperature or varying pH for the protein alpha-synuclein. From the variable temperature data, activation parameters DeltaG(double dagger), DeltaH(double dagger), and DeltaS(double dagger) are provided for nucleation and growth, and those values are compared to the available parameters reported in the previous literature determined using an empirical method. Our activation parameters suggest that nucleation and growth are energetically similar for alpha-synuclein aggregation (DeltaG(double dagger)(nucleation)=23(3) kcal/mol; DeltaG(double dagger)(growth)=22(1) kcal/mol at 37 degrees C). From the variable pH data, the F-W analyses show a maximal k(1) value at pH approximately 3, as well as minimal k(1) near the isoelectric point (pI) of alpha-synuclein. Since solubility and net charge are minimized at the pI, either or both of these factors may be important in determining the kinetics of the nucleation step. On the other hand, the k(2) values increase with decreasing pH (i.e., do not appear to have a minimum or maximum near the pI) which, when combined with the k(1) vs. pH (and pI) data, suggest that solubility and charge are less important factors for growth, and that charge is important in the k(1), nucleation step of alpha-synuclein. The chemically well-defined nucleation (k(1)) rate constants obtained from the F-W analysis are, as expected, different than the 1/lag-time empirical constants previously obtained. However, k(2)x[A](0) (where k(2) is the rate constant for autocatalytic growth and [A](0) is the initial protein concentration) is related to the empirical constant, k(app) obtained previously. Overall, the average nucleation and average growth rate constants for alpha-synuclein aggregation as a function of pH and variable temperature have been quantitated. Those values support the previously suggested formation of a partially folded intermediate that promotes aggregation under high temperature or acidic conditions.

    Biophysical chemistry 2009;140;1-3;9-15

  • Alpha-synuclein is part of a diverse and highly conserved interaction network that includes PARK9 and manganese toxicity.

    Gitler AD, Chesi A, Geddie ML, Strathearn KE, Hamamichi S, Hill KJ, Caldwell KA, Caldwell GA, Cooper AA, Rochet JC and Lindquist S

    Whitehead Institute for Biomedical Research and Howard Hughes Medical Institute, Cambridge, Massachusetts 02142, USA.

    Parkinson's disease (PD), dementia with Lewy bodies and multiple system atrophy, collectively referred to as synucleinopathies, are associated with a diverse group of genetic and environmental susceptibilities. The best studied of these is PD. alpha-Synuclein (alpha-syn) has a key role in the pathogenesis of both familial and sporadic PD, but evidence linking it to other predisposition factors is limited. Here we report a strong genetic interaction between alpha-syn and the yeast ortholog of the PD-linked gene ATP13A2 (also known as PARK9). Dopaminergic neuron loss caused by alpha-syn overexpression in animal and neuronal PD models is rescued by coexpression of PARK9. Further, knockdown of the ATP13A2 ortholog in Caenorhabditis elegans enhances alpha-syn misfolding. These data provide a direct functional connection between alpha-syn and another PD susceptibility locus. Manganese exposure is an environmental risk factor linked to PD and PD-like syndromes. We discovered that yeast PARK9 helps to protect cells from manganese toxicity, revealing a connection between PD genetics (alpha-syn and PARK9) and an environmental risk factor (PARK9 and manganese). Finally, we show that additional genes from our yeast screen, with diverse functions, are potent modifiers of alpha-syn-induced neuron loss in animals, establishing a diverse, highly conserved interaction network for alpha-syn.

    Funded by: Howard Hughes Medical Institute; NIH HHS: 1-DP2-OD004417-01, DP2 OD004417; NINDS NIH HHS: NS038372, NS049221, P50 NS038372, P50 NS038372-100005, R01 NS049221

    Nature genetics 2009;41;3;308-15

  • Changes in adult olfactory bulb neurogenesis in mice expressing the A30P mutant form of alpha-synuclein.

    Marxreiter F, Nuber S, Kandasamy M, Klucken J, Aigner R, Burgmayer R, Couillard-Despres S, Riess O, Winkler J and Winner B

    Department of Neurology, University of Regensburg, Universitätsstrasse 84, 93053 Regensburg, Germany.

    In familial and sporadic forms of Parkinson's disease (PD), alpha-synuclein pathology is present in the brain stem nuclei and olfactory bulb (OB) long before Lewy bodies are detected in the substantia nigra. The OB is an active region of adult neurogenesis, where newly generated neurons physiologically integrate. While accumulation of wild-type alpha-synuclein is one of the pathogenic hallmarks of non-genetic forms of PD, the A30P alpha-synuclein mutation results in an earlier disease onset and a severe clinical phenotype. Here, we study the regulation of adult neurogenesis in the subventricular zone (SVZ)/OB system in a tetracycline-suppressive (tet-off) transgenic model of synucleinopathies, expressing human mutant A30P alpha-synuclein under the control of the calcium/calmodulin-dependent protein kinase II alpha (CaMK) promoter. In A30P transgenic mice alpha-synuclein was abundant at the site of integration in the glomerular cell layer of the OB. Without changes in proliferation in the SVZ, significantly fewer newly generated neurons were observed in the OB granule cell and glomerular layers of A30P transgenic mice than in controls, most probably due to increased cell death. By tetracycline-dependent abrogation of A30P alpha-synuclein expression, OB neurogenesis and programmed cell death was restored to control levels. Our results indicate that, using A30P conditional (tet-off) mice, A30P alpha-synuclein has a negative impact on olfactory neurogenesis and suppression of A30P alpha-synuclein enhances survival of newly generated neurons. This finding suggests that interfering with alpha-synuclein pathology can rescue newly generated neurons, possibly leading to new targets for therapeutic interventions in synucleinopathies.

    The European journal of neuroscience 2009;29;5;879-90

  • Fluorescence microscopy and 3D image reconstruction of cytokine initiated disruption of the Parkinson disease associated proteins alpha-synuclein, tau and ubiquitin in cultured glial cells.

    Dinh K, Poindexter BJ, Barnes JL, Schiess MC and Bick RJ

    Department of Pathology and Laboratory Medicine, University of Texas Medical School at Houston, 6431 Fannin Street, MSB 2.288, Houston, TX 77030, USA. kha.dinh@uth.tmc.edu

    Human derived glioblastoma cells were cultured and treated with cytokines interleukin-6 (IL6), tumor necrosis factor alpha (TNF) and interferon-gamma (IFN) and imaged by fluorescence deconvolution microscopy to localize alpha-synuclein, tau and ubiquitin. Exposures were for short (2 h) and prolonged times (up to 96 h), with doses at both low (10 ng/ml), and high (100 ng/ml) concentrations. Further experiments used additive doses up to 200 ng/ml (2 x 100 ng), mimicking a super-infection state. Single, low doses of the cytokines initiated changes in levels of intracellular proteins, but these changes, be they increases or decreases, were not sustained, so we added higher doses of cytokine to the culture medium or fresh aliquots of cytokines over time. Finally, we treated cells with high, single doses of cytokine (200 ng/ml), to try to sustain perturbations of the proteins with cytokines. IFN caused a disruption and reduction of peripheral synuclein, TNF treatment resulted in increased levels of ubiquitin and IL6 disrupted and appeared to fragment tau. Of note, each of the proteins was found in a specific locale, tau being perinuclear, ubiquitin residing in the cytoplasm, and alpha-synuclein occupying the tips of cellular processes, exhibiting the characteristics of an adhesion protein/molecule [Word count=198].

    Cytokine 2009;45;3;179-83

  • Microarray expression analysis of human dopaminergic neuroblastoma cells after RNA interference of SNCA--a key player in the pathogenesis of Parkinson's disease.

    Häbig K, Walter M, Stappert H, Riess O and Bonin M

    Department of Medical Genetics, Institute of Human Genetics, University of Tübingen, Germany.

    The pre-synaptic protein alpha-synuclein is a key player in the pathogenesis of Parkinson's disease (PD). Misfolded alpha-synuclein protofibrils accumulate and serve as seed structures that cause numerous proteins in the cytoplasm of neuronal cells to aggregate into so-called Lewy bodies. Furthermore, both missense mutations and multiplications of the SNCA gene lead to autosomal dominant forms of familial PD. However, the exact biological role of alpha-synuclein in normal brains remains elusive. To gain more insight into the normal function of this protein, we evaluated changes in whole genome expression in dopaminergic neuroblastoma cells (SH-SY5Y) caused by reductions of 90% in alpha-synuclein RNA levels and of 59% in alpha-synuclein protein levels as a result of RNA interference. The expression of 361 genes was altered at least+/-1.5-fold by the RNA interference, with 82 up-regulated and 279 down-regulated. The differentially expressed gene products are involved in the regulation of transcription, cell cycle, protein degradation, apoptosis, neurogenesis, and lipid metabolism. To examine the influence of SNCA down-regulation by RNAi on apoptosis, we performed cell death assays using different stress triggers. The changes observed in the expression profile of dopaminergic neuronal cells following reduction of SNCA expression warrant studies to investigate the role of signaling cascades in familial and idiopathic PD.

    Brain research 2009;1256;19-33

  • Inhibition of alpha-synuclein fibril assembly by small molecules: analysis using epitope-specific antibodies.

    Masuda M, Hasegawa M, Nonaka T, Oikawa T, Yonetani M, Yamaguchi Y, Kato K, Hisanaga S and Goedert M

    Department of Molecular Neurobiology, Tokyo Institute of Psychiatry, 2-1-8 Kamikitazawa, Setagaya-ku, Tokyo 156-8585, Japan.

    The conversion of soluble peptides and proteins into amyloid fibrils and/or intermediate oligomers is believed to be the central event in the pathogenesis of most human neurodegenerative diseases. Existing treatments are at best symptomatic. Accordingly, small molecule inhibitors of amyloid fibril formation and their mechanisms are of great interest. Here we report that the conformational changes undergone by alpha -synuclein as it assembles into amyloid fibrils can be detected by epitope-specific antibodies. We show that the conformations of polyphenol-bound alpha-synuclein monomers and dimers differ from those of unbound monomers and resemble amyloid fibrils. This strongly suggests that small molecule inhibitors bind and stabilize intermediates of amyloid fibril formation, consistent with the view that inhibitor-bound molecular species are on-pathway intermediates.

    Funded by: Medical Research Council: MC_U105184291

    FEBS letters 2009;583;4;787-91

  • Evidence that alpha-synuclein does not inhibit phospholipase D.

    Rappley I, Gitler AD, Selvy PE, LaVoie MJ, Levy BD, Brown HA, Lindquist S and Selkoe DJ

    Department of Neurology, Harvard Medical School and Brigham & Women's Hospital, Boston, Massachusetts 02115, USA.

    Alpha-synuclein (alphaSyn) is a small cytosolic protein of unknown function, which is highly enriched in the brain. It is genetically linked to Parkinson's disease (PD) in that missense mutations or multiplication of the gene encoding alphaSyn causes early onset familial PD. Furthermore, the neuropathological hallmarks of both sporadic and familial PD, Lewy bodies and Lewy neurites, contain insoluble aggregates of alphaSyn. Several studies have reported evidence that alphaSyn can inhibit phospholipase D (PLD), which hydrolyzes phosphatidylcholine to form phosphatidic acid and choline. Although various hypotheses exist regarding the roles of alphaSyn in health and disease, no other specific biochemical function for this protein has been reported to date. Because PLD inhibition could represent an important function of alphaSyn, we sought to extend existing reports on this interaction. Using purified proteins, we tested the ability of alphaSyn to inhibit PLD activity in cell-free assays. We also examined several cell lines and transfection conditions to assess whether alphaSyn inhibits endogenous or overexpressed PLD in cultured mammalian cells. In yeast, we extended our previous report of an interaction between alphaSyn and PLD-dependent phenotypes, for which PLD activity is absolutely necessary. Despite testing a range of experimental conditions, including those previously published, we observed no significant inhibition of PLD by alphaSyn in any of these systems. We propose that the previously reported effects of alphaSyn on PLD activity could be due to increased endoplasmic reticulum-related stress associated with alphaSyn overexpression in cells, but are not likely due to a specific and direct interaction between alphaSyn and PLD.

    Funded by: NINDS NIH HHS: NS038375, P50 NS038375, P50 NS038375-090002

    Biochemistry 2009;48;5;1077-83

  • Cerebrospinal fluid alpha-synuclein in neurodegenerative disorders-a marker of synapse loss?

    Ohrfelt A, Grognet P, Andreasen N, Wallin A, Vanmechelen E, Blennow K and Zetterberg H

    Institute of Neuroscience and Physiology, Department of Psychiatry and Neurochemistry, The Sahlgrenska Academy at University of Gothenburg, Mölndal, Sweden.

    The association of alpha-synuclein (alpha-syn) neuropathology with Parkinson's disease (PD) and several related disorders has led to an intense research effort to develop cerebrospinal fluid (CSF)- or blood-based alpha-syn biomarkers for these types of diseases. Recent studies show that alpha-syn is present in CSF and possible to measure using enzyme-linked immunosorbent assay (ELISA). Here, we describe a novel ELISA that allows for quantification of alpha-syn in CSF down to 50pg/mL. The diagnostic value of the test was assessed using CSF samples from 66 Alzheimer's disease (AD) patients, 15PD patients, 15 patients with dementia with Lewy bodies (DLB) and 55 cognitively normal controls. PD and DLB patients and controls displayed similar CSF alpha-syn levels. AD patients had significantly lower alpha-syn levels than controls (median [inter-quartile range] 296 [234-372] and 395 [298-452], respectively, p<0.001). Moreover, AD patients with mini-mental state examination (MMSE) scores below 20 had significantly lower alpha-syn than AD patients with MMSE scores of 20 or higher (p=0.02). There was also a tendency towards a negative correlation between alpha-syn levels and disease duration in the AD group (r=-0.247, p=0.06). Altogether, our results speak against CSF alpha-syn as a reliable biomarker for PD and DLB. The lower alpha-syn levels in AD, as well as the association of alpha-syn reduction with AD severity, approximated by MMSE, suggests that it may be a general marker of synapse loss, a hypothesis that warrants further investigation.

    Neuroscience letters 2009;450;3;332-5

  • Alpha-synuclein and polyunsaturated fatty acids promote clathrin-mediated endocytosis and synaptic vesicle recycling.

    Ben Gedalya T, Loeb V, Israeli E, Altschuler Y, Selkoe DJ and Sharon R

    Department of Cellular Biochemistry and Human Genetics, Faculty of Medicine, Hebrew University, Ein-Kerem, Jerusalem 91120, Israel.

    Alpha-synuclein (alphaS) is an abundant neuronal cytoplasmic protein implicated in Parkinson's disease (PD), but its physiological function remains unknown. Consistent with its having structural motifs shared with class A1 apolipoproteins, alphaS can reversibly associate with membranes and help regulate membrane fatty acid composition. We previously observed that variations in alphaS expression level in dopaminergic cultured cells or brains are associated with changes in polyunsaturated fatty acid (PUFA) levels and altered membrane fluidity. We now report that alphaS acts with PUFAs to enhance the internalization of the membrane-binding dye, FM 1-43. Specifically, alphaS expression coupled with exposure to physiological levels of certain PUFAs enhanced clathrin-mediated endocytosis in neuronal and non-neuronal cultured cells. Moreover, alphaS expression and PUFA-enhanced basal and -evoked synaptic vesicle (SV) endocytosis in primary hippocampal cultures of wild type (wt) and genetically depleted alphaS mouse brains. We suggest that alphaS and PUFAs normally function in endocytic mechanisms and are specifically involved in SV recycling upon neuronal stimulation.

    Funded by: NINDS NIH HHS: R01 NS051318, R01 NS051318-03, R01 NS051318-04

    Traffic (Copenhagen, Denmark) 2009;10;2;218-34

  • Association of alpha-synuclein immunoreactivity with inflammatory activity in multiple sclerosis lesions.

    Lu JQ, Fan Y, Mitha AP, Bell R, Metz L, Moore GR and Yong VW

    Department of Pathology and Laboratory Medicine, University of Calgary, Alberta, Canada.

    Multiple sclerosis (MS) has neurodegenerative features including neuronal and axonal loss and widespread atrophy of the brain and spinal cord. The cause of this neurodegeneration has been largely attributed to inflammation, but other mechanisms, including those associated with classic neurodegenerative diseases such as the alpha-synucleinopathies, might also be involved in MS pathogenesis. In this study, 96 brain lesions containing varying degrees of inflammatory activity from 12 autopsied MS cases were compared with corresponding regions from 6 neuropathologically normal controls; 2 cerebral biopsy lesions from an MS patient were also studied. We found alpha-synuclein immunoreactivity in the cytoplasm of cells in MS lesions with inflammatory activity but not in control samples. alpha-Synuclein-immunoreactive cells were identified in active (15/15 lesions in the brainstem, 9/13 in cerebral hemispheres) and chronic active (14/15 in the brainstem, 12/22 in cerebral hemispheres) lesions but were absent in chronic inactive lesions (0/31); the greater immunoreactivity in brainstem compared with cerebral hemisphere lesions was significant (p < 0.05). Double-immunofluorescence staining revealed localization of alpha-synuclein immunoreactivity mostly in neurons, microglia/macrophages, and oligodendrocytes, and only rarely in astrocytes. The results suggest that alpha-synuclein expression regulated by inflammatory signals may contribute to neurodegenerative processes in MS lesions.

    Journal of neuropathology and experimental neurology 2009;68;2;179-89

  • Proteomic profiling of phosphoproteins and glycoproteins responsive to wild-type alpha-synuclein accumulation and aggregation.

    Kulathingal J, Ko LW, Cusack B and Yen SH

    Department of Neuroscience, Mayo Clinic, Jacksonville, FL 32224, USA.

    A tetracycline inducible transfectant cell line (3D5) capable of producing soluble and sarkosyl-insoluble assemblies of wild-type human alpha-synuclein (alpha-Syn) upon differentiation with retinoic acid was used to study the impact of alpha-Syn accumulation on protein phosphorylation and glycosylation. Soluble proteins from 3D5 cells, with or without the induced alpha-Syn expression were analyzed by two-dimensional gel electrophoresis and staining of gels with dyes that bind to proteins (Sypro ruby), phosphoproteins (Pro-Q diamond) and glycoproteins (Pro-Q emerald). Phosphoproteins were further confirmed by binding to immobilized metal ion affinity column. alpha-Syn accumulation caused differential phosphorylation and glycosylation of 16 and 12, proteins, respectively, whose identity was revealed by mass spectrometry. These proteins, including HSP90, have diverse biological functions including protein folding, signal transduction, protein degradation and cytoskeletal regulation. Importantly, cells accumulating alpha-Syn assemblies with different abilities to bind thioflavin S displayed different changes in phosphorylation and glycosylation. Consistent with the cell-based studies, we demonstrated a reduced level of phosphorylated HSP90 alpha/beta in the substantia nigra of subjects with Parkinson's disease as compared to normal controls. Together, the results indicate that alpha-Syn accumulation causes complex cellular responses, which if persist may compromise cell viability.

    Funded by: NINDS NIH HHS: P50 NS040256-100006, P50-NS40256

    Biochimica et biophysica acta 2009;1794;2;211-24

  • Expression of Lewy body protein septin 4 in postmortem brain of Parkinson's disease and control subjects.

    Shehadeh L, Mitsi G, Adi N, Bishopric N and Papapetropoulos S

    Department of Molecular and Cellular Pharmacology, University of Miami Miller School of Medicine, Miami, Florida 33136, USA.

    In Parkinson's disease (PD) neuronal degeneration is associated with abnormal protein aggregation in various forms including Lewy bodies (LBs). A major component of LBs is alpha-synuclein; septin 4 (SEPT4), a polymerizing GTP-binding protein that serves as scaffold for diverse molecules has been found to colocalize with alpha-synuclein in LBs. The central role of SEPT4 in the etiopathogenesis of PD has been suggested since SEPT4 also shows a physiological association with alpha-synuclein and serves as a substrate for parkin. To this end, we studied the expression of septin 4 and alpha-synuclein in postmortem human substantia nigra (SN) and amygdala from patients with PD and healthy controls. Twenty patients (14 men : 6 women, onset 63.0 +/- 11.4 years, age 77.3 +/- 7.6 years, Hoehn and Yahr 4.05/5) and 9 neurologically healthy controls (4 men/5 women, age at death 80.1 +/- 8.6 years) were studied. Sporadic PD cases showed a statistically significant decrease of the fold change (FC) of SNCA (FC = 0.31, P = 0.00001) and SEPT4 (FC = 0.67, P = 0.054) gene expressions in the SN and the amygdala (SNCA: FC = 0.49, P = 0.02; SEPT4: FC = 0.32, P = 0.007) versus healthy controls. However, an increase of both proteins in PD versus control subjects was observed with immunoblotting. The semi-quantitative protein ratio calculations revealed more than 10-fold increases for both SEPT4 and alpha-synuclein in PD versus control subjects. We present for the first time similar signal expression patterns and parallel accumulation of SEPT4 and alpha-synuclein in well-characterized postmortem PD brain. Considering the heterogeneous etiology of sporadic PD and the variability of individual human samples, SEPT4 accumulation may be regarded as one of the common pathological changes in PD and should therefore be further explored.

    Funded by: NHLBI NIH HHS: F32 HL083673, R01 HL071094; NIMH NIH HHS: 5R03MH074059-02

    Movement disorders : official journal of the Movement Disorder Society 2009;24;2;204-10

  • Genotypic and phenotypic characteristics of Dutch patients with early onset Parkinson's disease.

    Macedo MG, Verbaan D, Fang Y, van Rooden SM, Visser M, Anar B, Uras A, Groen JL, Rizzu P, van Hilten JJ and Heutink P

    Section of Medical Genomics, Department of Clinical Genetics, VU University Medical Center, Amsterdam, The Netherlands. m.macedo@vumc.nl

    Early onset Parkinson's disease (EOPD) has been associated with mutations in the Parkin, DJ-1, PINK1, LRRK2, and SNCA genes. The aim of this study is to assess the contribution of these genes in a Dutch EOPD cohort and the phenotypic characteristics of the mutation carriers. A total of 187 unrelated Dutch EOPD patients (age at onset < or = 50 years) were phenotyped and screened for mutations in all exons of Parkin, DJ-1, and PINK1 by direct sequencing and gene dosage analysis. Additionally, analysis of the A30P mutation and exon dosage of SNCA and sequencing of exons 19,31,35,38,41, and 48 of LRRK2 was performed. Pathogenic variations could explain disease in 4% (7 of 187) of the patients including five patients carrying homozygous or compound heterozygous mutations in Parkin, one with a novel homozygous deletion in DJ-1 (P158Del) and one with a heterozygous mutation in LRRK2 (T2356I). We found seven novel mutations. The phenotypic characteristics of mutation carriers varied widely, comparable to the variability seen in sporadic EOPD. Parkin is the most frequently mutated gene in this EOPD cohort, followed by DJ-1, PINK1 and LRRK2. The low overall mutation frequency indicates that the extrapolation of mutation frequencies from other populations should be applied with caution.

    Movement disorders : official journal of the Movement Disorder Society 2009;24;2;196-203

  • Polo-like kinase 2 (PLK2) phosphorylates alpha-synuclein at serine 129 in central nervous system.

    Inglis KJ, Chereau D, Brigham EF, Chiou SS, Schöbel S, Frigon NL, Yu M, Caccavello RJ, Nelson S, Motter R, Wright S, Chian D, Santiago P, Soriano F, Ramos C, Powell K, Goldstein JM, Babcock M, Yednock T, Bard F, Basi GS, Sham H, Chilcote TJ, McConlogue L, Griswold-Prenner I and Anderson JP

    Elan Pharmaceuticals Inc, South San Francisco, CA 94080, USA.

    Several neurological diseases, including Parkinson disease and dementia with Lewy bodies, are characterized by the accumulation of alpha-synuclein phosphorylated at Ser-129 (p-Ser-129). The kinase or kinases responsible for this phosphorylation have been the subject of intense investigation. Here we submit evidence that polo-like kinase 2 (PLK2, also known as serum-inducible kinase or SNK) is a principle contributor to alpha-synuclein phosphorylation at Ser-129 in neurons. PLK2 directly phosphorylates alpha-synuclein at Ser-129 in an in vitro biochemical assay. Inhibitors of PLK kinases inhibited alpha-synuclein phosphorylation both in primary cortical cell cultures and in mouse brain in vivo. Finally, specific knockdown of PLK2 expression by transduction with short hairpin RNA constructs or by knock-out of the plk2 gene reduced p-Ser-129 levels. These results indicate that PLK2 plays a critical role in alpha-synuclein phosphorylation in central nervous system.

    The Journal of biological chemistry 2009;284;5;2598-602

  • CSF alpha-synuclein levels in dementia with Lewy bodies and Alzheimer's disease.

    Noguchi-Shinohara M, Tokuda T, Yoshita M, Kasai T, Ono K, Nakagawa M, El-Agnaf OM and Yamada M

    Department of Neurology and Neurobiology of Aging, Kanazawa University Graduate School of Medical Science, Kanazawa, Japan.

    Dementia with Lewy bodies (DLB) is characterized by widespread depositions of alpha-synuclein, which are described as Lewy bodies. Recently, it was shown that neuronal cells in culture constitutively release alpha-synuclein into the culture medium and that alpha-synuclein is normally present in human cerebrospinal fluid (CSF). The aim of the present study was to evaluate the diagnostic value of CSF alpha-synuclein levels in discriminating DLB from Alzheimer's disease (AD). Alpha-synuclein was measured in CSF from 16 patients with DLB and 21 patients with AD. Iodine-123 metaiodobenzylguanidine cardiac scintigraphy was also performed to assess Lewy body pathology. CSF alpha-synuclein levels did not differ significantly between DLB and AD patients. However, the duration of illness was associated with lower alpha-synuclein levels (p<0.05) in DLB, while no such association was found in AD. The present data show CSF alpha-synuclein levels are not sensitive diagnostic markers to discriminate DLB from AD. However, the lower alpha-synuclein levels in DLB patients with longer duration suggest a reduction in CSF alpha-synuclein in association with increased severity of alpha-synucleinopathy in the brain.

    Brain research 2009;1251;1-6

  • The lipid peroxidation metabolite 4-oxo-2-nonenal cross-links alpha-synuclein causing rapid formation of stable oligomers.

    Näsström T, Wahlberg T, Karlsson M, Nikolajeff F, Lannfelt L, Ingelsson M and Bergström J

    Department of Public Health and Caring Sciences, Rudbeck Laboratory, Uppsala University, Uppsala, Sweden.

    Recently, the aldehyde 4-oxo-2-nonenal (ONE) was identified as a product of lipid peroxidation and found to be an effective protein modifier. In this in vitro study we investigated structural implications of the interaction between ONE and alpha-synuclein, a protein which forms intraneuronal inclusions in neurodegenerative disorders such as Parkinson's disease and dementia with Lewy bodies. Our results demonstrate that ONE induced an almost complete conversion of monomeric alpha-synuclein into 40-80 nm wide and 6-8 nm high soluble beta-sheet-rich oligomers with a molecular weight of approximately 2000 kDa. Furthermore, the ONE-induced alpha-synuclein oligomers displayed a high stability and were not sensitive to treatment with sodium dodecyl sulfate, indicating that ONE stabilized the oligomers by cross-linking individual alpha-synuclein molecules. Despite prolonged incubation the oligomers did not continue to aggregate into a fibrillar state, thus suggesting that these alpha-synuclein species were not on a fibrillogenic pathway.

    Biochemical and biophysical research communications 2009;378;4;872-6

  • Aging and alpha-synuclein affect synaptic plasticity in the dentate gyrus.

    Gureviciene I, Gurevicius K and Tanila H

    Department of Neurobiology, University of Kuopio, Neulaniementie 2, P.O. Box 1627, 70211, Kuopio, Finland. Irina.Gureviciene@uku.fi

    Although intracellular accumulation of alpha-synuclein (alpha-syn) is a characteristic pathological change in Parkinson's disease, Lewy body dementia and Alzheimer's disease, the normal function of this presynaptic protein is still unknown. To assess the contribution of alpha-syn to synaptic plasticity as well as to age-related synaptic degeneration in mice, we compared adult and aged mice overexpressing mutated (A30P) human alpha-syn with their nontransgenic littermates using behavioral tests and electrophysiological measures in the dentate gyrus. We found decreased basal synaptic transmission and paired-pulse facilitation in the perforant path-dentate granule cell synapses of aged mice. In addition, alpha-syn accumulation in aged A30P mice but not in aged wild-type mice led to long-term depression of synaptic transmission after a stimulation protocol that normally induces long-term potentiation. These findings suggest that overexpression of mutated alpha-syn exacerbates the aging process and leads to impaired synaptic plasticity.

    Journal of neural transmission (Vienna, Austria : 1996) 2009;116;1;13-22

  • Alpha-synuclein gene rearrangements in dominantly inherited parkinsonism: frequency, phenotype, and mechanisms.

    Ibáñez P, Lesage S, Janin S, Lohmann E, Durif F, Destée A, Bonnet AM, Brefel-Courbon C, Heath S, Zelenika D, Agid Y, Dürr A, Brice A and French Parkinson's Disease Genetics Study Group

    Institut National de la Santé et de la Recherche Médicale (INSERM), Unité Mixte de Recherche en Santé (UMR)_S679 Neurologie & Thérapeutique Expérimentale, F-75013, Paris, France.

    Objective: Genomic multiplications of the alpha-synuclein gene (SNCA) cause autosomal dominant Parkinson disease (ADPD). The aim of this study was to assess the frequency and phenotype of SNCA rearrangements in a large series of families with typical or atypical AD parkinsonism.

    Design: Patients were screened by the exon dosage of the SNCA gene. The genotype of patients and relatives carrying SNCA rearrangements, the size of the multiplied regions, and the centromeric and telomeric breakpoints were determined by microsatellite dosage and 250K Affymetrix Single Polymorphism Nucleotide microarrays (Affymetrix, Santa Clara, California).

    Subjects: Index cases and, whenever appropriate, relatives of 286 mainly European families with ADPD were screened.

    Results: Four of 264 families (1.5%) with typical ADPD carried duplications and 1 of 22 families (4.5%) with atypical AD parkinsonism carried a triplication of SNCA. Genotyping and dosage analyses showed that the multiplied regions were variable in size (0.42-5.29 megabase pairs), suggesting that SNCA multiplications occurred independently. Phenotype analyses showed that the severity of the disease correlated with the SNCA copy number, but not with the minimal number of multiplied genes (1 to 33). Haplotype analysis of polymorphic markers suggested that multiplication of the SNCA gene occurred by both interchromosomal and intrachromosomal rearrangement.

    Conclusions: Our results suggest that SNCA rearrangements may be more frequent than point mutations in ADPD. Furthermore, our results indicate that the phenotype associated with SNCA multiplications correlates with the number of copies of the gene and provides the first insight into the mechanisms underlying SNCA multiplication.

    Archives of neurology 2009;66;1;102-8

  • Genomewide association study for susceptibility genes contributing to familial Parkinson disease.

    Pankratz N, Wilk JB, Latourelle JC, DeStefano AL, Halter C, Pugh EW, Doheny KF, Gusella JF, Nichols WC, Foroud T, Myers RH and PSG-PROGENI and GenePD Investigators, Coordinators and Molecular Genetic Laboratories

    Indiana University School of Medicine, Health Information and Translational Sciences Building, Indianapolis, IN 46202-3002, USA. npankrat@iupui.edu

    Five genes have been identified that contribute to Mendelian forms of Parkinson disease (PD); however, mutations have been found in fewer than 5% of patients, suggesting that additional genes contribute to disease risk. Unlike previous studies that focused primarily on sporadic PD, we have performed the first genomewide association study (GWAS) in familial PD. Genotyping was performed with the Illumina HumanCNV370Duo array in 857 familial PD cases and 867 controls. A logistic model was employed to test for association under additive and recessive modes of inheritance after adjusting for gender and age. No result met genomewide significance based on a conservative Bonferroni correction. The strongest association result was with SNPs in the GAK/DGKQ region on chromosome 4 (additive model: p = 3.4 x 10(-6); OR = 1.69). Consistent evidence of association was also observed to the chromosomal regions containing SNCA (additive model: p = 5.5 x 10(-5); OR = 1.35) and MAPT (recessive model: p = 2.0 x 10(-5); OR = 0.56). Both of these genes have been implicated previously in PD susceptibility; however, neither was identified in previous GWAS studies of PD. Meta-analysis was performed using data from a previous case-control GWAS, and yielded improved p values for several regions, including GAK/DGKQ (additive model: p = 2.5 x 10(-7)) and the MAPT region (recessive model: p = 9.8 x 10(-6); additive model: p = 4.8 x 10(-5)). These data suggest the identification of new susceptibility alleles for PD in the GAK/DGKQ region, and also provide further support for the role of SNCA and MAPT in PD susceptibility.

    Funded by: NINDS NIH HHS: R01 NS036711, R01 NS036711-09, R01 NS037167-10, R01 NS37167; PHS HHS: HHSN268200782096C; Telethon: GTB07001

    Human genetics 2009;124;6;593-605

  • Alpha-Synuclein conformation affects its tyrosine-dependent oxidative aggregation.

    Ruf RA, Lutz EA, Zigoneanu IG and Pielak GJ

    Department of Chemistry, University of North Carolina, Chapel Hill, North Carolina 27599, USA.

    Oxidative stress and aggregation of the protein alpha-synuclein are thought to be key factors in Parkinson's disease. Previous work shows that cytochrome c with H(2)O(2) causes tyrosine-dependent in vitro peroxidative aggregation of proteins, including alpha-synuclein. Here, we examine the role of each of alpha-synuclein's four tyrosine residues and how the protein's conformation affects covalent oxidative aggregation. When alpha-synuclein adopts a collapsed conformation, tyrosine 39 is essential for wild-type-like covalent aggregation. This lone N-terminal tyrosine, however, is not required for wild-type-like covalent aggregation in the presence of a denaturant or when alpha-synuclein is present in noncovalent fibrils. We also show that preformed oxidative aggregates are not incorporated into noncovalent fibrils. These data provide insight into how dityrosine may be formed in Lewy bodies seen in Parkinson's disease.

    Funded by: NIGMS NIH HHS: R01 GM042501-09; NIH HHS: 5DP1OD783, DP1 OD000783-01, DP1 OD000783-02, DP1 OD000783-03

    Biochemistry 2008;47;51;13604-9

  • Is olfactory impairment in Parkinson disease related to phenotypic or genotypic characteristics?

    Verbaan D, Boesveldt S, van Rooden SM, Visser M, Marinus J, Macedo MG, Fang Y, Heutink P, Berendse HW and van Hilten JJ

    Department of Neurology, K5Q-92, Leiden University Medical Center, P.O. Box 9600, NL- 2300 RC Leiden, the Netherlands. D.Verbaan@lumc.nl

    Objective: To evaluate the relation between olfactory impairment (OI) and other impairment domains in Parkinson disease (PD) and the characteristics of OI in patients with certain genotypic characteristics.

    Methods: In 295 nondemented patients with PD and 150 controls with a similar overall age and sex distribution, olfactory function was evaluated with the identification (ID) and discrimination (DIS) tests of the Sniffin' Sticks. In patients, demographic and clinical characteristics were evaluated, and genetic analyses were performed.

    Results: Of all patients, 61% had an impaired ID and 43% had an impaired DIS. No significant correlations >0.4 were found between olfactory scores and other demographic or clinical variables. Age and sex accounted for the 22% explained variance of the ID score regression model, whereas age, sex, and disease duration accounted for the 15% explained variance of the DIS score regression model. Parkin and DJ-1 mutation carriers (homozygous or heterozygous compound, n = 6) had normal ID scores. APOE epsilon2 or APOE epsilon4 carriers had no significantly different olfactory scores than noncarriers. The allele distribution of the alpha-synuclein (SNCA)-REP1 polymorphism in groups with an impaired or normal ID or DIS was comparable.

    Conclusions: Olfactory impairment (OI) in Parkinson disease (PD) may be unrelated to other impairment domains of the disease, which may indicate that olfaction is an independent feature of PD. Parkin and DJ-1 mutation carriers had normal identification scores but the number of mutation carriers is too small to draw conclusions. The APOE genotype (APOE epsilon2 or APOE epsilon4 alleles) and SNCA-REP1 polymorphism do not seem to influence olfaction in PD.

    Neurology 2008;71;23;1877-82

  • The effect of alpha-synuclein knockdown on MPP+ toxicity in models of human neurons.

    Fountaine TM, Venda LL, Warrick N, Christian HC, Brundin P, Channon KM and Wade-Martins R

    Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 3QX, UK.

    The protein alpha-synuclein is central to the pathophysiology of Parkinson's disease (PD) but its role in the development of neurodegeneration remains unclear. alpha-Synuclein-knockout mice develop without gross abnormality and are resistant to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a mitochondrial inhibitor widely used to model parkinsonism. Here we show that differentiated human dopaminergic neuron-like cells also have increased resistance to 1-methyl-4-phenylpyridine (MPP+), the active metabolite of MPTP, when alpha-synuclein is knocked down using RNA interference. In attempting to understand how this occurred we found that lowering alpha-synuclein levels caused changes to intracellular vesicles, dopamine transporter (DAT) and vesicular monoamine transporter (VMAT2), each of which is known to be an important component of the early events leading to MPP+ toxicity. Knockdown of alpha-synuclein reduced the availability of DAT on the neuronal surface by 50%, decreased the total number of intracellular vesicles by 37% but increased the density of VMAT2 molecules per vesicle by 2.8-fold. However, these changes were not associated with any reduction in MPP+ -induced superoxide production, suggesting that alpha-synuclein knockdown may have other downstream effects which are important. We then showed that alpha-synuclein knockdown prevented MPP+ -induced activation of nitric oxide synthase (NOS). Activation of NOS is an essential step in MPTP toxicity and increasing evidence points to nitrosative stress as being important in neurodegeneration. Overall, these results show that as well as having a number of effects on cellular events upstream of mitochondrial dysfunction alpha-synuclein affects pathways downstream of superoxide production, possibly involving regulation of NOS activity.

    Funded by: Wellcome Trust: 073141

    The European journal of neuroscience 2008;28;12;2459-73

  • Enduring involvement of tau, beta-amyloid, alpha-synuclein, ubiquitin and TDP-43 pathology in the amyotrophic lateral sclerosis/parkinsonism-dementia complex of Guam (ALS/PDC).

    Miklossy J, Steele JC, Yu S, McCall S, Sandberg G, McGeer EG and McGeer PL

    Kinsmen Laboratory of Neurological Research, The University of British Columbia, 2255 Wesbrook Mall, Vancouver, BC, V6T 1Z3, Canada.

    Guam ALS/PDC is a severe tangle forming disorder endemic to Guam with features overlapping such neurodegenerative disorders as Alzheimer disease (AD), Parkinson disease (PD), progressive supranuclear palsy (PSP), ALS, corticobasal degeneration (CBD) and pallido-ponto-nigral degeneration (PPND). Since the prevalence is declining, we examined brain tissue from 35 clinically diagnosed Chamorro patients with ALS/PDC and two Chamorro controls autopsied between 1946 and 2006, to determine if distinct variations in the pathology could be identified up to this time. Although the age at autopsy increased by 4.5-5 years per decade, we identified no qualitative differences in pathological deposits with antibodies against tau, ubiquitin, A beta, alpha-synuclein and TDP-43, indicating that these more recently identified proteins have been involved in the neuropathogenesis over the past 6 decades. Tau and TDP-43 positive neuronal, oligodendroglial and astrocytic inclusions involving multiple nerve fiber tracts occurred in both the ALS and PDC types, reinforcing the concept that these forms are part of the same disorder. The results obtained may help to define the commonality of the Guam disease with other tangle forming disorders and may help in monitoring the epidemiological changes that are taking place.

    Acta neuropathologica 2008;116;6;625-37

  • Targeted overexpression of human alpha-synuclein triggers microglial activation and an adaptive immune response in a mouse model of Parkinson disease.

    Theodore S, Cao S, McLean PJ and Standaert DG

    Center for Neurodegeneration and Experimental Therapeutics, The University of Alabama at Birmingham, Alabama 35294-0021, USA.

    Microglial activation and adaptive immunity have been implicated in the neurodegenerative processes in Parkinson disease. It has been proposed that these responses may be triggered by modified forms of alpha-synuclein (alpha-SYN), particularly nitrated species, which are released as a consequence of dopaminergic neurodegeneration. To examine the relationship between alpha-SYN, microglial activation, and adaptive immunity, we used a mouse model of Parkinson disease in which human alpha-SYN is overexpressed by a recombinant adeno-associated virus vector, serotype 2 (AAV2-SYN); this overexpression leads to slow degeneration of dopaminergic neurons. Microglial activation and components of the adaptive immune response were assessed using immunohistochemistry; quantitative polymerase chain reaction was used to examine cytokine expression. Four weeks after injection, there was a marked increase in CD68-positive microglia and greater infiltration of B and T lymphocytes in the substantia nigra pars compacta of the AAV2-SYN group than in controls. At 12 weeks, CD68 staining declined, but B- and T-cell infiltration persisted. Expression of proinflammatory cytokines was enhanced, whereas markers of alternative activation (i.e. arginase I and interleukins 4 and 13) were not altered. Increased immunoreactivity for mouse immunoglobulin was detected at all time points in the AAV2-SYN animals. These data show that overexpression of alpha-SYN alone, in the absence of overt neurodegeneration, is sufficient to trigger neuroinflammation with both microglial activation and stimulation of adaptive immunity.

    Funded by: NINDS NIH HHS: P30 NS057098, P30 NS057098-03, P50 NS038372, P50 NS038372-080006, P50 NS38372

    Journal of neuropathology and experimental neurology 2008;67;12;1149-58

  • Interactions between Hsp70 and the hydrophobic core of alpha-synuclein inhibit fibril assembly.

    Luk KC, Mills IP, Trojanowski JQ and Lee VM

    Center for Neurodegenerative Disease Research, Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-4283, USA.

    Molecular chaperones of the heat shock protein 70 (Hsp70) family counteract protein misfolding in a variety of neurodegenerative disease models. To determine whether human Hsp70 exerts similar effects on the aggregation of alpha-synuclein (alpha-Syn), the key component of insoluble fibrils present in Parkinson's disease, we investigated alpha-Syn fibril assembly in the presence of Hsp70. We found in vitro assembly was efficiently inhibited by substoichiometric concentrations of purified Hsp70 in the absence of cofactors. Experiments using alpha-Syn deletion mutants indicated that interactions between the Hsp70 substrate binding domain and the alpha-Syn core hydrophobic region underlie assembly inhibition. This assembly process was inhibited prior to the elongation stage as we failed to detect any fibrils by electron microscopy. In addition, fluorescence polarization and binding assays suggest that Hsp70 recognizes soluble alpha-Syn species in a highly dynamic and reversible manner. Together, these results provide novel insights into how Hsp70 suppresses alpha-Syn aggregation. Furthermore, our findings suggest that this critical step in Parkinson's disease pathogenesis may be subject to modulation by a common molecular chaperone.

    Funded by: NIA NIH HHS: AG09215, P01 AG009215, P01 AG009215-080006; NINDS NIH HHS: NS044233, NS053488, P01 NS044233, P50 NS053488, P50 NS053488-020003

    Biochemistry 2008;47;47;12614-25

  • Cardiac sympathetic denervation in Parkinson's disease linked to SNCA duplication.

    Orimo S, Uchihara T, Nakamura A, Mori F, Ikeuchi T, Onodera O, Nishizawa M, Ishikawa A, Kakita A, Wakabayashi K and Takahashi H

    Acta neuropathologica 2008;116;5;575-7

  • High level of alpha-synuclein mRNA in peripheral lymphocytes of patients with alcohol dependence syndrome.

    Taraskina AE, Filimonov VA, Kozlovskaya YA, Morozova MN, Gaschin DV and Schwarzman AL

    St. Petersburg Institute of Nuclear Physics named after B. P. Konstantinov, Russian Academy of Sciences, Leningrad Region, Gatchina, Russia. ataraskina@mail.ru

    The content of mRNA for alpha-synuclein (a key protein of the dopaminergic system) was elevated in the peripheral lymphocytes of patients with alcohol dependence syndrome. Increased level of alpha-synuclein mRNA was not associated with changes in the expression of NR4A2 gene encoding Nurrl, one of the main transcription factors of dopaminergic neurons.

    Bulletin of experimental biology and medicine 2008;146;5;609-11

  • Multiple alpha-synuclein gene polymorphisms are associated with Parkinson's disease in a Norwegian population.

    Myhre R, Toft M, Kachergus J, Hulihan MM, Aasly JO, Klungland H and Farrer MJ

    Department of Laboratory Medicine, Children's and Women's Health, Norwegian University of Science and Technology, Trondheim, Norway. ronny.myhre@ntnu.no

    Objectives: Previous studies have found associations between Parkinson's disease (PD) and polymorphisms located within both the alpha-synuclein gene (SNCA) promoter and other gene regions. Our aim was to study SNCA gene markers in a closely matched Norwegian PD population to examine the genetic relationship between different polymorphisms associated with the disease.

    Methods: We genotyped seven single nucleotide polymorphisms (SNPs) located in the SNCA promoter and two SNPs in the 3' gene region and seven microsatellite markers located across the gene in a closely matched series of 236 PD patients and 236 controls. Linkage disequilibrium (LD) structure was examined, and association of single markers and gene haplotypes analyzed.

    Results: Several markers located across the SNCA gene were associated with PD, including marker alleles associated with disease in previous studies (Rep1 263-bp allele, rs356165 and rs356219).

    Conclusion: LD between associated marker alleles located across the SNCA gene suggests that a single genetic effect might explain the previous reported association in the promoter and 3' regions.

    Acta neurologica Scandinavica 2008;118;5;320-7

  • Proteomics analysis identifies phosphorylation-dependent alpha-synuclein protein interactions.

    McFarland MA, Ellis CE, Markey SP and Nussbaum RL

    National Institute of Mental Health, National Institutes of Health, Bethesda, Maryland 20891, USA.

    Mutations and copy number variation in the SNCA gene encoding the neuronal protein alpha-synuclein have been linked to familial Parkinson disease (Thomas, B., and Beal, M. F. (2007) Parkinson's disease. Hum. Mol. Genet. 16, R183-R194). The carboxyl terminus of alpha-synuclein can be phosphorylated at tyrosine 125 and serine 129, although only a small fraction of the protein is phosphorylated under normal conditions (Okochi, M., Walter, J., Koyama, A., Nakajo, S., Baba, M., Iwatsubo, T., Meijer, L., Kahle, P. J., and Haass, C. (2000) Constitutive phosphorylation of the Parkinson's disease associated alpha-synuclein. J. Biol. Chem. 275, 390-397). Under pathological conditions, such as in Parkinson disease, alpha-synuclein is a major component of Lewy bodies, a pathological hallmark of Parkinson disease, and is mostly phosphorylated at Ser-129 (Anderson, J. P., Walker, D. E., Goldstein, J. M., de Laat, R., Banducci, K., Caccavello, R. J., Barbour, R., Huang, J. P., Kling, K., Lee, M., Diep, L., Keim, P. S., Shen, X. F., Chataway, T., Schlossmacher, M. G., Seubert, P., Schenk, D., Sinha, S., Gai, W. P., and Chilcote, T. J. (2006) Phosphorylation of Ser-129 is the dominant pathological modification of alpha-synuclein in familial and sporadic Lewy body disease. J. Biol. Chem. 281, 29739-29752). Controversy exists over the extent to which phosphorylation of alpha-synuclein and/or the visible protein aggregation in Lewy bodies are steps in disease pathogenesis, are protective, or are neutral markers for the disease process. Here we used the combination of peptide pulldown assays and mass spectrometry to identify and compare protein-protein interactions of phosphorylated and non-phosphorylated alpha-synuclein. We showed that non-phosphorylated alpha-synuclein carboxyl terminus pulled down protein complexes that were highly enriched for mitochondrial electron transport proteins, whereas alpha-synuclein carboxyl terminus phosphorylated on either Ser-129 or Tyr-125 did not. Instead the set of proteins pulled down by phosphorylated alpha-synuclein was highly enriched in certain cytoskeletal proteins, in vesicular trafficking proteins, and in a small number of enzymes involved in protein serine phosphorylation. This targeted comparative proteomics approach for unbiased identification of protein-protein interactions suggests that there are functional consequences when alpha-synuclein is phosphorylated.

    Funded by: Intramural NIH HHS; NIMH NIH HHS: Z01 MH000279

    Molecular & cellular proteomics : MCP 2008;7;11;2123-37

  • Structural characteristics of alpha-synuclein oligomers stabilized by the flavonoid baicalein.

    Hong DP, Fink AL and Uversky VN

    Department of Chemistry and Biochemistry, University of California at Santa Cruz, Santa Cruz, CA 95064, USA.

    The flavonoid baicalein inhibits fibrillation of alpha-synuclein, which is a major component of Lewy bodies in Parkinson's disease. It has been known that baicalein induces the formation of alpha-synuclein oligomers and consequently prevents their fibrillation. In order to evaluate the structural properties of baicalein-stabilized oligomers, we purified oligomer species by HPLC and examined their stability and structure by CD, Fourier transform infrared spectroscopy, size exclusion chromatography HPLC, small-angle X-ray scattering, and atomic force microscopy. Baicalein-stabilized oligomers are beta-sheet-enriched according to CD and Fourier transform infrared spectroscopy analyses. They did not form fibrils even after very prolonged incubation. From small-angle X-ray scattering data and atomic force microscopy images, the oligomers were characterized as quite compact globular species. Oligomers were extremely stable, with a GdmCl C(m)=3.3 M. This high stability explains the previously observed inhibition properties of baicalein against alpha-synuclein fibrillation. These baicalein-stabilized oligomers, added to the solution of aggregating alpha-synuclein, were able to noticeably inhibit its fibrillation. After prolonged coincubation, short fibrils were formed, suggesting an effective interaction of oligomers with monomeric alpha-synuclein. Membrane permeability tests suggested that the baicalein-stabilized oligomers had a mild effect on the integrity of the membrane surface. This effect was rather similar to that of the monomeric protein, suggesting that targeted stabilization of certain alpha-synuclein oligomers might offer a potential strategy for the development of novel Parkinson's disease therapies.

    Funded by: NIGMS NIH HHS: GM071714-01A2, R01 GM071714-01A2; NINDS NIH HHS: R01 NS039985-02S1, R01 NS39985; NLM NIH HHS: R01 LM007688-01A1, R01 LM007688-01A1S1

    Journal of molecular biology 2008;383;1;214-23

  • N-terminal region of alpha-synuclein is essential for the fatty acid-induced oligomerization of the molecules.

    Karube H, Sakamoto M, Arawaka S, Hara S, Sato H, Ren CH, Goto S, Koyama S, Wada M, Kawanami T, Kurita K and Kato T

    Department of Neurology, Yamagata University, 2-2-2 Iida-Nishi, Yamagata 990-9585, Japan.

    Exposure of alpha-synuclein (alphaS), a major component of Lewy bodies in Parkinson's disease, to polyunsaturated fatty acids (PUFAs) triggers the formation of soluble alphaS oligomers. Here, we demonstrate that PUFA binds recombinant alphaS protein through its N-terminal region (residues 2-60). In HEK293 cells, alphaS mutants lacking the N-terminal region failed to form oligomers in the presence of PUFA. The PUFA-induced alphaS oligomerization was accelerated by C-terminal truncation or Ser129 phosphorylation of alphaS; however, this effect was abolished by deletion of the N-terminus. The results indicate that the N-terminus of alphaS is essential for the PUFA-induced alphaS oligomerization.

    FEBS letters 2008;582;25-26;3693-700

  • Prominent psychiatric symptoms and glucose hypometabolism in a family with a SNCA duplication.

    Uchiyama T, Ikeuchi T, Ouchi Y, Sakamoto M, Kasuga K, Shiga A, Suzuki M, Ito M, Atsumi T, Shimizu T and Ohashi T

    Department of Neurology, Seirei Hamamatsu General Hospital, Japan.

    Neurology 2008;71;16;1289-91

  • Genetic association between alpha-synuclein and idiopathic Parkinson's disease.

    Kay DM, Factor SA, Samii A, Higgins DS, Griffith A, Roberts JW, Leis BC, Nutt JG, Montimurro JS, Keefe RG, Atkins AJ, Yearout D, Zabetian CP and Payami H

    New York State Department of Health, Division of Genetic Disorders, Wadsworth Center, Albany, New York 12208, USA.

    Point mutations and copy number variations in SNCA, the gene encoding alpha-synuclein, cause familial Parkinson's disease (PD). A dinucleotide polymorphism (REP1) in the SNCA promoter may be a risk factor for common forms of PD. We studied 1,802 PD patients and 2,129 controls from the NeuroGenetics Research Consortium, using uniform, standardized protocols for diagnosis, subject recruitment, data collection, genotyping, and data analysis. Three common REP1 alleles (257, 259, and 261 bp, with control frequencies of 0.28, 0.65, and 0.06) and several rare alleles (combined frequency <0.01) were detected. We confirmed association of REP1 with PD risk [odds ratio (OR) = 0.86, P = 0.006 for 257-carriers; OR = 1.25, P = 0.022 for 261-carriers]. Using a normalization procedure, we showed that the 257 and 261 alleles are both independently associated with PD risk (for 257, P = 0.002 in overall data, 0.003 in non-familial PD, 0.001 in early-onset PD; for 261, P = 0.056 in overall data, 0.024 in non-familial PD, 0.052 in early-onset PD). The 257-associated risk was consistent with a dominant model [hazard ratio (HR) = 0.99, P = 0.91 for 257/257 vs. 257/X where X denotes all other common alleles; HR = 1.16, P = 0.004 for X/X vs. 257/X]. The 261-associated risk was consistent with a recessive model (HR = 1.89, P = 0.026 for 261/261 vs. 261/X; HR = 0.95, P = 0.42 for X/X vs. 261/X). Genotype-specific mean onset ages (+/-SD) ranged from 54.8 +/- 12.1 for 261/261 to 59.4 +/- 11.5 for 257/257, displaying a trend of decreasing onset age with increasing allele size (P = 0.055). Genetic variation in SNCA and its regulatory regions play an important role in both familial and sporadic PD.

    Funded by: NIA NIH HHS: AG 08017; NINDS NIH HHS: K08-NS044138, NS R01-36960

    American journal of medical genetics. Part B, Neuropsychiatric genetics : the official publication of the International Society of Psychiatric Genetics 2008;147B;7;1222-30

  • A systematic RNAi screen reveals involvement of endocytic pathway in neuronal dysfunction in alpha-synuclein transgenic C. elegans.

    Kuwahara T, Koyama A, Koyama S, Yoshina S, Ren CH, Kato T, Mitani S and Iwatsubo T

    Department of Neuropathology and Neuroscience, Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo, Japan.

    Mutations or multiplications in alpha-synuclein gene cause familial forms of Parkinson disease or dementia with Lewy bodies (LB), and the deposition of wild-type alpha-synuclein as LB occurs as a hallmark lesion of these disorders, collectively referred to as synucleinopathies, implicating alpha-synuclein in the pathogenesis of synucleinopathy. To identify modifier genes of alpha-synuclein-induced neurotoxicity, we conducted an RNAi screen in transgenic C. elegans (Tg worms) that overexpress human alpha-synuclein in a pan-neuronal manner. To enhance the RNAi effect in neurons, we crossed alpha-synuclein Tg worms with an RNAi-enhanced mutant eri-1 strain. We tested RNAi of 1673 genes related to nervous system or synaptic functions, and identified 10 genes that, upon knockdown, caused severe growth/motor abnormalities selectively in alpha-synuclein Tg worms. Among these were four genes (i.e. apa-2, aps-2, eps-8 and rab-7) related to the endocytic pathway, including two subunits of AP-2 complex. Consistent with the results by RNAi, crossing alpha-synuclein Tg worms with an aps-2 mutant resulted in severe growth arrest and motor dysfunction. alpha-Synuclein Tg worms displayed a decreased touch sensitivity upon RNAi of genes involved in synaptic vesicle endocytosis, and they also showed impaired neuromuscular transmission, suggesting that overexpression of alpha-synuclein caused a failure in uptake or recycling of synaptic vesicles. Furthermore, knockdown of apa-2, an AP-2 subunit, caused an accumulation of phosphorylated alpha-synuclein in neuronal cell bodies, mimicking synucleinopathy. Collectively, these findings raise a novel pathogenic link between endocytic pathway and alpha-synuclein-induced neurotoxicity in synucleinopathy.

    Human molecular genetics 2008;17;19;2997-3009

  • alpha-synuclein degradation by autophagic pathways: a potential key to Parkinson's disease pathogenesis.

    Xilouri M, Vogiatzi T, Vekrellis K and Stefanis L

    Division of Basic Neurosciences, Biomedical Research Foundation of the Academy of Athens, Athens, Greece.

    The neuronal protein alpha-synuclein is thought to be central in the pathogenesis of Parkinson's disease (PD). Excessive wild type alpha-synuclein levels can lead to PD in select familial cases and alpha-synuclein protein accumulation occurs in sporadic PD. Therefore, elucidation of the mechanisms that control alpha-synuclein levels is critical for PD pathogenesis and potential therapeutics. The subject of alpha-synuclein degradation has been controversial. Previous work shows that, in an assay with isolated liver lysosomes, purified wild type alpha-synuclein is degraded by the process of chaperone-mediated autophagy (CMA). Whether this actually occurs in a cellular context has been unclear. In our most recent work, we find that wild type alpha-synuclein, but not the closely related protein beta-synuclein, is indeed degraded by CMA in neuronal cells, including primary postnatal ventral midbrain neurons. Macroautophagy, but not the proteasome, also contributes to alpha-synuclein degradation. Therefore, two separate lysosomal pathways, CMA and macroautophagy, degrade wild type alpha-synuclein in neuronal cells. It is hypothesized that impairment of either of these two pathways, or of more general lysosomal function, may be an initiating factor in alpha-synuclein accumulation and sporadic PD pathogenesis.

    Funded by: NINDS NIH HHS: R21 NS055693

    Autophagy 2008;4;7;917-9

  • Analysis of PARK genes in a Korean cohort of early-onset Parkinson disease.

    Choi JM, Woo MS, Ma HI, Kang SY, Sung YH, Yong SW, Chung SJ, Kim JS, Shin HW, Lyoo CH, Lee PH, Baik JS, Kim SJ, Park MY, Sohn YH, Kim JH, Kim JW, Lee MS, Lee MC, Kim DH and Kim YJ

    Department of Neurology, Hallym University Sacred Heart Hospital, ILSONG Institute of Life Science, Hallym University, Dongan-gu, Anyang-si, Gyeonggi-do, Korea.

    Mutations in five PARK genes (SNCA, PARKIN, DJ-1, PINK1, and LRRK2) are well-established genetic causes of Parkinson disease (PD). Recently, G2385R substitution in LRRK2 has been determined as a susceptibility allele in Asian PD. The objective of this study is to determine the frequency of mutations in these PARK genes in a Korean early-onset Parkinson disease (EOPD) cohort. The authors sequenced 35 exons in SNCA, PARKIN, DJ-1, PINK1, and LRRK2 in 72 unrelated EOPD (age-at-onset <or=50) recruited from ten movement disorders clinics in South Korea. Gene dosage change of the aforementioned genes was studied using multiple ligation-dependent probe amplification. We found four patients with PARKIN mutations, which were homozygous deletion of exon 4, compound heterozygous deletion of exon 2 and exon 4, heterozygous deletion of exon 4, and heterozygous nonsense mutation (Q40X). Four patients had PINK1 mutations; a compound heterozygous mutation (N367S and K520RfsX522) and three heterozygous mutations (G32R, R279H, and F385L). A missense mutation of SNCA (A53T) was found in a familial PD with autosomal dominant inheritance. Nine patients (12.5%) had heterozygous G2385R polymorphism of LRRK2, whereas none had G2019S mutation. However, no mutations were detected in DJ-1 and UCHL1 in our series. We identified genetic variants in PARKIN, PINK1, LRRK2, and SNCA as a cause or genetic risk factors for PD in 25% of Korean EOPD, and mutation of PARKIN was the most common genetic cause.

    Neurogenetics 2008;9;4;263-9

  • Cerebellar alpha-synuclein levels are decreased in Parkinson's disease and do not correlate with SNCA polymorphisms associated with disease in a Swedish material.

    Westerlund M, Belin AC, Anvret A, Håkansson A, Nissbrandt H, Lind C, Sydow O, Olson L and Galter D

    Department of Neuroscience, Retzius väg 8, Karolinska Institutet, 171 77 Stockholm, Sweden.

    Alterations of brain and plasma alpha-synuclein levels and SNCA gene variability have been implicated in the pathogenesis of Parkinson's disease (PD). We therefore measured alpha-synuclein protein levels in postmortem PD and control cerebellum tissue using Western blot and investigated whether the levels correlated to SNCA genotype. We found markedly decreased alpha-synuclein levels in PD patients (n=16) compared to gender- and age-matched controls (n=14; P=0.004) normalized to alpha-tubulin. We also performed an association study of the noncoding polymorphisms rs2737029 (A/G) and rs356204 (A/G) (intron 4), and of rs356219 (T/C) (3'-region) of SNCA in a Swedish PD case-control material. Using a two-sided chi(2) test, we found significant association of rs2737029 (P=0.003; chi(2)=9.07) and rs356204 (P=0.048; chi(2)=3.91) with disease, strengthening the involvement of SNCA polymorphisms in sporadic PD. Stratification of the human postmortem brain material by genotype of the three investigated polymorphisms, did not indicate any influence of genotype on alpha-synuclein protein levels when comparing PD with controls. Taken together, our findings demonstrate that the investigated Parkinson patients have markedly reduced levels of alpha-synuclein in cerebellum, and that this reduction is general, rather then correlated to the investigated polymorphisms, although two of the polymorphisms also associated with disease in a Swedish material.

    FASEB journal : official publication of the Federation of American Societies for Experimental Biology 2008;22;10;3509-14

  • Direct quantification of CSF alpha-synuclein by ELISA and first cross-sectional study in patients with neurodegeneration.

    Mollenhauer B, Cullen V, Kahn I, Krastins B, Outeiro TF, Pepivani I, Ng J, Schulz-Schaeffer W, Kretzschmar HA, McLean PJ, Trenkwalder C, Sarracino DA, Vonsattel JP, Locascio JJ, El-Agnaf OM and Schlossmacher MG

    Center for Neurologic Diseases, Department of Neurology, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA.

    Because accumulation of alpha-synuclein (alphaS) in the brain is a hallmark of Parkinson disease (PD) and related disorders, we examined its occurrence in human cerebrospinal fluid (CSF). Following affinity enrichment and trypsin digestion of CSF collected from a neurologically healthy donor, we identified several alphaS-derived peptides by mass spectrometry. The concentration of alphaS amounted to <0.001% of the CSF proteome. We then built, validated and optimized a sandwich-type, enzyme-linked immunoadsorbent assay (ELISA) to measure total alphaS levels in unconcentrated CSF. In a cross-sectional study of 100 living donors, we examined cell-free CSF samples from subjects clinically diagnosed with advanced PD, dementia with Lewy bodies (DLB), Alzheimer disease (AD), and a group of non-neurodegenerative disease controls (NCO). In these four groups the CSF alphaS concentrations ranged from 0.8 to 16.2 pg/microl. Mean CSF alphaS values were lower in donors with a primary synucleinopathy (PD, DLB: n=57) than in the other two groups (AD, NCO: n=35; p=0.025). By contrast, living Creutzfeldt-Jakob disease patients showed markedly elevated CSF alphaS levels (n=8; mean, 300 pg/microl; p<0.001). Our results unequivocally confirm the presence of alphaS in adult human CSF. In a first feasibility study employing a novel ELISA, we found relatively low CSF alphaS concentrations in subjects with parkinsonism linked to synucleinopathy, PD and DLB. In definite prion disease cases, we recorded a marked rise in total CSF alphaS resulting from rapid cell death. Our results will likely aid future biomarker explorations in neurodegenerative conditions and facilitate target validation studies.

    Funded by: NINDS NIH HHS: 5P50-NS038375, P50 NS038375-06A10002

    Experimental neurology 2008;213;2;315-25

  • Impact of Tyr to Ala mutations on alpha-synuclein fibrillation and structural properties.

    Ulrih NP, Barry CH and Fink AL

    Department of Chemistry and Biochemistry, University of California, Santa Cruz, CA 95964, USA. natasa.poklar@bf.uni-lj.si

    Substantial evidence suggests that the fibrillation of alpha-synuclein is a critical step in the development of Parkinson's disease. In vitro, alpha-synuclein forms fibrils with morphologies and a staining characteristic similar to those extracted from disease-affected brain. Monomeric alpha-synuclein is an intrinsically disordered protein, with three Tyr residues in the C-terminal region, one in the N-terminus, and lacking Trp. It is thought that interactions between the C-terminus and the central portion of the molecule may prevent or minimize aggregation/fibrillation. To test this hypothesis we examined the importance of the Tyr residues on the propensity for alpha-synuclein to fibrillate in vitro. Fibril formation of alpha-synuclein was completely inhibited, in the timescale over which measurements were made, by replacing the three C-terminal Tyr residues with Ala. In addition, substitution of Tyr133 by Ala also resulted in the absence of fibrillation, whereas the individual Y125A and Y136A mutants showed limited inhibition. Replacement of Tyr39 by Ala also resulted in substantial inhibition of fibrillation. Structural analysis showed that the Y133A mutant had a substantially different conformation, rich in alpha-helical secondary structure, as compared with the wild-type and other mutants, although the formation of any tertiary structure has not been observed as can be judged from near-UV-CD spectra. These observations suggest that the long-range intramolecular interactions between the N- and C-termini of alpha-synuclein are likely to be crucial to the fibrillation process.

    Biochimica et biophysica acta 2008;1782;10;581-5

  • Phosphorylation, lipid raft interaction and traffic of alpha-synuclein in a yeast model for Parkinson.

    Zabrocki P, Bastiaens I, Delay C, Bammens T, Ghillebert R, Pellens K, De Virgilio C, Van Leuven F and Winderickx J

    Laboratory of Functional Biology, Kasteelpark Arenberg 31, 3001 Heverlee, Belgium.

    Parkinson's disease is a neurodegenerative disorder characterized by the formation of Lewy bodies containing aggregated alpha-synuclein. We used a yeast model to screen for deletion mutants with mislocalization and enhanced inclusion formation of alpha-synuclein. Many of the mutants were affected in functions related to vesicular traffic but especially mutants in endocytosis and vacuolar degradation combined inclusion formation with enhanced alpha-synuclein-mediated toxicity. The screening also allowed for identification of casein kinases responsible for alpha-synuclein phosphorylation at the plasma membrane as well as transacetylases that modulate the alpha-synuclein membrane interaction. In addition, alpha-synuclein was found to associate with lipid rafts, a phenomenon dependent on the ergosterol content. Together, our data suggest that toxicity of alpha-synuclein in yeast is at least in part associated with endocytosis of the protein, vesicular recycling back to the plasma membrane and vacuolar fusion defects, each contributing to the obstruction of different vesicular trafficking routes.

    Biochimica et biophysica acta 2008;1783;10;1767-80

  • Cathepsin D is the main lysosomal enzyme involved in the degradation of alpha-synuclein and generation of its carboxy-terminally truncated species.

    Sevlever D, Jiang P and Yen SH

    Department of Neuroscience, Mayo Clinic College of Medicine, 4500 San Pablo Road, Jacksonville, Florida 32224, USA. sevlever.daniel@mayo.edu

    Alpha-synuclein is likely to play a key role in the development of Parkinson's disease as well as other synucleinopathies. In animal models, overexpression of full-length or carboxy-terminally truncated alpha-synuclein has been shown to produce pathology. Although the proteosome and lysosome have been proposed to play a role in the degradation of alpha-synuclein, the enzyme(s) involved in alpha-synuclein clearance and generation of its carboxy-terminally truncated species have not been identified. In this study, the role of cathepsin D and calpain I in these processes was analyzed. In vitro experiments, using either recombinant or endogenous alpha-synuclein as substrates and purified cathepsin D or lysosomes, demonstrated that cathepsin D degraded alpha-synuclein very efficiently, and that limited proteolysis resulted in the generation of carboxy-terminally truncated species. Purified calpain I also cleaved alpha-synuclein, but carboxy-terminally truncated species were not the main cleavage products, and calpain I activity present in cellular lysates was not able to degrade the protein. Knockdown of cathepsin D in cells overexpressing wild-type alpha-synuclein increased total alpha-synuclein levels by 28% and lysosomal alpha-synuclein by 2-fold. In in vitro experiments, pepstatin A completely blocked the degradation of alpha-synuclein in purified lysosomes. Furthermore, lysosomes isolated from cathepsin D knockdown cells showed a marked reduction in alpha-synuclein degrading activity, indicating that cathepsin D is the main lysosomal enzyme involved in alpha-synuclein degradation. Our findings suggest that upregulation of cathepsin D could be an additional therapeutic strategy to lessen alpha-synuclein burden in synucleinopathies.

    Funded by: NINDS NIH HHS: P50 NS040256, P50 NS040256-100006, P50-NS40256

    Biochemistry 2008;47;36;9678-87

  • Hsp104 antagonizes alpha-synuclein aggregation and reduces dopaminergic degeneration in a rat model of Parkinson disease.

    Lo Bianco C, Shorter J, Régulier E, Lashuel H, Iwatsubo T, Lindquist S and Aebischer P

    Wallenberg Neuroscience Center, Division of Neurobiology, Department of Experimental Medical Science, Lund University, Lund, Sweden.

    Parkinson disease (PD) is characterized by dopaminergic neurodegeneration and intracellular inclusions of alpha-synuclein amyloid fibers, which are stable and difficult to dissolve. Whether inclusions are neuroprotective or pathological remains controversial, because prefibrillar oligomers may be more toxic than amyloid inclusions. Thus, whether therapies should target inclusions, preamyloid oligomers, or both is a critically important issue. In yeast, the protein-remodeling factor Hsp104 cooperates with Hsp70 and Hsp40 to dissolve and reactivate aggregated proteins. Metazoans, however, have no Hsp104 ortholog. Here we introduced Hsp104 into a rat PD model. Remarkably, Hsp104 reduced formation of phosphorylated alpha-synuclein inclusions and prevented nigrostriatal dopaminergic neurodegeneration induced by PD-linked alpha-synuclein (A30P). An in vitro assay employing pure proteins revealed that Hsp104 prevented fibrillization of alpha-synuclein and PD-linked variants (A30P, A53T, E46K). Hsp104 coupled ATP hydrolysis to the disassembly of preamyloid oligomers and amyloid fibers composed of alpha-synuclein. Furthermore, the mammalian Hsp70 and Hsp40 chaperones, Hsc70 and Hdj2, enhanced alpha-synuclein fiber disassembly by Hsp104. Hsp104 likely protects dopaminergic neurons by antagonizing toxic alpha-synuclein assemblies and might have therapeutic potential for PD and other neurodegenerative amyloidoses.

    Funded by: NIH HHS: DP2 OD002177, DP2OD002177

    The Journal of clinical investigation 2008;118;9;3087-97

  • Neurofibrillary tau pathology modulated by genetic variation of alpha-synuclein.

    Peuralinna T, Oinas M, Polvikoski T, Paetau A, Sulkava R, Niinistö L, Kalimo H, Hernandez D, Hardy J, Singleton A, Tienari PJ and Myllykangas L

    Molecular Neurology Programme, Biomedicum, University of Helsinki, Helsinki, Finland.

    We analyzed whether genetic variation of alpha-synuclein modulates the extent of neuropathological changes in a population-based autopsied sample of 272 elderly Finns. None of the 11 markers was associated with the extent of neocortical beta-amyloid pathology. The intron 4 marker rs2572324 was associated with the extent of neurofibrillary pathology (p = 0.0006, permuted p = 0.004; Braak stages IV-VI vs 0-II). The same variant also showed a trend for association with neocortical Lewy-related pathology. These results suggest for the first time that variation of alpha-synuclein modulates neurofibrillary tau pathology and support the recent observations of an interaction of alpha-synuclein and tau in neurodegeneration.

    Funded by: Medical Research Council: G0701075; Parkinson's UK: G-0907

    Annals of neurology 2008;64;3;348-52

  • Wild type alpha-synuclein is degraded by chaperone-mediated autophagy and macroautophagy in neuronal cells.

    Vogiatzi T, Xilouri M, Vekrellis K and Stefanis L

    Division of Basic Neurosciences, Biomedical Research Foundation of the Academy of Athens, Soranou Efesiou 4, Athens, Greece.

    Alpha-synuclein (ASYN) is crucial in Parkinson disease (PD) pathogenesis. Increased levels of wild type (WT) ASYN expression are sufficient to cause PD in humans. The manner of post-transcriptional regulation of ASYN levels is controversial. Previously, we had shown that WT ASYN can be degraded by chaperone-mediated autophagy (CMA) in isolated liver lysosomes. Whether this occurs in a cellular and, in particular, in a neuronal cell context is unclear. Using a mutant ASYN form that lacks the CMA recognition motif and RNA interference against the rate-limiting step in the CMA pathway, Lamp2a, we show here that CMA is indeed involved in WT ASYN degradation in PC12 and SH-SY5Y cells, and in primary cortical and midbrain neurons. However, the extent of involvement varies between cell types, potentially because of differences in compensatory mechanisms. CMA inhibition leads to an accumulation of soluble high molecular weight and detergent-insoluble species of ASYN, suggesting that CMA dysfunction may play a role in the generation of such aberrant species in PD. ASYN and Lamp2a are developmentally regulated in parallel in cortical neuron cultures and in vivo in the central nervous system, and they physically interact as indicated by co-immunoprecipitation. In contrast to previous reports, inhibition of macroautophagy, but not the proteasome, also leads to WT ASYN accumulation, suggesting that this lysosomal pathway is also involved in normal ASYN turnover. These results indicate that CMA and macroautophagy are important pathways for WT ASYN degradation in neurons and underline the importance of CMA as degradation machinery in the nervous system.

    Funded by: NINDS NIH HHS: R21 NS 055693

    The Journal of biological chemistry 2008;283;35;23542-56

  • Serine 129 phosphorylation of alpha-synuclein induces unfolded protein response-mediated cell death.

    Sugeno N, Takeda A, Hasegawa T, Kobayashi M, Kikuchi A, Mori F, Wakabayashi K and Itoyama Y

    Department of Neurology, Tohoku University School of Medicine, 1-1 Seiryomachi, Aobaku, Sendai, Miyagi 980-8574, Japan.

    alpha-Synuclein is a major protein component deposited in Lewy bodies and Lewy neurites that is extensively phosphorylated at Ser(129), although its role in neuronal degeneration is still elusive. In this study, several apoptotic pathways were examined in alpha-synuclein-overexpressing SH-SY5Y cells. Following the treatment with rotenone, a mitochondrial complex I inhibitor, wild type alpha-synuclein-overexpressing cells demonstrated intracellular aggregations, which shared a number of features with Lewy bodies, although cells overexpressing the S129A mutant, in which phosphorylation at Ser(129) was blocked, showed few aggregations. In wild typealpha-synuclein cells treated with rotenone, the proportion of phosphorylated alpha-synuclein was about 1.6 times higher than that of untreated cells. Moreover, induction of unfolded protein response (UPR) markers was evident several hours before the induction of mitochondrial disruption and caspase-3 activation. Eukaryotic initiation factor 2alpha, a member of the PERK pathway family, was remarkably activated at early phases. On the other hand, the S129A mutant failed to activate UPR. Casein kinase 2 inhibitor, which decreased alpha-synuclein phosphorylation, also reduced UPR activation. The alpha-synuclein aggregations were colocalized with a marker for the endoplasmic reticulum-Golgi intermediate compartment. Taken together, it seems plausible that alpha-synuclein toxicity is dependent on the phosphorylation at Ser(129) that induces the UPRs, possibly triggered by the disturbed endoplasmic reticulum-Golgi trafficking.

    The Journal of biological chemistry 2008;283;34;23179-88

  • Microglial phagocytosis is enhanced by monomeric alpha-synuclein, not aggregated alpha-synuclein: implications for Parkinson's disease.

    Park JY, Paik SR, Jou I and Park SM

    Chronic Inflammatory Disease Research Center, Ajou University School of Medicine, Suwon, Korea.

    Gathering evidence has associated activation of microglia with the pathogenesis of numerous neurodegenerative diseases of the central nervous system (CNS) such as Alzheimer's disease and Parkinson's disease. Microglia are the resident macrophages of the CNS whose functions include chemotaxis, phagocytosis, and secretion of a variety of cytokines and proteases. In this study, we examined the possibility that alpha-synuclein (alpha-syn), which is associated with the pathogenesis of Parkinson's disease, may affect the phagocytic function of microglia. We found that extracellular monomeric alpha-syn enhanced microglial phagocytosis in both a dose- and time-dependent manner, but beta- and gamma- syn did not. We also found that the N-terminal and NAC region of alpha-syn, especially the NAC region, might be responsible for the effect of alpha-syn on microglial phagocytosis. In contrast to monomeric alpha-syn, aggregated alpha-syn actually inhibited microglial phagocytosis. The different effects of monomeric and aggregated alpha-syn on phagocytosis might be related to their localization in cells. This study indicates that alpha-syn can modulate the function of microglia and influence inflammatory changes such as those seen in neurodegenerative disorders.

    Glia 2008;56;11;1215-23

  • Clearance and deposition of extracellular alpha-synuclein aggregates in microglia.

    Lee HJ, Suk JE, Bae EJ and Lee SJ

    Department of Biomedical Science and Technology, RCTC, IBST, Konkuk University, 1 Hwayang-dong, Gwangjin-gu, Seoul 143-701, Republic of Korea.

    Abnormal deposition of alpha-synuclein in neurons and glia is implicated in many neurological diseases, such as Parkinson's disease and Dementia with Lewy bodies. Recently, evidence has emerged that this protein and its aggregates are secreted from neuronal cells, and this extracellular protein may contribute to the pathogenic process. Here, we show that all the major brain cell types (neurons, astrocytes, and microglia) are capable of clearing the extracellular alpha-synuclein aggregates by internalization and degradation. Among these cell types, microglia showed the highest rate of degradation. Upon activation by lipopolysaccharide, the degradation of the internalized alpha-synuclein aggregates was slowed, causing protein accumulation in the microglial cytoplasm. These results suggest that microglia may be the major scavenger cells for extracellular alpha-synuclein aggregates in brain parenchyma, and that clearance may be regulated by the activation state of these cells.

    Biochemical and biophysical research communications 2008;372;3;423-8

  • Calbindin 1, fibroblast growth factor 20, and alpha-synuclein in sporadic Parkinson's disease.

    Mizuta I, Tsunoda T, Satake W, Nakabayashi Y, Watanabe M, Takeda A, Hasegawa K, Nakashima K, Yamamoto M, Hattori N, Murata M and Toda T

    Division of Clinical Genetics, Department of Medical Genetics, Osaka University Graduate School of Medicine, 2-2-B9 Yamadaoka, Suita, Osaka, 565-0871, Japan.

    Parkinson's disease (PD), one of the most common human neurodegenerative disorders, is characterized by the loss of dopaminergic neurons in the substantia nigra of the midbrain. Our recent case-control association study of 268 SNPs in 121 candidate genes identified alpha-synuclein (SNCA) as a susceptibility gene for sporadic PD (P = 1.7 x 10(-11)). We also replicated the association of fibroblast growth factor 20 (FGF20) with PD (P = 0.0089). To find other susceptibility genes, we added 34 SNPs to the previous screen. Of 302 SNPs in a total 137 genes, but excluding SNCA, SNPs in NDUFV2, FGF2, CALB1 and B2M showed significant association (P < 0.01; 882 cases and 938 control subjects). We replicated the association analysis for these SNPs in a second independent sample set (521 cases and 1,003 control subjects). One SNP, rs1805874 in calbindin 1 (CALB1), showed significance in both analyses (P = 7.1 x 10(-5); recessive model). When the analysis was stratified relative to the SNCA genotype, the odds ratio of CALB1 tended to increase according to the number of protective alleles in SNCA. In contrast, FGF20 was significant only in the subgroup of SNCA homozygote of risk allele. CALB1 is a calcium-binding protein that widely is expressed in neurons. A relative sparing of CALB1-positive dopaminergic neurons is observed in PD brains, compared with CALB1-negative neurons. Our genetic analysis suggests that CALB1 is associated with PD independently of SNCA, and that FGF20 is associated with PD synergistically with SNCA.

    Human genetics 2008;124;1;89-94

  • Tissue transglutaminase modulates alpha-synuclein oligomerization.

    Segers-Nolten IM, Wilhelmus MM, Veldhuis G, van Rooijen BD, Drukarch B and Subramaniam V

    Biophysical Engineering Group, MESA+ Institute for Nanotechnology and Institute for Biomedical Technology, University of Twente, 7500 AE Enschede, The Netherlands.

    We have studied the interaction of the enzyme tissue transglutaminase (tTG), catalyzing cross-link formation between protein-bound glutamine residues and primary amines, with Parkinson's disease-associated alpha-synuclein protein variants at physiologically relevant concentrations. We have, for the first time, determined binding affinities of tTG for wild-type and mutant alpha-synucleins using surface plasmon resonance approaches, revealing high-affinity nanomolar equilibrium dissociation constants. Nanomolar tTG concentrations were sufficient for complete inhibition of fibrillization by effective alpha-synuclein cross-linking, resulting predominantly in intramolecularly cross-linked monomers accompanied by an oligomeric fraction. Since oligomeric species have a pathophysiological relevance we further investigated the properties of the tTG/alpha-synuclein oligomers. Atomic force microscopy revealed morphologically similar structures for oligomers from all alpha-synuclein variants; the extent of oligomer formation was found to correlate with tTG concentration. Unlike normal alpha-synuclein oligomers the resultant structures were extremely stable and resistant to GdnHCl and SDS. In contrast to normal beta-sheet-containing oligomers, the tTG/alpha-synuclein oligomers appear to be unstructured and are unable to disrupt phospholipid vesicles. These data suggest that tTG binds equally effective to wild-type and disease mutant alpha-synuclein variants. We propose that tTG cross-linking imposes structural constraints on alpha-synuclein, preventing the assembly of structured oligomers required for disruption of membranes and for progression into fibrils. In general, cross-linking of amyloid forming proteins by tTG may prevent the progression into pathogenic species.

    Protein science : a publication of the Protein Society 2008;17;8;1395-402

  • Neuroinflammation and oxidation/nitration of alpha-synuclein linked to dopaminergic neurodegeneration.

    Gao HM, Kotzbauer PT, Uryu K, Leight S, Trojanowski JQ and Lee VM

    Department of Pathology and Laboratory Medicine, Center for Neurodegenerative Disease Research, Institute on Aging, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA.

    alpha-Synuclein (SYN) is the major component of Lewy bodies, the neuropathological hallmarks of Parkinson's disease (PD). Missense mutations and multiplications of the SYN gene cause autosomal dominant inherited PD. Thus, SYN is implicated in the pathogenesis of PD. However, the mechanism whereby SYN promotes neurodegeneration remains unclear. Familial PD with SYN gene mutations are rare because the majority of PD is sporadic and emerging evidence indicates that sporadic PD may result from genetic and environmental risk factors including neuroinflammation. Hence, we examined the relationship between SYN dysfunction and neuroinflammation in mediating dopaminergic neurodegeneration in mice and dopaminergic neuronal cultures derived from wild-type SYN and mutant A53T SYN transgenic mice in a murine SYN-null (SYNKO) background (M7KO and M83KO, respectively). Stereotaxic injection of an inflammagen, lipopolysaccharide, into substantia nigra of these SYN genetically engineered mice induced similar inflammatory reactions. In M7KO and M83KO, but not in SYNKO mice, the neuroinflammation was associated with dopaminergic neuronal death and the accumulation of insoluble aggregated SYN as cytoplasmic inclusions in nigral neurons. Nitrated/oxidized SYN was detected in these inclusions and abatement of microglia-derived nitric oxide and superoxide provided significant neuroprotection in neuron-glia cultures from M7KO mice. These data suggest that nitric oxide and superoxide released by activated microglia may be mediators that link inflammation and abnormal SYN in mechanisms of PD neurodegeneration. This study advances understanding of the role of neuroinflammation and abnormal SYN in the pathogenesis of PD and opens new avenues for the discovery of more effective therapies for PD.

    Funded by: NIA NIH HHS: AG-P01-09215, AG-T32-00255, T32 AG000255-11A1

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2008;28;30;7687-98

  • Solid-state NMR reveals structural differences between fibrils of wild-type and disease-related A53T mutant alpha-synuclein.

    Heise H, Celej MS, Becker S, Riedel D, Pelah A, Kumar A, Jovin TM and Baldus M

    Department of NMR-Based Structural Biology, Max Planck Institute for Biophysical Chemistry, Solid-State NMR Group, Am Fassberg 11, D-37077 Göttingen, Germany. h.heise@fz-juelich.de

    Fibrils from the Parkinson's-disease-related A53T mutant of alpha-synuclein were investigated by solid-state NMR spectroscopy, electron microscopy, and atomic force microscopy. Sequential solid-state NMR resonance assignments were obtained for a large fraction of the fibril core. Experiments conducted above and below the freezing point suggest that the fibrils contain regions with increased mobility and structural elements different from beta-strand character, in addition to the rigid beta-sheet-rich core region. As in earlier studies on wild-type alpha-synuclein, the C-terminus was found to be flexible and unfolded, whereas the main core region was highly rigid and rich in beta-sheets. Compared to fibrils from wild-type alpha-synuclein, the well-ordered beta-sheet region extends to at least L38 and L100. These results demonstrate that a disease-related mutant of alpha-synuclein differs in both aggregation kinetics and fibril structure.

    Journal of molecular biology 2008;380;3;444-50

  • Differential expression of alpha-synuclein, parkin, and synphilin-1 isoforms in Lewy body disease.

    Beyer K, Domingo-Sàbat M, Humbert J, Carrato C, Ferrer I and Ariza A

    Department of Pathology, Hospital Germans Trias i Pujol, Autonomous University of Barcelona, Barcelona, Spain. katrinbeyer@hotmail.com

    Alpha-synuclein, parkin, and synphilin-1 are proteins mainly involved in the pathogenesis of Lewy body (LB) diseases. mRNAs of all three undergo alternative splicing, so that the existence of various isoforms has been described. Since increasing evidence supports the importance of differential isoform-expression changes in disease development, we have established isoform-expression profiles in frontal cortices of LB disease brains in comparison with those of Alzheimer disease (AD) and control frontal cortices. The differential expression of four alpha-synuclein, seven parkin, and four synphilin-1 isoforms was ascertained by the use of isoform-specific primers and relative expression analysis with SybrGreen and beta-actin as an internal standard. The establishment of isoform-expression profiles revealed that these are disease specific. Moreover, isoform-expression deregulation of mainly one gene in each disease could be observed. All four alpha-synuclein isoforms were affected in the case of the pure form of dementia with LB, most parkin transcript variants in common LB disease, and all synphilin-1 isoforms in Parkinson disease. Only minor involvement was detected in AD. Finally, the existence of a proprietary isoform-expression profile in common LB disease indicates that this disease develops as a result of its own molecular mechanisms, and so, at the molecular level, it does not exactly share changes found in pure dementia with LB and AD. In conclusion, isoform-expression profiles in LB diseases represent additional evidence for the direct involvement of isoform-expression deregulation in the development of neurodegenerative disorders.

    Neurogenetics 2008;9;3;163-72

  • FK506 binding protein 12 differentially accelerates fibril formation of wild type alpha-synuclein and its clinical mutants A30P or A53T.

    Gerard M, Debyser Z, Desender L, Baert J, Brandt I, Baekelandt V and Engelborghs Y

    Laboratory of Biomolecular Dynamics, K. U. Leuven, Flanders, Belgium.

    Aggregation of alpha-synuclein (alpha-SYN) plays a key role in Parkinson's disease. We have previously shown that aggregation of alpha-SYN in vitro is accelerated by addition of FK506 binding proteins (FKBP) and that this effect can be counteracted by FK506, a specific inhibitor of these enzymes. In this paper, we investigated in detail the effect of FKBP12 on early aggregation and on fibril formation of wild-type, A53T and A30P alpha-SYN. FKBP12 has a much smaller effect on the fibril formation of these two clinical mutants alpha-SYN. Using an inactive enzyme, we were able to discriminate between catalytic and non-catalytic effects that differentially influence the two processes. A model explaining non-linear concentration dependencies is proposed.

    Journal of neurochemistry 2008;106;1;121-33

  • Molecular understanding of copper and iron interaction with alpha-synuclein by fluorescence analysis.

    Bharathi and Rao KS

    Department of Biochemistry and Nutrition, Central Food Technological Research Institute, Mysore, India.

    Alpha-synuclein aggregation is a hallmark pathological feature in Parkinson's disease (PD). The conversion of alpha-synuclein from a soluble monomer to an insoluble fibril may underlie the neurodegeneration associated with PD. Redox-active metal ions such as iron (Fe) and copper (Cu) are known to enhance alpha-synuclein fibrillogenesis. In the present investigation, we analyzed the binding efficiency of Cu and Fe to alpha-synuclein by fluorescence studies. It is interesting to note that Cu and Fe showed differential binding pattern toward alpha-synuclein (wild type and A30P, A53T, and E46K mutant forms) as revealed by intrinsic tyrosine fluorescence, thioflavin-T fluorescence, 1-anilino-8-naphthalenesulfonate-binding studies, and scatchard plot analysis. The experimental data might prove useful in understanding the hierarchy of metals binding to alpha-synuclein and its role in neurodegeneration.

    Journal of molecular neuroscience : MN 2008;35;3;273-81

  • Olfactory deficits in mice overexpressing human wildtype alpha-synuclein.

    Fleming SM, Tetreault NA, Mulligan CK, Hutson CB, Masliah E and Chesselet MF

    Department of Neurology and Neurobiology, The David Geffen School of Medicine at UCLA, 710 Westwood Plaza, Los Angeles, CA 90095-1769, USA. sfleming@ucla.edu

    Accumulation of alpha-synuclein in neurons of the central and peripheral nervous system is a hallmark of sporadic Parkinson's disease (PD) and mutations that increase alpha-synuclein levels cause familial PD. Transgenic mice overexpressing alpha-synuclein under the Thy1 promoter (Thy1-aSyn) have high levels of alpha-synuclein expression throughout the brain but no loss of nigrostriatal dopamine neurons up to 8 months, suggesting that they may be useful to model pre-clinical stages of PD. Olfactory dysfunction often precedes the onset of the cardinal motor symptoms of PD by several years and includes deficits in odor detection, discrimination and identification. In the present study, we measured olfactory function in 3- and 9-month-old male Thy1-aSyn mice with a buried pellet test based on latency to find an exposed or hidden odorant, a block test based on exposure to self and non-self odors, and a habituation/dishabituation test based on exposure to non-social odors. In a separate group of mice, alpha-synuclein immunoreactivity was assessed in the olfactory bulb. Compared with wildtype littermates, Thy1-aSyn mice could still detect and habituate to odors but showed olfactory impairments in aspects of all three testing paradigms. Thy1-aSyn mice also displayed proteinase K-resistant alpha-synuclein inclusions throughout the olfactory bulb. These data indicate that overexpression of alpha-synuclein is sufficient to cause olfactory deficits in mice similar to that observed in patients with PD. Furthermore, the buried pellet and block tests provided sufficient power for the detection of a 50% drug effect, indicating their usefulness for testing novel neuroprotective therapies.

    Funded by: NINDS NIH HHS: P50 NS038367-07, P50NS38367

    The European journal of neuroscience 2008;28;2;247-56

  • CHIP targets toxic alpha-Synuclein oligomers for degradation.

    Tetzlaff JE, Putcha P, Outeiro TF, Ivanov A, Berezovska O, Hyman BT and McLean PJ

    Department of Neurology, MassGeneral Institute for Neurodegenerative Disease, Massachusetts General Hospital, Charlestown, MA 02129, USA.

    alpha-Synuclein (alphaSyn) can self-associate, forming oligomers, fibrils, and Lewy bodies, the pathological hallmark of Parkinson disease. Current dogma suggests that oligomeric alphaSyn intermediates may represent the most toxic alphaSyn species. Here, we studied the effect of a potent molecular chaperone, CHIP (carboxyl terminus of Hsp70-interacting protein), on alphaSyn oligomerization using a novel bimolecular fluorescence complementation assay. CHIP is a multidomain chaperone, utilizing both a tetratricopeptide/Hsp70 binding domain and a U-box/ubiquitin ligase domain to differentially impact the fate of misfolded proteins. In the current study, we found that co-expression of CHIP selectively reduced alphaSyn oligomerization and toxicity in a tetratricopeptide domain-dependent, U-box-independent manner by specifically degrading toxic alphaSyn oligomers. We conclude that CHIP preferentially recognizes and mediates degradation of toxic, oligomeric forms of alphaSyn. Further elucidation of the mechanisms of CHIP-induced degradation of oligomeric alphaSyn may contribute to the successful development of drug therapies that target oligomeric alphaSyn by mimicking or enhancing the powerful effects of CHIP.

    Funded by: NIA NIH HHS: P50 AG005134; NINDS NIH HHS: P50 NS038372, P50 NS038372-030001, P50 NS038372-080001, R01 NS063963, R01 NS063963-01A1

    The Journal of biological chemistry 2008;283;26;17962-8

  • Cu2+ binding modes of recombinant alpha-synuclein--insights from EPR spectroscopy.

    Drew SC, Leong SL, Pham CL, Tew DJ, Masters CL, Miles LA, Cappai R and Barnham KJ

    Department of Pathology, The University of Melbourne, Victoria 3010, Australia.

    The interaction of the small (140 amino acid) protein, alpha-synuclein (alphaS), with Cu(2+) has been proposed to play a role in Parkinson's disease (PD). While some insight from truncated model complexes has been gained, the nature of the corresponding Cu(2+) binding modes in the full length protein remains comparatively less well characterized. This work examined the Cu(2+) binding of recombinant human alphaS using Electron Paramagnetic Resonance (EPR) spectroscopy. Wild type (wt) alphaS was shown to bind stoichiometric Cu(2+) via two N-terminal binding modes at physiological pH. An H50N mutation isolated one binding mode, whose g parallel, A parallel, and metal-ligand hyperfine parameters correlated well with a {NH2, N(-), beta-COO(-), H2O} mode previously identified in truncated model fragments. Electron spin-echo envelope modulation (ESEEM) studies of wt alphaS confirmed the second binding mode at pH 7.4 involved coordination of His50 and its g parallel and A parallel parameters correlated with either {NH2, N(-), beta-COO(-), N(Im)} or {N(Im), 2 N(-)} coordination observed in alphaS fragments. At pH 5.0, His50-anchored Cu(2+) binding was greatly diminished, while {NH2, N(-), beta-COO(-), H2O} binding persisted in conjunction with another two binding modes. Metal-ligand hyperfine interactions from one of these indicated a 1N3O coordination sphere, which was ascribed to a {NH2, CO} binding mode. The other was characterized by a spectrum similar to that previously observed for diethylpyrocarbonate-treated alphaS and was attributed to C-terminal binding centered on Asp121. In total, four Cu(2+) binding modes were identified within pH 5.0-7.4, providing a more comprehensive picture of the Cu(2+) binding properties of recombinant alphaS.

    Journal of the American Chemical Society 2008;130;24;7766-73

  • The reaction of alpha-synuclein with tyrosinase: possible implications for Parkinson disease.

    Tessari I, Bisaglia M, Valle F, Samorì B, Bergantino E, Mammi S and Bubacco L

    Department of Biology, University of Padova, Padova 35121, Italy.

    Oxidative stress appears to be directly involved in the pathogenesis of Parkinson disease. Several different pathways have been identified for the production of oxidative stress conditions in nigral dopaminergic neurons, including a pathological accumulation of cytosolic dopamine with the subsequent production of toxic reactive oxygen species or the formation of highly reactive quinone species. On these premises, tyrosinase, a key copper enzyme known for its role in the synthesis of melanin in skin and hair, has been proposed to take part in the oxidative chemistry related to Parkinson disease. A study is herein presented of the in vitro reactivity of tyrosinase with alpha-synuclein, aimed at defining the molecular basis of their synergistic toxic effect. The results presented here indicate that, in conformity with the stringent specificity of tyrosinase, the exposed tyrosine side-chains are the reactive centers of alpha-synuclein. The reactivity of alpha-synuclein depends on whether it is free or membrane bound, and the chemical modifications on the tyrosinase-treated alpha-synuclein strongly influence its aggregation properties. On the basis of our results, we propose a cytotoxic model which includes a possible new toxic role for alpha-synuclein exacerbated by its direct chemical modification by tyrosinase.

    The Journal of biological chemistry 2008;283;24;16808-17

  • Phosphorylation at Ser-129 but not the phosphomimics S129E/D inhibits the fibrillation of alpha-synuclein.

    Paleologou KE, Schmid AW, Rospigliosi CC, Kim HY, Lamberto GR, Fredenburg RA, Lansbury PT, Fernandez CO, Eliezer D, Zweckstetter M and Lashuel HA

    Laboratory of Molecular Neurobiology and Neuroproteomics, Brain Mind Institute, Ecole Polytechnique Federale de Lausanne, CH-1015 Lausanne, Switzerland.

    alpha-Synuclein (alpha-syn) phosphorylation at serine 129 is characteristic of Parkinson disease (PD) and related alpha-synulceinopathies. However, whether phosphorylation promotes or inhibits alpha-syn aggregation and neurotoxicity in vivo remains unknown. This understanding is critical for elucidating the role of alpha-syn in the pathogenesis of PD and for development of therapeutic strategies for PD. To better understand the structural and molecular consequences of Ser-129 phosphorylation, we compared the biochemical, structural, and membrane binding properties of wild type alpha-syn to those of the phosphorylation mimics (S129E, S129D) as well as of in vitro phosphorylated alpha-syn using a battery of biophysical techniques. Our results demonstrate that phosphorylation at Ser-129 increases the conformational flexibility of alpha-syn and inhibits its fibrillogenesis in vitro but does not perturb its membrane-bound conformation. In addition, we show that the phosphorylation mimics (S129E/D) do not reproduce the effect of phosphorylation on the structural and aggregation properties of alpha-syn in vitro. Our findings have significant implications for current strategies to elucidate the role of phosphorylation in modulating protein structure and function in health and disease and provide novel insight into the underlying mechanisms that govern alpha-syn aggregation and toxicity in PD and related alpha-synulceinopathies.

    Funded by: NIA NIH HHS: AG019391, R01 AG019391-08, R01 AG025440-03; NIGMS NIH HHS: GM66354

    The Journal of biological chemistry 2008;283;24;16895-905

  • Mitochondrial translocation of alpha-synuclein is promoted by intracellular acidification.

    Cole NB, Dieuliis D, Leo P, Mitchell DC and Nussbaum RL

    Genetic Diseases Research Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland 20892, USA. ncole@mail.nih.gov

    Mitochondrial dysfunction plays a central role in the selective vulnerability of dopaminergic neurons in Parkinson's disease (PD) and is influenced by both environmental and genetic factors. Expression of the PD protein alpha-synuclein or its familial mutants often sensitizes neurons to oxidative stress and to damage by mitochondrial toxins. This effect is thought to be indirect, since little evidence physically linking alpha-synuclein to mitochondria has been reported. Here, we show that the distribution of alpha-synuclein within neuronal and non-neuronal cells is dependent on intracellular pH. Cytosolic acidification induces translocation of alpha-synuclein from the cytosol onto the surface of mitochondria. Translocation occurs rapidly under artificially-induced low pH conditions and as a result of pH changes during oxidative or metabolic stress. Binding is likely facilitated by low pH-induced exposure of the mitochondria-specific lipid cardiolipin. These results imply a direct role for alpha-synuclein in mitochondrial physiology, especially under pathological conditions, and in principle, link alpha-synuclein to other PD genes in regulating mitochondrial homeostasis.

    Funded by: Intramural NIH HHS: Z99 HL999999

    Experimental cell research 2008;314;10;2076-89

  • Alpha-synuclein A30P point-mutation generates age-dependent nigrostriatal deficiency in mice.

    Plaas M, Karis A, Innos J, Rebane E, Baekelandt V, Vaarmann A, Luuk H, Vasar E and Koks S

    Department of Physiology, Center of Molecular and Clinical Medicine, University of Tartu, Tartu, Estonia.

    Lewy bodies are mainly composed of alpha-synuclein (SNCA) and specific mutations in SNCA gene are related to familial forms of Parkinson's disease (PD). The purpose of our study was to generate a mouse line with A30P knock-in point mutation in SNCA gene and to test if a single point-mutation is able to turn otherwise normal SNCA into a toxic form. The behavioral profile of SNCA A30P mice was followed for 16 months. Generally, these mice are healthy and viable without any obvious abnormalities. Starting from the age of 13 months mice developed a significant deficit in motor performance tests related to nigrostriatal function (ink-test and beam walk). In other tests (motility boxes, rotarod) mice continuously performed normally. Moreover, SNCA A30P mice expressed the altered sensitivity to VMAT2 inhibitor reserpine, possibly reflecting a functional deficiency of dopamine. Indeed, mice at 15 months of age had significantly reduced levels of dopamine and its major metabolite DOPAC in the striatum, and reduced levels of dopamine in the mesolimbic system. The present study confirms that SNCA plays an important role in the development of PD and an insertion of a single point mutation is sufficient to generate age-related decline in specific motor performance. The generated mouse line has a potential to become a model for PD with comparable time course and phenotype.

    Journal of physiology and pharmacology : an official journal of the Polish Physiological Society 2008;59;2;205-16

  • Genomic investigation of alpha-synuclein multiplication and parkinsonism.

    Ross OA, Braithwaite AT, Skipper LM, Kachergus J, Hulihan MM, Middleton FA, Nishioka K, Fuchs J, Gasser T, Maraganore DM, Adler CH, Larvor L, Chartier-Harlin MC, Nilsson C, Langston JW, Gwinn K, Hattori N and Farrer MJ

    Division of Neurogenetics, Department of Neuroscience, College of Medicine, Mayo Clinic, Jacksonville, FL 32224, USA.

    Objective: Copy number variation is a common polymorphic phenomenon within the human genome. Although the majority of these events are non-deleterious they can also be highly pathogenic. Herein we characterize five families with parkinsonism that have been identified to harbor multiplication of the chromosomal 4q21 locus containing the alpha-synuclein gene (SNCA).

    Methods: A methodological approach using fluorescent in situ hybridization and Affymetrix (Santa Clara, CA) 250K SNP microarrays was used to characterize the multiplication in each family and to identify the genes encoded within the region. The telomeric and centromeric breakpoints of each family were further narrowed using semiquantitative polymerase chain reaction with microsatellite markers and then screened for transposable repeat elements.

    Results: The severity of clinical presentation is correlated with SNCA dosage and does not appear to be overtly affected by the presence of other genes in the multiplicated region. With the exception of the Lister kindred, in each family the multiplication event appears de novo. The type and position of Alu/LINE repeats are also different at each breakpoint. Microsatellite analysis demonstrates two genomic mechanisms are responsible for chromosome 4q21 multiplications, including both SNCA duplication and recombination.

    Interpretation: SNCA dosage is responsible for parkinsonism, autonomic dysfunction, and dementia observed within each family. We hypothesize dysregulated expression of wild-type alpha-synuclein results in parkinsonism and may explain the recent association of common SNCA variants in sporadic Parkinson's disease. SNCA genomic duplication results from intraallelic (segmental duplication) or interallelic recombination with unequal crossing over, whereas both mechanisms appear to be required for genomic SNCA triplication.

    Funded by: NIA NIH HHS: P01 AG017216, P01 AG017216-05, P01 AG017216-09, P01 AG017216-090005, P01 AG17216; NINDS NIH HHS: N01-NS-2-2349, P50 #NS40256

    Annals of neurology 2008;63;6;743-50

  • Abnormal colonic motility in mice overexpressing human wild-type alpha-synuclein.

    Wang L, Fleming SM, Chesselet MF and Taché Y

    CURE/Digestive Diseases Center and Center for Neurobiology of Stress, David Geffen School of Medicine, University of California at Los Angeles and VAGLAHS, Los Angeles, California, USA. lixinw@ucla.edu

    The presynaptic protein alpha-synuclein (alphaSyn) has been implicated in both familial and sporadic forms of Parkinson's disease. We examined whether human alphaSyn-overexpressing mice under Thy1 promoter (Thy1-alphaSyn) display alterations of colonic function. Basal fecal output was decreased in Thy1-alphaSyn mice fed ad libitum. Fasted/refed Thy1-alphaSyn mice had a slower distal colonic transit than the wild-type mice, as monitored by 2.2-fold increase in time to expel an intracolonic bead and 2.9-fold higher colonic fecal content. By contrast, Thy1-alphaSyn mice had an increased fecal response to novelty stress and corticotropin releasing factor injected intraperipherally. These results indicate that Thy1-alphaSyn mice display altered basal and stress-stimulated propulsive colonic motility and will be a useful model to study gut dysfunction associated with Parkinson's disease.

    Funded by: NIDDK NIH HHS: R01 DK 57238, R01 DK057238; NINDS NIH HHS: P50 NS038367, P50 NS038367-07, P50NS38367; PHS HHS: 41301

    Neuroreport 2008;19;8;873-6

  • Nurr1 transcriptionally regulates the expression of alpha-synuclein.

    Yang YX and Latchman DS

    Medical Molecular Biology Unit, Institute of Child Health, University College London, UK. y.yang@ich.ucl.ac.uk

    Parkinson's disease is one of the most common neurodegenerative disorders and still remains incurable. The condition is linked to mutations and alterations in expression in several genes, in particular that encoding alpha-synuclein. Mutations in Nurr1 leading to a reduction in expression were also found to lead to Parkinson's disease. In view of the importance of gene regulation in Parkinson's disease, we examined the effect of changes in Nurr1 expression on alpha-synuclein expression. Nurr1 was shown to be involved in the regulation of alpha-synuclein, as decreased expression of Nurr1, which has been found in Parkinson's disease patients with Nurr1 mutations, was shown to transcriptionally increase alpha-synuclein expression.

    Funded by: Parkinson's UK: G-4051

    Neuroreport 2008;19;8;867-71

  • Characterization of conformational and dynamic properties of natively unfolded human and mouse alpha-synuclein ensembles by NMR: implication for aggregation.

    Wu KP, Kim S, Fela DA and Baum J

    Department of Chemistry and Chemical Biology, Rutgers University, 610 Taylor Road, Piscataway, NJ 08854, USA.

    Conversion of human alpha-synuclein (aS) from the free soluble state to the insoluble fibrillar state has been implicated in the etiology of Parkinson's disease. Human aS is highly homologous in amino acid sequence to mouse aS, which contains seven substitutions including the A53T that has been linked to familial Parkinson's disease, and including five substitutions in the C-terminal region. It has been shown that the rate of fibrillation is highly dependent on the exact sequence of the protein, and mouse aS is reported to aggregate more rapidly than human aS in vitro. Nuclear magnetic resonance experiments of mouse and human aS at supercooled temperatures (263 K) are used to understand the effect of sequence on conformational fluctuations in the disordered ensembles and to relate these to differences in propensities to aggregate. We show that both aS are natively unfolded at low temperature with different propensities to secondary structure, backbone dynamics and long-range contacts across the protein. Mouse aS exhibits a higher propensity to helical conformation around the C-terminal substitutions as well as the loss of transient long-range contacts from the C- to the N-terminal end and hydrophobic central regions of the protein relative to human aS. Lack of back-folding from the C-terminal end of mouse aS exposes the N-terminal region, which is shown, by (15)N relaxation experiments, to be very restricted in mobility relative to human aS. We propose that the restricted mobility in the N-terminal region may arise from transient interchain interactions, suggesting that the N-terminal KTK(E/Q)GV repeats may serve as initiation sites for aggregation in mouse aS. These transient interchain interactions coupled with a non-A beta amyloid component (NAC) region that is both more exposed and has a higher propensity to beta structure may accelerate the rate of fibril formation of aS.

    Funded by: NIGMS NIH HHS: R01 GM087012, R01 GM087012-02

    Journal of molecular biology 2008;378;5;1104-15

  • Characterization of fibrillation process of alpha-synuclein at the initial stage.

    Tashiro M, Kojima M, Kihara H, Kasai K, Kamiyoshihara T, Uéda K and Shimotakahara S

    Department of Chemistry, College of Science and Technology, Meisei University, Hino, Tokyo 191-8506, Japan.

    alpha-Synuclein is the major component of the filamentous Lewy bodies and Lewy-related neurites, neuropathological hallmarks of Parkinson's disease. Although numerous studies on alpha-synuclein fibrillation have been reported, the molecular mechanisms of aggregation and fibrillation at the initial stage are still unclear. In the present study, structural properties and propensities to form fibrils of alpha-synuclein at the initial stage were investigated using 2D (1)H-(15)N NMR spectroscopy, electron microscope, and small angle X-ray scattering (SAXS). Observation of the 2D (1)H-(15)N HSQC spectra indicated significant attenuation of many cross peak intensities in the regions of KTKEGV-type repeats and the non-Abeta component of Alzheimer's disease amyloid (NAC), suggesting that these regions contributed fibril formation. Oligomerization comprising heptamer was successfully monitored at the initial stage using the time-dependent SAXS measurements.

    Biochemical and biophysical research communications 2008;369;3;910-4

  • Alpha-synuclein activates stress signaling protein kinases in THP-1 cells and microglia.

    Klegeris A, Pelech S, Giasson BI, Maguire J, Zhang H, McGeer EG and McGeer PL

    Kinsmen Laboratory of Neurological Research, University of British Columbia, 2255 Wesbrook Mall, Vancouver, BC V6T 1Z3, Canada.

    Here we show that alpha-synuclein, a major constituent of Lewy bodies, induces inflammation in human microglial and human THP-1 cells. Secretions from such stimulated THP-1 cells contain increased levels of IL-1beta and TNF-alpha. When stimulated by alpha-synuclein in combination with IFN-gamma, secretions from the cells also become toxic towards SH-SY5Y neuroblastoma cells. The A30P, E46K and A53T alpha-synuclein mutations, which induce Parkinson's disease, are more potent than normal alpha-synuclein in the induction of such cytotoxicity. To investigate the signaling mechanisms evoked, protein phosphorylation profiling was applied. At least 81 target phospho-sites were identified. Large increases were induced in the three major mitogen-activated protein (MAP) kinase pathways: p38 MAP kinase, extracellular regulated protein-serine kinase (ERK)1/2 and c-Jun-N-terminal kinase (JNK). Upregulation occurred within minutes following exposure to alpha-synuclein, which is consistent with a receptor-mediated effect. These findings demonstrate that alpha-synuclein acts as a potent inflammatory stimulator of microglial cells, and that inhibitors of such stimulation might be beneficial in the treatment of Parkinson's disease and other synucleinopathies.

    Neurobiology of aging 2008;29;5;739-52

  • Genetic variability in the SNCA gene influences alpha-synuclein levels in the blood and brain.

    Fuchs J, Tichopad A, Golub Y, Munz M, Schweitzer KJ, Wolf B, Berg D, Mueller JC and Gasser T

    University of Tübingen, Hertie-Institute for Clinical Brain Research, Department of Neurodegenerative Diseases, Hoppe-Seyler Str. 3, 72076 Tübingen, Germany.

    Genetic variability in the promoter and 3' region of the SNCA gene coding alpha-synuclein modulates the risk to develop sporadic Parkinson's disease (PD). Whether this is mediated by regulating alpha-synuclein expression levels remains unknown. Therefore, we analyzed levels of alpha-synuclein in blood and human post mortem brain tissue including the substantia nigra using quantitative real-time reverse transcriptase-polymerase chain reaction and enzyme linked immunosorbent assay in vivo. Single nucleotide polymorphism (SNP) rs356219, a tagging SNP for a disease-associated haplotype in the 3' region of the SNCA gene, has a significant effect on SNCA mRNA levels in the substantia nigra and the cerebellum. Further, the "protective" genotype 259/259 of the PD-associated promoter repeat NACP-Rep1 is associated with lower protein levels in blood than genotypes 261/261, 259/261, and 259/263. In conclusion, we provide evidence that alpha-synuclein levels are influenced by genetic variability in the promoter and 3' region of the SNCA gene in vivo.

    Funded by: NIMH NIH HHS: R24 MH068855

    FASEB journal : official publication of the Federation of American Societies for Experimental Biology 2008;22;5;1327-34

  • Molecular determinants of the aggregation behavior of alpha- and beta-synuclein.

    Rivers RC, Kumita JR, Tartaglia GG, Dedmon MM, Pawar A, Vendruscolo M, Dobson CM and Christodoulou J

    Department of Chemistry, University of Cambridge, Cambridge CB2 1EW, United Kingdom.

    Alpha- and beta-synuclein are closely related proteins, the first of which is associated with deposits formed in neurodegenerative conditions such as Parkinson's disease while the second appears to have no relationship to any such disorders. The aggregation behavior of alpha- and beta-synuclein as well as a series of chimeric variants were compared by exploring the structural transitions that occur in the presence of a widely used lipid mimetic, sodium dodecyl sulfate (SDS). We found that the aggregation rates of all these protein variants are significantly enhanced by low concentrations of SDS. In particular, we inserted the 11-residue sequence of mainly hydrophobic residues from the non-amyloid-beta-component (NAC) region of alpha-synuclein into beta-synuclein and show that the fibril formation rate of this chimeric protein is only weakly altered from that of beta-synuclein. These intrinsic propensities to aggregate are rationalized to a very high degree of accuracy by analysis of the sequences in terms of their associated physicochemical properties. The results begin to reveal that the differences in behavior are primarily associated with a delicate balance between the positions of a range of charged and hydrophobic residues rather than the commonly assumed presence or absence of the highly aggregation-prone region of the NAC region of alpha-synuclein. This conclusion provides new insights into the role of alpha-synuclein in disease and into the factors that regulate the balance between solubility and aggregation of a natively unfolded protein.

    Funded by: Wellcome Trust

    Protein science : a publication of the Protein Society 2008;17;5;887-98

  • Recent advances in the genetics of dementia with lewy bodies.

    Bonifati V

    Department of Clinical Genetics, Erasmus MC, PO Box 2040, 3000 CA, Rotterdam, The Netherlands. v.bonifati@erasmusmc.nl

    In the past few years, mutations have been identified in the genes encoding alpha-synuclein, leucine-rich repeat kinase 2, and glucocerebrosidase in some patients with dementia with Lewy bodies (DLB). Furthermore, a novel locus for familial DLB has been mapped to chromosome 2q35-q36. Collectively, these discoveries highlight a substantial overlap between the known genetic determinants of Parkinson's disease and DLB, as well as the presence of profound etiologic heterogeneity in Lewy body disorders.

    Current neurology and neuroscience reports 2008;8;3;187-9

  • Specificity and regulation of casein kinase-mediated phosphorylation of alpha-synuclein.

    Waxman EA and Giasson BI

    Department of Pharmacology, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6084, USA.

    alpha-Synuclein (alpha-syn) is the major component of pathologic inclusions that characterize neurodegenerative disorders such as Parkinson disease, dementia with Lewy body disease, and multiple system atrophy. The present study uses novel phospho-specific antibodies to assess the presence and regulation of phosphorylated Ser87 and Ser129 in alpha-syn in human brain samples and in a transgenic mouse model of alpha-synucleinopathies. By immunohistochemistry, alpha-syn phosphorylated at Ser129, but not at Ser87, was abundant in alpha-syn inclusions. Under normal conditions, Ser129 phosphorylation, but not Ser87 phosphorylation, was detected at low levels in the soluble biochemical fractions in human alpha-syn transgenic mice and stably transfected cultured cells. Therefore, a role for Ser87 phosphorylation in alpha-synucleinopathies is unlikely, and in vitro assays showed that phosphorylation at this site would inhibit polymerization. In vitro studies also indicated that hyperphosphorylation of Ser129 alpha-syn in pathologic inclusions may be due in part to the intrinsic properties of aggregated alpha-syn to act as substrates for kinases but not phosphatases. Further studies in transgenic mice and cultured cells suggest that cellular toxicity, including proteasomal dysfunction, increases casein kinase 2 activity, which results in elevated Ser129 alpha-syn phosphorylation. These data provide novel explanations for the presence of hyperphosphorylated Ser129 alpha-syn in pathologic inclusions.

    Funded by: NIA NIH HHS: AG09215, P01 AG009215, P01 AG009215-18, T32 AG000255-12, T32 AG00255; NINDS NIH HHS: NS053488

    Journal of neuropathology and experimental neurology 2008;67;5;402-16

  • Single particle characterization of iron-induced pore-forming alpha-synuclein oligomers.

    Kostka M, Högen T, Danzer KM, Levin J, Habeck M, Wirth A, Wagner R, Glabe CG, Finger S, Heinzelmann U, Garidel P, Duan W, Ross CA, Kretzschmar H and Giese A

    CNS Research, Boehringer Ingelheim Pharma GmbH & Co. KG, CNS Research, Birkendorferstrasse 65, 88397 Biberach, Germany. Marcus.Kostka@boehringer-ingelheim.com

    Aggregation of alpha-synuclein is a key event in several neurodegenerative diseases, including Parkinson disease. Recent findings suggest that oligomers represent the principal toxic aggregate species. Using confocal single-molecule fluorescence techniques, such as scanning for intensely fluorescent targets (SIFT) and atomic force microscopy, we monitored alpha-synuclein oligomer formation at the single particle level. Organic solvents were used to trigger aggregation, which resulted in small oligomers ("intermediate I"). Under these conditions, Fe(3+) at low micromolar concentrations dramatically increased aggregation and induced formation of larger oligomers ("intermediate II"). Both oligomer species were on-pathway to amyloid fibrils and could seed amyloid formation. Notably, only Fe(3+)-induced oligomers were SDS-resistant and could form ion-permeable pores in a planar lipid bilayer, which were inhibited by the oligomer-specific A11 antibody. Moreover, baicalein and N'-benzylidene-benzohydrazide derivatives inhibited oligomer formation. Baicalein also inhibited alpha-synuclein-dependent toxicity in neuronal cells. Our results may provide a potential disease mechanism regarding the role of ferric iron and of toxic oligomer species in Parkinson diseases. Moreover, scanning for intensely fluorescent targets allows high throughput screening for aggregation inhibitors and may provide new approaches for drug development and therapy.

    The Journal of biological chemistry 2008;283;16;10992-1003

  • Alpha-synuclein alters Notch-1 expression and neurogenesis in mouse embryonic stem cells and in the hippocampus of transgenic mice.

    Crews L, Mizuno H, Desplats P, Rockenstein E, Adame A, Patrick C, Winner B, Winkler J and Masliah E

    Departments of Neurosciences, University of California, San Diego, La Jolla, California 92093-0624, USA.

    Altered expression and mutations in alpha-synuclein (alpha-syn) have been linked to Parkinson's disease (PD) and related disorders. The neurological alterations in PD patients have been associated with degeneration of dopaminergic cells and other neuronal populations. Moreover, recent studies in murine models have shown that alterations in neurogenesis might also contribute to the neurodegenerative phenotype. However, the mechanisms involved and the effects of alpha-syn expression on neurogenesis are not yet clear. To this end, murine embryonic stem (mES) cells were infected with lentiviral (LV) vectors expressing wild-type (WT) and mutant alpha-syn. Compared with mES cells infected with LV-green fluorescent protein (GFP), cells expressing WT and mutant alpha-syn showed reduced proliferation as indicated by lower 5-bromo-2'-deoxyuridine uptake, increased apoptosis, and reduced expression of neuronal markers such as neuron specific enolase and beta-III tubulin. The alterations in neurogenesis in alpha-syn-expressing mES cells were accompanied by a reduction in Notch-1 and Hairy and enhancer of split-5 (Hes-5) mRNA and protein levels. Moreover, levels of total Notch-1 and Notch intracellular domain (NICD) were lower in mES cells expressing WT and mutant alpha-syn compared with GFP controls. The reduced survival of alpha-syn-expressing mES cells was reverted by overexpressing constitutively active NICD. Similarly, in alpha-syn transgenic mice, the alterations in neurogenesis in the hippocampal subgranular zone were accompanied by decreased Notch-1, NICD, and Hes-5 expression. Together, these results suggest that accumulation of alpha-syn might impair survival of NPCs by interfering with the Notch signaling pathway. Similar mechanisms could be at play in PD and Lewy body disease.

    Funded by: NIA NIH HHS: AG022074, AG10435, AG18440, AG5131, P01 AG010435-120006, P01 AG022074-010004, P50 AG005131-140023, R37 AG018440-06

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2008;28;16;4250-60

  • Oxidative-stress-induced apoptosis in PBLs of two patients with Parkinson disease secondary to alpha-synuclein mutation.

    Battisti C, Formichi P, Radi E and Federico A

    Department of Neurological and Behavioural Sciences, O.U. of Neurometabolic Disease, University of Siena, Siena, Italy.

    Alpha-synuclein has been implicated in the pathology of certain neurodegenerative diseases, including Parkinson disease (PD). Although the precise physiological and pathological role of alpha-synuclein is unclear, overexpression of the protein or its mutants may reduce cell viability. In this study we evaluated the apoptotic response to oxidative stress induced by 2-deoxy-d-ribose (dRib) in peripheral blood lymphocytes (PBLs) of two siblings with Parkinson disease secondary to A53T alpha-synuclein mutation. PBLs exposed to oxidative stress showed a higher percentage of apoptotic cells in PD patients than in controls. However in cells of PD patients, the increase of apoptotic response was lower than in controls, suggesting that cells of PD patients have greater "resistance" to oxidative stress. We conclude that other environmental agents could play a key role in inducing programmed cell death in cells of PD patients with mutant alpha-synuclein.

    Journal of the neurological sciences 2008;267;1-2;120-4

  • Alpha-synuclein, pesticides, and Parkinson disease: a case-control study.

    Brighina L, Frigerio R, Schneider NK, Lesnick TG, de Andrade M, Cunningham JM, Farrer MJ, Lincoln SJ, Checkoway H, Rocca WA and Maraganore DM

    Department of Neurology, Mayo Clinic College of Medicine, 200 First Street SW, Rochester, MN 55905, USA.

    Background: Aggregation and fibrillization of the alpha-synuclein protein (encoded by the SNCA gene) may represent key events in the pathogenesis of Parkinson disease (PD). Variability in the length of a dinucleotide repeat sequence (REP1) within the SNCA promoter confers susceptibility to sporadic PD. Pesticide exposures may also confer susceptibility to PD. Our objective was to test possible joint effects of SNCA REP1 genotypes and pesticide exposures on the risk of PD.

    Methods: This was a case-control study. Cases were recruited prospectively from the Department of Neurology of the Mayo Clinic, Rochester, MN, after June 1, 1996. The control subjects included unaffected siblings of cases and unrelated population control subjects. We assessed pesticide exposures by telephone interview and genotyped SNCA REP1. Odds ratios (ORs) and 95% CIs were determined using conditional logistic regression models.

    Results: There were 833 case-control pairs. We observed an increased risk of PD with increasing SNCA REP1 bp length (OR, 1.18 for each score unit; 95% CI, 1.02-1.37; p = 0.03). Pesticide exposures were associated with PD in younger subjects only (lowest quartile of age at study, <or=59.8 years; OR, 1.80; 95% CI, 1.12-2.87; p = 0.01 for all pesticides; OR, 2.46; 95% CI, 1.34-4.52; p = 0.004 for herbicides). In multivariate analyses, both SNCA REP1 score and pesticide exposures were significantly associated with PD in younger subjects, but there were no pairwise interactions.

    Conclusions: Our findings suggest that SNCA REP1 genotype and herbicides have independent effects on risk of Parkinson disease, primarily in younger subjects.

    Funded by: NIEHS NIH HHS: ES 10751, R01 ES010751; NINDS NIH HHS: NS 33978, R01 NS033978

    Neurology 2008;70;16 Pt 2;1461-9

  • Alpha-synuclein aggregation alters tyrosine hydroxylase phosphorylation and immunoreactivity: lessons from viral transduction of knockout mice.

    Alerte TN, Akinfolarin AA, Friedrich EE, Mader SA, Hong CS and Perez RG

    Department of Neurology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15260, United States.

    Tyrosine hydroxylase (TH), the rate limiting enzyme in catecholamine synthesis, is frequently used as a marker of dopaminergic neuronal loss in animal models of Parkinson's disease (PD). We have been exploring the normal function of the PD-related protein alpha-synuclein (alpha-Syn) with regard to dopamine synthesis. TH is activated by the phosphorylation of key seryl residues in the TH regulatory domain. Using in vitro models, our laboratory discovered that alpha-Syn inhibits TH by acting to reduce TH phosphorylation, which then reduces dopamine synthesis [X.-M. Peng, R. Tehranian, P. Dietrich, L. Stefanis, R.G. Perez, Alpha-synuclein activation of protein phosphatase 2A reduces tyrosine hydroxylase phosphorylation in dopaminergic cells, J. Cell. Sci. 118 (2005) 3523-3530; R.G. Perez, J.C. Waymire, E. Lin, J.J. Liu, F. Guo, M.J. Zigmond, A role for alpha-synuclein in the regulation of dopamine biosynthesis, J. Neurosci. 22 (2002) 3090-3099]. We recently began exploring the impact of alpha-Syn on TH in vivo, by transducing dopaminergic neurons in alpha-Syn knockout mouse (ASKO) olfactory bulb using wild type human alpha-Syn lentivirus. At 3.5-21 days after viral delivery, alpha-Syn expression was transduced primarily in periglomerular dopaminergic neurons. Cells with modest levels of alpha-Syn consistently co-labeled for Total-TH. However, cells bearing aggregated alpha-Syn, as revealed by proteinase K or Thioflavin-S treatment had significantly reduced Total-TH immunoreactivity, but high phosphoserine-TH labeling. On immunoblots, we noted that Total-TH immunoreactivity was equivalent in all conditions, although tissues with alpha-Syn aggregates again had higher phosphoserine-TH levels. This suggests that aggregated alpha-Syn is no longer able to inhibit TH. Although the reason(s) underlying reduced Total-TH immunoreactivity on tissue sections await(s) confirmation, the dopaminergic phenotype was easily verified using phosphorylation-state-specific TH antibodies. These findings have implications not only for normal alpha-Syn function in TH regulation, but also for measuring cell loss that is associated with synucleinopathy.

    Funded by: NINDS NIH HHS: NS42094, R01 NS042094, R01 NS042094-01A1, R01 NS042094-02, R01 NS042094-03, R01 NS042094-03S1, R01 NS042094-04, R01 NS042094-05

    Neuroscience letters 2008;435;1;24-9

  • Sequence determinants regulating fibrillation of human alpha-synuclein.

    Koo HJ, Lee HJ and Im H

    Department of Molecular Biology, Sejong University, 98 Gunja-dong, Kwangjin-gu, Seoul 143-747, Republic of Korea.

    alpha-Synuclein is a neural protein that comprises the fibrillar core of Lewy bodies, a histologically defining lesion of Parkinson's disease. To investigate the role of each specific residue of the alpha-synuclein molecule in fibril formation, amino acid substitutions were introduced throughout the molecule. Incorporation of proline, especially in the region spanning residues 37-89, drastically retarded fibril formation. Substitutions with polar residues showed that the hydrophobicity of the central hydrophobic region is also important in fibrillation regulation. In the N-terminal repeated region, increasing the number of negative charges interfered with fibrillation. In contrast, single amino acid substitutions in the C-terminal acidic region of alpha-synuclein had only minimal effects on fibrillation. More than 20 different single amino acid substitutions that were sufficient to prevent fibrillation of alpha-synuclein were obtained, and most of them were impaired in both nucleation and fibril elongation. Identification of sequence determinants regulating fibrillation of amyloidogenic proteins may provide valuable information for designing peptide analog drugs to prevent protein amyloidosis.

    Biochemical and biophysical research communications 2008;368;3;772-8

  • Alpha-synuclein promoter haplotypes and dementia in Parkinson's disease.

    De Marco EV, Tarantino P, Rocca FE, Provenzano G, Civitelli D, De Luca V, Annesi F, Carrideo S, Cirò Candiano IC, Romeo N, Nicoletti G, Marconi R, Novellino F, Morelli M, Quattrone A and Annesi G

    Institute of Neurological Sciences, National Research Council, Mangone (Cosenza), Italy. v.demarco@isn.cnr.it

    Dementia is a common complication of Parkinson's disease (PD). It correlates significantly with the presence of cortical, limbic or nigral Lewy bodies, mainly constituted of alpha-synuclein. Mutations of the alpha-synuclein gene (SNCA) have been linked to rare familial forms of PD, while association studies on the promoter polymorphisms have given conflicting results in sporadic patients. We have performed a case control study to investigate whether genetic variability in the promoter of the alpha-synuclein gene could predispose to dementia in PD. A total of 114 demented patients and 114 non-demented patients with sporadic PD were included in the study. Six polymorphic loci (including the Rep1 microsatellite) in the promoter of the SNCA gene were examined. Each marker, taken individually, did not show association to dementia and no significant differences were observed in the inferred haplotype frequencies of demented and non-demented patients. Our data suggest the lack of involvement of the SNCA promoter in the pathogenesis of dementia in PD. Further studies in other populations are needed to confirm these results.

    American journal of medical genetics. Part B, Neuropsychiatric genetics : the official publication of the International Society of Psychiatric Genetics 2008;147;3;403-7

  • Mitochondrial import and accumulation of alpha-synuclein impair complex I in human dopaminergic neuronal cultures and Parkinson disease brain.

    Devi L, Raghavendran V, Prabhu BM, Avadhani NG and Anandatheerthavarada HK

    Department of Animal Biology, School of Veterinary Medicine, University of Pennsylvania, 3800 Spruce Street, Philadelphia, PA 19104, USA.

    Alpha-synuclein, a protein implicated in the pathogenesis of Parkinson disease (PD), is thought to affect mitochondrial functions, although the mechanisms of its action remain unclear. In this study we show that the N-terminal 32 amino acids of human alpha-synuclein contain cryptic mitochondrial targeting signal, which is important for mitochondrial targeting of alpha-synuclein. Mitochondrial imported alpha-synuclein is predominantly associated with the inner membrane. Accumulation of wild-type alpha-synuclein in the mitochondria of human dopaminergic neurons caused reduced mitochondrial complex I activity and increased production of reactive oxygen species. However, these defects occurred at an early time point in dopaminergic neurons expressing familial alpha-synuclein with A53T mutation as compared with wild-type alpha-synuclein. Importantly, alpha-synuclein that lacks mitochondrial targeting signal failed to target to the mitochondria and showed no detectable effect on complex I function. The PD relevance of these results was investigated using mitochondria of substantia nigra, striatum, and cerebellum of postmortem late-onset PD and normal human brains. Results showed the constitutive presence of approximately 14-kDa alpha-synuclein in the mitochondria of all three brain regions of normal subjects. Mitochondria of PD-vulnerable substantia nigra and striatum but not cerebellum from PD subjects showed significant accumulation of alpha-synuclein and decreased complex I activity. Analysis of mitochondria from PD brain and alpha-synuclein expressing dopaminergic neuronal cultures using blue native gel electrophoresis and immunocapture technique showed the association of alpha-synuclein with complex I. These results provide evidence that mitochondrial accumulated alpha-synuclein may interact with complex I and interfere with its functions.

    Funded by: NIA NIH HHS: AG 021920

    The Journal of biological chemistry 2008;283;14;9089-100

  • DJ-1 modulates alpha-synuclein aggregation state in a cellular model of oxidative stress: relevance for Parkinson's disease and involvement of HSP70.

    Batelli S, Albani D, Rametta R, Polito L, Prato F, Pesaresi M, Negro A and Forloni G

    Department of Neuroscience, Mario Negri Institute for Pharmacological Research, Milan, Italy.

    Background: Parkinson's disease (PD) is a neurodegenerative pathology whose molecular etiopathogenesis is not known. Novel contributions have come from familial forms of PD caused by alterations in genes with apparently unrelated physiological functions. The gene coding for alpha-synuclein (alpha-syn) (PARK1) has been investigated as alpha-syn is located in Lewy bodies (LB), intraneuronal inclusions in the substantia nigra (SN) of PD patients. A-syn has neuroprotective chaperone-like and antioxidant functions and is involved in dopamine storage and release. DJ-1 (PARK7), another family-PD-linked gene causing an autosomal recessive form of the pathology, shows antioxidant and chaperone-like activities too.

    The present study addressed the question whether alpha-syn and DJ-1 interact functionally, with a view to finding some mechanism linking DJ-1 inactivation and alpha-syn aggregation and toxicity. We developed an in vitro model of alpha-syn toxicity in the human neuroblastoma cell line SK-N-BE, influencing DJ-1 and alpha-syn intracellular concentrations by exogenous addition of the fusion proteins TAT-alpha-syn and TAT-DJ-1; DJ-1 was inactivated by the siRNA method. On a micromolar scale TAT-alpha-syn aggregated and triggered neurotoxicity, while on the nanomolar scale it was neuroprotective against oxidative stress (induced by H(2)O(2) or 6-hydroxydopamine). TAT-DJ-1 increased the expression of HSP70, while DJ-1 silencing made SK-N-BE cells more susceptible to oxidative challenge, rendering TAT-alpha-syn neurotoxic at nanomolar scale, with the appearance of TAT-alpha-syn aggregates.

    DJ-1 inactivation may thus promote alpha-syn aggregation and the related toxicity, and in this model HSP70 is involved in the antioxidant response and in the regulation of alpha-syn fibril formation.

    PloS one 2008;3;4;e1884

  • alpha-Synuclein pathology in the neostriatum in Parkinson's disease.

    Mori F, Tanji K, Zhang H, Kakita A, Takahashi H and Wakabayashi K

    Department of Neuropathology, Institute of Brain Science, Hirosaki University Graduate School of Medicine, Hirosaki, 036-8562, Japan. neuropal@cc.hirosaki-u.ac.jp

    We immunohistochemically examined the neostriatum from 25 patients with symptomatic and presymptomatic Parkinson's disease (PD) with various degrees of Lewy body pathology, using anti-phosphorylated alpha-synuclein (alphaS) antibody. These patients were classified according to the PD staging proposed by Braak et al. (Neurobiol Aging 24:197-211, 2003): stage II (alphaS pathology confined to the medulla oblongata and pontine tegmentum), stage III (alphaS pathology confined to the brainstem), stage IV (limbic stage), and stages V and VI (neocortical stage). alphaS immunohistochemistry revealed neuronal and glial cytoplasmic inclusions and neuritic changes in the neostriatum. alphaS inclusions were found in the medium-sized neurons (GABAergic neurons that project to the globus pallidus) and large neurons (cholinergic interneurons); the former began to appear at stage III and the latter was noted at stages V and VI. Neuritic changes and glial inclusions also began to appear at stage III. The numbers of neuronal and glial inclusions, and the extent of neuritic changes, correlated with the PD stage (P < 0.001). These findings suggest that intrinsic neostriatal neurons degenerate through alphaS aggregation during PD progression.

    Acta neuropathologica 2008;115;4;453-9

  • Alpha-synuclein transgenic mice exhibit reduced anxiety-like behaviour.

    George S, van den Buuse M, San Mok S, Masters CL, Li QX and Culvenor JG

    Department of Pathology, The Centre for Neuroscience, The University of Melbourne, Parkville, Victoria, 3010, Australia.

    Up to 40% of Parkinson's disease patients suffer from anxiety, but little is known about the mechanisms involved. We used the elevated plus maze and open field test to evaluate groups of young adult mice expressing different levels of alpha-synuclein, including mice transgenic for human alpha-synuclein with the A53T mutation. Compared to alpha-synuclein knock-out mice and wild-type controls, alpha-synuclein A53T transgenic mice exhibited reduced anxiety-like behaviour by spending markedly greater amounts of time on the maze open arms and by a higher proportion of entries to the open arms. In the open field, transgenic mice showed a trend towards reduced locomotor habituation and increased thigmotaxis. These results indicate a possible role for alpha-synuclein in anxiety-like behaviours.

    Experimental neurology 2008;210;2;788-92

  • [Blockade of the aberrant aggregation of alpha-synuclein in HEK293 cells induced by overexpression of wild-type alpha-synuclein by RNA interference].

    Chen T, Tang BS and Liao XP

    Department of Neurology, the Affiliated Xiangya Hospital, Central South University, Changsha, Hunan, 410008 P. R. China.

    Objective: To construct specific and effective RNA interference(RNAi) plasmid for alpha-synuclein gene and investigate RNAi blockade of the aberrant aggregation of alpha-synuclein in HEK293 cells induced by overexpression of wild-type alpha-synuclein.

    Methods: Hairpin RNAs for four target sites were designed to construct four RNAi plasmids pSYNi-1, pSYNi-2, pSYNi-3 and pSYNi-4, using plasmid pBSHH1 vector under the control of the H1 promoter. Western blot and reverse transcription-PCR(RT-PCR) were performed to screen the most specific and effective RNAi plasmid. After confirming the sequences of the plasmids, they were co-transfected into HEK293 cells with the recombinant plasmids alpha-synuclein-pEGFP by using lipofectamin 2000. The aberrant aggregation of alpha-synuclein was measured by EGFP fluorescence and immunocytochemistry for alpha-synuclein. The inclusions in the cultured cells were identified with HE staining.

    Results: By Western blot and RT-PCR, pSYNi-1 showed the most effective RNAi gene silencing effect (69.6%). After transfecting the RNAi plasmid, the aberrant aggregation of alpha-synuclein in HEK293 cells induced by overexpression of wild-type alpha-synuclein was inhibited. The Lewy body-like inclusions were found in cytoplasm of cultured cells in control group, but disappeared in HEK293 cells cotransfected by pSYNi-1 and alpha-synuclein-pEGFP plasmid.

    Conclusion: RNAi can block the aberrant aggregation and Lewy body-like inclusion formation in cytoplasm of HEK293 cell induced by overexpression of wild-type alpha-synuclein.

    Zhonghua yi xue yi chuan xue za zhi = Zhonghua yixue yichuanxue zazhi = Chinese journal of medical genetics 2008;25;2;128-31

  • Conformational characteristics of unstructured peptides: alpha-synuclein.

    Yoon J, Park J, Jang S, Lee K and Shin S

    School of Chemistry, Seoul National University, Seoul 151-747, Korea.

    We have performed replica-exchange molecular dynamics simulations on 41 residue peptides containing NAC region of alpha-synuclein in various force fields and solvent conditions. Alpha-synuclein is known to be the major cause of Parkinson's disease by amyloid-like aggregation, and one of the natively unfolded proteins. To investigate conformational characteristics of intrinsically unstructured peptides, we carried out structural analysis by introducing 'representative structure' for ensemble of structures occurring during the overall trajectory. Representative structures may be defined by using either coordinate averaging or distance averaging. When applied to the natively folded proteins such as villin headpiece, structural analysis based on representative structure was found to yield consistent results with those obtained from conventional analysis. Individual conformations obtained from the simulations of NAC peptide for various conditions show flexible structures close to random coil. Secondary structure contents and free energy surfaces showed dependency on solvent conditions, which may be interpreted as another manifestation of structural diversity. It is found that representative structures can provide useful information about structural characteristics of intrinsically unstructured proteins.

    Journal of biomolecular structure & dynamics 2008;25;5;505-15

  • Dimeric structures of alpha-synuclein bind preferentially to lipid membranes.

    Giannakis E, Pacífico J, Smith DP, Hung LW, Masters CL, Cappai R, Wade JD and Barnham KJ

    The Howard Florey Institute of Medical Research, Australia.

    There is substantial evidence which implicates alpha-synuclein and its ability to aggregate and bind vesicle membranes as critical factors in the development of Parkinson's disease. In order to investigate the interaction between alpha-synuclein wild type (Wt) and its familial mutants, A53T and A30P with lipid membranes, we developed a novel lipid binding assay using surface enhanced laser desorption/ionisation-time of flight-mass spectrometry (SELDI-TOF MS). Wt and A53T exhibited similar lipid binding profiles; monomeric species and dimers bound with high relative affinity to the lipid surface, the latter of which exhibited preferential binding. Wt and A53T trimers and tetramers were also detected on the lipid surface. A30P exhibited a unique lipid binding profile; monomeric A30P bound with a low relative affinity, however, the dimeric species of A30P exhibited a higher binding ability. Larger order A30P oligomers were not detected on the lipid surface. Tapping mode atomic force microscopy (AFM) imaging was conducted to further examine the alpha-synuclein-lipid interaction. AFM analysis revealed Wt and its familial mutants can penetrate lipid membranes or disrupt the lipid and bind the hydrophobic alkyl self-assembled monolayer (SAM) used to form the lipid layer. The profile of these studied proteins revealed the presence of 'small features' consistent with the presence of monomeric and dimeric forms of the protein. These data collectively indicate that the dimeric species of Wt and its mutants can bind and cause membrane perturbations.

    Funded by: Wellcome Trust: WT069851MA

    Biochimica et biophysica acta 2008;1778;4;1112-9

  • Exploring gene-environment interactions in Parkinson's disease.

    McCulloch CC, Kay DM, Factor SA, Samii A, Nutt JG, Higgins DS, Griffith A, Roberts JW, Leis BC, Montimurro JS, Zabetian CP and Payami H

    Applied Statistics Laboratory, General Electric Global Research Center, Niskayuna, NY, USA.

    The objective of this study was to explore combined effects of four candidate susceptibility genes and two exposures on Parkinson's disease (PD) risk; namely, alpha-synuclein (SNCA) promoter polymorphism REP1, microtubule-associated protein tau (MAPT) H1/H2 haplotypes, apolipoprotein E (APOE) epsilon2/epsilon3/epsilon4 polymorphism, ubiquitin carboxy-terminal esterase L1 (UCHL1) S18Y variant, cigarette smoking and caffeinated coffee consumption. 932 PD patients and 664 control subjects from the NeuroGenetics Research Consortium, with complete data on all six factors, were studied. Uniform protocols were used for diagnosis, recruitment, data collection and genotyping. A logistic regression model which included gene-exposure interactions was applied. Likelihood ratio tests (LRTs) were used for significance testing and Bayesian inference was used to estimate odds ratios (ORs). MAPT (P = 0.007), SNCA REP1 (P = 0.012), smoking (P = 0.001), and coffee (P = 0.011) were associated with PD risk. Two novel interactions were detected: APOE with coffee (P = 0.005), and REP1 with smoking (P = 0.021). While the individual main effects were modest, each yielding OR < 1.6, the effects were cumulative, with some combinations reaching OR = 12.6 (95% CI: 5.9-26.8). This study provides evidence for the long-held notion that PD risk is modulated by cumulative and interactive effects of genes and exposures. Furthermore, the study demonstrates that while interaction studies are useful for exploring risk relationships that might otherwise go undetected, results should be interpreted with caution because of the inherent loss of power due to multiple testing. The novel findings of this study that warrant replication are the evidence for interaction of coffee with APOE, and of smoking with REP1 on PD risk.

    Funded by: NINDS NIH HHS: K08-NS044138, NS R01-36960

    Human genetics 2008;123;3;257-65

  • Mitochondrial association of alpha-synuclein causes oxidative stress.

    Parihar MS, Parihar A, Fujita M, Hashimoto M and Ghafourifar P

    Department of Surgery, Davis Heart and Lung Research Institute, Ohio State University, 460 West 12th Avenue, Columbus, 43210 OH, USA.

    Alpha-synuclein is a neuron-specific protein that contributes to the pathology of Parkinson's disease via mitochondria-related mechanisms. The present study investigated possible interaction of alpha-synuclein with mitochondria and consequences of such interaction. Using SHSY cells overexpressing alpha-synuclein A53T mutant or wild-type, as well as isolated rat brain mitochondria, the present study shows that alpha-synuclein localizes at the mitochondrial membrane. In both SHSY cells and isolated mitochondria, interaction of alpha-synuclein with mitochondria causes release of cytochrome c, increase of mitochondrial calcium and nitric oxide, and oxidative modification of mitochondrial components. These findings suggest a pivotal role for mitochondria in oxidative stress and apoptosis induced by alpha-synuclein.

    Funded by: NIA NIH HHS: AG 023264-02

    Cellular and molecular life sciences : CMLS 2008;65;7-8;1272-84

  • Patients homozygous and heterozygous for SNCA duplication in a family with parkinsonism and dementia.

    Ikeuchi T, Kakita A, Shiga A, Kasuga K, Kaneko H, Tan CF, Idezuka J, Wakabayashi K, Onodera O, Iwatsubo T, Nishizawa M, Takahashi H and Ishikawa A

    Department of Molecular Neuroscience, Brain Research Institute, Niigata University, 1 Asahimachi, Niigata 951-8585, Japan. ikeuchi@bri.niigata-u.ac.jp

    Background: Multiplication of the alpha-synuclein gene (SNCA) (OMIM 163890) has been identified as a causative mutation in hereditary Parkinson disease or dementia with Lewy bodies.

    Objective: To determine the genetic, biochemical, and neuropathologic characteristics of patients with autopsy-confirmed autosomal dominant Lewy body disease, with particular reference to the dosage effects of SNCA.

    Design: Four-generation family study.

    Setting: Academic research. Patients We fractionated samples extracted from frozen brain tissues of 4 patients for biochemical characterization, followed by immunoblot analysis.

    We determined the dosages of SNCA and its surrounding genes by quantitative polymerase chain reaction analysis.

    Results: Quantitative polymerase chain reaction analysis revealed that 3 patients were heterozygous for SNCA duplication and 1 patient was homozygous for SNCA duplication. The homozygous patient showed earlier age at onset and earlier death, with more severe cognitive impairment than the heterozygous patients. Biochemical analysis revealed that phosphorylated alpha-synuclein accumulated in the sarkosyl-insoluble urea-extracted fraction of the brains of the patients.

    Conclusions: Pathologically confirmed Lewy body disease clinically characterized by progressive parkinsonism and cognitive dysfunction is caused by SNCA duplication. The homozygous patient demonstrated the most severe phenotype, suggesting that SNCA dosage has a considerable effect on disease phenotype even within a family. SNCA duplication results in the hyperaccumulation of phosphorylated alpha-synuclein in the brains of patients.

    Archives of neurology 2008;65;4;514-9

  • Selective loss of nigral dopamine neurons induced by overexpression of truncated human alpha-synuclein in mice.

    Wakamatsu M, Ishii A, Iwata S, Sakagami J, Ukai Y, Ono M, Kanbe D, Muramatsu S, Kobayashi K, Iwatsubo T and Yoshimoto M

    Medicinal Research Laboratories, Taisho Pharmaceutical Co., Ltd., Yoshino-cho 1-403, Kita-ku, Saitama-shi, Saitama 331-9530, Japan.

    Parkinson's disease is characterized by loss of nigral dopaminergic neurons and presence of Lewy bodies, whose major component is alpha-synuclein. In the present study, we generated transgenic mice termed Syn130m that express truncated human alpha-synuclein (amino acid residue number: 1-130) in dopaminergic neurons. Notably, dopaminergic neurons were selectively diminished in the substantia nigra pars compacta of Syn130m, while transgenic mice that expressed comparable amount of full-length human alpha-synuclein did not develop such pathology. Therefore, the truncation of human alpha-synuclein seems to be primarily responsible for the loss of nigral dopaminergic neurons. The nigral pathology resulted in impairment of axon terminals in the striatum and concomitant decrease in striatal dopamine content. Behaviorally, spontaneous locomotor activities of Syn130m were reduced, but the abnormality was ameliorated by treatment with L-DOPA. The loss of nigral dopaminergic neurons was not progressive and seemed to occur during embryogenesis along with the onset of expression of the transgene. Our results indicate that truncated human alpha-synuclein is deleterious to the development and/or survival of nigral dopaminergic neurons.

    Neurobiology of aging 2008;29;4;574-85

  • Patterns and stages of alpha-synucleinopathy: Relevance in a population-based cohort.

    Zaccai J, Brayne C, McKeith I, Matthews F, Ince PG and MRC Cognitive Function, Ageing Neuropathology Study

    Neuropathology, E Floor, Royal Hallamshire Hospital, Sheffield UK.

    Background: It is proposed that alpha-synucleinopathy (AS) initially affects the medulla oblongata and progresses to more rostral brain areas in a hierarchical sequence ("Braak hypothesis"). Predominant involvement of the amygdala is also described. This study examines the applicability of these patterns, and their relationship to Alzheimer disease (AD) pathology, in brains of a population-based donor cohort.

    Methods: Brains donated in two of six Cognitive Function and Ageing Study cohorts (Cambridgeshire and Nottingham) were examined. More than 80% were older than 80 years at death. The respondents were evaluated prospectively in life for cognitive decline and dementia. Immunocytochemistry for tau and alpha-synuclein was carried out in 208 brains to establish Braak stage and the pattern and severity of AS.

    Results: Seventy-six brains showed Lewy bodies. Half (51%) conformed to the Braak hypothesis while 17% had pathology in a higher region which was absent in a lower region. A further 29% showed amygdala-predominant pathology. Six brains showed predominant neocortical pathology with minimal pathology in amygdala or substantia nigra. The stage of AD pathology was not associated with particular patterns of AS.

    Conclusion: alpha-Synucleinopathy (AS) is common in older people, and frequently associated with Alzheimer disease-type pathology. Although half of brains corresponded to the Braak hypothesis, and 29% to amygdala-predominant AS, there were a high proportion of cases which did not fit a staging system. An unexpectedly high proportion with a cortical form of Lewy body disease was identified.

    Neurology 2008;70;13;1042-8

  • Ubiquitination of alpha-synuclein by Siah-1 promotes alpha-synuclein aggregation and apoptotic cell death.

    Lee JT, Wheeler TC, Li L and Chin LS

    Department of Pharmacology, Center for Neurodegenerative Disease, Emory University School of Medicine, Atlanta, GA 30322-3090, USA.

    Point mutations and gene multiplication of alpha-synuclein cause autosomal dominant familial Parkinson's disease (PD). Moreover, alpha-synuclein- and ubiquitin-positive inclusion bodies are the pathological hallmarks of PD and several other neurodegenerative diseases, such as dementia with Lewy bodies and multiple system atrophy. Despite the presence of ubiquitinated alpha-synuclein species in Lewy bodies, the regulation of alpha-synuclein ubiquitination and its role in Lewy body formation and neurodegeneration remain poorly understood. Here, we report that alpha-synuclein interacts and colocalizes with mammalian seven in absentia homologue-1 (Siah-1), a RING-type E3 ubiquitin-protein ligase. Siah-1 binds the brain-enriched E2 ubiquitin-conjugating enzyme UbcH8 and facilitates mono- and di-ubiquitination of alpha-synuclein in vivo. The ubiquitination of alpha-synuclein by Siah-1 is disrupted by the PD-linked A30P mutation but not by A53T mutation. We find that Siah-1-mediated ubiquitination does not target alpha-synuclein for degradation by the proteasome, but rather, it promotes alpha-synuclein aggregation and enhances alpha-synuclein toxicity. Our findings suggest that Siah-1-mediated alpha-synuclein ubiquitination may play a critical role in Lewy body formation and PD pathogenesis.

    Funded by: NIA NIH HHS: AG021489; NINDS NIH HHS: NS047199, NS050650

    Human molecular genetics 2008;17;6;906-17

  • alpha-Synuclein aggregates interfere with Parkin solubility and distribution: role in the pathogenesis of Parkinson disease.

    Kawahara K, Hashimoto M, Bar-On P, Ho GJ, Crews L, Mizuno H, Rockenstein E, Imam SZ and Masliah E

    Department of Neurosciences, School of Medicine, University of California at San Diego, La Jolla, California 92039-0624, USA.

    Parkinson disease (PD) belongs to a heterogeneous group of neurodegenerative disorders with movement alterations, cognitive impairment, and alpha-synuclein accumulation in cortical and subcortical regions. Jointly, these disorders are denominated Lewy body disease. Mutations in the parkin gene are the most common cause of familial parkinsonism, and a growing number of studies have shown that stress factors associated with sporadic PD promote parkin accumulation in the insoluble fraction. alpha-Synuclein and parkin accumulation and mutations in these genes have been associated with familial PD. To investigate whether alpha-synuclein accumulation might be involved in the pathogenesis of these disorders by interfering with parkin solubility, synuclein-transfected neuronal cells were transduced with lentiviral vectors expressing parkin. Challenging neurons with proteasome inhibitors or amyloid-beta resulted in accumulation of insoluble parkin and, to a lesser extent, alpha-tubulin. Similarly to neurons in the brains of patients with Lewy body disease, in co-transduced cells alpha-synuclein and parkin colocalized and co-immunoprecipitated. These effects resulted in decreased parkin and alpha-tubulin ubiquitination, accumulation of insoluble parkin, and cytoskeletal alterations with reduced neurite outgrowth. Taken together, accumulation of alpha-synuclein might contribute to the pathogenesis of PD and other Lewy body diseases by promoting alterations in parkin and tubulin solubility, which in turn might compromise neural function by damaging the neuronal cytoskeleton. These studies provide a new perspective on the potential nature of pathogenic alpha-synuclein and parkin interactions in Parkinson disease.

    Funded by: NIA NIH HHS: AG10435, AG18440, AG22074

    The Journal of biological chemistry 2008;283;11;6979-87

  • Analyses of copy number and mRNA expression level of the alpha-synuclein gene in multiple system atrophy.

    Jin H, Ishikawa K, Tsunemi T, Ishiguro T, Amino T and Mizusawa H

    Department of Neurology and Neurological Science, Graduate School, Tokyo Medical and Dental University, Yushima 1-5-45, Bunkyo-ku 113-8519, Tokyo, Japan.

    Multiple system atrophy (MSA) is a sporadic neurodegenerative disease manifested clinically by progressive ataxia, parkinsonism, and autonomic dysfunction. Its cause is unknown, and there is no curative therapy. Alpha-synuclein is an important protein forming aggregations called glial cytoplasmic inclusions (GCIs) in oligodendroglia; these aggregations are considered important in MSA pathogenesis. Overexpression of the human alpha-synuclein gene in mice induces the formation of GCI-like aggregations in oligodendrocytes, leading mice to exhibit neurological signs similar to those in MSA patients. However, previous studies have excluded mutations within the coding region of the alpha-synuclein gene in MSA patients. To determine whether alteration in the expression level of the alpha-synuclein gene is associated with MSA pathogenesis, we used TaqMan quantitative PCR assay to analyze the alpha-synuclein gene copy number in patients' genomes. We also used quantitative RT-PCR and in situ hybridization to analyze alpha-synuclein mRNA expression in MSA patients' brain tissues. We found no alteration in the alpha-synuclein gene copy number in the patients' genomes (n = 50). Quantitative analysis for alpha-synuclein mRNA by the TaqMan method showed that alpha-synuclein mRNA levels were comparable between control (n = 3) and MSA (n = 3) cerebella. On in situ hybridization, the number of neurons with alpha-synuclein mRNA expression was no greater in the cerebella of MSA patients (n = 3) than in the controls (n = 3). However, GCIs were seen in these MSA specimens on immunohistochemistry for alpha-synuclein. These results suggest that alpha-synuclein gene expression is not the fundamental cause of MSA.

    Journal of medical and dental sciences 2008;55;1;145-53

  • C. elegans model identifies genetic modifiers of alpha-synuclein inclusion formation during aging.

    van Ham TJ, Thijssen KL, Breitling R, Hofstra RM, Plasterk RH and Nollen EA

    Department of Genetics, University Medical Centre Groningen and University of Groningen, Groningen, The Netherlands.

    Inclusions in the brain containing alpha-synuclein are the pathological hallmark of Parkinson's disease, but how these inclusions are formed and how this links to disease is poorly understood. We have developed a C. elegans model that makes it possible to monitor, in living animals, the formation of alpha-synuclein inclusions. In worms of old age, inclusions contain aggregated alpha- synuclein, resembling a critical pathological feature. We used genome-wide RNA interference to identify processes involved in inclusion formation, and identified 80 genes that, when knocked down, resulted in a premature increase in the number of inclusions. Quality control and vesicle-trafficking genes expressed in the ER/Golgi complex and vesicular compartments were overrepresented, indicating a specific role for these processes in alpha-synuclein inclusion formation. Suppressors include aging-associated genes, such as sir-2.1/SIRT1 and lagr-1/LASS2. Altogether, our data suggest a link between alpha-synuclein inclusion formation and cellular aging, likely through an endomembrane-related mechanism. The processes and genes identified here present a framework for further study of the disease mechanism and provide candidate susceptibility genes and drug targets for Parkinson's disease and other alpha-synuclein related disorders.

    PLoS genetics 2008;4;3;e1000027

  • Nitrated alpha-synuclein-activated microglial profiling for Parkinson's disease.

    Reynolds AD, Glanzer JG, Kadiu I, Ricardo-Dukelow M, Chaudhuri A, Ciborowski P, Cerny R, Gelman B, Thomas MP, Mosley RL and Gendelman HE

    Center for Neurovirology and Neurodegenerative Disorders, University of Nebraska Medical Center, Omaha, Nebraska 68198-5880, USA.

    Microglial neuroinflammatory processes play a primary role in dopaminergic neurodegeneration for Parkinson's disease (PD). This can occur, in part, by modulation of glial function following activation by soluble or insoluble modified alpha-synuclein (alpha-syn), a chief component of Lewy bodies that is released from affected dopaminergic neurons. alpha-Syn is nitrated during oxidative stress responses and in its aggregated form, induces inflammatory microglial functions. Elucidation of these microglial function changes in PD could lead to new insights into disease mechanisms. To this end, PD-associated inflammation was modeled by stimulation of microglia with aggregated and nitrated alpha-syn. These activated microglia were ameboid in morphology and elicited dopaminergic neurotoxicity. A profile of nitrated, aggregated alpha-syn-stimulated microglia was generated using combinations of genomic (microarrays) and proteomic (liquid chromatography-tandem mass spectrometry, differential gel electrophoresis, and protein array) assays. Genomic studies revealed a substantive role for nuclear factor-kappa B transcriptional activation. Qualitative changes in the microglial proteome showed robust increases in inflammatory, redox, enzyme, and cytoskeletal proteins supporting the genomic tests. Autopsy brain tissue acquired from substantia nigra and basal ganglia of PD patients demonstrated that parallel nuclear factor-kappa B-related inflammatory processes were, in part, active during human disease. Taken together, the transcriptome and proteome of nitrated alpha-syn activated microglia, shown herein, provide new potential insights into disease mechanisms.

    Funded by: NIMH NIH HHS: P01 MH64570, R01 MH79886, U01 MH083545; NINDS NIH HHS: 1R21 NS049264, 1T32 NS07488, 2R37 NS36136, P01 NS43985, R24 NS45491

    Journal of neurochemistry 2008;104;6;1504-25

  • Monoubiquitylation of alpha-synuclein by seven in absentia homolog (SIAH) promotes its aggregation in dopaminergic cells.

    Rott R, Szargel R, Haskin J, Shani V, Shainskaya A, Manov I, Liani E, Avraham E and Engelender S

    Department of Pharmacology, The B. Rappaport Faculty of Medicine and Institute of Medical Research, Technion-Israel Institute of Technology, Haifa 31096, Israel.

    alpha-Synuclein plays a major role in Parkinson disease. Unraveling the mechanisms of alpha-synuclein aggregation is essential to understand the formation of Lewy bodies and their involvement in dopaminergic cell death. alpha-Synuclein is ubiquitylated in Lewy bodies, but the role of alpha-synuclein ubiquitylation has been mysterious. We now report that the ubiquitin-protein isopeptide ligase seven in absentia homolog (SIAH) directly interacts with and monoubiquitylates alpha-synuclein and promotes its aggregation in vitro and in vivo, which is toxic to cells. Mass spectrometry analysis demonstrates that SIAH monoubiquitylates alpha-synuclein at lysines 12, 21, and 23, which were previously shown to be ubiquitylated in Lewy bodies. SIAH ubiquitylates lysines 10, 34, 43, and 96 as well. Suppression of SIAH expression by short hairpin RNA to SIAH-1 and SIAH-2 abolished alpha-synuclein monoubiquitylation in dopaminergic cells, indicating that endogenous SIAH ubiquitylates alpha-synuclein. Moreover, SIAH co-immunoprecipitated with alpha-synuclein from brain extracts. Inhibition of proteasomal, lysosomal, and autophagic pathways, as well as overexpression of a ubiquitin mutant less prone to deubiquitylation, G76A, increased monoubiquitylation of alpha-synuclein by SIAH. Monoubiquitylation increased the aggregation of alpha-synuclein in vitro. At the electron microscopy level, monoubiquitylated alpha-synuclein promoted the formation of massive amounts of amorphous aggregates. Monoubiquitylation also increased alpha-synuclein aggregation in vivo as observed by increased formation of alpha-synuclein inclusion bodies within dopaminergic cells. These inclusions are toxic to cells, and their formation was prevented when endogenous SIAH expression was suppressed. Our data suggest that monoubiquitylation represents a possible trigger event for alpha-synuclein aggregation and Lewy body formation.

    The Journal of biological chemistry 2008;283;6;3316-28

  • Formation of a high affinity lipid-binding intermediate during the early aggregation phase of alpha-synuclein.

    Smith DP, Tew DJ, Hill AF, Bottomley SP, Masters CL, Barnham KJ and Cappai R

    Department of Pathology, The University of Melbourne, Victoria, 3010, Australia.

    The alpha-synuclein (alpha-syn) protein is clearly implicated in Parkinson's disease (PD). Mutations or triplication of the alpha-syn gene leads to early onset PD, possibly by accelerating alpha-syn oligomerization. alpha-syn interacts with lipids, and this membrane binding activity may relate to its toxic activity. To understand how the alpha-syn aggregation state affects its lipid binding activity we used surface plasmon resonance to study the interaction of wild-type and mutant alpha-syn with a charged phospholipid membrane, as a function of its aggregation state. Apparent dissociation constants for alpha-syn indicated that an intermediate species, present during the lag phase of amyloid formation, binds with an increased affinity to the membrane surface. Formation of this species was dependent upon the rate of fibril formation. Fluorescence anisotropy studies indicate that only upon the formation of amyloid material can alpha-syn perturb the acyl-chain region of the lipid bilayer. Circular dichroism spectroscopy showed that upon aging, both wild-type and mutant alpha-syn lose their ability to form lipid-bound alpha-helical species once they become fibrillar. These results indicate that alpha-syn forms a high affinity lipid binding intermediate species during fibril formation. Oligomeric alpha-syn is known to be toxic, and it is feasible that the high affinity binding species described here may correspond to a toxic species involved in PD.

    Funded by: Wellcome Trust: WT069851MA

    Biochemistry 2008;47;5;1425-34

  • Identification and characterization of a new alpha-synuclein isoform and its role in Lewy body diseases.

    Beyer K, Domingo-Sábat M, Lao JI, Carrato C, Ferrer I and Ariza A

    Department of Pathology, Hospital Universitari Germans Trias i Pujol, Autonomous University of Barcelona, Barcelona, Spain. katrinbeyer@hotmail.com

    Alternative splicing is an important mechanism to generate a large number of mRNAs, thus increasing proteome diversity and tissue specificity. Three transcript variants of alpha-synuclein, a neuronal protein mainly involved in synapses, have been described so far. Whereas alpha-synuclein 140 is the whole and main transcript, alpha-synuclein 112 and 126 are short proteins that result from in-frame deletions of exons 3 and 5, respectively. Because the aforesaid alpha-synuclein isoforms show differential expression changes in Lewy body diseases (LBDs), in the present work, we searched for a fourth alpha-synuclein isoform and studied its expression levels in LBD brains. By using isoform-specific primers, isoform co-amplification and direct sequencing, we identified alpha-synuclein 98, which lacks exons 3 and 5. mRNA expression analyses in non-neuronal tissue revealed that alpha-synuclein 98 is a brain-specific splice variant with varying expression levels in different areas of fetal and adult brain. Additionally, we studied alpha-synuclein 98 expression levels by real-time semi-quantitative RT-PCR in the frontal cortices of LBD patients and compared them with those of Alzheimer disease (AD) patients and control subjects. Overexpression of alpha-synuclein 98 in LBD and AD brains would indicate its specific involvement in the pathogenesis of these neurodegenerative disorders.

    Neurogenetics 2008;9;1;15-23

  • Risk and protective haplotypes of the alpha-synuclein gene associated with Parkinson's disease differentially affect cognitive sequence learning.

    Kéri S, Nagy H, Myers CE, Benedek G, Shohamy D and Gluck MA

    Department of Psychiatry and Psychotherapy, Semmelweis University, Budapest, Hungary. szkeri2000@yahoo.com

    Alpha-synuclein (SNCA) is a key factor in the regulation of dopaminergic transmission and is related to Parkinson's disease. In this study, we investigated the effects of risk and protective SNCA haplotypes associated with Parkinson's disease on cognitive sequence learning in 204 healthy volunteers. We found that the 3'-block risk SNCA haplotypes are associated with less effective stimulus-reward learning of sequences and with superior context representation of sequences. In contrast, participants with protective haplotypes exhibit better stimulus-reward learning and worse context representation, which suggest that these functions are inversely affected by risk and protective haplotypes. The Rep1 promoter polymorphism does not influence cognitive sequence learning. Because stimulus-reward learning may be mediated by the basal ganglia and context learning may be related to the medial temporal lobe, our data raise the possibility that dopaminergic signals regulated by SNCA inversely affect these memory systems.

    Genes, brain, and behavior 2008;7;1;31-6

  • Dopamine differentially induces aggregation of A53T mutant and wild type alpha-synuclein: insights into the protein chemistry of Parkinson's disease.

    Moussa CE, Mahmoodian F, Tomita Y and Sidhu A

    Department of Biochemistry, Molecular and Cell Biology, and Georgetown University, Washington, DC 20007, USA.

    Aggregation of alpha-synuclein is known to be a causal factor in the genesis of Parkinson's disease and Dementia with Lewy bodies. Duplication and/or triplication and mutation of the alpha-synuclein gene are associated with sporadic and familial Parkinson's disease. Synucleinopathies appear to primarily affect dopaminergic neurons. The present studies investigate the role of dopamine in alpha-synuclein aggregation through NMR. Dopamine causes aggregation of both wild type and A53T mutant alpha-synuclein in a temperature-dependent manner, but the mutant A53T shows a greater propensity to aggregate in the presence of dopamine only at 37 degrees C. A single point mutation in the alpha-synuclein A53T mutant gene results in a structural change in the protein and drastically increases its propensity to aggregate in the presence of dopamine. The present data indicate that mutation in the alpha-synuclein gene may predispose the protein to dopamine-induced aggregation, thereby contributing to disease pathogenesis.

    Funded by: NIA NIH HHS: R01 AG028108-01A2; NIMH NIH HHS: R01 MH075020-01A2; NINDS NIH HHS: NS04326, R01 NS045326-01A1; PHS HHS: AGO28108

    Biochemical and biophysical research communications 2008;365;4;833-9

  • The phosphorylation state of Ser-129 in human alpha-synuclein determines neurodegeneration in a rat model of Parkinson disease.

    Gorbatyuk OS, Li S, Sullivan LF, Chen W, Kondrikova G, Manfredsson FP, Mandel RJ and Muzyczka N

    Department of Molecular Genetics and Microbiology, University of Florida College of Medicine, Gainesville, FL 32610, USA.

    Studies have shown that alpha-synuclein (alpha-syn) deposited in Lewy bodies in brain tissue from patients with Parkinson disease (PD) is extensively phosphorylated at Ser-129. We used recombinant Adeno-associated virus (rAAV) to overexpress human wild-type (wt) alpha-syn and two human alpha-syn mutants with site-directed replacement of Ser-129 to alanine (S129A) or to aspartate (S129D) in the nigrostriatal tract of the rat to investigate the effect of Ser-129 phosphorylation state on dopaminergic neuron pathology. Rats were injected with rAAV2/5 vectors in the substantia nigra pars compacta (SNc) on one side of the brain; the other side remained as a nontransduced control. The level of human wt or mutant alpha-syn expressed on the injected side was about four times the endogenous rat alpha-syn. There was a significant reduction of dopaminergic neurons in the SNc and dopamine (DA) and tyrosine hydroxylase (TH) levels in the striatum of all S129A-treated rats as early as 4 wk postinjection. Nigral DA pathology occurred more slowly in the wt-injected animals, but by 26 wk the wt alpha-syn group lost nigral TH neurons equivalent to the mutated S129A group at 8 wk. In stark contrast, we did not observe any pathological changes in S129D-treated animals. Therefore, the nonphosphorylated form of S129 exacerbates alpha-syn-induced nigral pathology, whereas Ser-129 phosphorylation eliminates alpha-syn-induced nigrostriatal degeneration. This suggests possible new therapeutic targets for Parkinson Disease.

    Funded by: NHLBI NIH HHS: P01 HL059412; NINDS NIH HHS: P01 NS036302, P01 NS36302

    Proceedings of the National Academy of Sciences of the United States of America 2008;105;2;763-8

  • Clinicopathologic study of a SNCA gene duplication patient with Parkinson disease and dementia.

    Obi T, Nishioka K, Ross OA, Terada T, Yamazaki K, Sugiura A, Takanashi M, Mizoguchi K, Mori H, Mizuno Y and Hattori N

    Department of Neurology, Shizuoka Institute of Epilepsy and Neurological Disorders, Tokyo, Japan.

    Neurology 2008;70;3;238-41

  • Hypothesis-based RNAi screening identifies neuroprotective genes in a Parkinson's disease model.

    Hamamichi S, Rivas RN, Knight AL, Cao S, Caldwell KA and Caldwell GA

    Department of Biological Sciences, University of Alabama, Tuscaloosa, AL 35487, USA.

    Genomic multiplication of the locus-encoding human alpha-synuclein (alpha-syn), a polypeptide with a propensity toward intracellular misfolding, results in Parkinson's disease (PD). Here we report the results from systematic screening of nearly 900 candidate genetic targets, prioritized by bioinformatic associations to existing PD genes and pathways, via RNAi knockdown. Depletion of 20 gene products reproducibly enhanced misfolding of alpha-syn over the course of aging in the nematode Caenorhabditis elegans. Subsequent functional analysis of seven positive targets revealed five previously unreported gene products that significantly protect against age- and dose-dependent alpha-syn-induced degeneration in the dopamine neurons of transgenic worms. These include two trafficking proteins, a conserved cellular scaffold-type protein that modulates G protein signaling, a protein of unknown function, and one gene reported to cause neurodegeneration in knockout mice. These data represent putative genetic susceptibility loci and potential therapeutic targets for PD, a movement disorder affecting approximately 2% of the population over 65 years of age.

    Proceedings of the National Academy of Sciences of the United States of America 2008;105;2;728-33

  • The Parkinson's disease protein alpha-synuclein disrupts cellular Rab homeostasis.

    Gitler AD, Bevis BJ, Shorter J, Strathearn KE, Hamamichi S, Su LJ, Caldwell KA, Caldwell GA, Rochet JC, McCaffery JM, Barlowe C and Lindquist S

    Whitehead Institute for Biomedical Research and Howard Hughes Medical Institute, Cambridge, MA 02142, USA.

    alpha-Synuclein (alpha-syn), a protein of unknown function, is the most abundant protein in Lewy bodies, the histological hallmark of Parkinson's disease (PD). In yeast alpha-syn inhibits endoplasmic reticulum (ER)-to-Golgi (ER-->Golgi) vesicle trafficking, which is rescued by overexpression of a Rab GTPase that regulates ER-->Golgi trafficking. The homologous Rab1 rescues alpha-syn toxicity in dopaminergic neuronal models of PD. Here we investigate this conserved feature of alpha-syn pathobiology. In a cell-free system with purified transport factors alpha-syn inhibited ER-->Golgi trafficking in an alpha-syn dose-dependent manner. Vesicles budded efficiently from the ER, but their docking or fusion to Golgi membranes was inhibited. Thus, the in vivo trafficking problem is due to a direct effect of alpha-syn on the transport machinery. By ultrastructural analysis the earliest in vivo defect was an accumulation of morphologically undocked vesicles, starting near the plasma membrane and growing into massive intracellular vesicular clusters in a dose-dependent manner. By immunofluorescence/immunoelectron microscopy, these clusters were associated both with alpha-syn and with diverse vesicle markers, suggesting that alpha-syn can impair multiple trafficking steps. Other Rabs did not ameliorate alpha-syn toxicity in yeast, but RAB3A, which is highly expressed in neurons and localized to presynaptic termini, and RAB8A, which is localized to post-Golgi vesicles, suppressed toxicity in neuronal models of PD. Thus, alpha-syn causes general defects in vesicle trafficking, to which dopaminergic neurons are especially sensitive.

    Funded by: NCRR NIH HHS: S10 RR019409-01, S10 RR021023-01, S10 RR022588, S10 RR022588-01, S10 RR023454, S10 RR023454-01; NIGMS NIH HHS: GM52549, R01 GM052549, R37 GM052549; NINDS NIH HHS: NS049221, NS38372, P50 NS038372, R01 NS049221

    Proceedings of the National Academy of Sciences of the United States of America 2008;105;1;145-50

  • alpha-Synuclein gene duplication is present in sporadic Parkinson disease.

    Ahn TB, Kim SY, Kim JY, Park SS, Lee DS, Min HJ, Kim YK, Kim SE, Kim JM, Kim HJ, Cho J and Jeon BS

    Department of Neurology, Kyung Hee University College of Medicine, Korea.

    Objective: alpha-Synuclein gene (SNCA) multiplication was found in familial Parkinson disease (PD). We examined SNCA multiplication in patients with familial and sporadic PD and multiple system atrophy (MSA).

    Methods: We screened 1,106 patients with parkinsonism (PD = 906, MSA = 200) for SNCA multiplication by multiplex PCR. Fluorescent in situ hybridization was done to confirm the multiplication. [(123)I]N-omega-Fluoropropyl-2 beta-carbomethoxy-3beta-(4-iodophenyl)-tropane ([(123)I]FP-CIT) SPECT was done in the patients with SNCA multiplication and their family members.

    Results: Three patients were identified as having SNCA duplication. One patient had a positive family history, and two patients were sporadic. Each patient had asymptomatic carriers in their families. The familial case had early onset parkinsonism with rapidly progressive course, cognitive impairment, and dysautonomia. Sporadic cases were more typical of PD. [(123)I]FP-CIT SPECT was abnormal in the patients and normal in the asymptomatic carriers.

    Conclusion: SNCA multiplication is present in sporadic Parkinson disease (PD) and needs to be screened. Low penetrance, clinical heterogeneity, and normal dopamine transporter imaging in asymptomatic carriers may suggest the presence of other genetic modifiers or environmental triggers that play a role in the pathogenesis of PD due to SNCA duplication.

    Neurology 2008;70;1;43-9

  • Cell systems and the toxic mechanism(s) of alpha-synuclein.

    Cookson MR and van der Brug M

    Laboratory of Neurogenetics, National Institute on Aging, NIH, Bethesda, MD 20982-3707, USA. Cookson@mail.nih.gov

    Mutations in the SNCA gene are causal for familial Parkinson disease/Lewy body disease. alpha-Synuclein is a small acidic protein that binds loosely to the surface of vesicles and may play a role in synaptic dynamics, although its normal function remains somewhat unclear. What is clear is that point mutations or increased expression of wild type alpha-synuclein causes disease. A great deal of literature supports the overall hypothesis that alpha-synuclein is damaging to neurons because it is inherently prone to aggregation; mutations or increased concentration of the protein both increase this tendency. An unproven, but popular, contention is that the toxic species are small oligomers that are relatively soluble, which may react with membranes to damage key processes within the cell. The details of this process, especially in determining the order of events and the requirement of particular processes in cell death, are unclear. Derangements in vesicle processing, including synaptic function, protein turnover, mitochondrial function and oxidative stress, have all been suggested to occur. Whether there is a sequence of events or whether these are interacting effects is unclear, but the outcome is to trigger cell death, by both apoptotic and non-apoptotic mechanisms depending on the system studied. In this article, we develop a framework for thinking about alpha-synuclein in terms of initiating events and secondary processes that are required to trigger neuronal dysfunction and cell death.

    Funded by: NIA NIH HHS: Z01 AG000953-06

    Experimental neurology 2008;209;1;5-11

  • Human-based studies on alpha-synuclein deposition and relationship to Parkinson's disease symptoms.

    Halliday GM and McCann H

    Prince of Wales, Medical Research Institute, Randwick, NSW, Australia. G.Halliday@unsw.edu.au

    This article reviews the current knowledge on alpha-synuclein and its cellular locations in studies using human brain tissue. Alterations in the conformation and distribution of alpha-synuclein are examined in Parkinson's disease and the relationship between clinical symptoms and pathology explored. alpha-Synuclein as a molecular chaperone has several isoforms and is known to have different environment-dependent conformations. Processing methods for studying human brain tissue significantly impact on the conformational type of alpha-synuclein analysed, and antibody species used for the in situ detection of alpha-synuclein give variable results depending on the epitope visualised. Human studies show that alpha-synuclein is not isolated to neurons, but is also found in glia, making the interpretation of studies using brain tissue homogenates less clearly related to neurons. These methodological issues impact significantly on our understanding of the form, location, and therefore function of alpha-synuclein in normal human brain tissue. There are less methodological issues regarding highly aggregated alpha-synuclein found in the major hallmark of Parkinson's disease, the Lewy body. However, it remains unclear whether these alpha-synuclein inclusions are harmful to host neurons or provide protection. Several correlations exist between the clinical symptoms of Parkinson's disease and the distribution of Lewy pathology, the strongest being the association between limbic and cortical Lewy bodies and well-formed visual hallucinations. Further correlation studies in prospectively-followed patients and, perhaps more importantly, controls are required in order to determine normal versus pathologic alpha-synuclein and how to detect such differences in clinical situations.

    Experimental neurology 2008;209;1;12-21

  • Inhibition of vesicular monoamine transporter-2 activity in alpha-synuclein stably transfected SH-SY5Y cells.

    Guo JT, Chen AQ, Kong Q, Zhu H, Ma CM and Qin C

    Institute of Laboratory Animal Sciences, Chinese Academy of Medical Sciences, Peking Union Medical College, 5 Panjiayuannanli, Beijing, 100021, China.

    alpha-Synuclein plays a key role in the pathological neurodegeneration in Parkinson's disease. Although its contribution to normal physiology remains elusive, the selective degeneration of alpha-synuclein-containing dopaminergic neurons in Parkinson's disease may be linked to abnormal alpha-synuclein induced toxicity. In the present study, a complex of alpha-synuclein and vesicular monoamine transporter-2 was identified by GST-Pull Down experiment. In wild-type alpha-synuclein stably transfected SH-SY5Y cell lines, the activity of vesicular monoamine transporter-2 decreased by 31% as determined by [(3)H] dopamine uptake, and its expression also decreased in both protein and mRNA levels using western and northern blot analysis. Overexpression of wild-type alpha-synuclein did not induce cell death or apoptosis, but significantly enhanced the intracellular reactive oxygen species level as assayed by flow cytometry. These data suggest that Up-regulated alpha-synuclein expression inhibits the activity of vesicular monoamine transporter-2, thereby interrupting dopamine homeostasis and resulting in dopaminergic neuron injury in Parkinson's disease.

    Cellular and molecular neurobiology 2008;28;1;35-47

  • Overexpressed alpha-synuclein regulated the nuclear factor-kappaB signal pathway.

    Yuan Y, Jin J, Yang B, Zhang W, Hu J, Zhang Y and Chen NH

    Department of Pharmacology, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Key Laboratory of Bioactive Substances and Resources Utilization, Ministry of Education, Beijing, 100050, PR China.

    Alpha-synuclein is a presynaptic protein which is implicated in some neurodegenerative disorders including Parkinson's disease, dementia with Lewy bodies, multiple systems atrophy, and Hallervorden-Spatz disease, and its overexpression contributes to the loss of dopaminergic neurons. Although the role of alpha-synuclein in these paradigms has been widely documented, its exact function is still elusive. And the dysfunction of the transcription factor nuclear factor (NF-kappaB) also exists in many neurodegenerative diseases. In this reason the purpose of this study was to investigate the molecular mechanism of alpha-synuclein's toxicity and its association with NF-kappaB by MTT assay, Western blot method, and luciferase assay. Results showed that overexpressed alpha-synuclein and 1-methyl-4-phenylpyridinium (MPP(+)) suppressed the SH-SY5Y cell viability and attenuate NF-kappaB-mediated luciferase expression significantly. Moreover, the impairment function was enhanced with the increase of alpha-synuclein protein level. We also found that overexpressed alpha-synuclein localized both in the cytoplasms and nuclei, down-regulated the anti-apoptotic Bcl-2 expression and up-regulated the pro-apoptotic glycogen synthase kinase 3beta (GSK3beta) protein level. In conclusion, all these findings mentioned above suggested that alpha-synuclein shared some toxic functional homology with neurotoxin MPP(+), and the proapoptotic effects of alpha-synuclein might be mediated at least in part by the impairment of NF-kappaB signaling pathway which involves GSK3beta.

    Cellular and molecular neurobiology 2008;28;1;21-33

  • Synphilin-1 isoforms in Parkinson's disease: regulation by phosphorylation and ubiquitylation.

    Szargel R, Rott R and Engelender S

    Department of Pharmacology, The B. Rappaport Institute of Medical Research, Technion-Israel Institute of Technology, Haifa, 31096, Israel.

    Parkinson's disease (PD) is characterized by the death of dopaminergic neurons and the presence of Lewy bodies in the substantia nigra pars compacta. The mechanisms involved in the death of neurons as well as the role of Lewy bodies in the pathogenesis of the disease are still unclear. Lewy bodies are made of aggregated proteins, in which alpha-synuclein represents their major component. Alpha-synuclein interacts with synphilin-1, a protein that is also present in Lewy bodies. When expressed in cells, synphilin-1 forms inclusions together with alpha-synuclein that resemble Lewy bodies. Synphilin-1 is ubiquitylated by various E3 ubiquitin-ligases, such as SIAH, parkin and dorfin. Ubiquitylation of synphilin-1 by SIAH is essential for its aggregation into inclusions. We recently identified a new synphilin-1 isoform, synphilin-1A, that is toxic to neurons, aggregation-prone and accumulates in detergent-insoluble fractions of brains from alpha-synucleinopathy patients. Synphilin-1A inclusions recruit both alpha-synuclein and synphilin-1. Aggregation of synphilin-1 and synphilin-1A seems to be protective to cells. We now discuss several aspects of the neurobiology and pathology of synphilin-1 isoforms, focusing on possible implications for PD.

    Cellular and molecular life sciences : CMLS 2008;65;1;80-8

  • Analysis of Parkinson disease patients from Portugal for mutations in SNCA, PRKN, PINK1 and LRRK2.

    Bras J, Guerreiro R, Ribeiro M, Morgadinho A, Januario C, Dias M, Calado A, Semedo C, Oliveira C, Hardy J and Singleton A

    Center for Neurosciences and Cell Biology, Faculty of Medicine, University of Coimbra, Coimbra, Portugal. brasj@nia.nih.gov

    Background: Mutations in the genes PRKN and LRRK2 are the most frequent known genetic lesions among Parkinson's disease patients. We have previously reported that in the Portuguese population the LRRK2 c.6055G > A; p.G2019S mutation has one of the highest frequencies in Europe.

    Methods: Here, we follow up on those results, screening not only LRRK2, but also PRKN, SNCA and PINK1 in a cohort of early-onset and late-onset familial Portuguese Parkinson disease patients. This series comprises 66 patients selected from a consecutive series of 132 patients. This selection was made in order to include only early onset patients (age at onset below 50 years) or late-onset patients with a positive family history (at least one affected relative). All genes were sequenced bi-directionally, and, additionally, SNCA, PRKN and PINK1 were subjected to gene dosage analysis.

    Results: We found mutations both in LRRK2 and PRKN, while the remaining genes yielded no mutations. Seven of the studied patients showed pathogenic mutations, in homozygosity or compound heterozygosity for PRKN, and heterozygosity for LRRK2.

    Conclusion: Mutations are common in Portuguese patients with Parkinson's disease, and these results clearly have implications not only for the genetic diagnosis, but also for the genetic counseling of these patients.

    BMC neurology 2008;8;1

  • Assembly-dependent endocytosis and clearance of extracellular alpha-synuclein.

    Lee HJ, Suk JE, Bae EJ, Lee JH, Paik SR and Lee SJ

    Institute of Biomedical Science and Technology, Konkuk University, Seoul 143-701, Republic of Korea.

    Abnormal folding and accumulation of alpha-synuclein is implicated in several neurological disorders including Parkinson's disease. Although alpha-synuclein is a typical cytoplasmic protein, a small amount of both monomeric and aggregated forms is secreted from cells and is present in human body fluids, such as cerebrospinal fluid. Extracellular alpha-synuclein aggregates have been shown to be neurotoxic, posing a challenge to any cell exposed to them. Here, we examine the internalization of various forms of extracellular alpha-synuclein, including fibrils, oligomers, and monomer, into neuronal cells and their subsequent degradation. Internalization of fibrillar alpha-synuclein could be inhibited by low temperature or the expression of a dominant-negative mutant dynamin-1 K44A, suggesting the endocytosis-mediated internalization. The internalized fibrils moved through the endosomal pathway and were degraded in the lysosome, which ultimately resulted in the clearance of the alpha-synuclein aggregates from the culture medium. Non-fibrillar oligomeric aggregates were also internalized via endocytosis and degraded by the lysosome. In contrast to aggregate uptake, the internalization of monomeric alpha-synuclein was unaffected by cold temperature and the expression of dynamin-1 K44A, consistent with direct translocation across the plasma membrane. Internalized monomers rapidly pass the plasma membrane, escaping the cells before being degraded by the cellular proteolytic systems. These results suggest that only aggregated forms of extracellular alpha-synuclein can be cleared by cell-mediated uptake and degradation, and this might represent a mechanism of preventing neurons from exposure to potentially toxic alpha-synuclein.

    The international journal of biochemistry & cell biology 2008;40;9;1835-49

  • Inhibition of alpha-synuclein fibrillization by dopamine is mediated by interactions with five C-terminal residues and with E83 in the NAC region.

    Herrera FE, Chesi A, Paleologou KE, Schmid A, Munoz A, Vendruscolo M, Gustincich S, Lashuel HA and Carloni P

    International School for Advanced Studies (SISSA), INFM DEMOCRITOS, Trieste, Italy.

    The interplay between dopamine and alpha-synuclein (AS) plays a central role in Parkinson's disease (PD). PD results primarily from a severe and selective devastation of dopaminergic neurons in substantia nigra pars compacta. The neuropathological hallmark of the disease is the presence of intraneuronal proteinaceous inclusions known as Lewy bodies within the surviving neurons, enriched in filamentous AS. In vitro, dopamine inhibits AS fibril formation, but the molecular determinants of this inhibition remain obscure. Here we use molecular dynamic (MD) simulations to investigate the binding of dopamine and several of its derivatives onto conformers representative of an NMR ensemble of AS structures in aqueous solution. Within the limitations inherent to MD simulations of unstructured proteins, our calculations suggest that the ligands bind to the (125)YEMPS(129) region, consistent with experimental findings. The ligands are further stabilized by long-range electrostatic interactions with glutamate 83 (E83) in the NAC region. These results suggest that by forming these interactions with AS, dopamine may affect AS aggregation and fibrillization properties. To test this hypothesis, we investigated in vitro the effects of dopamine on the aggregation of mutants designed to alter or abolish these interactions. We found that point mutations in the (125)YEMPS(129) region do not affect AS aggregation, which is consistent with the fact that dopamine interacts non-specifically with this region. In contrast, and consistent with our modeling studies, the replacement of glutamate by alanine at position 83 (E83A) abolishes the ability of dopamine to inhibit AS fibrillization.

    Funded by: Telethon: GGP06268

    PloS one 2008;3;10;e3394

  • Leucine-rich repeat kinase 2 colocalizes with alpha-synuclein in Parkinson's disease, but not tau-containing deposits in tauopathies.

    Perry G, Zhu X, Babar AK, Siedlak SL, Yang Q, Ito G, Iwatsubo T, Smith MA and Chen SG

    Department of Pathology, Case Western Reserve University, Cleveland, Ohio, USA. george.perry@utsa.edu

    Background: Mutations in leucine-rich repeat kinase 2 (LRRK2) are thus far the most frequent genetic cause associated with autosomal dominant and idiopathic Parkinson's disease.

    Objective: To examine whether LRRK2 is directly associated with the pathological structures of Parkinson's disease, dementia with Lewy bodies, and other related disorders using highly specific antibodies to LRRK2.

    Results: LRRK2 antibodies strongly labeled brainstem and cortical Lewy bodies, the pathological hallmarks of Parkinson's disease and dementia with Lewy bodies, respectively. We found that 20-100% (mean 60%) of alpha-synuclein-positive Lewy bodies contained LRRK2. While antibodies raised against various regions of LRRK2 were previously shown to label recombinant LRRK2 on Western blots, only antibodies raised against the N- and C-termini, but not the regions containing folded protein domains of LRRK2, immunolabeled Lewy bodies. In Alzheimer's disease, Hirano bodies were found to contain LRRK2 and the neurofibrillary tangles in progressive supranuclear palsy remained unlabeled.

    Conclusions: Information on the cellular localization of LRRK2 under normal and pathological conditions will deepen our understanding of its functions and molecular pathways relevant to the progression of Parkinson's disease and related disorders.

    Funded by: NIA NIH HHS: R01 AG024028-03

    Neuro-degenerative diseases 2008;5;3-4;222-4

  • Red blood cells are the major source of alpha-synuclein in blood.

    Barbour R, Kling K, Anderson JP, Banducci K, Cole T, Diep L, Fox M, Goldstein JM, Soriano F, Seubert P and Chilcote TJ

    Elan Pharmaceuticals, Inc., South San Francisco, Calif 94080, USA. robin.barbour@elan.com

    Background: Alpha-synuclein has been directly linked to Parkinson's disease etiology by mutations in and multiplication of its gene that result in a familial form of Parkinson's disease. Alpha-synuclein has been detected in blood, and was found to be elevated in the blood of those individuals with the alpha-synuclein gene multiplication.

    Objective: A complete analysis of the level of alpha-synuclein in blood has not been performed. In this report, we determine the quantitative distribution of alpha-synuclein in the plasma and different cellular fractions of human blood. The levels of alpha-synuclein in human and mouse blood are compared.

    Methods: Alpha-synuclein levels in the different fractions of blood were quantified by a sandwich ELISA with purified recombinant alpha-synuclein as an assay standard. Samples were further characterized by Western immunoblot analysis.

    Results: More than 99% of the alpha-synuclein resides in the red blood cells (RBCs) with less than 1% of the total detected in the plasma, platelets and peripheral blood mononuclear cells.

    Conclusions: More than 99% of the alpha-synuclein in human blood is present in the peripheral blood cells, with the remainder in plasma. Fractionation of peripheral blood cells from human blood and quantification of alpha-synuclein revealed that only a very small amount of the total alpha-synuclein is present in peripheral blood mononuclear cells, and platelets, with the majority of alpha-synuclein in blood being present in RBCs. Considering the abundance and fragility of RBCs, alpha-synuclein levels in these other blood fractions or other bodily fluids such as cerebrospinal fluid may be artificially elevated by contamination with intact or lysed RBCs.

    Neuro-degenerative diseases 2008;5;2;55-9

  • Helical alpha-synuclein forms highly conductive ion channels.

    Zakharov SD, Hulleman JD, Dutseva EA, Antonenko YN, Rochet JC and Cramer WA

    Departments of Biological Sciences, Purdue University, West Lafayette, Indiana 47907-2054, USA.

    Alpha-synuclein (alphaS) is a cytosolic protein involved in the etiology of Parkinson's disease (PD). Disordered in an aqueous environment, alphaS develops a highly helical conformation when bound to membranes having a negatively charged surface and a large curvature. It exhibits a membrane-permeabilizing activity that has been attributed to oligomeric protofibrillar forms. In this study, monomeric wild-type alphaS and two mutants associated with familial PD, E46K and A53T, formed ion channels with well-defined conductance states in membranes containing 25-50% anionic lipid and 50% phosphatidylethanolamine (PE) in the presence of a trans-negative potential. Another familial mutant, A30P, known to have a lower membrane affinity, did not form ion channels. Ca2+ prevented channel formation when added to membranes before alphaS and decreased channel conductance when added to preformed channels. In contrast to the monomer, membrane permeabilization by oligomeric alphaS was not characterized by formation of discrete channels, a requirement for PE lipid, or a membrane potential. Channel activity, alpha-helical content, thermal stability of membrane-bound alphaS determined by far-UV CD, and lateral mobility of alphaS bound to planar membranes measured by fluorescence correlation spectroscopy were correlated. It was inferred that discrete ion channels with well-defined conductance states were formed in the presence of a membrane potential by one or several molecules of monomeric alphaS in an alpha-helical conformation and that such channels may have a role in the normal function and/or pathophysiology of the protein.

    Funded by: FIC NIH HHS: TW01235; NIGMS NIH HHS: GM-18457; NINDS NIH HHS: NS-049221

    Biochemistry 2007;46;50;14369-79

  • Monitoring the prevention of amyloid fibril formation by alpha-crystallin. Temperature dependence and the nature of the aggregating species.

    Rekas A, Jankova L, Thorn DC, Cappai R and Carver JA

    Department of Chemistry, University of Wollongong, Australia.

    The molecular chaperone, alpha-crystallin, has the ability to prevent the fibrillar aggregation of proteins implicated in human diseases, for example, amyloid beta peptide and alpha-synuclein. In this study, we examine, in detail, two aspects of alpha-crystallin's fibril-suppressing ability: (a) its temperature dependence, and (b) the nature of the aggregating species with which it interacts. First, the efficiency of alpha-crystallin to suppress fibril formation in kappa-casein and alpha-synuclein increases with temperature, despite their rate of fibrillation also increasing in the absence of alpha-crystallin. This is consistent with an increased chaperone ability of alpha-crystallin at higher temperatures to protect target proteins from amorphous aggregation [GB Reddy, KP Das, JM Petrash & WK Surewicz (2000) J Biol Chem275, 4565-4570]. Second, dual polarization interferometry was used to monitor real-time alpha-synuclein aggregation in the presence and absence of alphaB-crystallin. In contrast to more common methods for monitoring the time-dependent formation of amyloid fibrils (e.g. the binding of dyes like thioflavin T), dual polarization interferometry data did not reveal any initial lag phase, generally attributed to the formation of prefibrillar aggregates. It was shown that alphaB-crystallin interrupted alpha-synuclein aggregation at its earliest stages, most likely by binding to partially folded monomers and thereby preventing their aggregation into fibrillar structures.

    The FEBS journal 2007;274;24;6290-304

  • Ultra-high field NMR studies of antibody binding and site-specific phosphorylation of alpha-synuclein.

    Sasakawa H, Sakata E, Yamaguchi Y, Masuda M, Mori T, Kurimoto E, Iguchi T, Hisanaga S, Iwatsubo T, Hasegawa M and Kato K

    Department of Structural Biology and Biomolecular Engineering, Graduate School of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabe-dori, Mizuho-ku, Nagoya 467-8603, Japan.

    Although biological importance of intrinsically disordered proteins is becoming recognized, NMR analyses of this class of proteins remain as tasks with more challenge because of poor chemical shift dispersion. It is expected that ultra-high field NMR spectroscopy offers improved resolution to cope with this difficulty. Here, we report an ultra-high field NMR study of alpha-synuclein, an intrinsically disordered protein identified as the major component of the Lewy bodies. Based on NMR spectral data collected at a 920 MHz proton frequency, we performed epitope mapping of an anti-alpha-synuclein monoclonal antibody, and furthermore, characterized conformational effects of phosphorylation at Ser129 of alpha-synuclein.

    Biochemical and biophysical research communications 2007;363;3;795-9

  • Role of different regions of alpha-synuclein in the assembly of fibrils.

    Qin Z, Hu D, Han S, Hong DP and Fink AL

    Department of Chemistry & Biochemistry, University of California, Santa Cruz, California 95064, USA.

    Elucidating the details of the assembly of amyloid fibrils is a key step to understanding the mechanism of amyloid deposition diseases including Parkinson's disease. Although several models have been proposed, based on analyses of polypeptides and short peptides, a detailed understanding of the structure and mechanism of alpha-synuclein fibrillation remains elusive. In this study, we used trypsin and endoproteinase GluC to digest intact alpha-synuclein fibrils and to analyze the detailed morphology of the resultant fibrils/remnants. We also created three mutants of alpha-synuclein, in which the N-terminal and C-terminal regions were removed, both individually and in combination, and investigated the detailed morphology of the fibrils from these mutants. Our results indicate that the assembly of mature alpha-synuclein fibrils is hierarchical: protofilaments --> protofibrils --> mature fibrils. There is a core region of approximately 70 amino acids, from residues approximately 32 to 102, which comprises the beta-rich core of the protofilaments and fibrils. In contrast, the two terminal regions show no evidence of participating in the assembly of the protofilament core but play a key role in the interactions between the protofilaments, which is necessary for the fibril maturation.

    Funded by: NINDS NIH HHS: NS39985

    Biochemistry 2007;46;46;13322-30

  • Microglial activation mediates neurodegeneration related to oligodendroglial alpha-synucleinopathy: implications for multiple system atrophy.

    Stefanova N, Reindl M, Neumann M, Kahle PJ, Poewe W and Wenning GK

    Clinical Neurobiology Unit, Neurodegeneration Research Laboratory, Department of Neurology, Innsbruck Medical University, Innsbruck, Austria.

    The role of microglial activation in multiple system atrophy (MSA) was investigated in a transgenic mouse model featuring oligodendroglial alpha-synuclein inclusions and loss of midbrain dopaminergic neurons by means of histopathology and morphometric analysis. Our findings demonstrate early progressive microglial activation in substantia nigra pars compacta (SNc) associated with increased expression of iNOS and correlating with dopaminergic neuronal loss. Suppression of microglial activation by early long-term minocycline treatment protected dopaminergic SNc neurons. The results suggest that oligodendroglial overexpression of alpha-synuclein may induce neuroinflammation related to nitrosive stress which is likely to contribute to neurodegeneration in MSA. Further, we detected increased toll-like receptor 4 immunoreactivity in both transgenic mice and MSA brains indicating a possible signaling pathway in MSA which needs to be further studied as a candidate target for neuroprotective interventions.

    Funded by: Austrian Science Fund FWF: P 19989-B05

    Movement disorders : official journal of the Movement Disorder Society 2007;22;15;2196-203

  • Neuronal and glial accumulation of alpha- and beta-synucleins in human lipidoses.

    Suzuki K, Iseki E, Togo T, Yamaguchi A, Katsuse O, Katsuyama K, Kanzaki S, Shiozaki K, Kawanishi C, Yamashita S, Tanaka Y, Yamanaka S and Hirayasu Y

    Department of Psychiatry, Yokohama City University School of Medicine, 3-9 Fukuura, Kanazawa-ku, Yokohama, 236-0004, Japan. ksuzuki@med.yokohama-cu.ac.jp

    A number of the lysosomal storage diseases that have now been characterized are associated with intra-lysosomal accumulation of lipids, caused by defective lysosomal enzymes. We have previously reported neuronal accumulation of both alpha- and beta-synucleins in brain tissue of a GM2 gangliosidosis mouse model. Although alpha-synuclein has been implicated in several neurodegenerative disorders including Parkinson's disease, dementia with Lewy bodies and multiple system atrophy, its functions remain largely unclear. In our present study, we have examined a cohort of human lipidosis cases, including Sandhoff disease, Tay-Sachs disease, metachromatic leukodystrophy, beta-galactosialidosis and adrenoleukodystrophy, for the expression of alpha- and beta-synucleins and the associated lipid storage levels. The accumulation of alpha-synuclein was found in brain tissue in not only cases of lysosomal storage diseases, but also in instances of adrenoleukodystrophy, which is a peroxisomal disease. alpha-synuclein was detected in both neurons and glial cells of patients with these two disorders, although its distribution was found to be disease-dependent. In addition, alpha-synuclein-positive neurons were also found to be NeuN-positive, whereas NeuN-negative neurons did not show any accumulation of this protein. By comparison, the accumulation of beta-synuclein was detectable only in the pons of Sandhoff disease cases. This differential accumulation of alpha- and beta-synucleins in human lipidoses may be related to functional differences between these two proteins. In addition, the accumulation of alpha-synuclein may also be a condition that is common to lysosomal storage diseases and adrenoleukodystrophies that show an enhanced expression of this protein upon the elevation of stored lipids.

    Acta neuropathologica 2007;114;5;481-9

  • alpha-Synuclein and Parkinson disease susceptibility.

    Winkler S, Hagenah J, Lincoln S, Heckman M, Haugarvoll K, Lohmann-Hedrich K, Kostic V, Farrer M and Klein C

    Department of Neurology, University of Lübeck; Ratzeburger Allee 160, 23538 Lübeck, Germany.

    Background: Mutations in the alpha-synuclein (SNCA) gene have been shown to be responsible for a rare familial form of Parkinson disease (PD). Furthermore, polymorphic variants in multiple regions of the gene have been associated with susceptibility to idiopathic PD in different populations.

    Objective: To evaluate and to confirm the role of SNCA variants in PD pathogenesis.

    Methods: We included 667 subjects (397 cases with idiopathic PD and 270 healthy, ethnically matched controls) of Northern Central and Southeastern European origin. We analyzed genotypes at 14 markers spanning the SNCA locus and its major haplotype blocks and conducted a haplotype analysis for four promoter markers including the microsatellite marker Rep1.

    Results: The three single nucleotide polymorphisms (SNPs) of the promoter region (rs2583988, rs2619364, rs2619363) and a SNP in the 3'UTR (rs356165) of the SNCA gene showed the greatest evidence for an association with PD (p <or= 0.003), with significant pairwise values for linkage disequilibrium (D' >or= 0.74, r (2) >or= 0.29). The promoter haplotype "261-T-G-T" (Rep1-rs2583988-rs2619364-rs2619363) was associated with disease (p = 0.032). The most significant association with PD was generated by excluding Rep1 (p = 0.008). This association remained significant when analyzing the Serbian patients separately and was of borderline significance for the German patients.

    Conclusions: Our findings confirm that genetic variability within the SNCA locus is associated with susceptibility to idiopathic Parkinson disease (PD). We found evidence for disease association with single nucleotide polymorphisms at both the 5' and the 3' end of the gene with pairwise linkage disequilibrium between them. The association was independent of the Rep1 status, and one major SNCA promoter haplotype class seems to be associated with PD susceptibility.

    Funded by: NINDS NIH HHS: P50 NS40256

    Neurology 2007;69;18;1745-50

  • Cellular oligomerization of alpha-synuclein is determined by the interaction of oxidized catechols with a C-terminal sequence.

    Mazzulli JR, Armakola M, Dumoulin M, Parastatidis I and Ischiropoulos H

    Joseph Stokes Jr. Research Institute, Children's Hospital of Philadelphia and University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.

    The mechanisms that govern the formation of alpha-synuclein (alpha-syn) aggregates are not well understood but are considered a central event in the pathogenesis of Parkinson's disease (PD). A critically important modulator of alpha-syn aggregation in vitro is dopamine and other catechols, which can prevent the formation of alpha-syn aggregates in cell-free and cellular model systems. Despite the profound importance of this interaction for the pathogenesis of PD, the processes by which catechols alter alpha-syn aggregation are unclear. Molecular and biochemical approaches were employed to evaluate the mechanism of catechol-alpha-syn interactions and the effect on inclusion formation. The data show that the intracellular inhibition of alpha-syn aggregation requires the oxidation of catechols and the specific noncovalent interaction of the oxidized catechols with residues (125)YEMPS(129) in the C-terminal region of the protein. Cell-free studies using novel near infrared fluorescence methodology for the detection of covalent protein-ortho-quinone adducts showed that although covalent modification of alpha-syn occurs, this does not affect alpha-syn fibril formation. In addition, oxidized catechols are unable to prevent both thermal and acid-induced protein aggregation as well as fibrils formed from a protein that lacks a YEMPS amino acid sequence, suggesting a specific effect for alpha-syn. These results suggest that inappropriate C-terminal cleavage of alpha-syn, which is known to occur in vivo in PD brain or a decline of intracellular catechol levels might affect disease progression, resulting in accelerated alpha-syn inclusion formation and dopaminergic neurodegeneration.

    Funded by: NIA NIH HHS: AG13966; NIEHS NIH HHS: ES013508

    The Journal of biological chemistry 2007;282;43;31621-30

  • Casein kinase 2 is the major enzyme in brain that phosphorylates Ser129 of human alpha-synuclein: Implication for alpha-synucleinopathies.

    Ishii A, Nonaka T, Taniguchi S, Saito T, Arai T, Mann D, Iwatsubo T, Hisanaga S, Goedert M and Hasegawa M

    Department of Molecular Neurobiology, Tokyo Institute of Psychiatry, 2-1-8 Kamikitazawa, Setagaya-ku, Tokyo 156-8585, Japan.

    In Lewy body diseases and multiple system atrophy, alpha-synuclein is hyperphosphorylated at Ser129, suggesting a role in pathogenesis. Here, we report purification of the protein kinase in rat brain that phosphorylates Ser129 and its identification as casein kinase-2 (CK2). We show that most of the activity can be inhibited by heparin, an inhibitor of CK2. Phosphorylated Ser129 was detected in primary cultured neurons and inhibited by CK2 inhibitors. In some cases of Lewy body disease, CK2-like immunoreactivity was recovered in the sarkosyl-insoluble fraction, which was enriched in phosphorylated alpha-synuclein. Taken together, these findings suggest that CK2 may be involved in the hyperphosphorylation of alpha-synuclein in alpha-synucleinopathies.

    FEBS letters 2007;581;24;4711-7

  • The Lewy body in Parkinson's disease: molecules implicated in the formation and degradation of alpha-synuclein aggregates.

    Wakabayashi K, Tanji K, Mori F and Takahashi H

    Department of Neuropathology, Institute of Brain Science, Hirosaki University School of Medicine, Hirosaki, Japan. koichi@cc.hirosaki-u.ac.jp

    The histological hallmark of Parkinson's disease (PD) is the presence of fibrillar aggregates called Lewy bodies (LBs). LB formation has been considered to be a marker for neuronal degeneration, because neuronal loss is found in the predilection sites for LBs. To date, more than 70 molecules have been identified in LBs, in which alpha-synuclein is a major constituent of LB fibrils. Alpha-synuclein immunohistochemistry reveals that diffuse cytoplasmic staining develops into pale bodies via compaction, and that LBs arise from the peripheral portion of pale bodies. This alpha-synuclein abnormality is found in 10% of pigmented neurons in the substantia nigra and more than 50% of those in the locus ceruleus in PD. Recent studies have suggested that oligomers and protofibrils of alpha-synuclein are cytotoxic, and that LBs may represent a cytoprotective mechanism in PD.

    Neuropathology : official journal of the Japanese Society of Neuropathology 2007;27;5;494-506

  • Alpha-synuclein protects cerebellar granule neurons against 6-hydroxydopamine-induced death.

    Monti B, Polazzi E, Batti L, Crochemore C, Virgili M and Contestabile A

    Department of Biology, University of Bologna, Bologna, Italy.

    The physiological role of alpha-synuclein, a protein found enriched in intraneuronal deposits characterizing Parkinson's disease, is debated. While its aggregation is usually considered linked to neuropathology, its normal function may be related to fundamental processes of synaptic transmission and plasticity. By using antisense oligonucleotide strategy, we report in this study that alpha-synuclein silencing in cultured cerebellar granule cells results in widespread death of these neurons, thus demonstrating an essential pro-survival role of the protein towards primary neurons. To study alpha-synuclein expression and processing in a Parkinson's disease model of neurotoxicity, we exposed differentiated cultures of cerebellar granule neurons to toxic concentrations of 6-hydroxydopamine (6-OHDA). This resulted in neuronal death accompanied by a decrease of the monomeric form of alpha-synuclein, which was due to both decreased synthesis of the protein and its increased mono-ubiquitination accompanied by nuclear translocation. The essential neuroprotective role of alpha-synuclein was confirmed by the fact that subchronic valproate treatment, which increases alpha-synuclein expression and prevents its nuclear translocation in cerebellar granule cells exposed to 6-OHDA, significantly protected these neurons from 6-OHDA insult. In agreement with the pro-survival role of alpha-synuclein in this model, subtoxic concentrations of alpha-synuclein antisense oligonucleotides, aggravated 6-OHDA toxicity towards granule neurons. Our results demonstrate that normal alpha-synuclein expression is essential for the viability of primary neurons and that its pro-survival role is abolished in 6-OHDA neurotoxic challenge. These results are relevant to more precisely define the role of alpha-synuclein in neuronal cells and to better understand its putative involvement in neurodegeneration.

    Journal of neurochemistry 2007;103;2;518-30

  • Multiple system atrophy: alpha-synuclein and neuronal degeneration.

    Yoshida M

    Department of Neuropathology, Institute for Medical Science of Aging, Aichi Medical University, Aichi, Japan. myoshida@aichi-med-u.ac.jp

    Multiple system atrophy (MSA) is a sporadic neurodegenerative disorder that encompasses olivopontocerebellar atrophy (OPCA), striatonigral degeneration (SND) and Shy-Drager syndrome (SDS). The histopathological hallmark is the formation of alpha-synuclein-positive glial cytoplasmic inclusions (GCIs) in oligodendroglia. alpha-synuclein aggregation is also found in glial nuclear inclusions, neuronal cytoplasmic inclusions (NCIs), neuronal nuclear inclusions (NNIs) and dystrophic neurites. We evaluated the pathological features of 102 MSA cases, and presented the pathological spectrum of MSA and initial features of alpha-synuclein accumulation. We found that 39% of the 102 cases showed equivalent SND and OPCA pathologies, 33% showed OPCA- and 22% showed SND-predominant pathology, whereas 6% showed extremely mild changes. Our pathological analysis indicated that OPCA-type was relatively more frequent and SND-type was less frequent in Japanese MSA cases, compared to the relatively high frequency of SND-type in Western countries, suggesting that different phenotypic patterns of MSA may exist between races. In the early stage, in addition to GCIs, NNIs and diffuse homogenous alpha-synuclein staining in neuronal nuclei and cytoplasm were observed in lesions in the pontine nuclei, putamen, substantia nigra, locus ceruleus, inferior olivary nucleus, intermediolateral column of thoracic spinal cord, lower motor neurons and cortical pyramidal neurons. A subgroup of MSA cases with severe temporal atrophy showed numerous NCIs, particularly in the limbic system. These findings suggest that primary non-fibrillar and fibrillar alpha-synuclein aggregation also occur in neurons. The oligo-myelin-axon-neuron complex mechanism, along with the direct involvement of neurons themselves, may synergistically accelerate the degenerative process of MSA.

    Neuropathology : official journal of the Japanese Society of Neuropathology 2007;27;5;484-93

  • Pathological biochemistry of alpha-synucleinopathy.

    Iwatsubo T

    Department of Neuropathology and Neuroscience, Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo, Japan. iwatsubo@mol.f.u-tokyo.ac.jp

    Lewy bodies (LBs) are hallmark lesions in the brains of patients with Parkinson's disease (PD) and dementia with Lewy bodies (DLB). We raised a monoclonal antibody LB509 against purified LBs from the brains of patients with DLB that strongly immunolabled LBs, and found that alpha-synuclein is one of the major components of LBs. Thus, the deposition of alpha-synuclein, an abundant presynaptic brain protein, as fibrillary aggregates in affected neurons or glial cells, was highlighted as a hallmark lesion of a subset of neurodegenerative disorders, including PD, DLB and multiple system atrophy collectively referred to as synucleinopathies. Importantly, the identification of missense mutations in and multiplication of alpha-synuclein gene in some pedigrees of familial PD has strongly implicated alpha-synuclein in the pathogenesis of PD and other synucleinopathies. We then examined the specific posttranslational modifications that characterize and underlie the aggregation of alpha-synuclein in synucleinopathy brains by mass spectrometry and using a specific antibody, and found that serine 129 of alpha-synuclein deposited in synucleinopathy lesions is selectively and extensively phosphorylated. Furthermore we generated transgenic C. elegans overexpressing alpha-synuclein in neurons, and found that overexpression of familial PD-linked mutant form of alpha-synuclein impairs functions of dopamine neurons. These findings collectively underscore the importance of deposition of alpha-synuclein as well as its phosphorylation in the pathogenesis of alpha-synucleinopathies.

    Neuropathology : official journal of the Japanese Society of Neuropathology 2007;27;5;474-8

  • Residual structure, backbone dynamics, and interactions within the synuclein family.

    Sung YH and Eliezer D

    Department of Biochemistry and Program in Structural Biology, Weill Cornell Medical College, 1300 York Avenue, New York, NY 10021, USA.

    The human synuclein protein family includes alpha-synuclein, which has been linked to both familial and sporadic Parkinson's disease, and the highly homologous beta and gamma-synuclein. Mutations in alpha-synuclein cause autosomal dominant early onset Parkinson's, and the protein is found deposited in a fibrillar form in hereditary and idiopathic forms of the disease. No genetic link between beta and gamma-synuclein, and any neurodegenerative disease has been established, and it is generally considered that these proteins are not highly pathogenic. In addition, beta and gamma-synuclein are reported to aggregate less readily than alpha-synuclein in vitro. Indeed, beta-synuclein has been reported to protect against alpha-synuclein aggregation in vitro, as well as alpha-synuclein-mediated toxicity in vivo. Earlier, we compared the structural properties of the highly helical states adopted by all three synucleins in association with detergent micelles in an attempt to delineate the basis for functional differences between the three proteins. Here, we report a comparison of the structural and dynamic properties of the free states of all three proteins in order to shed light on differences that may help to explain their different propensities to aggregate, which in turn may underlie their differing contributions to the etiology of Parkinson's disease. We find that gamma-synuclein closely resembles alpha-synuclein in its free-state residual secondary structure, consistent with the more similar propensities of the two proteins to aggregate in vitro. beta-Synuclein, however, differs significantly from alpha-synuclein, exhibiting a lower predisposition towards helical structure in the second half of its lipid-binding domain, and a higher preference for extended structures in its C-terminal tail. Both beta and gamma-synuclein show less extensive transient long-range structure than that observed in alpha-synuclein. These results raise questions regarding the role of secondary structure propensities and transient long-range contacts in directing synuclein aggregation reactions.

    Funded by: NIA NIH HHS: AG019391, R01 AG019391, R01 AG019391-07, R01 AG025440, R01 AG025440-01A1, R37 AG019391; NIGMS NIH HHS: GM66254, P41 GM066354

    Journal of molecular biology 2007;372;3;689-707

  • An investigation into the lipid-binding properties of alpha-, beta- and gamma-synucleins in human brain and cerebrospinal fluid.

    Salem SA, Allsop D, Mann DM, Tokuda T and El-Agnaf OM

    Biomedical Sciences Unit, Department of Biological Sciences, Lancaster University, Lancaster LA1 4YQ, UK.

    Parkinson's disease (PD) and dementia with Lewy bodies (DLB) are both characterized by the formation and intraneuronal accumulation of fibrillar aggregates of alpha-synuclein (alpha-syn) protein in affected brain regions. alpha-Syn has biochemical properties and a structural motif characteristic of fatty acid binding proteins. Using the fatty acid binding resin Lipidex-1000, we investigated the capture of alpha-, beta-, and gamma-syn proteins as lipid-associated proteins from normal and DLB brain lysates, and from normal human cerebrospinal fluid (CSF). These were eluted from Lipidex-1000 and analyzed by SDS-NuPAGE followed by Western blotting. Using this methodology, we have been able to extract full-length and truncated forms of alpha-syn from brain lysates. We also extracted low levels of beta-syn from DLB brains, but failed to extract any gamma-syn. We were able to capture only full-length monomeric alpha-syn from normal human CSF. Our data confirm the fatty acid binding properties of alpha-syn, and to a lesser extent beta-syn, but suggest that gamma-syn does not share this same characteristic.

    Brain research 2007;1170;103-11

  • Tilted properties of the 67-78 fragment of alpha-synuclein are responsible for membrane destabilization and neurotoxicity.

    Crowet JM, Lins L, Dupiereux I, Elmoualija B, Lorin A, Charloteaux B, Stroobant V, Heinen E and Brasseur R

    Gembloux Agricultural University, Centre de Biophysique Moléculaire Numérique, 2 Passage des Déportés, B-5030 Gembloux, Belgium. crowet.jm@fsagx.ac.be

    Alpha-synuclein is a 140 residue protein associated with Parkinson's disease. Intraneural inclusions called Lewy bodies and Lewy neurites are mainly composed of alpha-synuclein aggregated into amyloid fibrils. Other amyloidogenic proteins, such as the beta amyloid peptide involved in Alzheimer's disease and the prion protein (PrP) associated with Creuztfeldt-Jakob's disease, are known to possess "tilted peptides". These peptides are short protein fragments that adopt an oblique orientation at a hydrophobic/hydrophilic interface, which enables destabilization of the membranes. In this paper, sequence analysis and molecular modelling predict that the 67-78 fragment of alpha-synuclein is a tilted peptide. Its destabilizing properties were tested experimentally. The alpha-synuclein 67-78 peptide is able to induce lipid mixing and leakage of unilamellar liposomes. The neuronal toxicity, studied using human neuroblastoma cells, demonstrated that the alpha-synuclein 67-78 peptide induces neurotoxicity. A mutant designed by molecular modelling to be amphipathic was shown to be significantly less fusogenic and toxic than the wild type. In conclusion, we have identified a tilted peptide in alpha-synuclein, which could be involved in the toxicity induced during amyloidogenesis of alpha-synuclein.

    Proteins 2007;68;4;936-47

  • Quantitative proteomics identifies surfactant-resistant alpha-synuclein in cerebral cortex of Parkinsonism-dementia complex of Guam but not Alzheimer's disease or progressive supranuclear palsy.

    Yang W, Woltjer RL, Sokal I, Pan C, Wang Y, Brodey M, Peskind ER, Leverenz JB, Zhang J, Perl DP, Galasko DR and Montine TJ

    Department of Pathology, University of Washington, Harborview Medical Center, Box 359791, Seattle, WA 98104, USA.

    Parkinsonism-dementia complex (PDC) remains a significant health burden to the Chamorro population. We tested the hypothesis that quantitative proteomics might provide fresh insight into this enigmatic illness by analyzing proteins resistant to surfactant extraction from patients with Alzheimer's disease (AD) or PDC and their matched controls using isobaric tags for relative and absolute quantification. In addition to the expected increase in abnormal frontal cortical Abeta peptides, tau, ubiquitin, and apolipoprotein E in AD, and tau in PDC, we identified alpha-synuclein (SNCA) as a major abnormal protein in PDC but not AD. We confirmed our isobaric tags for relative and absolute quantification findings by enzyme-linked immunosorbent assay in frontal and temporal cortices. We extended our assays to include a limited number of cases of progressive supranuclear palsy (PSP) and dementia with Lewy bodies; we observed increased abnormal tau but not SNCA in PSP, and abnormal SNCA in dementia with Lewy bodies that was quantitatively similar to PDC. Finally, soluble Abeta oligomers were selectively increased in AD but not PDC or PSP. These results show that frontal and temporal cortex in PDC is distinguished from AD and PSP by its accumulation of abnormal SNCA and suggest that PDC be considered a synucleinopathy as well as a tauopathy.

    Funded by: NIA NIH HHS: AG24011, R01 AG024011; NINDS NIH HHS: NS48595, R01 NS048595

    The American journal of pathology 2007;171;3;993-1002

  • Investigation of alpha-synuclein fibril structure by site-directed spin labeling.

    Chen M, Margittai M, Chen J and Langen R

    Department of Molecular Pharmacology and Toxicology, School of Pharmacy, University of Southern California, Los Angeles, California 90033, USA.

    The misfolding and fibril formation of alpha-synuclein plays an important role in neurodegenerative diseases such as Parkinson disease. Here we used electron paramagnetic resonance spectroscopy, together with site-directed spin labeling, to investigate the structural features of alpha-synuclein fibrils. We generated fibrils from a total of 83 different spin-labeled derivatives and observed single-line, exchange-narrowed EPR spectra for the majority of all sites located within the core region of alpha-synuclein fibrils. Such exchange narrowing requires the orbital overlap between multiple spin labels in close contact. The core region of alpha-synuclein fibrils must therefore be arranged in a parallel, in-register structure wherein same residues from different molecules are stacked on top of each other. This parallel, in-register core region extends from residue 36 to residue 98 and is tightly packed. Only a few sites within the core region, such as residues 62-67 located at the beginning of the NAC region, as well as the N- and C-terminal regions outside the core region, are significantly less ordered. Together with the accessibility measurements that suggest the location of potential beta-sheet regions within the fibril, the data provide significant structural constraints for generating three-dimensional models. Furthermore, the data support the emerging view that parallel, in-register structure is a common feature shared by a number of naturally occurring amyloid fibrils.

    Funded by: NIA NIH HHS: P50 AG05142

    The Journal of biological chemistry 2007;282;34;24970-9

  • Association of alpha-synuclein gene haplotypes with Parkinson's disease.

    Parsian AJ, Racette BA, Zhao JH, Sinha R, Patra B, Perlmutter JS and Parsian A

    Department of Pediatrics, College of Medicine, University of Arkansas for Medical Sciences, ACHRI, 1120 Marshall Street, Little Rock, AR 72202, USA. parsianabbas@uams.edu

    In a previous study, we detected an association between a dinucleotide repeat (Rep1) in the alpha-Synuclein (SNCA) gene and sporadic Parkinson's disease (PD). To extend our previous finding in a larger sample and further determine the role of SNCA in the development of PD, we screened a sample of 194 familial PD (FPD), 327 sporadic PD (SPD), and 215 controls with the Rep1 marker and 2 single nucleotide polymorphisms (SNPs) (770 and int4) in the SNCA gene. There was significant difference in allele frequency between African American and American Indian groups for Rep1 marker (p=0.03). These two samples were excluded from further analysis because of sample size. Comparison of allele frequency differences between PD and controls for the single-locus was significant only for Rep1 and SPD (p=0.017). The global case control association was highly significant for the three loci haplotypes comparisons. Our results indicate that Rep1 locus may be in linkage disequilibrium (LD) with a mutation in the gene or itself could be a risk factor for SPD.

    Parkinsonism & related disorders 2007;13;6;343-7

  • Oxidative stress-induced phosphorylation, degradation and aggregation of alpha-synuclein are linked to upregulated CK2 and cathepsin D.

    Takahashi M, Ko LW, Kulathingal J, Jiang P, Sevlever D and Yen SH

    Department of Neuroscience, Mayo Clinic College of Medicine, 4500 San Pablo Road, Jacksonville, FL 32224, USA.

    Intracellular accumulation of alpha-synuclein (alpha-Syn) as filamentous aggregates is a pathological feature shared by Parkinson's disease, dementia with Lewy bodies and multiple system atrophy, referred to as synucleinopathies. To understand the mechanisms underlying alpha-Syn aggregation, we established a tetracycline-off inducible transfectant (3D5) of neuronal lineage overexpressing human wild-type alpha-Syn. Alpha-Syn aggregation was initiated by exposure of 3D5 cells to FeCl2. The exposure led to formation of alpha-Syn inclusions and oligomers of 34, 54, 68 kDa and higher molecular weights. The oligomers displayed immunoreactivity with antibodies to the amino-, but not to the carboxyl (C)-, terminus of alpha-Syn, indicating that C-terminally truncated alpha-Syn is a major component of oligomers. FeCl2 exposure also promoted accumulation of S129 phosphorylated monomeric alpha-Syn (P alpha-Syn) and casein kinase 2 (CK2); however, G-protein-coupled receptor kinase 2 was reduced. Treatment of FeCl2-exposed cells with CK2 inhibitors (DRB or TBB) led to decreased formation of alpha-Syn inclusions, oligomers and P alpha-Syn. FeCl2 exposure also enhanced the activity/level of cathepsin D. Treatment of the FeCl2-exposed cells with pepstatin A or NH4Cl led to reduced formation of oligomers/inclusions as well as of approximately 10 and 12 kDa truncated alpha-Syn. Our results indicate that alpha-Syn phosphorylation caused by FeCl2 is due to CK2 upregulation, and that lysosomal proteases may have a role in producing truncated alpha-Syn for oligomer assembly.

    Funded by: NINDS NIH HHS: P50-NS40256

    The European journal of neuroscience 2007;26;4;863-74

  • Quantitative PCR-based screening of alpha-synuclein multiplication in multiple system atrophy.

    Lincoln SJ, Ross OA, Milkovic NM, Dickson DW, Rajput A, Robinson CA, Papapetropoulos S, Mash DC and Farrer MJ

    Department of Neuroscience, Molecular Genetics Laboratory and Core, Morris K. Udall Parkinson's Disease Research Center of Excellence, Mayo Clinic, 4500 San Pablo Road, Jacksonville, FL 32224, USA.

    Multiple system atrophy (MSA) is by nature a 'sporadic' disease with no evidence of familial aggregation observed. However, the alpha-synuclein locus (SNCA) multiplication families have clinically displayed parkinsonism and autonomic dysfunction. The present study did not find any SNCA multiplications in a series of 58 pathologically confirmed MSA cases excluding this event as a common cause of MSA. The question of a genetic component in MSA remains to be answered.

    Funded by: NIMH NIH HHS: R03 MH074059, R03 MH074059-02; NINDS NIH HHS: P01 NS040256, P50 NS 40256, P50 NS040256, P50 NS040256-07, P50 NS040256-070005

    Parkinsonism & related disorders 2007;13;6;340-2

  • Tau and alpha-synuclein in susceptibility to, and dementia in, Parkinson's disease.

    Goris A, Williams-Gray CH, Clark GR, Foltynie T, Lewis SJ, Brown J, Ban M, Spillantini MG, Compston A, Burn DJ, Chinnery PF, Barker RA and Sawcer SJ

    Department of Clinical Neurosciences (Neurology Unit), University of Cambridge, Cambridge, United Kingdom. an.goris@med.kuleuven.be

    Objective: Parkinson's disease (PD) is a neurodegenerative condition that typically presents as a movement disorder but is known to be associated with variable degrees of cognitive impairment including dementia. We investigated the genetic basis of susceptibility to and cognitive heterogeneity of this disease.

    Methods: In 659 PD patients, 109 of which were followed up for 3.5 years from diagnosis, and 2,176 control subjects, we studied candidate genes involved in protein aggregation and inclusion body formation, the pathological hallmark of parkinsonism: microtubule-associated protein tau (MAPT), glycogen synthase kinase-3beta (GSK3B), and alpha-synuclein (SNCA).

    Results: We observed that cognitive decline and the development of PD dementia are strongly associated (p = 10(-4)) with the inversion polymorphism containing MAPT. We also found a novel synergistic interaction between the MAPT inversion polymorphism and the single nucleotide polymorphism rs356219 from the 3' region of SNCA. In our data, carrying a risk genotype at either of these loci marginally increases the risk for development of PD, whereas carrying the combination of risk genotypes at both loci approximately doubles the risk for development of the disease (p = 3 x 10(-6)).

    Interpretation: Our data support the hypothesis that tau and alpha-synuclein are involved in shared or converging pathways in the pathogenesis of PD, and suggest that the tau inversion influences the development of cognitive impairment and dementia in patients with idiopathic PD. These findings have potentially important implications for understanding the interface between tau and alpha-synuclein pathways in neurodegenerative disorders and for unraveling the biological basis for cognitive impairment and dementia in PD.

    Funded by: Parkinson's UK: G-4054

    Annals of neurology 2007;62;2;145-53

  • Thermodynamics imprinting reveals differential binding of metals to alpha-synuclein: relevance to Parkinson's disease.

    Bharathi and Rao KS

    Department of Biochemistry and Nutrition, Central Food Technological Research Institute, Mysore 570020, India.

    The aggregation of alpha-synuclein is a hallmark feature of Parkinson's disease (PD) and other synucleinopathies. Metals are the significant etiological factors in PD, and their interaction with alpha-synuclein affect dramatically the kinetics of fibrillation in vitro and are proposed to play an important and potential neurodegenerative role in vivo. In the present study, we investigated the stoichiometry of binding of copper [Cu (II)] and iron [Fe (III)] with alpha-synuclein (wild recombinant type and A30P, A53T, E46K mutant forms) using isothermal titration calorimetry (ITC). alpha-Synuclein monomer (wild and mutant forms) titrated by Cu (II), showed two binding sites, with an apparent K(B) of 10(5)M and 10(4)M, respectively. But, alpha-synuclein (wild type and mutant forms) titrated with Fe (III) revealed a K(B) of 10(5)M with single binding site. The present investigation uncovers the detailed binding propensities between metals and alpha-synuclein and has biological implications in PD.

    Biochemical and biophysical research communications 2007;359;1;115-20

  • The impact of the E46K mutation on the properties of alpha-synuclein in its monomeric and oligomeric states.

    Fredenburg RA, Rospigliosi C, Meray RK, Kessler JC, Lashuel HA, Eliezer D and Lansbury PT

    Center for Neurologic Diseases, Brigham and Women's Hospital and Department of Neurology, Harvard Medical School, 65 Landsdowne Street, Cambridge, Massachusetts 02139, USA.

    The third and most recently identified Parkinson's disease-linked variant of the neuronal protein alpha-synuclein to be identified (E46K) results in widespread brain pathology and early onset Parkinson symptoms (Zarranz et al. (2004) Ann. Neurol. 55, 164-173). Herein, we present biochemical and biophysical characterization of E46K alpha-synuclein in various states of aggregation. Circular dichroism and nuclear magnetic resonance spectroscopy illustrate that the E46K mutation results in subtle changes in the conformation of the monomeric protein both free in solution and in the presence of SDS micelles. However, it does not alter the overall helical propensity of the protein in the presence of phospholipids. E46K alpha-synuclein formed insoluble fibrils in vitro more rapidly than the wild type protein, and electron microscopy revealed that E46K alpha-synuclein fibrils possess a typical amyloid ultrastructure. E46K alpha-synuclein protofibrils, soluble aggregates that form during the transition from the monomeric form to the fibrillar form of alpha-synuclein, were characterized by electron microscopy and gel filtration and were found to include annular species. The unique ability of a subfraction of E46K and wild type alpha-synuclein protofibrils containing porelike species to permeabilize lipid vesicles was demonstrated in vitro using a real-time chromatographic method. In contrast to simplistic expectations, the total amount of protofibrils and the amount of permeabilizing activity per mole protein in the protofibril fraction were reduced by the E46K mutation. These results suggest that if the porelike activity of alpha-synuclein is important for neurotoxicity, there must be factors in the neuronal cytoplasm that reverse the trends in the intrinsic properties of E46K versus WT alpha-synuclein that are observed in vitro.

    Funded by: NIA NIH HHS: AG019391, AG08470, R01 AG019391, R01 AG019391-07, R01 AG025440, R01 AG025440-02, R37 AG019391; NINDS NIH HHS: NS038375

    Biochemistry 2007;46;24;7107-18

  • YGR198w (YPP1) targets A30P alpha-synuclein to the vacuole for degradation.

    Flower TR, Clark-Dixon C, Metoyer C, Yang H, Shi R, Zhang Z and Witt SN

    Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center, Shreveport, LA 71130, USA.

    Using a genetic screen we discovered that YGR198w (named YPP1), which is an essential Saccharomyces cerevisiae gene of unknown function, suppresses the toxicity of an alpha-synuclein (alpha-syn) mutant (A30P) that is associated with early onset Parkinson's disease. Here, we show that YPP1 suppresses lethality of A30P, but not of wild-type alpha-syn or the A53T mutant. The Ypp1 protein, when overexpressed, drives each of the three alpha-syns into vesicles that bud off the plasma membrane, but only A30P-containing vesicles traffick to and merge with the vacuole, where A30P is proteolytically degraded. We show that Ypp1p binds to A30P but not the other two alpha-syns; that YPP1 interacts with genes involved in endocytosis/actin dynamics (SLA1, SLA2, and END3), protein sorting (class E vps), and vesicle-vacuole fusion (MON1 and CCZ1) to dispose of A30P; and that YPP1 also participates in pheromone-triggered receptor-mediated endocytosis. Our data reveal that YPP1 mediates the trafficking of A30P to the vacuole via the endocytic pathway.

    Funded by: NINDS NIH HHS: R21 NS053678, R21NS053678

    The Journal of cell biology 2007;177;6;1091-104

  • Accumulation of phosphorylated alpha-synuclein in dopaminergic neurons of transgenic mice that express human alpha-synuclein.

    Wakamatsu M, Ishii A, Ukai Y, Sakagami J, Iwata S, Ono M, Matsumoto K, Nakamura A, Tada N, Kobayashi K, Iwatsubo T and Yoshimoto M

    Medicinal Research Laboratories, Taisho Pharmaceutical Co., Ltd., Saitama, Japan.

    Parkinson's disease is neuropathologically characterized by the presence of Lewy bodies, whose major component is alpha-synuclein. We had previously generated transgenic mice that expressed human alpha-synuclein carrying an Ala53Thr point mutation (halpha-syn140m) under the control of the rat tyrosine hydroxylase promoter and found that halpha-syn140m was localized not only in the cytoplasm but also in the nuclei of mesencephalic dopaminergic neurons. In the present study, we carried out immunohistochemical analysis of the brain of Tg mice using anti-PSer129, an antibody that specifically recognizes alpha-synuclein phosphorylated at Ser129. The antibody detected only phosphorylated halpha-syn140m, whereas phosphorylation of endogenous alpha-synuclein, if any, was below the detection limit of the method employed. The analysis showed that approximately one-third of the halpha-syn140m-positive neurons in the midbrain of heterozygous Tg mice were concomitantly reactive to anti-PSer129. The ratio almost doubled in homozygotes, indicating that the phosphorylation level depends directly on the amount of substrate. In addition, the ratio did not change at least up to 48 weeks of age. These data strongly suggest that halpha-syn140m underwent constitutive phosphorylation and that the phosphorylation level was maintained to a certain level until the aged stages. Remarkably, halpha-syn140m localized in the nuclei seemed to be preferentially phosphorylated compared with that in the cytoplasm. Among kinases that have been reported to be involved in the phosphorylation of alpha-synuclein, the beta subunit of casein kinase-2 was detected in the nuclei by immunohistochemistry. These data imply that at least casein kinase-2 is involved in the phosphorylation of halpha-syn140m in the Tg mice.

    Journal of neuroscience research 2007;85;8;1819-25

  • alpha-Synuclein and its disease-related mutants interact differentially with the microtubule protein tau and associate with the actin cytoskeleton.

    Esposito A, Dohm CP, Kermer P, Bähr M and Wouters FS

    Cell Biophysics Group at the European Neuroscience Institute-Göttingen, Waldweg, 33, 37073 Göttingen, Germany. aesposito@quantitative-microscopy.org

    alpha-Synuclein is a primarily neuronal protein that is enriched at the pre-synapse. alpha-Synuclein and the microtubule binding protein tau have been implicated in neurodegenerative diseases. alpha-Synuclein is known to associate with phospholipid vesicles, regulates dopamine metabolism and exhibits chaperone activity, but its main role remains largely unknown. Furthermore, knowledge on its interactions and post-translational modifications is essential for a molecular understanding of alpha-synucleinopathies. We investigated alpha-synuclein mutations, causative for autosomal dominant forms of Parkinson's disease (A30P, A53T and E46K), and phosphorylation mutants at serine 129 (S129A and S129D) using fluorescently labelled alpha-synuclein, actin and tau. The investigation of colocalization, and protein-protein interactions by Förster resonance energy transfer and fluorescence lifetime imaging showed that alpha-synuclein associates with the actin cytoskeleton and interacts with tau. The A30P mutation and cytoskeletal destabilization decreased this interaction. Given the concurrent loss of membrane binding by this mutation, we propose a membrane-bound functional complex with tau that might involve the actin cytoskeleton.

    Neurobiology of disease 2007;26;3;521-31

  • A variable poly-T sequence modulates alpha-synuclein isoform expression and is associated with aging.

    Beyer K, Humbert J, Ferrer A, Lao JI, Latorre P, Lopez D, Tolosa E, Ferrer I and Ariza A

    Department of Pathology, Hospital Germans Trias i Pujol, Autonomous University of Barcelona, Badalona, Spain. katrinbeyer@hotmail.com

    alpha-Synuclein, the main component of proteinaceous inclusions in synucleinopathies, is centrally involved in aggregation processes preceding Lewy body formation. Here we describe a new alpha-synuclein gene poly-T polymorphism that is situated upstream to exon 3 and consists of three different alleles. A correlation between poly-T length and expression of alpha-synuclein 126 mRNA, an isoform lacking exon 3, was detected in the human cerebral cortex. Specifically, when compared with the most frequent 7T/7T genotype, the shortest poly-T stretch (5T) was associated with the lowest alpha-synuclein 126 expression levels, whereas the longest poly-T stretch (12T) was accompanied by the highest alpha-synuclein 126 expression levels. Thus, three different expression-level-specific genotypes, with 5T+ genotypes as low alpha-synuclein 126 expression genotypes and 12T+ genotypes as high alpha-synuclein 126 expression genotypes, could be established. Poly-T genotype distributions were also analyzed in a healthy control population. Age-dependent variations in this distribution were observed and showed accumulation of low alpha-synuclein 126 expression genotypes at ages under 60 years and high alpha-synuclein 126 expression genotypes at ages over 80 years. To determine human specificity of the variable poly-T strech, the mouse alpha-synuclein gene sequence was analyzed. Although alpha-synuclein is very well conserved in vertebrates, the poly-T sequence was found to be absent in mice, and an alpha-synuclein 126 mouse homologue could not be detected. In conclusion, this newly identified poly-T polymorphism is a human-specific sequence; its length influences alpha-synuclein 126 expression levels; and, finally, it seems to exert a specific influence on normal aging.

    Journal of neuroscience research 2007;85;7;1538-46

  • Expression of alpha-synuclein mRNA in Parkinson's disease.

    Papapetropoulos S, Adi N, Mash DC, Shehadeh L, Bishopric N and Shehadeh L

    Funded by: NHLBI NIH HHS: F32 HL083673

    Movement disorders : official journal of the Movement Disorder Society 2007;22;7;1057-9; author reply 1057

  • Oligomeric alpha-synuclein inhibits tubulin polymerization.

    Chen L, Jin J, Davis J, Zhou Y, Wang Y, Liu J, Lockhart PJ and Zhang J

    Department of Pathology, University of Washington School of Medicine, Seattle, WA, USA.

    Earlier investigations have demonstrated that tubulin co-localizes with alpha-synuclein in Lewy bodies and influences the formation of alpha-synuclein aggregation. However, it is not clear whether aggregated alpha-synuclein has any effects on the function of tubulin, i.e. tubulin polymerization, a critical mechanism by which neurons maintain their morphology and execute functions. In this study, we evaluated the effects of aggregated alpha-synuclein on tubulin polymerization in dopaminergic neurons (MES cells), along with mitochondrial function, cell morphology, and viability. The results indicate that MES cells exposed to extracellular oligomeric alpha-synuclein exhibited decreased tubulin polymerization and mitochondrial function as well as morphological alternation long before cell death. Further investigation showed that internalization of oligomeric alpha-synuclein by neurons appeared to be critical in the process, although direct interaction between tubulin and intracellular oligomeric alpha-synuclein was not necessary. Finally, we demonstrated that neurotoxicity induced by oligomeric alpha-synuclein was largely prevented by overexpressing the neuroprotective protein, DJ-1.

    Funded by: NIA NIH HHS: R01AG025327; NIEHS NIH HHS: R01ES012703

    Biochemical and biophysical research communications 2007;356;3;548-53

  • The Ala53Thr mutation in the alpha-synuclein gene in a Korean family with Parkinson disease.

    Ki CS, Stavrou EF, Davanos N, Lee WY, Chung EJ, Kim JY and Athanassiadou A

    Clinical genetics 2007;71;5;471-3

  • Familial genes in sporadic disease: common variants of alpha-synuclein gene associate with Parkinson's disease.

    Ross OA, Gosal D, Stone JT, Lincoln SJ, Heckman MG, Irvine GB, Johnston JA, Gibson JM, Farrer MJ and Lynch T

    Department of Neuroscience, Mayo Clinic College of Medicine, Jacksonville, FL, USA. ross.owen@mayo.edu

    Genetic variation of the alpha-synuclein gene (SNCA) is known to cause familial parkinsonism, however the role of SNCA variants in sporadic Parkinson's disease (PD) remains elusive. The present study identifies an association of common SNCA polymorphisms with disease susceptibility in a series of Irish PD patients. There is evidence for association with alternate regions, of protection and risk which may act independently/synergistically, within the promoter region (Rep1; OR: 0.59, 95% CI: 0.37-0.84) and the 3'UTR of the gene (rs356165; OR: 1.67, 95% CI: 1.08-2.58). Given previous reports of association a collaborative effort is required which may exploit global linkage disequilibrium patterns for SNCA and standardise polymorphic markers used in each population. It is now crucial to identify the susceptibility allele and elucidate its functionality which may generate a therapeutic target for PD.

    Funded by: NINDS NIH HHS: P50 #NS40256, P50 NS040256, P50 NS040256-070004, P50 NS040256-079002

    Mechanisms of ageing and development 2007;128;5-6;378-82

  • Parkin polymorphisms and environmental exposure: decrease in age at onset of Parkinson's disease.

    Ghione I, Di Fonzo A, Saladino F, Del Bo R, Bresolin N, Comi GP and Rango M

    Centro per il Morbo di Parkinson, Centro Dino Ferrari, Department of Neurological Sciences, University of Milan, Fondazione I.R.C.C.S., Ospedale Maggiore Policlinico, Mangiagalli e Regina Elena, Milan, Italy.

    We tested the hypothesis that parkin polymorphisms (SNPs) and environmental exposure (EE) interact to reduce the age of onset of idiopathic Parkinson disease (PD). We prospectively and consecutively enrolled a total of 81 Italian PD patients. The diagnosis of PD was based on the UK Parkinson's Disease Society's brain bank criteria. Twenty-one patients with a positive family history for PD or tremor were excluded from the study. We collected information about medical history and EE. PARK1, PARK2 genes and PARK8 (exon 41) were screened. We detected one parkin mutation in a single patient and three parkin polymorphisms in a total of 25 patients; no alpha synuclein mutations, no common mutations of LRKK2 gene were found. The mutation-positive patient has been excluded from the study. The cohort of the remaining 59 patients has been divided into four subgroups, according to the presence/absence of parkin polymorphisms and the presence/absence of environmental factors-exposure. The age of onset of PD was significantly lower in patients with both SNPs and EE as compared to patients without (62.18+/-9.5 years versus 71.62+/-8 years, p=0.024; -13%). Patients with either SNPs or EE had an intermediate age of onset. The association of parkin polymorphisms and environmental exposure has a strong effect in lowering the age of onset of PD; the effect of environmental exposure or parkin polymorphisms alone seems to influence modestly the age of onset of PD. Individuals with environmental/occupational exposure should be screened for the presence of parkin SNPs.

    Neurotoxicology 2007;28;3;698-701

  • Association of alcohol craving with alpha-synuclein (SNCA).

    Foroud T, Wetherill LF, Liang T, Dick DM, Hesselbrock V, Kramer J, Nurnberger J, Schuckit M, Carr L, Porjesz B, Xuei X and Edenberg HJ

    Indiana University School of Medicine, Indianapolis, Indiana 46202-5251, USA. tforoud@iupui.edu

    Background: Studies have found that genomic variation in the gene SNCA, which encodes the protein alpha-synuclein, may contribute to the variation in alcohol consumption in an inbred rat model of alcohol preference. Studies in humans have provided support for an association between SNCA and craving for alcohol.

    Methods: To examine the role of this gene in alcohol dependence and related phenotypes, 30 single nucleotide polymorphisms (SNPs) were genotyped across the SNCA gene in a sample of 219 multiplex alcoholic families of European American descent. Two phenotypes, alcohol dependence and alcohol craving, were analyzed using the pedigree disequilibrium test.

    Results: There was no evidence of association between any of the SNCA SNPs and alcohol dependence (p>or=0.13). In contrast, 8 SNPs provided evidence of association (p<0.05) with the phenotype of alcohol craving. Haplotype analysis further supported evidence of an association with alcohol craving; a haplotype encompassing SNPs in intron 4 through the region downstream of the gene was overtransmitted to cravers and a second haplotype was overtransmitted to noncravers.

    Conclusions: These results suggest that variation in SNCA contributes to alcohol craving, a common, although not uniform, feature of alcohol dependence.

    Funded by: NIAAA NIH HHS: R01AA010707, U10AA008401

    Alcoholism, clinical and experimental research 2007;31;4;537-45

  • Linkage disequilibrium and association analysis of alpha-synuclein and alcohol and drug dependence in two American Indian populations.

    Clarimon J, Gray RR, Williams LN, Enoch MA, Robin RW, Albaugh B, Singleton A, Goldman D and Mulligan CJ

    Laboratory of Neurogenetics, National Institute on Aging, National Institutes of Health, Bethesda, Maryland, USA.

    Background: Alpha-synuclein is involved in dopaminergic neurotransmission and has been implicated in a number of neurodegenerative disorders, such as Parkinson's disease. Recent studies, in humans and in rat and monkey models, have suggested that alpha-synuclein may play a role in the development and maintenance of certain addictive disorders.

    Methods: Fifteen single-nucleotide polymorphisms (SNPs) in the alpha-synuclein gene (SNCA) and 1 upstream microsatellite repeat (NACP-REP1) were assayed in Southwest (SW; n=514) and Plains (n=420) American Indian populations. Patterns of linkage disequilibrium (LD) at SNCA were determined for the 2 populations and compared with Caucasian, African, and Asian populations in the HapMap database (http://www.hapmap.org). Assayed alleles and constructed haplotypes in the study populations were tested for association with 4 clinical phenotypes [alcohol dependence, alcohol use disorders, drug dependence, and drug use disorders (lifetime diagnoses)] as well as with 2 symptom count phenotypes (all 18 questions and the 8 questions diagnostic for alcohol dependence).

    Results: Patterns of LD at SNCA were similar in both Indian populations and were consistent with the LD structure in other populations as reflected in the HapMap database. Single allele tests revealed significant associations between 4 SNPs and drug dependence in the SW population and between 2 of those SNPs plus 2 other SNPs and drug dependence in SW males only. In the Plains population, a significant association was detected only in males between 2 SNPs and alcohol use disorders and between 1 SNP and alcohol dependence. In the SW population, 1 SNP was marginally significant with the total symptom count. However, in all cases, the support was modest and disappeared with correction for multiple comparisons. No association was found between constructed haplotypes and any of the phenotypes in either population.

    Conclusions: Despite modest support for association between multiple SNCA SNPs and several of the addictive disorders tested in this study, statistical significance disappeared after correction for multiple testing. Thus, our data do not support a role for a variant in the SNCA gene that contributes to alcohol or drug addiction in the 2 studied American Indian populations. Future research may focus on variants in the promoter region that could cause the changes in mRNA and protein levels observed in previous studies.

    Funded by: NIAAA NIH HHS: R03 AA 12906

    Alcoholism, clinical and experimental research 2007;31;4;546-54

  • Aggregated alpha-synuclein mediates dopaminergic neurotoxicity in vivo.

    Periquet M, Fulga T, Myllykangas L, Schlossmacher MG and Feany MB

    Department of Pathology, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA.

    Mutations in the synaptic protein alpha-synuclein cause rare genetic forms of Parkinson's disease. Alpha-synuclein is thought to play a critical role in more common sporadic cases of Parkinson's disease as well because the protein aggregates in the hallmark intraneuronal inclusions of the disorder, Lewy bodies. To test the role of protein aggregation in the pathogenesis of Parkinson's disease, we expressed a form of alpha-synuclein with a deletion of amino acids 71-82 that is unable to aggregate in vitro in a transgenic Drosophila model of the disorder. We found no evidence of large aggregates or oligomeric species of alpha-synuclein in these animals and no loss of tyrosine hydroxylase-positive neurons. We also expressed a truncated form of alpha-synuclein that has enhanced ability to aggregate in vitro. This truncated form of alpha-synuclein showed increased aggregation into large inclusions bodies, increased accumulation of high molecular weight alpha-synuclein species, and demonstrated enhanced neurotoxicity in vivo. Our findings thus support a critical role for aggregation of alpha-synuclein in mediating toxicity to dopaminergic neurons in vivo, although the precise role each aggregated form of alpha-synuclein plays in neurotoxicity remains to be determined.

    Funded by: NINDS NIH HHS: NS038375-07, NS041536

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2007;27;12;3338-46

  • Over-expression of alpha-synuclein in human neural progenitors leads to specific changes in fate and differentiation.

    Schneider BL, Seehus CR, Capowski EE, Aebischer P, Zhang SC and Svendsen CN

    Waisman Center and Department of Anatomy, University of Wisconsin, Madison, WI 53705, USA.

    Missense mutations and extra copies of the alpha-Synuclein gene result in Parkinson disease (PD). Human stem and progenitor cells can be expanded from embryonic tissues and provide a source of non-transformed neural cells to explore the effects of these pathogenic mutations specifically in human nervous tissue. We over-expressed the wild type, A53T and A30P forms of alpha-synuclein in expanded populations of progenitors derived from the human fetal cortex. The protein localized in the nucleus and around microvesicles. Only the A53T form was acutely toxic, suggesting a unique vulnerability of these progenitors to this mutation. Interestingly, constitutive over-expression of wild-type alpha-synuclein progressively impaired the innate ability of progenitors to switch toward gliogenesis at later passages. To explore the effect of alpha-synuclein on neuronal subtypes selectively affected in PD, such as dopaminergic neurons, alpha-synuclein and its mutations were also over-expressed in terminally differentiating neuroectodermal cultures derived from human embryonic stem cells (hESC). Alpha-synuclein induced acute cytotoxicity and reduced the number of neurons expressing either tyrosine hydroxylase or gamma-aminobutyric acid over time. Consistent with the selective vulnerability of ventral midbrain dopaminergic neurons, alpha-synuclein cytotoxicity appeared most pronounced following FGF8/SHH specification and was decreased by inhibition of dopamine synthesis. Together, these data show that alpha-synuclein over-expressed in human neural embryonic cells results in patterns of degeneration that in some cases match features of Parkinson Disease. Thus, neural cells derived from hESC provide a useful model system to understand the development of alpha-synuclein-related pathologies and allow therapeutic drug screening.

    Human molecular genetics 2007;16;6;651-66

  • Global DNA hypomethylation and DNA hypermethylation of the alpha synuclein promoter in females with anorexia nervosa.

    Frieling H, Gozner A, Römer KD, Lenz B, Bönsch D, Wilhelm J, Hillemacher T, de Zwaan M, Kornhuber J and Bleich S

    Molecular psychiatry 2007;12;3;229-30

  • Wild-type and mutant alpha-synuclein induce a multi-component gene expression profile consistent with shared pathophysiology in different transgenic mouse models of PD.

    Miller RM, Kiser GL, Kaysser-Kranich T, Casaceli C, Colla E, Lee MK, Palaniappan C and Federoff HJ

    Center for Aging and Developmental Biology, Aab Institute for Biomedical Research, Rochester, NY 14642, USA.

    The pathophysiological processes that cause Parkinson's disease (PD) affect dopamine neurons residing in the substantia nigra with devastating consequences for normal movement. One important gene involved in both familial and sporadic PD is alpha-synuclein. We have generated three strains of alpha-synuclein transgenic mice to study the pathologic consequences of the targeted expression of mutant or wild-type human alpha-synuclein in a model system. We have analyzed gene expression patterns in these mice using high throughput microarrays in anatomical regions implicated in disease (substantia nigra and brainstem). Our study reveals gene dosage-dependent dysregulation of several genes important for the dopaminergic phenotype in mice over-expressing wild-type human alpha-synuclein in the substantia nigra at time points preceding neuronal cell death. Analysis of mutant alpha-synuclein mice at a time point when pathology is advanced reveals several new candidate genes that may play a role in neuronal demise and/or protein accumulation.

    Funded by: NINDS NIH HHS: NS038065, NS038377

    Experimental neurology 2007;204;1;421-32

  • Dopamine affects the stability, hydration, and packing of protofibrils and fibrils of the wild type and variants of alpha-synuclein.

    Follmer C, Romão L, Einsiedler CM, Porto TC, Lara FA, Moncores M, Weissmüller G, Lashuel HA, Lansbury P, Neto VM, Silva JL and Foguel D

    Instituto de Bioquímica Médica, Programa de Biologia Estrutural, Universidade Federal do Rio de Janeiro, Rio de Janeiro 21941-590, Brazil.

    Parkinson's disease (PD) is characterized by the presence of cytoplasmic inclusions composed of alpha-synuclein (alpha-syn) in dopaminergic neurons. This suggests a pivotal role of dopamine (DA) on PD development. Here, we show that DA modulates differently the stability of protofibrils (PF) and fibrils (F) composed of wild type or variants of alpha-syn (A30P and A53T) as probed by high hydrostatic pressure (HHP). While in the absence of DA, all alpha-syn PF exhibited identical stability, in its presence, the variant-composed PF acquired a greater stability (DAPFwt < DAPFA30P = DAPFA53T), implying that they would last longer, which could shed light onto why these mutations are so aggressive. When alpha-syn was incubated for long times (18 days) in the presence of DA, we observed the formation of F by electronic microscopy, suggesting that the PF trapped in the presence of DA in short times can evolve into F. The stability of F was also altered by DA. DAFwt was more labile than Fwt, indicating that the former would be more susceptible to breakage. PFA30P and DAPFA30P, when added to mesencephalic and cortical neurons in culture, decreased the number and length of neurites and increased the number of apoptotic cells. Surprisingly, these toxic effects of PFA30P and DAPFA30P were practically abolished with HHP treatment, which was able to break the PF into smaller aggregates, as seen by atomic force microscopy. These results suggest that strategies aimed at breaking and/or clearing these aggregates is promising in alleviating the symptoms of PD.

    Biochemistry 2007;46;2;472-82

  • Isomers of human alpha-synuclein stabilized by disulfide bonds exhibit distinct structural and aggregative properties.

    Jiang C and Chang JY

    Research Center for Protein Chemistry, Brown Foundation Institute of Molecular Medicine for the Prevention of Human Diseases and Department of Biochemistry and Molecular Biology, The University of Texas, Houston, Texas 77030, USA.

    The discovery of three mutants in the -synuclein (alphaSyn) gene and the identification of alphaSyn as the major component of Lewy body have opened a new field for understanding the pathogenesis of Parkinson's disease (PD). AlphaSyn is a natively unfolded protein with unknown function and unspecified conformational heterogeneity. In this study, we introduce four Ser/Ala --> Cys mutations at positions 9, 42, 69, and 89 in human wild-type alphaSyn (wt-alphaSyn) and two PD-associated alphaSyn mutants, A30P-alphaSyn and A53T-alphaSyn. This allows expression of three alphaSyn mutants, wt-alphaSyn(4C), A30P-alphaSyn(4C), and A53T-Syn(4C). Subsequent oxidative folding enables each alphaSyn(4C) mutant to form three partially stabilized two-disulfide isomers, designated as alphaSyn(2SS), that are amenable to further isolation and characterization. These alphaSyn mutants exhibit the following properties. (a) A30P-alphaSyn(4C) exhibits a lower folding flexibility than wt-alphaSyn(4C) and A53T-alphaSyn(4C). (b) All three alphaSyn(4C) mutants, like wt-alphaSyn, exhibit a predominant structure of random coil. However, wt-alphaSyn(2SS) adopts an alpha-helical conformation, whereas A30P-alphaSyn(2SS) and A53T-alphaSyn(2SS) take on significant beta-sheet structure. (c) A30P-alphaSyn(2SS) shows a stronger tendency to aggregate than A53T-alphaSyn(2SS) and wt-alphaSyn(2SS). (d) Three isolated isomers of wt-alphaSyn(2SS) exhibit a propensity for forming oligomers different yet enhanced versus that for wt-alphaSyn. These data together substantiate the notion that under physiological conditions, human alphaSyn exists as diverse conformational isomers which exhibit distinct propensities for aggregation and fibril formation.

    Biochemistry 2007;46;2;602-9

  • [Frequency of the IVS4+66A-G polymorphism in the alpha-synuclein gene in patients with Parkinson's disease in north-western Mexico].

    Ramírez-Jirano LJ, Ruiz-Sandoval JL, Jiménez-Gil FJ, Ramírez-Vega J, Vargas-Frutos E and Gallegos-Arreola MP

    Laboratorio de Genética Molecular, División de Medicina Molecular, CIBO-IMSS, Jalisco, México.

    Introduction: Parkinson disease (PD) is the second most common neurodegenerative disease of adult onset. Is a progressive movement disorder including tremor, bradykinesia, rigidity and postural instability, with an age onset between 43 and 66 years. Histopathologically, is characterized by a severe loss of dopaminergic neurons in the substantia nigra and inclusions consisting of insoluble protein aggregates called Lewy bodies, this are comprised in part of alpha-synuclein. The etiology of PD is still not fully understood, but genetic analyses, epidemiologic studies and experimental models of PD are providing important new insights into the pathogenesis of PD.

    Aim: To determine allelic and genotypic frequencies of polymorphism IVS4+66A-G in the alpha-synuclein gene and to demonstrate its association with PD in northwest Mexican population.

    Genomic desoxyribonucleic acid (DNA) from 51 PD patients and 121 persons without PD were achieved by polymerase chain reaction and analyzed the allelic and genotypic distribution in IVS4+66A-G polymorphism of alpha-synuclein gene.

    Results: The genotypic frequency of IVS4+66AA was 43.1% in PD patients and 38.8% in control group; IVS4+66GG was 2% in PD patients and 4.1% in control group, whereas 54.9% in PD patients and 57.1% in control group were heterozygous. Statistical differences were not observed between groups (p<0.05).

    Conclusions: Association was not observed between the IVS4+66A-G polymorphism and PD.

    Revista de neurologia 2007;44;1;15-7

  • Age-associated increases of alpha-synuclein in monkeys and humans are associated with nigrostriatal dopamine depletion: Is this the target for Parkinson's disease?

    Chu Y and Kordower JH

    Department of Neurological Sciences, Rush University Medical Center, 1735 West Harrison Street, Chicago, IL 60612, USA.

    alpha-Synuclein is a synaptic protein that has been directly linked to both the etiology and pathogenesis of Parkinson's disease. We have previously shown that only nigral neurons in PD expressing alpha-synuclein inclusions display a loss dopaminergic phenotype. The present study tested the hypothesis that normal aging contributes to this effect. The relative abundance of alpha-synuclein protein within individual nigral neurons was quantified in eighteen normal humans between the age of 18 and 102 and twenty four rhesus monkeys between the age of 2 and 34. Optical densitometry revealed a robust age-related increase in alpha-synuclein protein within individual nigral neurons in both species. This effect was specific for nigral alpha-synuclein as no age-related changes were found in the ventral tegmental area nor were there changes in the nigra for non-pathogenic beta-synuclein. The age-related increases in nigral alpha-synuclein were non-aggregated and strongly associated with age-related decreases in tyrosine hydroxylase (TH), the rate limiting enzyme for dopamine production. In fact, only cells expressing alpha-synuclein displayed reductions in TH. We hypothesize that age-related increases in alpha-synuclein result in a subthreshold degeneration of nigrostriatal dopamine which, in PD, becomes symptomatic due to lysosomal failure resulting in protein misfolding and inclusion formation. We further hypothesize that preventing the age-related accumulation of non-aggregated alpha-synuclein might be a simple and potent therapeutic target for patients with PD.

    Neurobiology of disease 2007;25;1;134-49

  • Screening for SNCA and LRRK2 mutations in Greek sporadic and autosomal dominant Parkinson's disease: identification of two novel LRRK2 variants.

    Xiromerisiou G, Hadjigeorgiou GM, Gourbali V, Johnson J, Papakonstantinou I, Papadimitriou A and Singleton AB

    Neurogenetics Unit, Department of Neurology, Medical School, University of Thessaly, Larissa, Greece.

    Mutations in SNCA and LRRK2 genes, encoding alpha-synuclein and leucine-rich repeat kinase 2, respectively, cause autosomal dominant Parkinson's disease (AdPD). The LRRK2 G2019S (c.6055G > A) and R1441G (c.4321C > G) mutations have also been identified in sporadic PD (sPD). We studied 55 unrelated patients with AdPD, 235 patients with sPD, and 235 healthy age- and gender-matched controls all of Greek origin. Patients with AdPD were screened for SNCA and LRRK2 mutations by direct sequencing. SNCA gene dosage analysis was also performed for AdPD using quantitative duplex polymerase chain reaction of genomic DNA. In addition, we investigated the frequency of the LRRK2 G2019S mutation in sPD. We found no missense mutations or multiplications in the SNCA gene. Here we report two novel variants, A211V (c.632C > T) and K544E (c.1630A > G) in LRRK2 gene in two patients with AdPD that was not present in controls. We identified only one patient with sPD (1/235; 0.4%) carrying the G2019S mutation. LRRK2 mutations are present in AdPD and sPD patients of Greek origin.

    European journal of neurology 2007;14;1;7-11

  • The effect of truncated human alpha-synuclein (1-120) on dopaminergic cells in a transgenic mouse model of Parkinson's disease.

    Michell AW, Tofaris GK, Gossage H, Tyers P, Spillantini MG and Barker RA

    Department of Clinical Neuroscience, University of Cambridge and Cambridge Centre for Brain Repair, Cambridge, CB2 2PY, UK. awmichell@gmail.com

    Alpha-Synuclein is thought to play an important role in the pathology of Parkinson's disease (PD). Truncated forms of this protein can be found in PD brain extracts, and these species aggregate faster and are more susceptible to oxidative stress than the full-length protein. We investigated the effect of truncated alpha-synuclein on dopaminergic cells using a transgenic mouse expressing alpha-synuclein (1-120) driven by the rat tyrosine hydroxylase promoter on a mouse alpha-synuclein null background. We found a selective reduction in the yield of dopaminergic cells from transgenic embryonic ventral mesencephalic cell cultures. However, in vivo the substantia nigra/ventral tegmentum dopaminergic cell counts were not reduced in transgenics, although these mice are known to have reduced striatal dopamine. When transplanted to the striatum in the unilateral 6-hydroxydopamine-lesioned mouse model of PD, dopaminergic cells derived from transgenic embryonic ventral mesencephala were significantly smaller at 6 weeks, and showed a trend towards being less effective at ameliorating rotational asymmetry than those from control alpha-synuclein null mice. These results suggest that alpha-synuclein (1-120) renders dopaminergic cells more susceptible to stress, which may have important implications as to how this truncated protein might contribute to dopaminergic cell death in sporadic PD.

    Funded by: Parkinson's UK: G-4039

    Cell transplantation 2007;16;5;461-74

  • Determination of alpha-synuclein concentration in human plasma using ELISA.

    Fjorback AW, Varming K and Jensen PH

    Department of Neurology, Aalborg Hospital, Denmark.

    The nerve cell protein alpha-synuclein is important in Parkinson's disease and dementia with Lewy bodies, and its expression levels are directly linked to development of the diseases. Quantification of the plasma level of alpha-synuclein may therefore be important as a biomarker for disease susceptibility. We present a quantitative measurement of alpha-synuclein in the plasma of healthy control subjects in relation to their age using a novel enzyme-linked immunosorbent assay (ELISA). The plasma concentration among the 44 blood donors displayed a median of 5.6 microg/L (range 2.1-19.4 microg/L) with a narrow distribution (25 % and 75 % percentiles, 4.0 and 7.2 microg/L) and there was no correlation with age and gender. This narrow concentration range and the ease of measuring the quantitative ELISA support future investigations of plasma alpha-synuclein in relation to neurodegenerative diseases.

    Scandinavian journal of clinical and laboratory investigation 2007;67;4;431-5

  • Parkin disrupts the alpha-synuclein/dopamine transporter interaction: consequences toward dopamine-induced toxicity.

    Moszczynska A, Saleh J, Zhang H, Vukusic B, Lee FJ and Liu F

    Department of Neuroscience, Centre for Addiction and Mental Health, Clarke Division, 250 College Street, Toronto, ON M5T 1R8, Canada.

    Parkinson's disease is characterized by progressive neuronal degeneration of dopaminergic neurons in the substantia nigra. Many factors are thought to contribute to the neuronal cell death that occurs in Parkinson's disease, including alpha-synuclein-mediated toxicity. Previously, we have reported that alpha-synuclein directly couples to the carboxyl tail of the dopamine transporter (DAT) and that the alpha-synuclein/DAT protein complex formation accelerates DAT-mediated cellular dopamine (DA) uptake and DA-induced cellular apoptosis. In the present study, we report that parkin, an E2-dependent E3 protein ubiquitin ligase associated with recessive early onset Parkinson's disease, exerts a protective effect against DA-induced alpha-synuclein-dependent cell toxicity. Parkin impairs the alpha-synuclein/DAT coupling by interacting with the carboxyl-terminus of the DAT and blocks the alpha-synuclein-induced enhancement in both DAT cell surface expression and DAT-mediated DA uptake. Moreover, we have found that parkin protects against DA-induced cell toxicity in dopaminergic SK-N-SH cells. These findings will help identify the role of these proteins in the etiology and/or maintenance of Parkinson's disease.

    Journal of molecular neuroscience : MN 2007;32;3;217-27

  • Alpha-synuclein adopts an alpha-helical conformation in the presence of polyunsaturated fatty acids to hinder micelle formation.

    Broersen K, van den Brink D, Fraser G, Goedert M and Davletov B

    MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK.

    Alpha-synuclein is a small cytosolic protein involved in the pathogenesis of Parkinson's disease and other neurodegenerative disorders. Recent studies suggested a lipid-related function for this brain-enriched protein. Since the brain carries a high level of docosahexaenoic acid (DHA) and since the extent of alpha-synuclein gene expression increases in response to DHA intake, we have investigated the interaction of alpha-synuclein with this essential omega-3 fatty acid. We show that alpha-synuclein allows DHA to be present in a soluble rather than micellar form. Upon interaction with DHA, the normally unstructured alpha-synuclein rapidly adopts an alpha-helical conformation. Prolonged exposure to DHA, however, gradually converts alpha-synuclein into amyloid-like fibrils. These results identify a potential biological function for alpha-synuclein and define an omega-3-linked pathway leading to alpha-synuclein aggregation.

    Funded by: Medical Research Council: MC_U105178791, MC_U105184291; Parkinson's UK: G-4057, G-4066

    Biochemistry 2006;45;51;15610-6

  • Heat shock protein 70 inhibits alpha-synuclein fibril formation via interactions with diverse intermediates.

    Huang C, Cheng H, Hao S, Zhou H, Zhang X, Gao J, Sun QH, Hu H and Wang CC

    National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.

    alpha-Synuclein (AS) is a main component of Lewy bodies in midbrain dopamine neurons pathologically characteristic of Parkinson's disease. We show that heat shock protein (Hsp) 70 inhibits AS fibril formation via preventing the formation of prefibrillar AS (PreAS), binding with PreAS to impede nuclei formation, and binding with nuclei to retard fibril elongation. Also, Hsp70 suppresses the PreAS-induced permeabilization of vesicular membrane through interactions with PreAS. The substrate-binding domain alone is sufficient for Hsp70 to inhibit AS fibril formation. The binding of Hsp70 with PreAS only requires the substrate-binding subdomain, and the binding with AS nuclei requires the C-terminal lid subdomain as well. The results may form the molecular basis for elucidating the mechanism of AS fibril formation and the crucial roles of chaperones in protecting proteins from toxic conversion in many conformational diseases.

    Journal of molecular biology 2006;364;3;323-36

  • Quantitative morphological analysis reveals ultrastructural diversity of amyloid fibrils from alpha-synuclein mutants.

    van Raaij ME, Segers-Nolten IM and Subramaniam V

    MESA+ Institute for Nanotechnology, Faculty of Science and Technology, University of Twente, 7500 AE, Enschede, The Netherlands.

    High resolution atomic force microscopy is a powerful tool to characterize nanoscale morphological features of protein amyloid fibrils. Comparison of fibril morphological properties between studies has been hampered by differences in analysis procedures and measurement error determination used by various authors. We describe a fibril morphology analysis method that allows for quantitative comparison of features of amyloid fibrils of any amyloidogenic protein measured by atomic force microscopy. We have used tapping mode atomic force microscopy in liquid to measure the morphology of fibrillar aggregates of human wild-type alpha-synuclein and the disease-related mutants A30P, E46K, and A53T. Analysis of the images shows that fibrillar aggregates formed by E46K alpha-synuclein have a smaller diameter (9.0 +/- 0.8 nm) and periodicity (mode at 55 nm) than fibrils of wild-type alpha-synuclein (height 10.0 +/- 1.1 nm; periodicity has a mode at 65 nm). Fibrils of A30P have smaller diameter still (8.1 +/- 1.2 nm) and show a variety of periodicities. This quantitative analysis procedure enables comparison of the results with existing models for assembly of amyloid fibrils.

    Biophysical journal 2006;91;11;L96-8

  • Attenuation of the norepinephrine transporter activity and trafficking via interactions with alpha-synuclein.

    Wersinger C, Jeannotte A and Sidhu A

    Department of Biochemistry, Molecular and Cell Biology, Georgetown University, Washington, DC 20007, USA.

    Alpha-synuclein (alpha-Syn) has been studied in the context of Parkinson's disease, but its normative role remains elusive. We have shown that alpha-Syn regulates the homeostasis of dopaminergic and serotonergic synapses, through trafficking of the dopamine and serotonin transporter, respectively. In the present study we sought to determine if alpha-Syn could also modulate noradrenergic signaling, by studying its interactions with the norepinephrine transporter (NET). We co-transfected Ltk- cells with increasing amounts of alpha-Syn DNA and a constant amount of NET DNA, and observed a progressive decrease (68%) in [3H]-NE uptake in cells co-transfected with a ratio of 3:1 alpha-Syn:NET DNA. The Kd of transport did not change, but increasing alpha-Syn caused a decrease in the Vmax of the transporter, from 2.27+/-0.14 to 0.89+/-0.15 pmol/min/10(5) cells, with NET expression alone or 4:1 ratio of alpha-Syn:NET transfection, respectively. Decreases in surface biotinylation and [3H]-nisoxetine binding kinetics in intact cells revealed that NET cell surface expression was attenuated in correlation to the amount of alpha-Syn co-transfected into cells. The interaction between NET and alpha-Syn occurred via the NAC domain of alpha-Syn, the region directly responsible for self-aggregation. These findings are the first to show that alpha-Syn has a central role in the homeostasis of noradrenergic neurons. Together with our previous studies on dopamine and serotonin transporters, we propose that a primary physiological role of alpha-Syn may be to regulate the homeostasis of monoamines in synapses, through modulatory interactions of the protein with monoaminergic transporters.

    Funded by: NINDS NIH HHS: NS-34914, NS-41555

    The European journal of neuroscience 2006;24;11;3141-52

  • Identification of a risk haplotype of the alpha-synuclein gene in Japanese with sporadic Parkinson's disease.

    Kobayashi H, Ujike H, Hasegawa J, Yamamoto M, Kanzaki A and Sora I

    Department of Psychobiology, Tohoku University Graduate School of Medicine, Sendai, Japan.

    alpha-Synuclein is one of the main components of Lewy bodies, a pathological marker of Parkinson's disease (PD). Certain missense mutations of the alpha-synuclein gene cause familial PD, but the role of the gene in sporadic PD is still controversial. We scrutinized polymorphisms of the alpha-synuclein gene in a Japanese population and investigated their associations with sporadic cases of PD. The 5' flanking region to intron 2 of the alpha-synuclein gene (3.8 kb) and two polymorphisms in intron 4 previously reported in Caucasian sporadic cases of PD were analyzed in 185 sporadic PD and 191 controls. Five novel single nucleotide polymorphisms (SNPs), 16 reported SNPs, and one reported polynucleotide polymorphism (PNP) were found. Most of the polymorphisms examined were in linkage disequilibrium. Significant associations with PD were found in 15 of 21 SNPs, especially in intron 1 (IVS1+155 TmAn PNP and the IVS1+719 C>T SNP, P < 0.0001). Haplotype analysis showed that T10A7-A-A and T11A6-G-G haplotypes at three loci (IVS1+155 - IVS1+273 - IVS1+608) were strongly negative and positive risk factors of sporadic PD, respectively (odds ratios were 0.23 [95% confidence interval, 0.16-0.32] and 1.51 [95% confidence interval, 1.29-1.75]). In conclusion, our findings indicate that genetic variations of the alpha-synuclein gene affect the development of sporadic PD.

    Movement disorders : official journal of the Movement Disorder Society 2006;21;12;2157-64

  • Impairment of microtubule-dependent trafficking by overexpression of alpha-synuclein.

    Lee HJ, Khoshaghideh F, Lee S and Lee SJ

    The Parkinson's Institute, Sunnyvale, CA 94089, USA.

    Abnormal accumulation of alpha-synuclein (alpha-syn) has been linked to several neurological disorders, including Parkinson's disease (PD). However, the underlying mechanism by which alpha-syn accumulation affects neuronal function and survival remains unknown. Here, we provide data suggesting a possible effect of aggregated alpha-syn on the microtubule (MT) network. Consistent with the MT dysfunction, we also observed other degenerative changes, such as neuritic degeneration, trafficking defects, and Golgi fragmentation, which are common pathological features shared by many human neurodegenerative diseases. Neuritic degeneration and Golgi fragmentation were confirmed in primary cultures of dorsal root ganglia (DRG) neurons overexpressing alpha-syn. This effect of alpha-syn seems to have some selectivity to the MT system, as actin microfilaments and MT-independent trafficking remain unaffected. Within the degenerating neurites, we found numerous spherical co-aggregates of alpha-syn and tubulins, from which actin was excluded. These studies suggest that the MT system is a potential target of alpha-syn, and impairment of this system might have impacts on neuronal structure and function.

    The European journal of neuroscience 2006;24;11;3153-62

  • Behavioral effects of dopaminergic agonists in transgenic mice overexpressing human wildtype alpha-synuclein.

    Fleming SM, Salcedo J, Hutson CB, Rockenstein E, Masliah E, Levine MS and Chesselet MF

    Department of Neurology and Neurobiology, The David Geffen School of Medicine at the University of California Los Angeles, 710 Westwood Plaza, Los Angeles, CA 90095-1769, USA.

    Overexpression of alpha-synuclein causes familial Parkinson's disease and abnormal aggregates of the protein are present in sporadic cases of the disease. We have examined the behavioral effects of direct and indirect dopaminergic agonists in transgenic mice expressing human alpha-synuclein under the Thy-1 promoter (Thy1-aSyn, alpha-synuclein overexpressor), which exhibit progressive impairments in behavioral tests sensitive to nigrostriatal dopamine dysfunction. Male Thy1-aSyn and wild-type mice received vehicle, benserazide/L-DOPA (25 mg/kg, i.p.), high (2 mg/kg, s.c.) and low doses (0.125, 0.25, 0.5 mg/kg, s.c.) of apomorphine, and amphetamine (5 mg/kg, i.p.), beginning at 3 months of age, and were tested on the challenging beam, spontaneous activity, pole test, and gait. l-DOPA had a paradoxical effect and worsened the deficits in Thy1-aSyn mice compared with controls, whereas the high dose of apomorphine only produced few deficits above those already present in Thy1-aSyn. In contrast to wild-type mice, Thy1-aSyn mice did not show amphetamine-induced stereotypies. The results indicate that chronic overexpression of alpha-synuclein led to abnormal pharmacological responses in mice.

    Funded by: NIA NIH HHS: AG022074, AG18440, P01 AG022074, R01 AG018440, R37 AG018440; NIEHS NIH HHS: U54 ES012078, U54 ES012078-01, U54ES12078; NINDS NIH HHS: P50 NS038367, P50 NS038367-02, P50NS38367, T32 NS007449, T32 NS07449-05

    Neuroscience 2006;142;4;1245-53

  • Abnormal compartmentalization of norepinephrine in mouse dentate gyrus in alpha-synuclein knockout and A30P transgenic mice.

    Yavich L, Jäkälä P and Tanila H

    Department of Pharmacology and Toxicology, University of Kuopio, Finland. Leonid.Yavich@uku.fi

    In the dentate gyrus of the mouse hippocampus, presynaptic recruitment of norepinephrine in response to repeated-burst stimulation can be described in terms of an interaction between storage and readily releasable pools. The dynamics of this interaction depends on neuronal activity (bursting), so that the higher the demand for norepinephrine, the faster it is delivered from the storage pool. We also found that alpha-synuclein, a presynaptic protein that plays a crucial role in dopamine compartmentalization in the striatum, is also involved in the compartmentalization of norepinephrine in the dentate gyrus. Experiments in transgenic mice with modified or absent alpha-synuclein revealed that the familial Parkinson's disease-linked alpha-synuclein mutation A30P can cause selective changes in the function of noradrenergic terminals. Addition of mutated human alpha-synuclein abolished the normal norepinephrine mobilization. There were no compensatory mechanisms available in the norepinephrine presynaptic terminals. In contrast, deletion of mouse alpha-synuclein is compensated for by increased vesicle transport from the storage pool. The effects are essentially the same as previously reported for dopaminergic terminals in the striatum, indicating that the important role of alpha-synuclein in neurotransmitter mobilization is not limited to dopaminergic terminals.

    Journal of neurochemistry 2006;99;3;724-32

  • Alpha-synuclein induces hyperphosphorylation of Tau in the MPTP model of parkinsonism.

    Duka T, Rusnak M, Drolet RE, Duka V, Wersinger C, Goudreau JL and Sidhu A

    Department of Biochemistry, Molecular and Cellular Biology, Georgetown University, Washington, DC, USA.

    Many neurodegenerative diseases associated with functional Tau dysregulation, including Alzheimer's disease (AD) and other tauopathies, also show alpha-synuclein (alpha-Syn) pathology, a protein associated with Parkinson's disease (PD) pathology. Here we show that treatment of primary mesencephalic neurons (48 h) or subchronic treatment of wild-type (WT) mice with the Parkinsonism-inducing neurotoxin MPP+/MPTP, results in selective dose-dependent hyperphosphorylation of Tau at Ser396/404 (PHF-1-reactive Tau, p-Tau), with no changes in pSer202 but with nonspecific increases in pSer262 levels. The presence of alpha-Syn was absolutely mandatory to observe MPP+/MPTP-induced increases in p-Tau levels, since no alterations in p-Tau were seen in transfected cells not expressing alpha-Syn or in alpha-Syn-/- mice. MPP+/MPTP also induced a significant accumulation of alpha-Syn in both mesencephalic neurons and in WT mice striatum. MPTP/MPP+ lead to differential alterations in p-Tau and alpha-Syn levels in a cytoskeleton-bound, vs. a soluble, cytoskeleton-free fraction, inducing their coimmunoprecipitation in the cytoskeleton-free fraction and neuronal soma. Subchronic MPTP exposure increased sarkosyl-insoluble p-Tau in striatum of WT but not alpha-Syn-/- mice. These studies describe a novel mechanism for MPTP neurotoxicity, namely a MPTP-inducible, strictly alpha-Syn-dependent, increased formation of PHF-1-reactive Tau, suggesting convergent overlapping pathways in the genesis of clinically divergent diseases such as AD and PD.

    Funded by: NINDS NIH HHS: NS-34914, NS-41555, NS-45326

    FASEB journal : official publication of the Federation of American Societies for Experimental Biology 2006;20;13;2302-12

  • Dementia with Lewy bodies in an elderly Greek male due to alpha-synuclein gene mutation.

    Morfis L and Cordato DJ

    Department of Aged Care, St George Hospital, Gray St, Kogarah, Sydney, New South Wales 2217, Australia. morfisl@sesahs.nsw.gov.au

    We report the case of an elderly man of Greek background who presented with progressive cognitive decline and motor parkinsonism on a background of a strong family history of Parkinson's disease. Associated symptoms included visual hallucinations, excessive daytime drowsiness, recurrent falls, orthostatic hypotension and urinary incontinence. His major clinical symptoms and signs fulfilled consensus criteria for a clinical diagnosis of dementia with Lewy bodies. An alpha-synuclein gene mutation analysis for the G209A substitution was positive. We conclude that the alpha-synuclein (G209A) gene mutation genotype should be considered in the differential diagnosis of dementia with Lewy bodies, particularly in patients with European ancestry and a family history of Parkinson's disease.

    Journal of clinical neuroscience : official journal of the Neurosurgical Society of Australasia 2006;13;9;942-4

  • Alpha-synuclein acts in the nucleus to inhibit histone acetylation and promote neurotoxicity.

    Kontopoulos E, Parvin JD and Feany MB

    Department of Pathology, Brigham and Women's Hospital, Program in Neuroscience, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, MA 02115, USA.

    Alpha-synuclein is a neuronal protein implicated genetically in Parkinson's disease. alpha-synuclein localizes to the nucleus and presynaptic nerve terminals. Here we show that alpha-synuclein mediates neurotoxicity in the nucleus. Targeting of alpha-synuclein to the nucleus promotes toxicity, whereas cytoplasmic sequestration is protective in both cell culture and transgenic Drosophila. Toxicity of alpha-synuclein can be rescued by administration of histone deacetylase inhibitors in both cell culture and transgenic flies. Alpha-synuclein binds directly to histones, reduces the level of acetylated histone H3 in cultured cells and inhibits acetylation in histone acetyltransferase assays. Alpha-synuclein mutations that cause familial Parkinson's disease, A30P and A53T, exhibit increased nuclear targeting in cell culture. These findings implicate nuclear alpha-synuclein in promoting nigrostriatal degeneration in Parkinson's disease and encourage exploration of histone deacetylase inhibitors as potential therapies for the disorder.

    Funded by: NINDS NIH HHS: NS 049869, NS 41536

    Human molecular genetics 2006;15;20;3012-23

  • Beta-synuclein modulates alpha-synuclein neurotoxicity by reducing alpha-synuclein protein expression.

    Fan Y, Limprasert P, Murray IV, Smith AC, Lee VM, Trojanowski JQ, Sopher BL and La Spada AR

    Department of Laboratory Medicine, University of Washington Medical Center, Seattle, WA, USA.

    Parkinson's disease (PD) is a neurodegenerative disorder characterized by fibrillar aggregates of alpha-synuclein in characteristic inclusions known as "Lewy bodies". As mutations altering alpha-synuclein structure or increasing alpha-synuclein expression level can cause familial forms of PD or related Lewy body disorders, alpha-synuclein is believed to play a central role in the process of neuron toxicity, degeneration and death in "synucleinopathies". beta-synuclein is closely related to alpha-synuclein and has been shown to inhibit alpha-synuclein aggregation and ameliorate alpha-synuclein neurotoxicity. We generated beta-synuclein transgenic mice and observed a marked reduction in alpha-synuclein protein expression in the cortex of mice over-expressing beta-synuclein. This reduction in alpha-synuclein protein expression was not accompanied by decreases in alpha-synuclein mRNA expression. Using the prion protein promoter alpha-synuclein A53T mouse model of PD, we demonstrated that over-expression of beta-synuclein could retard the progression of impaired motor performance, reduce alpha-synuclein aggregation and extend survival in doubly transgenic mice. We attributed the amelioration of alpha-synuclein neurotoxicity in such bigenic mice to the ability of beta-synuclein to reduce alpha-synuclein protein expression based upon I(125) autoradiography quantification. Our findings indicate that increased expression of beta-synuclein protein results in a reduction of alpha-synuclein protein expression. As increased expression of alpha-synuclein may cause or contribute to PD pathogenesis in sporadic and familial forms of disease, this observation has important implications for the development of therapies for PD.

    Funded by: NIA NIH HHS: AG 09215, AG 10124

    Human molecular genetics 2006;15;20;3002-11

  • Secondary structural formation of alpha-synuclein amyloids as revealed by g-factor of solid-state circular dichroism.

    Lin XJ, Zhang F, Xie YY, Bao WJ, He JH and Hu HY

    Key Laboratory of Proteomics, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 320 Yue-yang Road, Shanghai 200031, China.

    Alpha-synuclein (alpha-Syn) has been identified as a component of intracellular fibrillar deposits in Parkinson's disease. Though the real pathogenesis is still unknown, many investigations have revealed that conformational alteration and fibril formation of alpha-Syn protein have an important role in causing the disease. In this work, we introduced the g-factor spectra of solid-state circular dichroism to estimate the secondary structure contents of alpha-Syn fragments in amyloids. Fourier-transform infrared (FTIR) was also applied to confirm the structural formation. The results suggest that the central hydrophobic region is critical for beta-sheet formation and the conformational alteration is the foundation of protein abnormal aggregation. The research provides a practical approach to estimate the secondary structure contents of protein amyloids and further insight into the relevance of structural transformation and amyloidogenesis.

    Biopolymers 2006;83;3;226-32

  • Decreased alpha-synuclein in cerebrospinal fluid of aged individuals and subjects with Parkinson's disease.

    Tokuda T, Salem SA, Allsop D, Mizuno T, Nakagawa M, Qureshi MM, Locascio JJ, Schlossmacher MG and El-Agnaf OM

    Department of Neurology, Kyoto Prefectural University of Medicine, Kyoto 602-0841, Japan, and Center for Neurologic Diseases, Brigham and Women's Hospital, Boston, MA 02115, USA.

    There is ample biochemical, pathological, and genetic evidence that the metabolism of alpha-synuclein (alpha-syn) plays a crucial role in the pathogenesis of Parkinson disease (PD). To examine whether quantification of alpha-syn in cerebrospinal fluid (CSF) is potentially informative in the diagnosis of PD, we developed a specific ELISA system and measured the concentration of alpha-syn in CSF from 33 patients with PD (diagnosed according to UK PD Society Brain Bank criteria) and 38 control subjects including 9 neurologically healthy individuals. We found that PD patients had significantly lower alpha-syn levels in their CSF than the control groups (p<0.0001) even after adjusting for gender and age. Age was independently associated with lower alpha-syn levels. Logistic regression analysis showed that reduction in CSF alpha-syn served as a significant predictor of PD beyond age and gender alone (area under ROC curve, c=0.882). Furthermore, we observed a close inverse correlation between alpha-syn levels in CSF and assigned Hoehn and Yahr score in this cohort of 71 living subjects (p<0.0001), even after adjusting for age. These findings identify in the quantification of alpha-syn from CSF a potential laboratory marker to aid the clinical diagnosis of PD.

    Biochemical and biophysical research communications 2006;349;1;162-6

  • Phosphorylation of Ser-129 is the dominant pathological modification of alpha-synuclein in familial and sporadic Lewy body disease.

    Anderson JP, Walker DE, Goldstein JM, de Laat R, Banducci K, Caccavello RJ, Barbour R, Huang J, Kling K, Lee M, Diep L, Keim PS, Shen X, Chataway T, Schlossmacher MG, Seubert P, Schenk D, Sinha S, Gai WP and Chilcote TJ

    Elan Pharmaceuticals, South San Francisco, California 94080, USA.

    A comprehensive, unbiased inventory of synuclein forms present in Lewy bodies from patients with dementia with Lewy bodies was carried out using two-dimensional immunoblot analysis, novel sandwich enzyme-linked immunosorbent assays with modification-specific synuclein antibodies, and mass spectroscopy. The predominant modification of alpha-synuclein in Lewy bodies is a single phosphorylation at Ser-129. In addition, there is a set of characteristic modifications that are present to a lesser extent, including ubiquitination at Lys residues 12, 21, and 23 and specific truncations at Asp-115, Asp-119, Asn-122, Tyr-133, and Asp-135. No other modifications are detectable by tandem mass spectrometry mapping, except for a ubiquitous N-terminal acetylation. Small amounts of Ser-129 phosphorylated and Asp-119-truncated alpha-synuclein are present in the soluble fraction of both normal and disease brains, suggesting that these Lewy body-associated forms are produced during normal metabolism of alpha-synuclein. In contrast, ubiquitination is only detected in Lewy bodies and is primarily present on phosphorylated synuclein; it therefore likely occurs after phosphorylated synuclein has deposited into Lewy bodies. This invariant pattern of specific phosphorylation, truncation, and ubiquitination is also present in the detergent-insoluble fraction of brain from patients with familial Parkinson's disease (synuclein A53T mutation) as well as multiple system atrophy, suggesting a common pathogenic pathway for both genetic and sporadic Lewy body diseases. These observations are most consistent with a model in which preferential accumulation of normally produced Ser-129 phosphorylated alpha-synuclein is the key event responsible for the formation of Lewy bodies in various Lewy body diseases.

    Funded by: NINDS NIH HHS: P50-NS40256

    The Journal of biological chemistry 2006;281;40;29739-52

  • Effect of reparation of repeat sequences in the human alpha-synuclein on fibrillation ability.

    Sode K, Ochiai S, Kobayashi N and Usuzaka E

    Department of Biotechnology, Graduate School of Engineering, Tokyo University of Agriculture & Technology, 2-2-4-16, Nakacho, Koganei, Tokyo 184-8588, Japan. sode@cc.tuat.ac.jp

    The aggregation and fibrillation of alpha-synuclein has been implicated as a causative factor in the Parkinson's disease. The hexamer motif KTKEGV is found in each of the seven imperfect repeat sequences in the N-terminal half of alpha-synuclein. The motif is not fully conserved in the sixth and seventh repeats. We created mutants in which the motif was repaired to be fully conserved in either (Rep6 and Rep7) or both (Rep67) of these two repeats. The Rep6 and Rep67 mutants showed a greatly reduced propensity to aggregate and fibrillate while all three mutants showed greater resistance to HFIP-induced formation of the alpha-helix intermediate. Resistance to formation in the partially folded intermediate may repress the folding of alpha-synuclein, consequently interfering with the aggregation and fibril formation. These results demonstrated that KTKEGV repeats may have a significant role in keeping native unfolded status of alpha-synuclein.

    International journal of biological sciences 2006;3;1;1-7

  • Alpha-synuclein and its disease-causing mutants induce ICAM-1 and IL-6 in human astrocytes and astrocytoma cells.

    Klegeris A, Giasson BI, Zhang H, Maguire J, Pelech S and McGeer PL

    Kinsmen Laboratory of Neurological Research, University of British Columbia, 2255 Wesbrook Mall, Vancouver, BC V6T 1Z3, Canada. aklegeri@interchange.ubc.ca

    Autosomal dominant Parkinson disease (PD) is caused by duplication or triplication of the alpha-synuclein gene as well as by the A30P, E46K, and A53T mutations. The mechanisms are unknown. Reactive astrocytes in the substantia nigra of PD and MPTP-treated monkeys display high levels of the inflammatory mediator intercellular adhesion molecule-1 (ICAM-1), indicating that chronic inflammation contributes to the degeneration. Here we report that alpha-synuclein strongly stimulates human astrocytes as well as human U-373 MG astrocytoma cells to up-regulate both interleukin (IL)-6 and ICAM-1 (ED50=5 microg ml(-1)). The mutated forms are more potent stimulators than wild-type (WT) alpha-synuclein in these assays. We demonstrate by immunoblotting analysis that this up-regulation is associated with activation of the major mitogen-activated protein kinase (MAPK) pathways. It is also attenuated by PD 98059, an inhibitor of the MAPK/extracellular-regulated kinase kinase MEK1/2, SP 600125, an inhibitor of c-Jun N-terminal kinase (JNK), and SB 202190, an inhibitor of p38 MAPK. The inhibitory effects on human astrocytes have IC50 values of 2, 5, and 1.5 microM respectively. We hypothesize that the neuroinflammation stimulated by release of an excess of normal alpha-synuclein or by release of its mutated forms can be involved in the pathobiology of PD.

    FASEB journal : official publication of the Federation of American Societies for Experimental Biology 2006;20;12;2000-8

  • alpha-Synuclein promoter RsaI T-to-C polymorphism and the risk of Parkinson's disease.

    Wang CK, Chen CM, Chang CY, Chang KH, Chen IC, Li ML, Lee-Chen GJ and Wu YR

    Department of Life Science, National Taiwan Normal University, Taipei, Taiwan.

    Increased alpha-synuclein expression may be involved in the pathogenesis of Parkinson's disease (PD). We investigated the association of Rep1 microsatellite and RsaI T-to-C substitution in the alpha-synuclein promoter region with the risk of PD by a case-control study. The RsaI C/C genotype and C allele were found less frequently in PD patients than in controls. A reduced risk of the Rep1-RsaI 0-C haplotype (OR = 0.57, 95% CI = 0.36-0.90) with PD was evident. The quantitative real-time PCR study showed that the alpha-synuclein mRNA expression was increased (although not significantly) in PD patients with RsaI T/T genotype or Rep1-RsaI 0-T haplotype as compared to T/C genotype or 0-C haplotype. Reporter constructs containing the RsaI C allele drove significantly lower transcriptional activity compared with the RsaI T allele in both IMR32 and 293 cells. The findings suggest that the RsaI T-to-C substitution may have a functional relevance to the susceptibility to PD.

    Journal of neural transmission (Vienna, Austria : 1996) 2006;113;10;1425-33

  • Detection of novel intracellular alpha-synuclein oligomeric species by fluorescence lifetime imaging.

    Klucken J, Outeiro TF, Nguyen P, McLean PJ and Hyman BT

    MassGeneral Institute for Neurodegenerative Disease, Alzheimer's Disease Research Unit, Massachusetts General Hospital, 114 16 St., Charlestown, MA 02129, USA.

    Oligomerization and aggregation of alpha-synuclein molecules are believed to play a major role in neuronal dysfunction and loss in Parkinson's disease (PD) and dementia with Lewy bodies. However, alpha-synuclein oligomerization and aggregation have been detected only indirectly in cells using detergent extraction methods. Here, we show for the first time intracellular alpha-synuclein oligomerization using fluorescence lifetime imaging (FLIM). Two forms of alpha-synuclein homomeric interactions were detected: an antiparallel amino terminus-carboxyl terminus interaction between alpha-synuclein molecules, and a close amino terminus-carboxy terminus interaction within single alpha-synuclein molecules. Coexpression of the chaperone protein Hsp70, which can block alpha-synuclein toxicity in several systems, causes alpha-synuclein to adopt a different, open conformation, but Hsp70 does not alter alpha-synuclein-alpha-synuclein interactions. Thus, the neuroprotective effect of Hsp70 can be explained by its chaperone activity on alpha-synuclein molecules, rather than alteration of alpha-synuclein-alpha-synuclein interactions.

    Funded by: NINDS NIH HHS: 5P50 NS38372A-06

    FASEB journal : official publication of the Federation of American Societies for Experimental Biology 2006;20;12;2050-7

  • Cytosolic catechols inhibit alpha-synuclein aggregation and facilitate the formation of intracellular soluble oligomeric intermediates.

    Mazzulli JR, Mishizen AJ, Giasson BI, Lynch DR, Thomas SA, Nakashima A, Nagatsu T, Ota A and Ischiropoulos H

    The Joseph Stokes Jr. Research Institute, The Children's Hospital of Philadelphia and The University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.

    Aberrant aggregation of alpha-synuclein (alpha-syn) to form fibrils and insoluble aggregates has been implicated in the pathogenic processes of many neurodegenerative diseases. Despite the dramatic effects of dopamine in inhibiting the formation of alpha-syn fibrils by stabilization of oligomeric intermediates in cell-free systems, no studies have examined the effects of intracellular dopamine on alpha-syn aggregation. To study this process and its association with neurodegeneration, intracellular catechol levels were increased to various levels by expressing different forms of tyrosine hydroxylase, in cells induced to form alpha-syn aggregates. The increase in the steady-state dopamine levels inhibited the formation of alpha-syn aggregates and induced the formation of innocuous oligomeric intermediates. Analysis of transgenic mice expressing the disease-associated A53T mutant alpha-syn revealed the presence of oligomeric alpha-syn in nondegenerating dopaminergic neurons that do contain insoluble alpha-syn. These data indicate that intraneuronal dopamine levels can be a major modulator of alpha-syn aggregation and inclusion formation, with important implications on the selective degeneration of these neurons in Parkinson's disease.

    Funded by: NIA NIH HHS: AG09215, AG13966; NINDS NIH HHS: NS45986

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2006;26;39;10068-78

  • Conformational properties of the SDS-bound state of alpha-synuclein probed by limited proteolysis: unexpected rigidity of the acidic C-terminal tail.

    de Laureto PP, Tosatto L, Frare E, Marin O, Uversky VN and Fontana A

    CRIBI Biotechnology Centre, University of Padua, Viale G. Colombo 3, 35121 Padua, Italy.

    Alpha-synuclein (alpha-syn) is a "natively unfolded" protein constituting the major component of intracellular inclusions in several neurodegenerative disorders. Here, we describe proteolysis experiments conducted on human alpha-syn in the presence of SDS micelles. Our aim was to unravel molecular features of micelle-bound alpha-syn using the limited proteolysis approach. The nonspecific proteases thermolysin and proteinase K, as well as the Glu-specific V8-protease, were used as proteolytic probes. While alpha-syn at neutral pH is easily degraded to a variety of relatively small fragments, in the presence of 10 mM SDS the proteolysis of the protein is rather selective. Complementary fragments 1-111 and 112-140, 1-113 and 114-140, and 1-123 and 124-140 are obtained when thermolysin, proteinase K, and V8 protease, respectively, are used. These results are in line with a conformational model of alpha-syn in which it acquires a folded helical structure in the N-terminal region in its membrane-bound state. At the same time, they indicate that the C-terminal portion of the molecule is rather rigid, as seen in its relative resistance to extensive proteolytic degradation. It is likely that, under the specific experimental conditions of proteolysis in the presence of SDS, the negatively charged C-terminal region can be rigidified by binding a calcium ion, as shown before with intact alpha-syn. In this study, some evidence of calcium binding properties of isolated C-terminal fragments 112-140, 114-140, and 124-140 was obtained by mass spectrometry measurements, since molecular masses for calcium-loaded fragments were obtained. Our results indicate that the C-terminal portion of the membrane-bound alpha-syn is quite rigid and structured, at variance from current models of the membrane-bound protein deduced mostly from NMR. Considering that the aggregation process of alpha-syn is modulated by its C-terminal tail, the results of this study may provide useful insights into the behavior of alpha-syn in a membrane-mimetic environment.

    Biochemistry 2006;45;38;11523-31

  • Calcium-triggered membrane interaction of the alpha-synuclein acidic tail.

    Tamamizu-Kato S, Kosaraju MG, Kato H, Raussens V, Ruysschaert JM and Narayanaswami V

    Children's Hospital Oakland Research Institute, 5700 Martin Luther King Jr. Way, Oakland, California 94609, USA.

    Alpha-synuclein (alpha-syn) is a 140-residue protein that aggregates in intraneuronal inclusions called Lewy bodies in Parkinson's disease (PD). It is composed of an N-terminal domain with a propensity to bind lipids and a C-terminal domain rich in acidic residues (the acidic tail). The objective of this study was to examine the effect of Ca(2+) on the acidic tail conformation in lipid-bound alpha-syn. We exploit the extreme sensitivity of the band III fluorescence emission peak of the pyrene fluorophore to the polarity of its microenvironment to monitor subtle conformational response of the alpha-syn acidic tail to Ca(2+). Using recombinant human alpha-syn bearing a pyrene to probe either the N-terminal domain or the acidic tail, we noted that lipid binding resulted in an increase in band III emission intensity in the pyrene probe tagging the N-terminal domain but not that in the acidic tail. This suggests that the protein is anchored to the lipid surface via the N-terminal domain. However, addition of Ca(2+) caused an increase in band III emission intensity in the pyrene tagging the acidic tail, with a corresponding increased susceptibility to quenching by quenchers located in the lipid milieu, indicative of lipid interaction of this domain. Taken together with the increased beta-sheet content of membrane-associated alpha-syn in the presence of Ca(2+), we propose a model wherein initial lipid interaction occurs via the N-terminal domain, followed by a Ca(2+)-triggered membrane association of the acidic tail as a potential mechanism leading to alpha-syn aggregation. These observations have direct implications in the role of age-related oxidative stress and the attendant cellular Ca(2+) dysregulation as critical factors in alpha-syn aggregation in PD.

    Biochemistry 2006;45;36;10947-56

  • Interaction between Abeta peptide and alpha synuclein: molecular mechanisms in overlapping pathology of Alzheimer's and Parkinson's in dementia with Lewy body disease.

    Mandal PK, Pettegrew JW, Masliah E, Hamilton RL and Mandal R

    Department of Psychiatry, Western Psychiatric Institute and Clinic, University of Pittsburgh Medical Center, 3811 O'Hara Street, Pittsburgh, PA 15213, USA. mandalp@upmc.edu

    Amyloidogenic proteins (Abeta peptide) in Alzheimer's disease (AD) and alpha-synuclein (alpha-Syn) in Parkinson's disease (PD) are typically soluble monomeric precursors, which undergo remarkable conformational changes and culminate in the form of aggregates in diseased condition. Overlap of clinical and neuropathological features of both AD and PD are observed in dementia with Lewy body (DLB) disease, the second most common form of dementia after AD. The identification of a 35-amino acid fragment of alpha-Syn in the amyloid plaques in DLB brain have raised the possibility that Abeta and alpha-Syn interact with each other. In this report, the molecular interaction of alpha-Syn with Abeta40 and/or Abeta42 are investigated using multidimensional NMR spectroscopy. NMR data in the membrane mimic environment indicate specific sites of interaction between membrane-bound alpha-Syn with Abeta peptide and vice versa. These Abeta-alpha-Syn interactions are demonstrated by reduced amide peak intensity or change in chemical shift of amide proton of the interacting proteins. Based on NMR results, the plausible molecular mechanism of overlapping pathocascade of AD and PD in DLB due to interactions between alpha-Syn and Abeta is described. To the best of our knowledge, it is the first report using multidimensional NMR spectroscopy that elucidates molecular interactions between Abeta and alpha-Syn which may lead to onset of DLB.

    Neurochemical research 2006;31;9;1153-62

  • Ventral tegmental area dopamine neurons are resistant to human mutant alpha-synuclein overexpression.

    Maingay M, Romero-Ramos M, Carta M and Kirik D

    Department of Experimental Medical Science, Section of Neuroscience, CNS Disease Modeling Unit, Lund University, Wallenberg Neuroscience Center, BMCA11, S-22184 Lund, Sweden. matthew.maingay@med.lu.se

    Parkinson's disease (PD) is characterized by the formation of intracytoplasmic inclusions, which contain alpha-synuclein (alpha-syn) protein. While most profound neurodegeneration is seen in the dopamine (DA) synthesizing neurons located in the ventral midbrain, it is unclear why some DA cell groups are more susceptible than others. In the midbrain, the degeneration of the substantia nigra (SN) DA neurons is severe, whereas the involvement of the ventral tegmental area (VTA) neurons is relatively spared. In the present study, we overexpressed human A53T alpha-syn in the VTA neurons and found that A53T toxicity did not affect their survival. There was, however, a mild functional impairment seen as altered open field locomotor activity. Overexpression of A53T in the SN, on the other hand, led to profound cell loss. These results suggest that the selective susceptibility of nigral DA neurons is at least in part associated with factor(s) involved in handling of alpha-syn that is not shared by the VTA neurons. Secondly, these results highlight the fact that impaired but surviving neurons can have a substantial impact on DA-dependent behavior and should therefore be considered as a critical part of animal models where novel therapeutic interventions are tested.

    Neurobiology of disease 2006;23;3;522-32

  • Collaborative analysis of alpha-synuclein gene promoter variability and Parkinson disease.

    Maraganore DM, de Andrade M, Elbaz A, Farrer MJ, Ioannidis JP, Krüger R, Rocca WA, Schneider NK, Lesnick TG, Lincoln SJ, Hulihan MM, Aasly JO, Ashizawa T, Chartier-Harlin MC, Checkoway H, Ferrarese C, Hadjigeorgiou G, Hattori N, Kawakami H, Lambert JC, Lynch T, Mellick GD, Papapetropoulos S, Parsian A, Quattrone A, Riess O, Tan EK, Van Broeckhoven C and Genetic Epidemiology of Parkinson's Disease (GEO-PD) Consortium

    Department of Neurology, Mayo Clinic College of Medicine, Rochester, Minn 55905, USA. dmaraganore@mayo.edu

    Context: Identification and replication of susceptibility genes for Parkinson disease at the population level have been hampered by small studies with potential biases. Alpha-synuclein (SNCA) has been one of the most promising susceptibility genes, but large-scale studies have been lacking.

    Objective: To determine whether allele-length variability in the dinucleotide repeat sequence (REP1) of the SNCA gene promoter is associated with Parkinson disease susceptibility, whether SNCA promoter haplotypes are associated with Parkinson disease, and whether REP1 variability modifies age at onset.

    We performed a collaborative analysis of individual-level data on SNCA REP1 and flanking markers in patients with Parkinson disease and controls. Study site recruitment, data collection, and analyses were performed between April 5, 2004, and December 31, 2005. Eighteen participating sites of a global genetics consortium provided clinical data. Genotyping was performed for SNCA REP1, -770, and -116 markers at individual sites; however, each site also provided 20 DNA samples for regenotyping centrally.

    Measures included estimations of Hardy-Weinberg equilibrium in controls; a test of heterogeneity; analyses for association of single variants or haplotypes; and survival analyses for age at onset.

    Results: Of the 18 sites, 11 met stringent criteria for concordance with Hardy-Weinberg equilibrium and low genotyping error rate. These 11 sites provided complete data for 2692 cases and 2652 controls. There was no heterogeneity across studies (P>.60). The SNCA REP1 alleles differed in frequency for cases and controls (P<.001). Genotypes defined by the 263 base-pair allele were associated with Parkinson disease (odds ratio, 1.43; 95% confidence interval, 1.22-1.69; P<.001 for trend). Multilocus haplotypes differed in frequency for cases and controls (global score statistic, P<.001). Two-loci haplotypes were associated with Parkinson disease only when they included REP1 as one of the loci. However, genotypes defined by REP1 alleles did not modify age at onset (P = .55).

    Conclusion: This large-scale collaborative analysis demonstrates that SNCA REP1 allele-length variability is associated with an increased risk of Parkinson disease.

    Funded by: NIAAA NIH HHS: AA09515; NIEHS NIH HHS: ES04696, ES10750, ES10751; NINDS NIH HHS: NS33978, NS39913, NS41509

    JAMA : the journal of the American Medical Association 2006;296;6;661-70

  • Interactions between fatty acids and alpha-synuclein.

    Lücke C, Gantz DL, Klimtchuk E and Hamilton JA

    Department of Physiology and Biophysics, Boston University School of Medicine, MA 02118, USA.

    alpha-Synuclein (alphaS) is an amyloidogenic neuronal protein associated with several neurodegenerative disorders. Although unstructured in solution, alphaS forms alpha-helices in the presence of negatively charged lipid surfaces. Moreover, alphaS was shown to interact with FAs in a manner that promotes protein aggregation. Here, we investigate whether alphaS has specific FA binding site(s) similar to fatty acid binding proteins (FABPs), such as the intracellular FABPs. Our NMR experiments reveal that FA addition results in i) the simultaneous loss of alphaS signal in both (1)H and (13)C spectra and ii) the appearance of a very broad FA (13)C-carboxyl signal. These data exclude high-affinity binding of FA molecules to specific alphaS sites, as in FABPs. One possible mode of binding was revealed by electron microscopy studies of oleic acid bilayers at pH 7.8; these high-molecular-weight FA aggregates possess a net negative surface charge because they contain FA anions, and they were easily disrupted to form smaller particles in the presence of alphaS, indicating a direct protein-lipid interaction. We conclude that alphaS is not likely to act as an intracellular FA carrier. Binding to negatively charged membranes, however, appears to be an intrinsic property of alphaS that is most likely related to its physiological role(s) in the cell.

    Funded by: NHLBI NIH HHS: HI-26335

    Journal of lipid research 2006;47;8;1714-24

  • Relationship among alpha-synuclein accumulation, dopamine synthesis, and neurodegeneration in Parkinson disease substantia nigra.

    Mori F, Nishie M, Kakita A, Yoshimoto M, Takahashi H and Wakabayashi K

    Department of Neuropathology, Institute of Brain Science, Hirosaki University School of Medicine, Japan.

    The histologic hallmark of Parkinson disease (PD) is loss of pigmented neurons in the substantia nigra (SN) and locus ceruleus (LC) with accumulation of alpha-synuclein (alphaS). It has been reported that tyrosine hydroxylase (TH)-negative pigmented neurons are present in these nuclei of patients with PD. However, the relationship between TH immunoreactivity and alphaS accumulation remains uncertain. We immunohistochemically examined the SN and LC from patients with PD (n = 10) and control subjects (n = 7). A correlation study indicated a close relationship among decreased TH immunoreactivity, alphaS accumulation, and neuronal loss. In addition, 10% of pigmented neurons in the SN and 54.9% of those in the LC contained abnormal alphaS aggregates. Moreover, 82.3% of pigmented neurons bearing alphaS aggregates in the SN and 39.2% of those in the LC lacked TH immunoreactivity, suggesting that pigmented neurons in the SN have a greater tendency to lack TH activity than those in the LC. Recent studies have shown that this decrease of TH activity leads to a decrease of cytotoxic substances and that decreased dopamine synthesis leads to a reduction of cytotoxic alphaS oligomers. Therefore, the decrease of TH immunoreactivity in pigmented neurons demonstrated here can be considered to represent a cytoprotective mechanism in PD.

    Journal of neuropathology and experimental neurology 2006;65;8;808-15

  • Alpha-synuclein blocks ER-Golgi traffic and Rab1 rescues neuron loss in Parkinson's models.

    Cooper AA, Gitler AD, Cashikar A, Haynes CM, Hill KJ, Bhullar B, Liu K, Xu K, Strathearn KE, Liu F, Cao S, Caldwell KA, Caldwell GA, Marsischky G, Kolodner RD, Labaer J, Rochet JC, Bonini NM and Lindquist S

    School of Biological Sciences, University of Missouri-Kansas City, Kansas City, MO 64110, USA.

    Alpha-synuclein (alphaSyn) misfolding is associated with several devastating neurodegenerative disorders, including Parkinson's disease (PD). In yeast cells and in neurons alphaSyn accumulation is cytotoxic, but little is known about its normal function or pathobiology. The earliest defect following alphaSyn expression in yeast was a block in endoplasmic reticulum (ER)-to-Golgi vesicular trafficking. In a genomewide screen, the largest class of toxicity modifiers were proteins functioning at this same step, including the Rab guanosine triphosphatase Ypt1p, which associated with cytoplasmic alphaSyn inclusions. Elevated expression of Rab1, the mammalian YPT1 homolog, protected against alphaSyn-induced dopaminergic neuron loss in animal models of PD. Thus, synucleinopathies may result from disruptions in basic cellular functions that interface with the unique biology of particular neurons to make them especially vulnerable.

    Funded by: NHGRI NIH HHS: R01-HG002923; NINDS NIH HHS: P50 NS038372

    Science (New York, N.Y.) 2006;313;5785;324-8

  • A novel mechanism of interaction between alpha-synuclein and biological membranes.

    Kim YS, Laurine E, Woods W and Lee SJ

    The Parkinson's Institute, Sunnyvale, CA 94089, USA.

    Conformational abnormalities and aggregation of alpha-synuclein (alpha-syn) have been linked to the pathogenesis of Parkinson's (PD) and related diseases. It has been shown that alpha-syn can stably bind artificial phospholipid vesicles through alpha-helix formation in its N-terminal repeat region. However, little is known about the membrane interaction in cells. In the current study, we determined the membrane-binding properties of alpha-syn to biological membranes by using bi-functional chemical crosslinkers, which allow the detection of transient, but specific, interactions. By utilizing various point mutations and deletions within alpha-syn, we demonstrated that the membrane interaction of alpha-syn in cells is also mediated by alpha-helix formation in the N-terminal repeat region. Moreover, the PD-linked A30P mutation causes reduced membrane binding, which is concordant with the artificial membrane studies. However, contrary to the interaction with artificial membranes, the interaction with biological membranes is rapidly reversible and is not driven by electrostatic attraction. Furthermore, the interaction of alpha-syn with cellular membranes occurs only in the presence of non-protein and non-lipid cytosolic components, which distinguishes it from the spontaneity of the interaction with artificial membranes. More interestingly, addition of the cytosolic preparation to artificial membranes resulted in the transient, charge-independent binding of alpha-syn similar to the interaction with biological membranes. These results suggest that in cells, alpha-syn is engaged in a fundamentally different mode of membrane interaction than the charge-dependent artificial membrane binding, and the mode of interaction is determined by the intrinsic properties of alpha-syn itself and by the cytoplasmic context.

    Journal of molecular biology 2006;360;2;386-97

  • Emerging evidence for the neuroprotective role of alpha-synuclein.

    Lee HG, Zhu X, Takeda A, Perry G and Smith MA

    Department of Pathology, Case Western Reserve University, Cleveland, OH 44106, USA.

    Experimental neurology 2006;200;1;1-7

  • Modulation of the trafficking of the human serotonin transporter by human alpha-synuclein.

    Wersinger C, Rusnak M and Sidhu A

    Department of Biochemistry, Molecular and Cell Biology, Georgetown University, The Research Building, Room W222, 3970 Reservoir Road, NW, Washington, DC 20007, USA.

    alpha-Synuclein (alpha-Syn), a protein primarily localized in the presynaptic compartment of neurons, is known to regulate dopaminergic neurotransmission by negatively modulating dopamine transporter activity and regulating its trafficking to or away from the cell surface. Given the considerable homology between dopamine transporters and the serotonin (5-HT) transporter (SERT), we examined whether alpha-Syn could similarly regulate SERT function. Increasing expression levels of human alpha-Syn gradually decreased [(3)H]5-HT uptake by human SERT in cotransfected Ltk(-) cells, by diminishing its V(max) without changing its K(m), as compared to cells expressing only SERT. Biotinylation studies to label cell-surface proteins showed that alpha-Syn decreased the levels of SERT present at the plasma membrane. alpha-Syn and SERT were able to coimmunoprecipitate (co-IP), suggesting heteromeric complexes between these two proteins through direct protein-protein interactions. The negative modulation of SERT activity by alpha-Syn occurred through the non-Abeta-amyloid component (NAC) domain of alpha-Syn (aa58-107); DNA constructs encoding this region mimicked the full-length alpha-Syn protein by decreasing [(3)H]5-HT uptake by the transporter. Furthermore, only the constructs encoding the NAC domain of alpha-Syn prevented the co-IPs between full-length alpha-Syn and SERT, in both transfected cells and in rat solubilized lysates isolated from the prefrontal cortex. These studies suggest a novel physiological role for alpha-Syn in regulating SERT activity and may be of relevance in certain mental illnesses and in depression, in which SERT function is believed to be dysregulated.

    Funded by: NINDS NIH HHS: NS-34914, NS-41555

    The European journal of neuroscience 2006;24;1;55-64

  • Transduced Tat-alpha-synuclein protects against oxidative stress in vitro and in vivo.

    Choi HS, Lee SH, Kim SY, An JJ, Hwang SI, Kim DW, Yoo KY, Won MH, Kang TC, Kwon HJ, Kang JH, Cho SW, Kwon OS, Choi JH, Park J, Eum WS and Choi SY

    Department of Biomedical Sciences, Hallym University, Chunchon, Korea. sychoi@hallym.ac.kr

    Parkinson's disease (PD) is a common neurodegenerative disorder and is characterized by the progressive loss of dopaminergic neurons in the substantia nigra. Although many studies showed that the aggregation of alpha-synuclein might be involved in the pathogenesis of PD, its protective properties against oxidative stress remain to be elucidated. In this study, human wild type and mutant alpha-synuclein genes were fused with a gene fragment encoding the nine amino acid transactivator of transcription (Tat) protein transduction domain of HIV-1 in a bacterial expression vector to produce a genetic in-frame WT Tat-alpha-synuclein (wild type) and mutant Tat-alpha-synucleins (mutants; A30P and A53T), respectively, and we investigated the protective effects of wild type and mutant Tat-alpha-synucleins in vitro and in vivo. WT Tat-alpha-synuclein rapidly transduced into an astrocyte cells and protected the cells against paraquat induced cell death. However, mutant Tat-alpha-synucleins did not protect at all. In the mice models exposed to the herbicide paraquat, the WT Tat-alpha-synuclein completely protected against dopaminergic neuronal cell death, whereas mutants failed in protecting against oxidative stress. We found that these protective effects were characterized by increasing the expression level of heat shock protein 70 (HSP70) in the neuronal cells and this expression level was dependent on the concentration of transduced WT Tat-alpha-synuclein. These results suggest that transduced Tat-alpha-synuclein might protect cell death from oxidative stress by increasing the expression level of HSP70 in vitro and in vivo and this may be of potential therapeutic benefit in the pathogenesis of PD.

    Journal of biochemistry and molecular biology 2006;39;3;253-62

  • Abnormal alpha-synuclein solubility, aggregation and nitration in the frontal cortex in Pick's disease.

    Dalfó E, Martinez A, Muntané G and Ferrer I

    Institut de Neuropatologia, Servei Anatomia Patològica, IDIBELL/Hospital Universitari de Bellvitge, carrer Feixa Llarga sn, 08907 Hospitalet de Llobregat, Spain.

    Abnormal solubility and aggregation of alpha-synuclein have been observed in the frontal cortex in three cases with Pick's disease (PiD) when compared with age-matched controls. Bands of 45 kDa and higher molecular weight were detected in the SDS-soluble fractions only in PiD. Patterns in PiD differed from that observed in the cerebral cortex in Lewy body diseases which were examined in parallel. Immunoblots to alpha-synuclein nitrated in tyrosines revealed bands of 45 and 60 kDa in Dxc- and SDS-soluble fractions in the frontal cortex (which is vulnerable to PiD) but not in the occipital cortex (which is resistant to this degenerative disease). Moreover, nitrated alpha-synuclein was found in Lewy bodies and neurites in synucleinopathies but diffusely in the cytoplasm of scattered neurons in PiD. These findings demonstrate abnormal and distinct alpha-synuclein solubility and aggregation, and alpha-synuclein nitration without formation of Lewy bodies in the frontal cortex in PiD.

    Neuroscience letters 2006;400;1-2;125-9

  • Heat shock proteins reduce alpha-synuclein aggregation induced by MPP+ in SK-N-SH cells.

    Fan GH, Zhou HY, Yang H and Chen SD

    Department of Neurology and Institute of Neurology, Ruijin Hospital affiliated to Shanghai Jiaotong University School of Medicine, No. 197 Rui jin er Road, Shanghai 200025, China.

    Alpha-synuclein has been implicated in the pathogenesis of Parkinson's disease (PD). Heat shock proteins (HSPs) can reduce protein misfolding and accelerate the degradation of misfolded proteins. 1-methyl-4-phenylpyridinium ion (MPP+) is the compound responsible for the PD-like neurodegeneration caused by MPTP. In this study, we found that MPP+ could increase the expression of alpha-synuclein mRNA but could not elevate proteasome activity sufficiently, leading to alpha-synuclein protein accumulation followed by aggregation. Both HSPs and HDJ-1, a homologue of human Hsp40, can inhibit MPP+-induced alpha-synuclein mRNA expression, promote ubiquitination and elevate proteasome activity. These findings suggest that HSPs may inhibit the MPP+-induced alpha-synuclein expression, accelerate alpha-synuclein degradation, thereby reducing the amount of alpha-synuclein protein and accordingly preventing its aggregation.

    FEBS letters 2006;580;13;3091-8

  • Characteristics of alpha-synucleinopathy in centenarians.

    Ding ZT, Wang Y, Jiang YP, Hashizume Y, Yoshida M, Mimuro M, Inagaki T and Iwase T

    Department of Neurology, Huashan Hospital, Fudan University, Shanghai, China.

    To investigate the characteristics of alpha-synucleinopathy in the brains of centenarians, the autopsied brains and spinal cords from 23 cases were studied. Coronal slices were prepared from a section of the cerebral hemisphere, following the guidelines of the Consortium to Establish a Registry for Alzheimer's Disease (AD) (CERAD) and the consensus guidelines for the clinical and pathologic diagnosis of dementia with Lewy bodies (DLB). Spinal cord specimens were prepared at each segment from the third cervical to the third sacral segment. In all cases, we performed standard stainings of hematoxylin-eosin, Klüver-Barrera, and Gallyas-Braak combined with Luxol fast blue/cresyl violet, and alpha-synuclein (AS), phosphorylated tau (AT8) and beta-amyloid protein immunostainings. One-way ANOVA analysis, Chi-square or Fisher exact test were used for statistical analysis. Overall, AS-positive structures were found in 8 (34.8%) of our 23 centenarians, 6 (35.3%) of 17 demented patients, and four (40%) out of ten AD patients. The frequencies of AS lesions in the brains with senile plaque (SP) stage 0-A, B, and C were 27.7, 33, and 50%, respectively. No statistical differences were found among the frequencies of AS lesions in the subgroups of NFT stages I-II, III-IV, and V-VI (P=0.478). Most cases showed a widespread distribution of AS-positive structures except for one patient, in whose brain only the medulla was involved. The distribution pattern of AS-positive lesions was similar to that in Parkinson's disease or DLB, but the pigmented neurons in substantia nigra were relatively well preserved. Our findings indicate that there is a high frequency of alpha-synucleinopathy in centenarians, SP-positive and AS-positive lesions may involve a synergistic interaction.

    Acta neuropathologica 2006;111;5;450-8

  • Characterization of tau pathologies in gray and white matter of Guam parkinsonism-dementia complex.

    Winton MJ, Joyce S, Zhukareva V, Practico D, Perl DP, Galasko D, Craig U, Trojanowski JQ and Lee VM

    The Center for Neurodegenerative Disease Research, Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA 19104-4283, USA.

    Guam parkinsonism-dementia complex (PDC) is a neurodegenerative tauopathy in ethnic Chamorro residents of the Mariana Islands that manifests clinically with parkinsonism as well as dementia and is characterized neuropathologically by prominent cortical neuron loss in association with extensive telencephalic neurofibrillary tau pathology. To further characterize cortical gray and white matter tau, alpha-synuclein and lipid peroxidation pathologies in Guam PDC, we examined the brains of 17 Chamorro PDC and control subjects using biochemical and immunohistological techniques. We observed insoluble tau pathology in both gray and white matter of PDC and Guam control cases, with frontal and temporal lobes being most severely affected. Using phosphorylation dependent anti-tau antibodies, abundant tau inclusions were detected by immunohistochemistry in both neuronal and glial cells of the neocortex, while less alpha-synuclein pathology was observed in more limited brain regions. Further, in sharp contrast to Alzheimer's disease (AD), levels of the lipid peroxidation product 8, 12-iso-iPF(2alpha)-VI isoprostane were not elevated in Guam PDC brains relative to controls. Thus, although the tau pathologies of Guam PDC share similarities with AD, the composite Guam PDC neuropathology profile of tau, alpha-synuclein and 8, 12-iso-iPF(2alpha)-VI isoprostane reported here more closely resembles that seen in other tauopathies including frontotemporal dementias (FTDs), which may imply that Guam PDC and FTD tauopathies share underlying mechanisms of neurodegeneration.

    Funded by: NIA NIH HHS: AG14382

    Acta neuropathologica 2006;111;5;401-12

  • Plasma membrane ion permeability induced by mutant alpha-synuclein contributes to the degeneration of neural cells.

    Furukawa K, Matsuzaki-Kobayashi M, Hasegawa T, Kikuchi A, Sugeno N, Itoyama Y, Wang Y, Yao PJ, Bushlin I and Takeda A

    Laboratory of Neurosciences, Gerontology Research Center, National Institute on Aging, National Institutes of Health, Baltimore, Maryland, USA. tatsfuru@hotmail.com

    Mutations in alpha-synuclein cause some cases of familial Parkinson's disease (PD), but the mechanism by which alpha-synuclein promotes degeneration of dopamine-producing neurons is unknown. We report that human neural cells expressing mutant alpha-synuclein (A30P and A53T) have higher plasma membrane ion permeability. The higher ion permeability caused by mutant alpha-synuclein would be because of relatively large pores through which most cations can pass non-selectively. Both the basal level of [Ca2+]i and the Ca2+ response to membrane depolarization are greater in cells expressing mutant alpha-synuclein. The membrane permeable Ca2+ chelator BAPTA-AM significantly protected the cells against oxidative stress, whereas neither L-type (nifedipine) nor N-type (omega-conotoxin-GVIA) Ca2+ channel blockers protected the cells. These findings suggest that the high membrane ion permeability caused by mutant alpha-synuclein may contribute to the degeneration of neurons in PD.

    Journal of neurochemistry 2006;97;4;1071-7

  • Small ubiquitin-like modifier (SUMO) modification of natively unfolded proteins tau and alpha-synuclein.

    Dorval V and Fraser PE

    Department of Medical Biophysics and Centre for Research in Neurodegenerative Diseases, University of Toronto, 6 Queen's Park Crescent West, Toronto, Ontario M5S 3H2, Canada.

    Sumoylation is an important post-translational modification that provides a rapid and reversible means for controlling the activity, subcellular localization, and stability of target proteins. We have examined the covalent attachment of the small ubiquitin-like modifier (SUMO) proteins to tau and alpha-synuclein, two natively unfolded proteins that define several neurodegenerative diseases. Both brain proteins were preferentially modified by SUMO1, as compared with SUMO2 or SUMO3. Tau contains two SUMO consensus sequences, and mutational analyses identified Lys(340) as the major sumoylation site. Although both tau and alpha-synuclein are targets for proteasomal degradation, only tau sumoylation was affected by inhibitors of the proteasome pathway. Tau is a microtubule-associated protein, whose ability to bind and stabilize microtubules is negatively regulated by phosphorylation. Treatment with the phosphatase inhibitor, okadaic acid, or the microtubule depolymerizing drug, colchicine, up-regulated tau sumoylation. This suggests that SUMO modification may preferentially target a free soluble pool of the substrate. These findings revealed a new, possibly regulatory, modification of tau and alpha-synuclein that may also have implications for their pathogenic roles in neurodegenerative diseases.

    The Journal of biological chemistry 2006;281;15;9919-24

  • Pathological changes in dopaminergic nerve cells of the substantia nigra and olfactory bulb in mice transgenic for truncated human alpha-synuclein(1-120): implications for Lewy body disorders.

    Tofaris GK, Garcia Reitböck P, Humby T, Lambourne SL, O'Connell M, Ghetti B, Gossage H, Emson PC, Wilkinson LS, Goedert M and Spillantini MG

    Centre for Brain Repair, Department of Clinical Neurosciences, University of Cambridge, Cambridge CB2 2PY, United Kingdom.

    Dysfunction of the 140 aa protein alpha-synuclein plays a central role in Lewy body disorders, including Parkinson's disease, as well as in multiple system atrophy. Here, we show that the expression of truncated human alpha-synuclein(1-120), driven by the rat tyrosine hydroxylase promoter on a mouse alpha-synuclein null background, leads to the formation of pathological inclusions in the substantia nigra and olfactory bulb and to a reduction in striatal dopamine levels. At the behavioral level, the transgenic mice showed a progressive reduction in spontaneous locomotion and an increased response to amphetamine. These findings suggest that the C-terminal of alpha-synuclein is an important regulator of aggregation in vivo and will help to understand the mechanisms underlying the pathogenesis of Lewy body disorders and multiple system atrophy.

    Funded by: Medical Research Council: MC_U105184291; NIA NIH HHS: P30 AG10133; Parkinson's UK: G-4039

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2006;26;15;3942-50

  • Alpha-synuclein pathology in the spinal cords of neurologically asymptomatic aged individuals.

    Klos KJ, Ahlskog JE, Josephs KA, Apaydin H, Parisi JE, Boeve BF, DeLucia MW and Dickson DW

    Department of Neurology, Mayo Clinic, Rochester, MN, USA.

    The authors assessed the frequency of spinal cord alpha-synuclein pathology in neurologically asymptomatic individuals older than 60 years of age (N = 106). Using alpha-synuclein immunohistochemistry, nine cases (8%) had incidental Lewy neurites in the intermediolateral column and at least some alpha-synuclein pathology in the dorsal motor nucleus of the vagus, locus ceruleus, and central raphe nucleus. Sparse alpha-synuclein pathology was also detected in the substantia nigra, basal forebrain, amygdala, or cortex in all but two cases.

    Funded by: NIA NIH HHS: P50 AG16754, U01 AG06786; NINDS NIH HHS: P50 NS40256

    Neurology 2006;66;7;1100-2

  • Synphilin-1A: an aggregation-prone isoform of synphilin-1 that causes neuronal death and is present in aggregates from alpha-synucleinopathy patients.

    Eyal A, Szargel R, Avraham E, Liani E, Haskin J, Rott R and Engelender S

    Department of Pharmacology, The B. Rappaport Institute of Medical Research, Technion-Israel Institute of Technology, Haifa 31096, Israel.

    alpha-Synucleinopathies are a group of neurological disorders characterized by the presence of intracellular inclusion bodies containing alpha-synuclein. We previously demonstrated that synphilin-1 interacts with alpha-synuclein, implying a role in Parkinson's disease. We now report the identification and characterization of synphilin-1A, an isoform of synphilin-1, which has enhanced aggregatory properties and causes neurotoxicity. The two transcripts encoding synphilin-1A and synphilin-1 originate from the SNCAIP gene but differ in both their exon organization and initial reading frames used for translation. Synphilin-1A binds to alpha-synuclein and induces the formation of intracellular aggregates in human embryonic kidney 293 cells, primary neuronal cultures, and human dopaminergic cells. Overexpression of synphilin-1A in neurons results in striking cellular toxicity that is attenuated by the formation of synphilin-1A inclusions, which recruit alpha-synuclein. Synphilin-1A is present in Lewy bodies of patients with Parkinson's disease and Diffuse Lewy Body disease, and is observed in detergent-insoluble fractions of brain protein samples obtained from Diffuse Lewy Body disease patients. These findings suggest that synphilin-1A may contribute to neuronal degeneration in alpha-synucleinopathies and also provide important insights into the role of inclusion bodies in neurodegenerative disorders.

    Proceedings of the National Academy of Sciences of the United States of America 2006;103;15;5917-22

  • Binding of alpha-synuclein affects the lipid packing in bilayers of small vesicles.

    Kamp F and Beyer K

    Laboratory of Alzheimer's and Parkinson's Disease Research, Department of Biochemistry, Ludwig Maximilian University, 80336 Munich, Germany.

    The intracellular deposition of fibrillar aggregates of alpha-synuclein is a characteristic feature of Parkinson disease. Alternatively, as a result of its unusual conformational plasticity, alpha-synuclein may fold into an amphipathic helix upon contact with a lipid-water interface. Using spin label ESR and fluorescence spectroscopy, we show here that alpha-synuclein affects the lipid packing in small unilamellar vesicles. The ESR hyperfine splittings of spin-labeled phospholipid probes revealed that alpha-synuclein induces chain ordering at carbon 14 of the acyl chains below the chain melting phase transition temperature but not in the liquid crystalline state of electroneutral vesicle membranes. Binding of alpha-synuclein leads to an increase in the temperature and cooperativity of the phase transition according to the fluorescence anisotropy of the hydrophobic polyene 1,6-diphenylhexatriene and of the fluorescence emission maxima of the amphiphilic probe 6-dodecanoyl-2-dimethylaminonaphthalene. Binding parameters were obtained from the fluorescence anisotropy measurements in combination with our previous determinations by titration calorimetry (Nuscher, B., Kamp, F., Mehnert, T., Odoy, S., Haass, C., Kahle, P. J., and Beyer, K. (2004) J. Biol. Chem. 279, 21966-21975). We also show that alpha-synuclein interacts with vesicle membranes containing sphingomyelin and cholesterol. We propose that the protein is capable of annealing defects in curved vesicle membranes, which may prevent synaptic vesicles from premature fusion.

    The Journal of biological chemistry 2006;281;14;9251-9

  • Alpha-synuclein facilitates the toxicity of oxidized catechol metabolites: implications for selective neurodegeneration in Parkinson's disease.

    Hasegawa T, Matsuzaki-Kobayashi M, Takeda A, Sugeno N, Kikuchi A, Furukawa K, Perry G, Smith MA and Itoyama Y

    Department of Neurology, Tohoku University School of Medicine, 1-1 Seiryomachi, Aobaku, Sendai, Miyagi 980-8574, Japan.

    Free radicals, including dopamine (DA)-oxidized metabolites, have long been implicated in pathogenesis of Parkinson's disease (PD). However, the relationships between such oxidative stresses and alpha-synuclein (alpha-S), a major constituent of Lewy bodies, remain unknown. In this study, we established neuronal cells that constitutively express alpha-S and tetracycline-regulated tyrosinase. While tyrosinase overexpression induced apoptosis, co-expression of wild type or A53T mutant human alpha-S with tyrosinase further exacerbated cell death. In this process, the formation of alpha-S oligomers and the reduction in mitochondrial membrane potential were demonstrated. This cellular model may reconstitute the pathological metabolism of alpha-S in the synucleinopathy and provide a useful tool to explore possible pathomechanisms of nigral degeneration in PD.

    FEBS letters 2006;580;8;2147-52

  • Multiple candidate gene analysis identifies alpha-synuclein as a susceptibility gene for sporadic Parkinson's disease.

    Mizuta I, Satake W, Nakabayashi Y, Ito C, Suzuki S, Momose Y, Nagai Y, Oka A, Inoko H, Fukae J, Saito Y, Sawabe M, Murayama S, Yamamoto M, Hattori N, Murata M and Toda T

    Division of Clinical Genetics, Department of Medical Genetics, Osaka University Graduate School of Medicine, Osaka, Japan.

    Parkinson's disease (PD), one of the most common human neurodegenerative diseases, is characterized by the loss of dopaminergic neurons in the substantia nigra of the midbrain. PD is a complex disorder with multiple genetic and environmental factors influencing disease risk. To identify susceptible genes for sporadic PD, we performed case-control association studies of 268 single nucleotide polymorphisms (SNPs) in 121 candidate genes. In two independent case-control populations, we found that a SNP in alpha-synuclein (SNCA), rs7684318, showed the strongest association with PD (P=5.0 x 10(-10)). Linkage disequilibrium (LD) analysis using 29 SNPs in a region around rs7684318 revealed that the entire SNCA gene lies within a single LD block (D'>0.9) spanning approximately 120 kb. A tight LD group (r2>0.85) of six SNPs, including rs7684318, associated most strongly with PD (P=2.0 x 10(-9)-1.7 x 10(-11)). Haplotype association analysis did not show lower P-values than any single SNP within this group. SNCA is a major component of Lewy bodies, the pathological hallmark of PD. Aggregation of SNCA is thought to play a crucial role in PD. SNCA expression levels tended to be positively correlated with the number of the associated allele in autopsied frontal cortices. These findings establish SNCA as a definite susceptibility gene for sporadic PD.

    Human molecular genetics 2006;15;7;1151-8

  • The alpha-synuclein gene in multiple system atrophy.

    Ozawa T, Healy DG, Abou-Sleiman PM, Ahmadi KR, Quinn N, Lees AJ, Shaw K, Wullner U, Berciano J, Moller JC, Kamm C, Burk K, Josephs KA, Barone P, Tolosa E, Goldstein DB, Wenning G, Geser F, Holton JL, Gasser T, Revesz T, Wood NW and European MSA study group

    Department of Molecular Neuroscience, Institute of Neurology, London, UK.

    Background: The formation of alpha-synuclein aggregates may be a critical event in the pathogenesis of multiple system atrophy (MSA). However, the role of this gene in the aetiology of MSA is unknown and untested.

    Method: The linkage disequilibrium (LD) structure of the alpha-synuclein gene was established and LD patterns were used to identify a set of tagging single nucleotide polymorphisms (SNPs) that represent 95% of the haplotype diversity across the entire gene. The effect of polymorphisms on the pathological expression of MSA in pathologically confirmed cases was also evaluated.

    In 253 Gilman probable or definite MSA patients, 457 possible, probable, and definite MSA cases and 1472 controls, a frequency difference for the individual tagging SNPs or tag-defined haplotypes was not detected. No effect was observed of polymorphisms on the pathological expression of MSA in pathologically confirmed cases.

    Funded by: Parkinson's UK: G-4029, G-4062

    Journal of neurology, neurosurgery, and psychiatry 2006;77;4;464-7

  • Amino acid sequence motifs and mechanistic features of the membrane translocation of alpha-synuclein.

    Ahn KJ, Paik SR, Chung KC and Kim J

    Department of Microbiology and Brain Korea 21 Projects For Medical Science, Yonsei University College of Medicine, Seoul, Korea.

    Many lines of evidence suggest that alpha-synuclein can be secreted from cells and can penetrate into them, although the detailed mechanism is not known. In this study, we investigated the amino acid sequence motifs required for the membrane translocation of alpha-synuclein, and the mechanistic features of the phenomenon. We first showed that not only alpha-synuclein but also beta- and gamma-synucleins penetrated into live cells, indicating that the conserved N-terminal region might be responsible for the membrane translocation. Using a series of deletion mutants, we demonstrated that the 11-amino acid imperfect repeats found in synuclein family members play a critical role in the membrane translocation of these proteins. We further demonstrated that fusion peptides containing the 11-amino acid imperfect repeats of alpha-synuclein can transverse the plasma membrane, and that the membrane translocation efficiency is optimal when the peptide contains two repeat motifs. alpha-Synuclein appeared to be imported rapidly and efficiently into cells, with detectable protein in the cytoplasm within 5 min after exogenous treatment. Interestingly, the import of alpha-synuclein at 4 degrees C was comparable with the import observed at 37 degrees C. Furthermore, membrane translocation of alpha-synuclein was not significantly affected by treatment with inhibitors of endocytosis. These results suggest that the internalization of alpha-synuclein is temperature-insensitive and occurs very rapidly via a mechanism distinct from normal endocytosis.

    Journal of neurochemistry 2006;97;1;265-79

  • Association of alpha-synuclein Rep1 polymorphism and Parkinson's disease: influence of Rep1 on age at onset.

    Hadjigeorgiou GM, Xiromerisiou G, Gourbali V, Aggelakis K, Scarmeas N, Papadimitriou A and Singleton A

    Neurogenetics Unit, Department of Neurology, Medical School, University of Thessaly, Larissa, Greece. gmhadji@med.uth.gr

    The alpha-synuclein Rep1 polymorphism was studied in patients and controls in an ethnic Greek population. There was an association of allele 2 with risk of Parkinson's disease (PD; adjusted odd ratio = 3.25; 95% CI = 1.80-5.87). Survival analyses (Cox proportional hazards models) were employed to explore the influence of genotypes on age at onset of PD. Age at onset of carriers of at least one Rep1 allele 2 was earlier (3.6 years) compared to noncarriers (adjusted hazard ratio = 2.21; 95% CI = 1.58-3.10). Kaplan-Meier analysis also supported a dosage effect of Rep1 allele 2 on age at onset. For Rep1 allele 1, there was neither association with risk of PD nor influence on age at onset. This is the first study showing an influence of Rep1 polymorphism on age at onset of PD.

    Movement disorders : official journal of the Movement Disorder Society 2006;21;4;534-9

  • Dopamine transporter-mediated cytotoxicity of 6-hydroxydopamine in vitro depends on expression of mutant alpha-synucleins related to Parkinson's disease.

    Lehmensiek V, Tan EM, Liebau S, Lenk T, Zettlmeisl H, Schwarz J and Storch A

    Department of Neurology, University of Ulm, Germany.

    6-Hydroxydopamine (6-OHDA) is widely used to produce animal models of Parkinson's disease (PD) by selectively destroying the nigro-striatal dopaminergic systems, but selective toxicity of 6-OHDA towards dopaminergic cells in vitro remains controversial. Mutant (A30P and A53T) alpha-synuclein isoforms cause increased vulnerability of cells towards various toxic insults and enhance dopamine transporter (DAT)-mediated toxicity of the selective dopaminergic neurotoxin and mitochondrial complex I inhibitor MPP(+) in vitro. Here we extend our recent studies on DAT-mediated toxicity to elucidate the mechanisms involved in selective dopaminergic toxicity of 6-OHDA. We studied the cytotoxicity as well as the toxic mechanisms of 6-OHDA in human embryonic kidney HEK-293 cells ectopically co-expressing mutant alpha-synucleins and the human DAT protein. 6-OHDA showed half-maximal toxic concentration (TC(50)) of 88 microM in HEK-hDAT cells without alpha-synuclein expression after 24 h, whereas the TC(50) values significantly decreased to 58 and 39 microM by expression of A30P and A53T alpha-synuclein, respectively. alpha-Synuclein expression did not affect 6-OHDA toxicity in HEK-293 cells not expressing the DAT. Analysis of intracellular parameters of cellular energy metabolism revealed that the co-expression of mutant alpha-synucleins in HEK-hDAT cells accelerates the reduction of intracellular net ATP levels and ATP/ADP ratios induced by 6-OHDA. Uptake function of the DAT was not altered by expression of alpha-synuclein isoforms. Our data suggest a mechanism of 6-OHDA-induced dopaminergic toxicity involving an interaction of mutant alpha-synucleins with the DAT molecule and subsequent acceleration of cellular energy depletion that might be relevant for the pathogenesis of PD.

    Neurochemistry international 2006;48;5;329-40

  • Gastric alpha-synuclein immunoreactive inclusions in Meissner's and Auerbach's plexuses in cases staged for Parkinson's disease-related brain pathology.

    Braak H, de Vos RA, Bohl J and Del Tredici K

    Institute for Clinical Neuroanatomy, J.W. Goethe University Clinic, Theodor Stern Kai 7, 60590 Frankfurt/Main, Germany. Braak@em.uni-frankfurt.de

    The progressive degenerative process associated with sporadic Parkinson's disease (sPD) is characterized by formation of alpha-synuclein-containing inclusion bodies in a few types of projection neurons in both the enteric and central nervous systems (ENS and CNS). In the brain, the process apparently begins in the brainstem (dorsal motor nucleus of the vagal nerve) and advances through susceptible regions of the basal mid-and forebrain until it reaches the cerebral cortex. Anatomically, all of the vulnerable brain regions are closely interconnected. Whether the pathological process begins in the brain or elsewhere in the nervous system, however, is still unknown. We therefore used immunocytochemisty to investigate the gastric myenteric and submucosal plexuses in 150 microm cryosections and 8 microm paraffin sections from five autopsy individuals, whose brains were also staged for Parkinson-associated synucleinopathy. alpha-synuclein immunoreactive inclusions were found in neurons of the submucosal Meissner plexus, whose axons project into the gastric mucosa and terminate in direct proximity to fundic glands. These elements could provide the first link in an uninterrupted series of susceptible neurons that extend from the enteric to the central nervous system. The existence of such an unbroken neuronal chain lends support to the hypothesis that a putative environmental pathogen capable of passing the gastric epithelial lining might induce alpha-synuclein misfolding and aggregation in specific cell types of the submucosal plexus and reach the brain via a consecutive series of projection neurons.

    Neuroscience letters 2006;396;1;67-72

  • The oxidation state of DJ-1 regulates its chaperone activity toward alpha-synuclein.

    Zhou W, Zhu M, Wilson MA, Petsko GA and Fink AL

    Department of Chemistry and Biochemistry, University of California, Santa Cruz, CA 95064, USA.

    DJ-1 has been reported to have chaperone activity by preventing the aggregation of some proteins, and by structural analogy to Hsp31. The L166P mutation has been linked to a familial early onset form of Parkinson's disease (PD). Since the aggregation of alpha-synuclein is believed to be a critical step in the etiology of PD, we have investigated the interaction of wild-type DJ-1 and its oxidized forms with alpha-synuclein. Native (unoxidized) DJ-1 did not inhibit alpha-synuclein fibrillation, and no evidence for stable interactions between alpha-synuclein and native DJ-1 was observed. However, DJ-1 is very susceptible to oxidation by the addition of two oxygen atoms to form the sulfinic acid of Cys106 (2O DJ-1) (no 1O oxidized state is detectable). 2O DJ-1 was readily prepared by the addition of H(2)O(2) at concentrations up to a 20-fold molar excess. The oxidation of Cys106 to the sulfinic acid had minimal effect on the structural properties of DJ-1. However, 2O DJ-1 was very effective in preventing the fibrillation of alpha-synuclein, and only this form of DJ-1 appears to have significant anti-aggregation properties against alpha-synuclein. Further oxidation of DJ-1 leads to loss of some secondary structure, and to loss of the ability to inhibit alpha-synuclein fibrillation. Our observations confirm the suggestion that DJ-1 may act as an oxidative-stress-induced chaperone to prevent alpha-synuclein fibrillation. Since oxidative stress has been associated with PD, this observation may explain why mutations of DJ-1 could be a contributing factor in PD, and also indicates that excess oxidative stress could also lead to enhanced alpha-synuclein aggregation and hence PD.

    Journal of molecular biology 2006;356;4;1036-48

  • The aggregation of alpha-synuclein is stimulated by FK506 binding proteins as shown by fluorescence correlation spectroscopy.

    Gerard M, Debyser Z, Desender L, Kahle PJ, Baert J, Baekelandt V and Engelborghs Y

    Laboratory of Biomolecular Dynamics, K.U. Leuven, Leuven, Belgium.

    Aggregation of alpha-synuclein (alpha-SYN) plays a key role in Parkinson's disease (PD). We have used fluorescence correlation spectroscopy (FCS) to study alpha-SYN aggregation in vitro and discovered that this process is clearly accelerated by addition of FK506 binding proteins (FKBPs). This effect was observed both with E. coli SlyD FKBP and with human FKBP12 and was counteracted by FK506, a specific inhibitor of FKBP. The alpha-SYN aggregates formed in the presence of FKBP12 showed fibrillar morphology. The rotamase activity of FKBP apparently accelerates the folding and subsequent aggregation of alpha-SYN. Since FK506 and other non-immunosuppressive FKBP inhibitors are known to display neuroregenerative and neuroprotective properties in disease models, the observed inhibition of rotamase activity and alpha-SYN aggregation, may explain their mode of action. Our results open perspectives for the treatment of PD with immunophilin ligands that inhibit a specific member of the FKBP family.

    FASEB journal : official publication of the Federation of American Societies for Experimental Biology 2006;20;3;524-6

  • Biochemical staging of synucleinopathy and amyloid deposition in dementia with Lewy bodies.

    Deramecourt V, Bombois S, Maurage CA, Ghestem A, Drobecq H, Vanmechelen E, Lebert F, Pasquier F and Delacourte A

    Inserm Unit 815, Lille, France.

    The primary feature of dementia with Lewy bodies (DLB) is the aggregation of alpha-synuclein into characteristic lesions: Lewy bodies (LBs) and Lewy neurites. However, in most of DLB cases, LBs are associated with neurofibrillary tangles and amyloid plaques (both Alzheimer disease [AD]-related lesions). We wanted to determine if this overlap of lesions is statistical, as a result of the late onset of both diseases, or results from a specific physiopathological synergy between synucleinopathy and either tauopathy or amyloid pathology. All patients with DLB from our prospective and multidisciplinary study were analyzed. These cases were compared with cases with pure AD and patients with Parkinson disease and controls. All cases were analyzed thoroughly at the neuropathologic and biochemical levels with a biochemical staging of aggregated alpha-synuclein, tau, and Abeta species. All sporadic cases of DLB were associated with abundant deposits of Abeta x-42 that were similar in quality and quantity to those of AD. Amyloid precursor protein (APP) dysfunction is a risk factor for AD as demonstrated by pathogenic mutations and Abeta accumulation. The constant and abundant Abeta x-42 deposition in sporadic DLB suggests that synucleinopathy is also promoted by APP dysfunction. Therefore, we conclude that APP is a therapeutic target for both AD and DLB.

    Journal of neuropathology and experimental neurology 2006;65;3;278-88

  • Dequalinium-induced protofibril formation of alpha-synuclein.

    Lee CH, Kim HJ, Lee JH, Cho HJ, Kim J, Chung KC, Jung S and Paik SR

    School of Chemical and Biological Engineering, College of Engineering, Seoul National University, San 56-1, Shillim-Dong, Kwanak-Ku, Seoul 151-744, Korea.

    alpha-Synuclein is the major constituent of Lewy bodies, a pathological signature of Parkinson disease, found in the degenerating dopaminergic neurons of the substantia nigra pars compacta. Amyloidosis generating the insoluble fibrillar protein deposition has been considered to be responsible for the cell death observed in the neurodegenerative disorder. In order to develop a controlling strategy toward the amyloid formation, 1,1'-(1,10-decanediyl)-bis-[4-a-mino-2-methylquinolinium] (dequalinium), was selected and examined in terms of its specific molecular interaction with alpha-synuclein. The protein was self-oligomerized by dequalinium, which gave rise to the ladder formation on N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine/SDS-PAGE in the presence of a coupling reagent of N-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline. The double-headed structure of dequalinium with the two cationic 4-aminoquinaldinium rings was demonstrated to be critical for the protein self-oligomerization. The dequalinium-binding site was located on the acidic C-terminal region of the protein with an approximate dissociation constant of 5.5 mum. The protein self-oligomerization induced by the compound has resulted in the protofibril formation of alpha-synuclein before it has developed into amyloids. The protofibrils were demonstrated to affect the membrane intactness of liposomes, and they have also been shown to influence cell viability of human neuroblastoma cells. In addition, dequalinium treatment of the alpha-synuclein-overexpressing cells exerted a significant cell death. Therefore, it is pertinent to consider that dequalinium could be used as a molecular probe to assess toxic mechanisms related to the amyloid formation of alpha-synuclein. Ultimately, the compound could be employed to develop therapeutic and preventive strategies toward alpha-synucleinopathies including Parkinson disease.

    The Journal of biological chemistry 2006;281;6;3463-72

  • Clinical heterogeneity of alpha-synuclein gene duplication in Parkinson's disease.

    Nishioka K, Hayashi S, Farrer MJ, Singleton AB, Yoshino H, Imai H, Kitami T, Sato K, Kuroda R, Tomiyama H, Mizoguchi K, Murata M, Toda T, Imoto I, Inazawa J, Mizuno Y and Hattori N

    Department of Neurology, Juntendo University School of Medicine, Hongo, Tokyo, Japan.

    Objective: Recently, genomic multiplications of alpha-synuclein gene (SNCA) have been reported to cause hereditary early-onset parkinsonism. The objective of this study was to assess the frequency of SNCA multiplications among autosomal dominant hereditary Parkinson's disease (ADPD).

    Methods: We screened 113 ADPD probands and 200 sporadic PD cases by quantitative polymerase chain reaction and confirmed SNCA multiplications by fluorescence in situ hybridization (FISH) and comparative genomic hybridization array.

    Results: Two families (two patients from Family A and one from Family B) with SNCA duplication were identified among ADPD patients. Even though they had the same SNCA duplication, one patient had dementia. Because there was exactly the same difference between the regions originated from each patient, the finding suggests that the phenotype of SNCA multiplication may be also influenced by the range of duplication region. We also detected asymptomatic carriers in the families of both patients. Interestingly, the penetrance ratio was 33.3% (2/6) in one kindred, indicating that the ratio was very much lower than expected.

    Interpretation: These two newly identified Japanese patients with SNCA duplication and the five previously identified American and European families with SNCA triplication or duplication mutations indicate that the incidence of SNCA multiplication may be more frequent than previously estimated.

    Annals of neurology 2006;59;2;298-309

  • Dopamine-related and caspase-independent apoptosis in dopaminergic neurons induced by overexpression of human wild type or mutant alpha-synuclein.

    Zhou ZD, Yap BP, Gung AY, Leong SM, Ang ST and Lim TM

    Department of Biological Science, National University of Singapore, 14 Science Drive 4, 117543, Singapore.

    Human wild type (WT) and mutant alpha-synuclein (alpha-syn) genes were overexpressed using a Tet-on expression system in stably transfected dopaminergic MN9D cells. Their overexpression induced caspase-independent and dopamine-related apoptosis not rescued by general caspase inhibitor Z-VAD-FMK. While apoptosis due to overexpression of WT alpha-syn was completely abrogated by a specific tyrosine hydroxylase (TH) inhibitor, alpha-methyl-p-tyrosine (alpha-MT), the inhibitor only partially rescued apoptosis caused by overexpression of alpha-syn mutants. In addition, overexpression of mutants enhanced the toxicity of 1-methyl-4-phenylpyridinium (MPP+) and 6-hydroxyldopamine (6-OHDA) to MN9D cells, whereas overexpression of WT protected MN9D cells against MPP+ toxicity, but not against 6-OHDA. We conclude that WT alpha-syn is beneficial to dopaminergic neurons but its overexpression in the presence of endogenous dopamine makes it a potential threat to the cells. In contrast, mutant alpha-syn not only caused the loss of WT protective function but also the gain-of-toxicity which becomes more serious in the presence of dopamine and neurotoxins.

    Experimental cell research 2006;312;2;156-70

  • 14-3-3eta is a novel regulator of parkin ubiquitin ligase.

    Sato S, Chiba T, Sakata E, Kato K, Mizuno Y, Hattori N and Tanaka K

    Department of Neurology, Juntendo University School of Medicine, Bunkyo, Tokyo, Japan.

    Mutation of the parkin gene, which encodes an E3 ubiquitin-protein ligase, is the major cause of autosomal recessive juvenile parkinsonism (ARJP). Although various substrates for parkin have been identified, the mechanisms that regulate the ubiquitin ligase activity of parkin are poorly understood. Here we report that 14-3-3eta, a chaperone-like protein present abundantly in neurons, could bind to parkin and negatively regulate its ubiquitin ligase activity. Furthermore, 14-3-3eta could bind to the linker region of parkin but not parkin with ARJP-causing R42P, K161N, and T240R mutations. Intriguingly, alpha-synuclein (alpha-SN), another familial Parkinson's disease (PD) gene product, abrogated the 14-3-3eta-induced suppression of parkin activity. alpha-SN could bind tightly to 14-3-3eta and consequently sequester it from the parkin-14-3-3eta complex. PD-causing A30P and A53T mutants of alpha-SN could not bind 14-3-3eta, and failed to activate parkin. Our findings indicate that 14-3-3eta is a regulator that functionally links parkin and alpha-SN. The alpha-SN-positive and 14-3-3eta-negative control of parkin activity sheds new light on the pathophysiological roles of parkin.

    The EMBO journal 2006;25;1;211-21

  • Parkinson's disease alpha-synuclein transgenic mice develop neuronal mitochondrial degeneration and cell death.

    Martin LJ, Pan Y, Price AC, Sterling W, Copeland NG, Jenkins NA, Price DL and Lee MK

    Division of Neuropathology, Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205-2196, USA. martinl@jhmi.edu

    Alpha-synuclein (alpha-Syn) is enriched in nerve terminals. Two mutations in the alpha-Syn gene (Ala53--> Thr and Ala30--> Pro) occur in autosomal dominant familial Parkinson's disease. Mice overexpressing the human A53T mutant alpha-Syn develop a severe movement disorder, paralysis, and synucleinopathy, but the mechanisms are not understood. We examined whether transgenic mice expressing human wild-type or familial Parkinson's disease-linked A53T or A30P mutant alpha-syn develop neuronal degeneration and cell death. Mutant mice were examined at early- to mid-stage disease and at near end-stage disease. Age-matched nontransgenic littermates were controls. In A53T mice, neurons in brainstem and spinal cord exhibited large axonal swellings, somal chromatolytic changes, and nuclear condensation. Spheroid eosinophilic Lewy body-like inclusions were present in the cytoplasm of cortical neurons and spinal motor neurons. These inclusions contained human alpha-syn and nitrated synuclein. Motor neurons were depleted (approximately 75%) in A53T mice but were affected less in A30P mice. Axonal degeneration was present in many regions. Electron microscopy confirmed the cell and axonal degeneration and revealed cytoplasmic inclusions in dendrites and axons. Some inclusions were degenerating mitochondria and were positive for humanalpha-syn. Mitochondrial complex IV and V proteins were at control levels, but complex IV activity was reduced significantly in spinal cord. Subsets of neurons in neocortex, brainstem, and spinal cord ventral horn were positive for terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling, cleaved caspase-3, and p53. Mitochondria in neurons had terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling-positive matrices and p53 at the outer membrane. Thus, A53T mutant mice develop intraneuronal inclusions, mitochondrial DNA damage and degeneration, and apoptotic-like death of neocortical, brainstem, and motor neurons.

    Funded by: NIA NIH HHS: AG16282, R01 AG016282; NINDS NIH HHS: NS34100, NS38065, NS38377, NS52098, P01 NS038065, P50 NS038377, R01 NS034100, R01 NS038065, R01 NS038065-05A2, R01 NS038065-06, R01 NS038065-07, R01 NS038065-08, R01 NS038065-09, R01 NS038065-10, R01 NS052098, R56 NS038065

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2006;26;1;41-50

  • Diversification of transcriptional modulation: large-scale identification and characterization of putative alternative promoters of human genes.

    Kimura K, Wakamatsu A, Suzuki Y, Ota T, Nishikawa T, Yamashita R, Yamamoto J, Sekine M, Tsuritani K, Wakaguri H, Ishii S, Sugiyama T, Saito K, Isono Y, Irie R, Kushida N, Yoneyama T, Otsuka R, Kanda K, Yokoi T, Kondo H, Wagatsuma M, Murakawa K, Ishida S, Ishibashi T, Takahashi-Fujii A, Tanase T, Nagai K, Kikuchi H, Nakai K, Isogai T and Sugano S

    Life Science Research Laboratory, Central Research Laboratory, Hitachi, Ltd., Kokubunji, Tokyo, 185-8601, Japan.

    By analyzing 1,780,295 5'-end sequences of human full-length cDNAs derived from 164 kinds of oligo-cap cDNA libraries, we identified 269,774 independent positions of transcriptional start sites (TSSs) for 14,628 human RefSeq genes. These TSSs were clustered into 30,964 clusters that were separated from each other by more than 500 bp and thus are very likely to constitute mutually distinct alternative promoters. To our surprise, at least 7674 (52%) human RefSeq genes were subject to regulation by putative alternative promoters (PAPs). On average, there were 3.1 PAPs per gene, with the composition of one CpG-island-containing promoter per 2.6 CpG-less promoters. In 17% of the PAP-containing loci, tissue-specific use of the PAPs was observed. The richest tissue sources of the tissue-specific PAPs were testis and brain. It was also intriguing that the PAP-containing promoters were enriched in the genes encoding signal transduction-related proteins and were rarer in the genes encoding extracellular proteins, possibly reflecting the varied functional requirement for and the restricted expression of those categories of genes, respectively. The patterns of the first exons were highly diverse as well. On average, there were 7.7 different splicing types of first exons per locus partly produced by the PAPs, suggesting that a wide variety of transcripts can be achieved by this mechanism. Our findings suggest that use of alternate promoters and consequent alternative use of first exons should play a pivotal role in generating the complexity required for the highly elaborated molecular systems in humans.

    Genome research 2006;16;1;55-65

  • NMR mapping of copper binding sites in alpha-synuclein.

    Sung YH, Rospigliosi C and Eliezer D

    Department of Biochemistry and Program in Structural Biology, Weill Medical College of Cornell University, 1300 York Avenue, New York, NY 10021, USA.

    Copper binding to the Parkinson disease-linked protein alpha-synuclein (aS) has been shown to accelerate its oligomerization in vitro and may therefore play a role in aS-mediated pathology in vivo. We use NMR spectroscopy to identify a number of independent copper binding sites in both the lipid-binding N-terminal domain and the highly acidic C-terminal domain of aS. Most of the sites appear to involve negatively charged amino acid side chains, but binding is also observed to the sole histidine residue located at position 50 and to the N-terminal amino group. Both the N-terminal and the histidine sites, as well as the sites in the C-terminal tail, can also bind copper in the more highly structured conformation adopted by aS upon binding to detergent micelles or lipid vesicles. There is no evidence for the formation of any sites requiring long-range order in the protein.

    Funded by: NIA NIH HHS: AG019391, R01 AG019391-06, R01 AG025440-01A1

    Biochimica et biophysica acta 2006;1764;1;5-12

  • A short overview on the role of alpha-synuclein and proteasome in experimental models of Parkinson's disease.

    Giorgi FS, Bandettini di Poggio A, Battaglia G, Pellegrini A, Murri L, Ruggieri S, Paparelli A and Fornai F

    Department of Neuroscience, Section of Neurology, University of Pisa, Italy.

    The Ubiquitin Proteasome System is a multi-enzymatic pathway which degrades polyubiquinated soluble cytoplasmic proteins. This biochemical machinery is impaired both in sporadic and inherited forms of Parkinsonism. In the present paper we focus on the role of the pre-synaptic protein alpha-synuclein in altering the proteasom based on the results emerging from experimental models showing a mechanistic chain of events between altered alpha-synuclein, proteasome impairment and formation of neuronal inclusions and catecholamine cell death.

    Journal of neural transmission. Supplementum 2006;70;105-9

  • alpha-Synuclein budding yeast model: toxicity enhanced by impaired proteasome and oxidative stress.

    Sharma N, Brandis KA, Herrera SK, Johnson BE, Vaidya T, Shrestha R and Debburman SK

    Biology Department, Lake Forest College, Lake Forest, IL 60045, USA.

    Parkinson's disease (PD) is a common neurodegenerative disorder that results from the selective loss of midbrain dopaminergic neurons. Misfolding and aggregation of the protein alpha-synuclein, oxidative damage, and proteasomal impairment are all hypotheses for the molecular cause of this selective neurotoxicity. Here, we describe a Saccharomyces cerevisiae model to evaluate the misfolding, aggregation, and toxicity-inducing ability of wild-type alpha-synuclein and three mutants (A30P, A53T, and A30P/A53T), and we compare regulation of these properties by dysfunctional proteasomes and by oxidative stress. We found prominent localization of wild-type and A53T alpha-synuclein near the plasma membrane, supporting known in vitro lipid-binding ability. In contrast, A30P was mostly cytoplasmic, whereas A30P/A53T displayed both types of fluorescence. Surprisingly, alpha-synuclein was not toxic to several yeast strains tested. When yeast mutants for the proteasomal barrel (doa3-1) were evaluated, delayed alpha-synuclein synthesis and membrane association were observed; yeast mutant for the proteasomal cap (sen3-1) exhibited increased accumulation and aggregation of alpha-synuclein. Both sen3-1and doa3-1 mutants exhibited synthetic lethality with alpha-synuclein. When yeasts were challenged with an oxidant (hydrogen peroxide), alpha-synuclein was extremely lethal to cells that lacked manganese superoxide dismutase Mn-SOD (sod2Delta) but not to cells that lacked copper, zinc superoxide dismutase Cu,Zn-SOD (sod1Delta). Despite the toxicity, sod2Delta cells never displayed intracellular aggregates of alpha-synuclein. We suggest that the toxic alpha-synuclein species in yeast are smaller than the visible aggregates, and toxicity might involve alpha-synuclein membrane association. Thus, yeasts have emerged effective organisms for characterizing factors and mechanisms that regulate alpha-synuclein toxicity.

    Funded by: NINDS NIH HHS: R15 NS048508

    Journal of molecular neuroscience : MN 2006;28;2;161-78

  • alpha-Synuclein fission yeast model: concentration-dependent aggregation without plasma membrane localization or toxicity.

    Brandis KA, Holmes IF, England SJ, Sharma N, Kukreja L and DebBurman SK

    Biology Department, Lake Forest College, Lake Forest, IL 60045, USA.

    Despite fission yeast's history of modeling salient cellular processes, it has not yet been used to model human neurodegeneration-linked protein misfolding. Because alpha-synuclein misfolding and aggregation are linked to Parkinson's disease (PD), here, we report a fission yeast (Schizosaccharomyces pombe) model that evaluates alpha-synuclein misfolding, aggregation, and toxicity and compare these properties with those recently characterized in budding yeast (Saccharomyces cerevisiae). Wild-type alpha-synuclein and three mutants (A30P, A53T, and A30P/A53T) were expressed with thiamine-repressible promoters (using vectors of increasing promoter strength: pNMT81, pNMT41, and pNMT1) to test directly in living cells the nucleation polymerization hypothesis for alpha-synuclein misfolding and aggregation. In support of the hypothesis, wild-type and A53T alpha-synuclein formed prominent intracellular cytoplasmic inclusions within fission yeast cells in a concentration- and time-dependent manner, whereas A30P and A30P/A53T remained diffuse throughout the cytoplasm. A53T alpha-synuclein formed aggregates faster than wild-type alpha-synuclein and at a lower alpha-synuclein concentration. Unexpectedly, unlike in budding yeast, wild-type and A53T alpha-synuclein did not target to the plasma membrane in fission yeast, not even at low alpha-synuclein concentrations or as a precursor step to forming aggregates. Despite alpha-synuclein's extensive aggregation, it was surprisingly nontoxic to fission yeast. Future genetic dissection might yield molecular insight into this protection against toxicity. We speculate that alpha-synuclein toxicity might be linked to its membrane binding capacity. To conclude, S. pombe and S. cerevisiae model similar yet distinct aspects of alpha-synuclein biology, and both organisms shed insight into alpha-synuclein's role in PD pathogenesis.

    Funded by: NINDS NIH HHS: R15 NS048508

    Journal of molecular neuroscience : MN 2006;28;2;179-91

  • Alpha-synuclein overexpression model.

    Mochizuki H, Yamada M and Mizuno Y

    Research Institute for Diseases of Old Age, Juntendo University, Bunkyo, Tokyo, Japan. hideki@med.juntendo.ac.jp

    Objectives: To elucidate the role of alpha-synuclein in the pathogenesis of Parkinson's disease (PD), both human alpha-synuclein transgenic mice and targeted overexpression of human alpha-synuclein in rat substantia nigra (SN) by viral vector-based methods have been studied, however little is known about the pathogenetic changes of dopaminergic neuron loss. Therefore, it is necessary to address whether the pathogenetic changes in the brains of patients with PD are recapitulated in these models.

    We used the recombinant adeno-associated viral (rAAV) vector system for human alpha-synuclein gene transfer to rat SN and observed approximately 50% loss of dopaminergic neurons in SN at 13 weeks after infection. In the slower progression of neurodegeneration, we identified several important features in common with the pathogenesis of PD, such as phosphorylation of alpha-synuclein at Ser129 and activation of caspase-9. Both findings were also evident in cortical tissues overexpressing alpha-synuclein via rAAV.

    Conclusions: Our results indicate that overexpression of alpha-synuclein via rAAV apparently recapitulates several important features of brains with PD and dementia with Lewy bodies (DLB), and thus alpha-synucleinopathy described here is likely to be an ideal model for the study of the pathogenesis of PD and DLB. This model is also useful for the gene therapy research.

    Journal of neural transmission. Supplementum 2006;70;281-4

  • Comparison of structure and dynamics of micelle-bound human alpha-synuclein and Parkinson disease variants.

    Ulmer TS and Bax A

    Laboratory of Chemical Physics, NIDDK, National Institutes of Health, Bethesda, Maryland 20892, USA. tulmer@usc.edu

    Three point mutations (A30P, E46K, and A53T) as well as gene triplication genetically link the 140-residue protein alpha-synuclein (aS) to the development of Parkinson disease. Here, the structure and dynamics of micelle-bound aS(A30P) and aS(A53T) are described and compared with wild-type aS, in addition to describing the aS-micelle interaction. A53T is sensed only by directly adjacent residues and leaves the backbone structure and dynamics indistinguishable from the wild type. A30P interrupts one helix turn (Val26-Ala29) and destabilizes the preceding one. A shift in helix register following A30P disturbs the canonical succession of polar and hydrophobic residues for at least two turns. The shortened helix-N adopts a slightly higher helical content and is less bent, indicating that strain was present in the micelle-bound helix. In the vicinity of the A30P-induced perturbations, the underlying micelle environment has rearranged, but nevertheless all aS variants maintain similar interrelationships with the micelle. Moreover, aS-micelle immersion correlates well with fast and slow aS backbone dynamics, allowing a rare insight into protein-micelle interplay.

    The Journal of biological chemistry 2005;280;52;43179-87

  • DJ-1 up-regulates glutathione synthesis during oxidative stress and inhibits A53T alpha-synuclein toxicity.

    Zhou W and Freed CR

    Division of Clinical Pharmacology and Toxicology, Department of Medicine, University Colorado Health Sciences Center, Denver, Colorado 80262, USA.

    DJ-1 is the third gene that has been linked to Parkinson disease. Mutations in the DJ-1 gene cause early onset PD with autosomal recessive inheritance. To clarify the mechanism of DJ-1 protection, we have overexpressed the gene in cultured dopaminergic cells that were then subjected to chemical stress. In the rat dopaminergic cell line, N27, and in primary dopamine neurons, overexpression of wild type DJ-1 protected cells from death induced by hydrogen peroxide and 6-hydroxydopamine. Overexpressing the L166P mutant DJ-1 had no protective effect. By contrast, knocking down endogenous DJ-1 with antisense DJ-1 rendered cells more susceptible to oxidative damage. We have found that DJ-1 improves survival by increasing cellular glutathione levels through an increase in the rate-limiting enzyme glutamate cysteine ligase. Blocking glutathione synthesis eliminated the beneficial effect of DJ-1. Protection could be restored by adding exogenous glutathione. Wild type DJ-1 reduced cellular reactive oxygen species and reduced the levels of protein oxidation caused by oxidative stress. By a separate mechanism, overexpressing wild type DJ-1 inhibited the protein aggregation and cytotoxicity usually caused by A53T human alpha-synuclein. Under these circumstances, DJ-1 increased the level of heat shock protein 70 but did not change the glutathione level. Our data indicate that DJ-1 protects dopaminergic neurons from oxidative stress through up-regulation of glutathione synthesis and from the toxic consequences of mutant humanalpha-synuclein through increased expression of heat shock protein 70. We conclude that DJ-1 has multiple specific mechanisms for protecting dopamine neurons from cell death.

    The Journal of biological chemistry 2005;280;52;43150-8

  • Similar patterns of mitochondrial vulnerability and rescue induced by genetic modification of alpha-synuclein, parkin, and DJ-1 in Caenorhabditis elegans.

    Ved R, Saha S, Westlund B, Perier C, Burnam L, Sluder A, Hoener M, Rodrigues CM, Alfonso A, Steer C, Liu L, Przedborski S and Wolozin B

    Deparment of Pharmacology, Boston University School of Medicine.

    How genetic and environmental factors interact in Parkinson disease is poorly understood. We have now compared the patterns of vulnerability and rescue of Caenorhabditis elegans with genetic modifications of three different genetic factors implicated in Parkinson disease (PD). We observed that expressing alpha-synuclein, deleting parkin (K08E3.7), or knocking down DJ-1 (B0432.2) or parkin produces similar patterns of pharmacological vulnerability and rescue. C. elegans lines with these genetic changes were more vulnerable than nontransgenic nematodes to mitochondrial complex I inhibitors, including rotenone, fenperoximate, pyridaben, or stigmatellin. In contrast, the genetic manipulations did not increase sensitivity to paraquat, sodium azide, divalent metal ions (Fe(II) or Cu(II)), or etoposide compared with the nontransgenic nematodes. Each of the PD-related lines was also partially rescued by the antioxidant probucol, the mitochondrial complex II activator, D-beta-hydroxybutyrate, or the anti-apoptotic bile acid tauroursodeoxycholic acid. Complete protection in all lines was achieved by combining d-beta-hydroxybutyrate with tauroursodeoxycholic acid but not with probucol. These results show that diverse PD-related genetic modifications disrupt the mitochondrial function in C. elegans, and they raise the possibility that mitochondrial disruption is a pathway shared in common by many types of familial PD.

    Funded by: NIA NIH HHS: AG/NS17485; NIEHS NIH HHS: R01 ES015567; NINDS NIH HHS: NS41786, R01 NS041786, R01 NS060872

    The Journal of biological chemistry 2005;280;52;42655-42668

  • Agrin binds alpha-synuclein and modulates alpha-synuclein fibrillation.

    Liu IH, Uversky VN, Munishkina LA, Fink AL, Halfter W and Cole GJ

    Biomedical/Biotechnology Research Institute, North Carolina Central University, Durham, NC 27707, USA.

    Recent studies have begun to investigate the role of agrin in brain and suggest that agrin's function likely extends beyond that of a synaptogenic protein. Particularly, it has been shown that agrin is associated with the pathological lesions of Alzheimer's disease (AD) and may contribute to the formation of beta-amyloid (Abeta) plaques in AD. We have extended the analysis of agrin's function in neurodegenerative diseases to investigate its role in Parkinson's disease (PD). Alpha-synuclein is a critical molecular determinant in familial and sporadic PD, with the formation of alpha-synuclein fibrils being enhanced by sulfated macromolecules. In the studies reported here, we show that agrin binds to alpha-synuclein in a heparan sulfate-dependent (HS-dependent) manner, induces conformational changes in this protein characterized by beta-sheet structure, and enhances insolubility of alpha-synuclein. We also show that agrin accelerates the formation of protofibrils by alpha-synuclein and decreases the half-time of fibril formation. The association of agrin with PD lesions was also explored in PD human brain, and these studies shown that agrin colocalizes with alpha-synuclein in neuronal Lewy bodies in the substantia nigra of PD brain. These studies indicate that agrin is capable of accelerating the formation of insoluble protein fibrils in a second common neurodegenerative disease. These findings may indicate shared molecular mechanisms leading to the pathophysiology in these two neurodegenerative disorders.

    Funded by: NINDS NIH HHS: NS 33981, NS 39985

    Glycobiology 2005;15;12;1320-31

  • Neurological and neurodegenerative alterations in a transgenic mouse model expressing human alpha-synuclein under oligodendrocyte promoter: implications for multiple system atrophy.

    Shults CW, Rockenstein E, Crews L, Adame A, Mante M, Larrea G, Hashimoto M, Song D, Iwatsubo T, Tsuboi K and Masliah E

    Department of Neurosciences, University of California, San Diego, La Jolla, California 92093-0624, USA.

    Multiple system atrophy (MSA) is a progressive, neurodegenerative disease characterized by parkinsonism, ataxia, autonomic dysfunction, and accumulation of alpha-synuclein (alpha-syn) in oligodendrocytes. To better understand the mechanisms of neurodegeneration and the role of alpha-syn accumulation in oligodendrocytes in the pathogenesis of MSA, we generated transgenic mouse lines expressing human (h) alpha-syn under the control of the murine myelin basic protein promoter. Transgenic mice expressing high levels of halpha-syn displayed severe neurological alterations and died prematurely at 6 months of age. Furthermore, mice developed progressive accumulation of halpha-syn-immunoreactive inclusions in oligodendrocytes along the axonal tracts in the brainstem, basal ganglia, cerebellum, corpus callosum, and neocortex. The inclusions also reacted with antibodies against phospho-serine (129) halpha-syn and ubiquitin, and halpha-syn was found in the detergent-insoluble fraction. In high-expresser lines, the white matter tracts displayed intense astrogliosis, myelin pallor, and decreased neurofilament immunostaining. Accumulation of halpha-syn in oligodendrocytes also leads to prominent neurodegenerative changes in the neocortex with decreased dendritic density and to loss of dopaminergic fibers in the basal ganglia. The oligodendrocytic inclusions were composed of fibrils and accompanied by mitochondrial alterations and disruption of the myelin lamina in the axons. Together, these studies support the contention that accumulation of alpha-syn in oligodendrocytes promotes neurodegeneration and recapitulates several of the key functional and neuropathological features of MSA.

    Funded by: NIA NIH HHS: AG022074, AG18440, AG5131; NINDS NIH HHS: P01 NS044233

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2005;25;46;10689-99

  • Alpha-synuclein cooperates with CSPalpha in preventing neurodegeneration.

    Chandra S, Gallardo G, Fernández-Chacón R, Schlüter OM and Südhof TC

    Center for Basic Neuroscience, Department of Molecular Genetics, Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA.

    Alpha-synuclein and cysteine-string protein-alpha (CSPalpha) are abundant synaptic vesicle proteins independently linked to neurodegeneration. Dominantly inherited mutations in alpha-synuclein cause Parkinson's disease, but the physiological role of alpha-synuclein remains unknown. Deletion of CSPalpha produces rapidly progressive neurodegeneration in mice, presumably because the cochaperone function of CSPalpha is essential for neuronal survival. Here, we report the surprising finding that transgenic expression of alpha-synuclein abolishes the lethality and neurodegeneration caused by deletion of CSPalpha. Conversely, ablation of endogenous synucleins exacerbates these phenotypes. Deletion of CSPalpha inhibits SNARE complex assembly; transgenic alpha-synuclein ameliorates this inhibition. In preventing neurodegeneration in CSPalpha-deficient mice, alpha-synuclein does not simply substitute for CSPalpha but acts by a downstream mechanism that requires phospholipid binding by alpha-synuclein. These observations reveal a powerful in vivo activity of alpha-synuclein in protecting nerve terminals against injury and suggest that this activity operates in conjunction with CSPalpha and SNARE proteins on the presynaptic membrane interface.

    Funded by: NINDS NIH HHS: 5 R01 NS40057-04

    Cell 2005;123;3;383-96

  • Locomotor activity and evoked dopamine release are reduced in mice overexpressing A30P-mutated human alpha-synuclein.

    Yavich L, Oksman M, Tanila H, Kerokoski P, Hiltunen M, van Groen T, Puoliväli J, Männistö PT, García-Horsman A, MacDonald E, Beyreuther K, Hartmann T and Jäkälä P

    Department of Pharmacology and Toxicology, University of Kuopio, FIN-70211 Kuopio, Finland. leonid.yavich@uku.fi

    We have generated a transgenic mouse line overexpressing mutated human A30P alpha-synuclein under the control of the prion-related protein promoter. Immunohistology revealed mutated human A30P alpha-synuclein protein in numerous brain areas, but no gross morphological changes, Lewy bodies, or loss of dopaminergic cell bodies. The transgenic mice displayed decreased locomotion, impaired motor coordination, and balance. In vivo voltammetry showed that A30P mice responded to longer stimulation of the ascending dopaminergic pathways with less dopamine release in striatum and had a slower rate of dopamine decline after repeated stimulations or after alpha-methyl-p-tyrosine-HCl treatment. However, dopamine re-uptake or transporter levels were similar in transgenic and control mice. Our data provide evidence that overexpression of mutated human A30P alpha-synuclein in mice leads to a reduced size of the dopamine storage pool. This is in agreement with the previously postulated involvement of alpha-synuclein in the turnover of transmitter vesicles and may explain the observed motor deficits in A30P mice.

    Neurobiology of disease 2005;20;2;303-13

  • Disruption of the interaction of alpha-synuclein with microtubules enhances cell surface recruitment of the dopamine transporter.

    Wersinger C and Sidhu A

    Department of Pediatrics, Georgetown University, Washington, DC 20007, USA.

    Mutations in alpha-synuclein have been implicated in the genesis of Parkinson's disease. A probable normative function of alpha-synuclein is the maintenance of dopamine homeostasis, partly through a negative modulation of dopamine transporter (DAT) activity, by reducing its level at the cell surface. To study the possible involvement of the microtubular network in the alpha-synuclein-dependent trafficking of DAT, we treated cotransfected cells and primary mesencephalic neurons with either colchicine, vinblastine, or nocodazole, each of which disrupts microtubules or affects microtubule dynamics. Treatment of both types of cells with vinblastine, colchicine, or nocodazole reversed alpha-synuclein-mediated inhibition of DAT activity, resulting in an increased rate of dopamine uptake and and increased level of extracellular dopamine-induced oxidative stress, with accelerated cell death. Treatment with these agents also reversed the alpha-synuclein-induced decrease in levels of cell surface-associated DAT. This effect of colchicine, vinblastine, or nocodazole was not linked to a disruption of formation of the alpha-synuclein-DAT complex but paradoxically caused an increased level of interaction between these proteins. Both alpha-synuclein and DAT co-immunoprecipitated with both alpha- and beta-tubulins, in both transfected cells and rat primary mesencephalic dopaminergic neurons, suggesting heteromeric complex formation between these various proteins. Treatment with the microtubule depolymerizing agents disrupted the heteromeric protein complex between either alpha-synuclein or the DAT, and alpha- or beta-tubulins. These results indicate a previously unappreciated role of microtubules in the modulation of DAT trafficking, and provide insight into a novel mechanism by which alpha-synuclein regulates DAT activity, by tethering the transporter to the microtubular network.

    Funded by: NINDS NIH HHS: NS-34914, NS-41555

    Biochemistry 2005;44;41;13612-24

  • Molecular motors implicated in the axonal transport of tau and alpha-synuclein.

    Utton MA, Noble WJ, Hill JE, Anderton BH and Hanger DP

    Department of Neuroscience, King's College London, Institute of Psychiatry, De Crespigny Park, London, SE5 8AF, UK.

    Tau and alpha-synuclein are both proteins implicated in the pathology of neurodegenerative disease. Here we have investigated the mechanisms of axonal transport of tau and alpha-synuclein, because failure of axonal transport has been implicated in the development of several neurodegenerative disorders. We found that the transport of both of these proteins depend on an intact microtubule- but not actin-cytoskeleton, and that tau and alpha-synuclein both move at overall slow rates of transport. We used time-lapse video microscopy to obtain images of live neurons that had been transfected with plasmids expressing proteins tagged with enhanced green fluorescent protein. We found that particulate structures containing tau or alpha-synuclein travel rapidly when moving along axons but spend the majority of the time paused, and these structures have similar characteristics to those previously observed for neurofilaments. The motile particles containing tau or alpha-synuclein colocalise with the fast-transporting molecular motor kinesin-1 in neurons. Co-immunoprecipitation experiments demonstrate that tau and alpha-synuclein are each associated with complexes containing kinesin-1, whereas only alpha-synuclein appears to interact with dynein-containing complexes. In vitro glutathione S-transferase-binding assays using rat brain homogenate or recombinant protein as bait reveals a direct interaction of kinesin-1 light chains 1 and 2 with tau, but not with alpha-synuclein. Our findings suggest that the axonal transport of tau occurs via a mechanism utilising fast transport motors, including the kinesin family of proteins, and that alpha-synuclein transport in neurons may involve both kinesin and dynein motor proteins.

    Journal of cell science 2005;118;Pt 20;4645-54

  • Engineered alpha-synuclein prevents wild type and familial Parkin variant fibril formation.

    Sode K, Usuzaka E, Kobayashi N and Ochiai S

    Department of Biotechnology, Faculty of Technology, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, 184-8588 Tokyo, Koganei, Japan. sode@cc.tuat.ac.jp

    Alpha-synuclein is a major component of several pathological lesions diagnostic of specific neurodegenerative disease such as Parkinson's disease. This study focuses on the non-amyloid beta component of Alzheimer's disease amyloid, a key region for the aggregation and fibril formation of alpha-synuclein. Several mutations were introduced in an attempt to repress beta-strand formation and hydrophobic interaction-based aggregation. Although reducing the hydrophobicity drastically decreased fibril formation, the Val70Thr and Val70Pro mutations resulted in an unstable secondary structure thereby increasing non-structural aggregation, instead of fibril formation. Therefore, the stabilization of non-structural natively unfolded status is important to prevent alpha-synuclein fibril formation. Mixing the Val70Thr/Val71Thr double mutant, which has inherently low potential, with the fibril forming alpha-synucleins, WT and Ala53Thr, greatly reduced their fibril formation and aggregation. This double mutant has great potential for further therapeutic approaches.

    Biochemical and biophysical research communications 2005;335;2;432-6

  • Wild-type alpha-synuclein interacts with pro-apoptotic proteins PKCdelta and BAD to protect dopaminergic neuronal cells against MPP+-induced apoptotic cell death.

    Kaul S, Anantharam V, Kanthasamy A and Kanthasamy AG

    Parkinson's Disorder Research Laboratory, Department of Biomedical Sciences, 2062, Veterinary Medicine Building, Iowa Sate University, Ames, IA 50011, USA.

    Alpha-synuclein is a pre-synaptic protein of unknown function that has been implicated in the pathogenesis of Parkinson's disease (PD). Recently, we demonstrated that 1-methyl-4-phenylpyridinium (MPP+) induces caspase-3-dependent proteolytic activation of PKCdelta, which subsequently contributes to neuronal apoptotic cell death in mesencephalic dopaminergic neuronal cells. In the present study, we examined whether PKCdelta interacts with alpha-synuclein to modulate MPP+-induced dopaminergic degeneration. Over-expression of wild-type human alpha-synuclein in mesencephalic dopaminergic neuronal cells (N27 cells) attenuated MPP+-induced (300 microM) cytotoxicity, release of mitochondrial cytochrome c, and subsequent caspase-3 activation, without affecting reactive oxygen species (ROS) generation. Wild-type alpha-synuclein over-expression also dramatically reduced MPP+-induced caspase-3-mediated proteolytic cleavage of PKCdelta, whereas over-expression of the mutant human alpha-synucleinA53T did not alter the PKCdelta cleavage under similar conditions. Immunoprecipitation-kinase assay revealed reduced PKCdelta kinase activity in wild-type alpha-synuclein over-expressing cells in response to MPP+ treatment. Wild-type alpha-synuclein over-expression also rescued mesencephalic dopaminergic neuronal cells from MPP+-induced apoptotic cell death, while alpha-synucleinA53T exacerbated the MPP+-induced DNA fragmentation. Furthermore, co-immunoprecipitation studies revealed that alpha-synuclein interacts with the pro-apoptotic proteins PKCdelta and BAD, but not with the anti-apoptotic protein Bcl-2 following MPP+ treatment. We also observed that the interaction between PKCdelta and alpha-synuclein does not involve direct phosphorylation. Together, our results demonstrate that wild-type alpha-synuclein interacts with the pro-apoptotic molecules BAD and PKCdelta to protect dopaminergic neuronal cells against neurotoxic insults.

    Funded by: NIEHS NIH HHS: ES10586; NINDS NIH HHS: NS 45133, NS38644

    Brain research. Molecular brain research 2005;139;1;137-52

  • A combinatorial code for the interaction of alpha-synuclein with membranes.

    Kubo S, Nemani VM, Chalkley RJ, Anthony MD, Hattori N, Mizuno Y, Edwards RH and Fortin DL

    Department of Neurology, Graduate Program in Biomedical Sciences, University of California, San Francisco, California 94143-2140, USA.

    Considerable genetic and pathological evidence has implicated the small, soluble protein alpha-synuclein in the pathogenesis of familial and sporadic forms of Parkinsons disease (PD). However, the precise role of alpha-synuclein in the disease process as well as its normal function remain poorly understood. We recently found that an interaction with lipid rafts is crucial for the normal, pre-synaptic localization of alpha-synuclein. To understand how alpha-synuclein interacts with lipid rafts, we have now developed an in vitro binding assay to rafts purified from native membranes. Recapitulating the specificity observed in vivo, recombinant wild type but not PD-associated A30P mutant alpha-synuclein binds to lipid rafts isolated from cultured cells and purified synaptic vesicles. Proteolytic digestion of the rafts does not disrupt the binding of alpha-synuclein, indicating an interaction with lipid rather than protein components of these membranes. We have also found that alpha-synuclein binds directly to artificial membranes whose lipid composition mimics that of lipid rafts. The binding of alpha-synuclein to these raft-like liposomes requires acidic phospholipids, with a preference for phosphatidylserine (PS). Interestingly, a variety of synthetic PS with defined acyl chains do not support binding when used individually. Rather, the interaction with alpha-synuclein requires a combination of PS with oleic (18:1) and polyunsaturated (either 20:4 or 22:6) fatty acyl chains, suggesting a role for phase separation within the membrane. Furthermore, alpha-synuclein binds with higher affinity to artificial membranes with the PS head group on the polyunsaturated fatty acyl chain rather than on the oleoyl side chain, indicating a stringent combinatorial code for the interaction of alpha-synuclein with membranes.

    Funded by: NCRR NIH HHS: RR014606

    The Journal of biological chemistry 2005;280;36;31664-72

  • Cocaine abuse elevates alpha-synuclein and dopamine transporter levels in the human striatum.

    Qin Y, Ouyang Q, Pablo J and Mash DC

    Department of Neurology, University of Miami School of Medicine, Miami, FL 33101, USA.

    Direct protein interactions between the dopamine transporter and alpha-synuclein demonstrate that dopamine uptake function is modulated by alpha-synuclein. We report here that chronic cocaine abuse results in an increase in alpha-synuclein expression in the human striatum. Immunoblot analysis in the ventral putamen showed that alpha-synuclein protein was increased in striatal synaptosomes from cocaine users compared with age-matched drug-free controls. [H]-Dopamine transporter uptake was increased in parallel with 2beta-carbomethoxy-3beta-(4-fluorophenyl)tropane binding to the dopamine transporter. The increase in alpha-synuclein protein was more marked in the ventromedial sectors of the striatum than in the dorsal caudate nucleus. These results demonstrate concomitant regulation of alpha-synuclein and dopamine transporter binding and function in human striatal synaptic terminals isolated from cocaine abusers. Overexpression of alpha-synuclein may play a role in cocaine-induced plasticity and regulation of dopamine synaptic tone.

    Funded by: NIDA NIH HHS: DA-06227

    Neuroreport 2005;16;13;1489-93

  • Familial mutants of alpha-synuclein with increased neurotoxicity have a destabilized conformation.

    Bertoncini CW, Fernandez CO, Griesinger C, Jovin TM and Zweckstetter M

    Department of Molecular Biology, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.

    A30P and A53T mutations of the presynaptic protein alpha-synuclein are associated with familial forms of Parkinson disease. NMR spectroscopy demonstrates that Parkinsonism-linked mutations greatly perturb specific tertiary interactions essential for the native state of alpha-synuclein. However, alpha-synuclein is not completely unfolded but exhibits structural fluctuations on the time scale of secondary structure formation and loses its native conformation gradually when protein stability decreases. The redistribution of the ensemble of alpha-synuclein conformers may underlie toxic gain-of-function by fostering self-association and altered binding affinity to ligands and receptors.

    The Journal of biological chemistry 2005;280;35;30649-52

  • Heat shock prevents alpha-synuclein-induced apoptosis in a yeast model of Parkinson's disease.

    Flower TR, Chesnokova LS, Froelich CA, Dixon C and Witt SN

    Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center, 1501 Kings Highway, Shreveport, LA 71130-3932, USA.

    We show that human wild-type alpha synuclein (WT alpha-syn), and the inherited mutants A53T or A30P, when expressed in the yeast Saccharomyces cerevisiae triggers events that are diagnostic of apoptosis: loss of membrane asymmetry due to the externalization of phosphatidylserine, accumulation of reactive oxygen species (ROS), and the release of cytochrome c from mitochondria. A brief heat shock was strikingly protective in that alpha-syn-expressing cells receiving a heat shock exhibited none of these apoptotic markers. Because the heat shock did not decrease the expression level of alpha-syn, a protective protein or proteins, induced by the heat shock, must be responsible for inhibition of alpha-syn-induced apoptosis. Using ROS accumulation as a marker of apoptosis, the role of various genes and various drugs in controlling alpha-syn-induced apoptosis was investigated. Treatment with geldanamycin or glutathione, overexpression of Ssa3 (Hsp70), or deletion of the yeast metacaspase gene YCA1 abolishes the ability of alpha-syn to induce ROS accumulation. Deletion of YCA1 also promotes vigorous growth of alpha-syn-expressing cells compared to cells that contain a functional copy of YCA1. These findings indicate that alpha-syn-induced ROS generation is mediated by the caspase, according to alpha-syn-->caspase-->ROS-->apoptosis. It is shown by co-immunoprecipitation that Ssa3 binds to alpha-syn in a nucleotide-dependent manner. Thus, we propose that Hsp70 chaperones inhibit this sequence of events by binding and sequestering alpha-syn.

    Journal of molecular biology 2005;351;5;1081-100

  • The role of alpha-synuclein gene multiplications in early-onset Parkinson's disease and dementia with Lewy bodies.

    Hofer A, Berg D, Asmus F, Niwar M, Ransmayr G, Riemenschneider M, Bonelli SB, Steffelbauer M, Ceballos-Baumann A, Haussermann P, Behnke S, Krüger R, Prestel J, Sharma M, Zimprich A, Riess O and Gasser T

    Department of Neurodegenerative Diseases, Hertie-Institute for Clinical Brain Research, University of Tübingen, Germany.

    Background: A triplication of the alpha-synuclein gene was found to cause autosomal dominant Lewy body disease in two distinct families.

    Method: We searched for alterations of alpha-synuclein gene dosage and analysed the entire coding region for point mutations in 54 dementia with Lewy body disease (DLB) and in 103 young onset Parkinson's disease (PD) patients from Central Europe.

    Results: We could not detect any quantitative alterations in the gene dosage of alpha-synuclein. Mutational screening of the entire coding region of alpha-synuclein revealed only one silent mutation V3V (adenine9guanine) in one case.

    Conclusions: Thus, this phenomenon appears not to be a major cause in the pathogenesis of sporadic DLB and young onset PD in this European population.

    Journal of neural transmission (Vienna, Austria : 1996) 2005;112;9;1249-54

  • Abundant neuritic inclusions and microvacuolar changes in a case of diffuse Lewy body disease with the A53T mutation in the alpha-synuclein gene.

    Yamaguchi K, Cochran EJ, Murrell JR, Polymeropoulos MH, Shannon KM, Crowther RA, Goedert M and Ghetti B

    Department of Pathology and Laboratory Medicine, Division of Neuropathology, Indiana University School of Medicine, Indianapolis, IN 46202-5120, USA. bghetti@iupui.edu

    We report here a case of diffuse Lewy body disease with the A53T mutation in the alpha-synuclein gene. The proband presented at the age of 41 years with parkinsonism that was poorly responsive to levodopa. She subsequently developed cognitive impairment and moderate dementia, and died at the age of 50. Her father, paternal grandfather and uncle were all reported to have suffered from Parkinson's disease. Staining of tissue sections from the proband's brain with hematoxylin-eosin and alpha-synuclein antibodies showed small numbers of Lewy bodies in a few brain regions. This contrasted with large numbers of Lewy neurites and neuroaxonal spheroids in many brain regions. By electron microscopy, Lewy neurites consisted of abnormal filaments and dense granular material. Isolated filaments resembled those previously described in idiopathic Parkinson's disease and dementia with Lewy bodies. They were decorated by antibodies specific for the N and C termini of alpha-synuclein, indicating the presence of the full-length protein. Nucleus accumbens and the lower layers in limbic areas of the cerebral cortex showed prominent vacuolation, with frequent clustering of microvacuoles around Lewy neurites. Nerve cell loss was most extensive in dorsal motor nucleus of the vagus nerve, substantia nigra and nucleus basalis of Meynert. Neurofibrillary tangles and senile plaques were not observed. However, in several brain regions, a few widely scattered tau-positive nerve cell bodies and neurites were present. By electron microscopy, Alzheimer-type paired helical and straight filaments were seen.

    Funded by: NIA NIH HHS: P30 AG10133

    Acta neuropathologica 2005;110;3;298-305

  • Alpha-synuclein and Parkinson's disease: implications from the screening of more than 1,900 patients.

    Berg D, Niwar M, Maass S, Zimprich A, Möller JC, Wuellner U, Schmitz-Hübsch T, Klein C, Tan EK, Schöls L, Marsh L, Dawson TM, Janetzky B, Müller T, Woitalla D, Kostic V, Pramstaller PP, Oertel WH, Bauer P, Krueger R, Gasser T and Riess O

    Institute for Medical Genetics, University of Tübingen, Germany.

    Data on the frequency of alpha-synuclein mutations in Parkinson's disease (PD) are limited. Screening the entire coding region in 1,921 PD patients with denaturing high performance liquid chromatography and subsequent sequencing we only detected silent mutations (g.2654A>G, g.10151G>A, and g.15986A>T) and the c.209G>A substitution corresponding to the p.A53T mutation. These results demonstrate that mutations in the alpha-synuclein gene are rare and suggest that other factors contribute to alpha-synuclein aggregation in the majority of PD patients.

    Funded by: NCRR NIH HHS: M01-RR00052; NINDS NIH HHS: P50-NS38377

    Movement disorders : official journal of the Movement Disorder Society 2005;20;9;1191-4

  • Alpha-synuclein alters proteasome function, protein synthesis, and stationary phase viability.

    Chen Q, Thorpe J and Keller JN

    Sanders-Brown Center on Aging, The Department of Anatomy and Neurobiology, University of Kentucky, Lexington, Kentucky 40536, USA.

    Alpha-synuclein appears to play a role in mediating neurotoxicity in a number of neurodegenerative disorders, collectively referred to as synucleinopathies. Most of these disorders are associated with aging and a probable impairment of the proteasome-proteolytic pathway, although the relationship between aging, proteasome inhibition, and alpha-synuclein toxicity has not been fully elucidated. Recent studies suggest that yeast may provide a useful system for studying the biology and toxicity of alpha-synuclein in mitotic cells, recapitulating many features observed in the various synucleinopathy disorders. Additional studies indicate that the stationary phase model of aging in yeast provides a useful system for understanding the biochemistry and regulation of aging in post-mitotic cells. In the present study we examined the effect of wild type and mutant alpha-synuclein (A30P) on multiple aspects of proteasome homeostasis, protein synthesis, as well as the ability of cells to survive stationary phase aging. These data demonstrate that alpha-synuclein alters proteasome composition, impairs proteasome-mediated protein degradation, impairs protein synthesis, and impairs the ability of cells to withstand stationary phase aging. Interestingly, alpha-synuclein had little effect on intracellular proteasome content or protein ubiquitination, and did not increase the vulnerability of cells to a variety of stressors. Together, these data suggest that yeast may be useful for understanding the ability of alpha-synuclein to impair proteasome-mediated protein degradation, as well as for understanding the basis for age-related alpha-synuclein cytotoxicity.

    Funded by: NIA NIH HHS: AG005119, AG018437

    The Journal of biological chemistry 2005;280;34;30009-17

  • Abeta deposition is associated with enhanced cortical alpha-synuclein lesions in Lewy body diseases.

    Pletnikova O, West N, Lee MK, Rudow GL, Skolasky RL, Dawson TM, Marsh L and Troncoso JC

    Department of Pathology (Neuropathology), Johns Hopkins University School of Medicine, Ross Building 558, Baltimore, MD 21205, USA.

    In order to understand better the neuropathological substrate of dementia in Parkinson's disease (PD) and to examine its interactions with Alzheimer's disease (AD), we examined autopsy brains from 21 cases of PD and Lewy body disease (LBD) with dementia. We separated brains in two groups according to the presence of Abeta deposits. In brains without Abeta, we found few or no Lewy bodies (LB) in the cerebral cortex. By contrast, in brains with Abeta, we observed significant increases in LB in the cerebral cortex (p < 0.01) and alpha-synuclein immunoreactive lesions in the cingulate cortex (p < 0.01). Immunoblots of alpha-synuclein from cingulate cortex in brains with Abeta showed significantly higher levels of insoluble alpha-synuclein compared to brains without Abeta. Our observations indicate that in cases of PD with dementia, the neocortex is not necessarily involved by LB. Furthermore, the presence of Abeta deposits in the cerebral cortex was associated with extensive alpha-synuclein lesions and higher levels of insoluble alpha-synuclein. This suggests that Abeta enhances the development of cortical alpha-synuclein lesions in cases of PD.

    Funded by: NIA NIH HHS: AG 05146; NINDS NIH HHS: NS038377, NS38065

    Neurobiology of aging 2005;26;8;1183-92

  • Altered alpha-synuclein homeostasis causing Parkinson's disease: the potential roles of dardarin.

    Singleton AB

    Molecular Genetics Unit, National Institute on Aging, National Institutes of Health, Porter Neuroscience Research Center, Room 1A1000, MSC3707, 35 Lincoln Drive, Bethesda, MD 20892, USA. singleta@mail.nih.gov

    The past decade has been a fruitful one for geneticists involved in Parkinson's disease (PD) research. The initial hurdle of identifying the first gene underlying parkinsonism was cleared with apparent ease in 1997 and four additional genes have since been found to contain mutations causing this disorder. Driving this research is the belief that these data will highlight disease mechanisms and directly implicate a pathway amenable to therapeutic intervention. This article will focus on recent genetic advances in the field, focusing on data that suggest alpha-synuclein expression is key in the etiology of PD. In addition, it will discuss the recent identification of LRRK2 mutation as a cause of PD and the potential of this finding to provide further insight into disease.

    Trends in neurosciences 2005;28;8;416-21

  • Proteolytic cleavage of extracellular secreted {alpha}-synuclein via matrix metalloproteinases.

    Sung JY, Park SM, Lee CH, Um JW, Lee HJ, Kim J, Oh YJ, Lee ST, Paik SR and Chung KC

    Department of Biology, College of Science, Yonsei University, Seoul 120-749, Korea.

    Although alpha-synuclein is the main structural component of the insoluble filaments that form Lewy bodies in Parkinson disease (PD), its physiological function and exact role in neuronal death remain poorly understood. In the present study, we examined the possible functional relationship between alpha-synuclein and several forms of matrix metalloproteinases (MMPs) in the human dopaminergic neuroblastoma (SK-N-BE) cell line. When SK-N-BE cells were transiently transfected with alpha-synuclein, it was secreted into the extracellular culture media, concomitantly with a significant decrease in cell viability. Also the addition of nitric oxide-generating compounds to the cells caused the secreted alpha-synuclein to be digested, producing a small fragment whose size was similar to that of the fragment generated during the incubation of alpha-synuclein with various MMPs in vitro. Among several forms of MMPs, alpha-synuclein was cleaved most efficiently by MMP-3, and MALDI-TOF mass spectra analysis showed that alpha-synuclein is cleaved from its C-terminal end with at least four cleavage sites within the non-Abeta component of AD amyloid sequence. Compared with the intact form, the protein aggregation of alpha-synuclein was remarkably facilitated in the presence of the proteolytic fragments, and the fragment-induced aggregates showed more toxic effect on cell viability. Moreover, the levels of MMP-3 were also found to be increased significantly in the rat PD brain model produced by the cerebral injection of 6-hydroxydopamine into the substantia nigra. The present study suggests that the extracellularly secreted alpha-synuclein could be processed via the activation of MMP-3 in a selective manner.

    The Journal of biological chemistry 2005;280;26;25216-24

  • A new method for purification of recombinant human alpha-synuclein in Escherichia coli.

    Huang C, Ren G, Zhou H and Wang CC

    The National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing.

    alpha-Synuclein (AS), a major component of Lewy body in Parkinson's disease patients, exists as a natively unfolded protein in physiological buffer. We recently found that the overexpressed AS in Escherichia coli bearing the cloned AS cDNA with no signal sequence was actually located inside the periplasm, but not in the cytoplasm as generally recognized. Therefore, a new protocol for preparing recombinant AS has been developed with only two steps: (1) osmotic shock for release of AS-containing periplasm fraction and (2) ion-exchange chromatography for further purification of AS. By using plasmids and E. coli strains commonly used the new protocol is much more convenient, faster, and cheaper compared to the current methods established since 1994. About 80 mg AS with 95% purity can be regularly prepared from a 1L culture in 3 days.

    Protein expression and purification 2005;42;1;173-7

  • The co-chaperone carboxyl terminus of Hsp70-interacting protein (CHIP) mediates alpha-synuclein degradation decisions between proteasomal and lysosomal pathways.

    Shin Y, Klucken J, Patterson C, Hyman BT and McLean PJ

    Alzheimer Disease Research Unit, Department of Neurology, Massachusetts General Hospital, Charlestown, Massachusetts 02129, USA.

    Alpha-synuclein is a major component of Lewy bodies, the pathological hallmark of Parkinson disease, dementia with Lewy bodies, and related disorders. Misfolding and aggregation of alpha-synuclein is thought to be a critical cofactor in the pathogenesis of certain neurodegenerative diseases. In the current study, we investigate the role of the carboxyl terminus of Hsp70-interacting protein (CHIP) in alpha-synuclein aggregation. We demonstrate that CHIP is a component of Lewy bodies in the human brain, where it colocalizes with alpha-synuclein and Hsp70. In a cell culture model, endogenous CHIP colocalizes with alpha-synuclein and Hsp70 in intracellular inclusions, and overexpression of CHIP inhibits alpha-synuclein inclusion formation and reduces alpha-synuclein protein levels. We demonstrate that CHIP can mediate alpha-synuclein degradation by two discrete mechanisms that can be dissected using deletion mutants; the tetratricopeptide repeat domain is critical for proteasomal degradation, whereas the U-box domain is sufficient to direct alpha-synuclein toward the lysosomal degradation pathway. Furthermore, alpha-synuclein, synphilin-1, and Hsp70 all coimmunoprecipitate with CHIP, raising the possibility of a direct alpha-synuclein-CHIP interaction. The fact that the tetratricopeptide repeat domain is required for the effects of CHIP on alpha-synuclein inclusion morphology, number of inclusions, and proteasomal degradation as well as the direct interaction of CHIP with Hsp70 implicates a cooperation of CHIP and Hsp70 in these processes. Taken together, these data suggest that CHIP acts a molecular switch between proteasomal and lysosomal degradation pathways.

    Funded by: NIA NIH HHS: AG05134-20; NIMH NIH HHS: 1R24MH68855; NINDS NIH HHS: NS 38372

    The Journal of biological chemistry 2005;280;25;23727-34

  • Common anti-apoptotic roles of parkin and alpha-synuclein in human dopaminergic cells.

    Machida Y, Chiba T, Takayanagi A, Tanaka Y, Asanuma M, Ogawa N, Koyama A, Iwatsubo T, Ito S, Jansen PH, Shimizu N, Tanaka K, Mizuno Y and Hattori N

    Department of Neurology, Juntendo University School of Medicine, Tokyo, Japan.

    Parkin, a product of the gene responsible for autosomal recessive juvenile parkinsonism (AR-JP), is an important player in the pathogenic process of Parkinson's disease (PD). Despite numerous studies including search for the substrate of parkin as an E3 ubiquitin-protein ligase, the mechanism by which loss-of-function of parkin induces selective dopaminergic neuronal death remains unclear. Related to this issue, here we show that antisense knockdown of parkin causes apoptotic cell death of human dopaminergic SH-SY5Y cells associated with caspase activation and accompanied by accumulation of oxidative dopamine (DA) metabolites due to auto-oxidation of DOPA and DA. Forced expression of alpha-synuclein (alpha-SN), another familial PD gene product, prevented accumulation of oxidative DOPA/DA metabolites and cell death caused by parkin loss. Our findings indicate that both parkin and alpha-SN share a common pathway in DA metabolism whose abnormality leads to accumulation of oxidative DA metabolites and subsequent cell death.

    Biochemical and biophysical research communications 2005;332;1;233-40

  • Intravesicular localization and exocytosis of alpha-synuclein and its aggregates.

    Lee HJ, Patel S and Lee SJ

    The Parkinson's Institute, Sunnyvale, California 94089, USA.

    Alpha-synuclein (alpha-syn), particularly in its aggregated forms, is implicated in the pathogenesis of Parkinson's disease and other related neurological disorders. However, the normal biology of alpha-syn and how it relates to the aggregation of the protein are not clearly understood. Because of the lack of the signal sequence and its predominant localization in the cytosol, alpha-syn is generally considered exclusively an intracellular protein. Contrary to this assumption, here, we show that a small percentage of newly synthesized alpha-syn is rapidly secreted from cells via unconventional, endoplasmic reticulum/Golgi-independent exocytosis. Consistent with this finding, we also demonstrate that a portion of cellular alpha-syn is present in the lumen of vesicles. Importantly, the intravesicular alpha-syn is more prone to aggregation than the cytosolic protein, and aggregated forms of alpha-syn are also secreted from cells. Furthermore, secretion of both monomeric and aggregated alpha-syn is elevated in response to proteasomal and mitochondrial dysfunction, cellular defects that are associated with Parkinson's pathogenesis. Thus, intravesicular localization and secretion are part of normal life cycle of alpha-syn and might also contribute to pathological function of this protein.

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2005;25;25;6016-24

  • A precipitating role for truncated alpha-synuclein and the proteasome in alpha-synuclein aggregation: implications for pathogenesis of Parkinson disease.

    Liu CW, Giasson BI, Lewis KA, Lee VM, Demartino GN and Thomas PJ

    Department of Physiology, University of Texas Southwestern Medical Center at Dallas, 75390, USA.

    Parkinson disease and other alpha-synucleinopathies are characterized by the deposition of intraneuronal alpha-synuclein (alphaSyn) inclusions. A significant fraction (about 15%) of alphaSyn in these pathological structures are truncated forms that have a much higher propensity than the full-length alphaSyn to form aggregates in vitro. However, little is known about the role of truncated alphaSyn species in pathogenesis or the means by which they are generated. Here, we have provided an in vitro mechanistic study demonstrating that truncated alphaSyns induce rapid aggregation of full-length protein at substoichiometric ratios. Co-overexpression of truncated alphaSyn with full-length protein increases cell vulnerability to oxidative stress in dopaminergic SH-SY5Y cells. These results suggest a precipitating role for truncated alphaSyn in the pathogenesis of diseases involving alphaSyn aggregation. In this regard, the A53T mutation found in some cases of familial Parkinson disease exacerbates the accumulation of insoluble alphaSyns that correlates with the onset of pathology in transgenic mice expressing human alphaSyn-A53T mutant. The caspase-like activity of the 20 S proteasome produces truncated fragments similar to those found in patients and animal models from degradation of unstructured alphaSyn. We propose a model in which incomplete degradation of alphaSyn, especially under overloaded proteasome capacity, produces highly amyloidogenic fragments that rapidly induce the aggregation of full-length protein. These aggregates in turn reduce proteasome activity, leading to further accumulation of fragmented and full-length alphaSyns, creating a vicious cycle of cytotoxicity. This model has parallels in other neurodegenerative diseases, such as Huntington disease, where coaggregation of poly(Q) fragments with full-length protein has been observed.

    Funded by: NIDDK NIH HHS: DK 49835, DK46818

    The Journal of biological chemistry 2005;280;24;22670-8

  • Alpha-synuclein phosphorylation enhances eosinophilic cytoplasmic inclusion formation in SH-SY5Y cells.

    Smith WW, Margolis RL, Li X, Troncoso JC, Lee MK, Dawson VL, Dawson TM, Iwatsubo T and Ross CA

    Department of Psychiatry, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.

    Parkinson's disease (PD) is a neurodegenerative disorder characterized by selective loss of dopaminergic neurons and the presence of Lewy bodies. Previous reports have shown that alpha-synuclein deposited in brain tissue from individuals with synucleinopathy is extensively phosphorylated at Ser-129. Here, we investigate the role of phosphorylation of alpha-synuclein in the formation of inclusions involving synphilin-1 and parkin using site-directed mutagenesis to change Ser-129 of alpha-synuclein to alanine (S129A) to abolish phosphorylation at this site. Coexpression of wild-type alpha-synuclein and synphilin-1 in human neuroblastoma SH-SY5Y cells yielded cytoplasmic eosinophilic inclusions with some features resembling Lewy bodies, whereas coexpression of S129A alpha-synuclein and synphlin-1 formed few or no inclusions. Moreover, coexpression of parkin with alpha-synuclein and synphilin-1 formed more ubiquitinated inclusions, but these inclusions decreased with expression of S129A alpha-synuclein instead of wild-type alpha-synuclein. Coimmunoprecipitation assays revealed a decreased interaction of S129A alpha-synuclein with synphilin-1 compared with wild-type alpha-synuclein. Expression of S129A alpha-synuclein instead of wild-type alpha-synuclein also decreased the association of synphilin-1 and parkin and subsequently reduced the parkin-mediated ubiquitination of synphilin-1 and the formation of ubiquitinated inclusions. Treatment of SH-SY5Y cells with H(2)O(2) increased alpha-synuclein phosphorylation and enhanced the formation of inclusions formed by coexpression of alpha-synuclein, synphilin-1, and parkin, whereas treatment with the casein kinase 2 inhibitor 5,6-dichloro-1-beta-d-ribofuranosylbenzimidazole had the opposite affect. These results indicate that phosphorylation of alpha-synuclein at S129 may be important for the formation of inclusions in PD and related alpha synucleinopathies.

    Funded by: NINDS NIH HHS: NS38377

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2005;25;23;5544-52

  • Reversible inhibition of alpha-synuclein fibrillization by dopaminochrome-mediated conformational alterations.

    Norris EH, Giasson BI, Hodara R, Xu S, Trojanowski JQ, Ischiropoulos H and Lee VM

    Center for Neurodegenerative Disease Research, Department of Pathology and Laboratory Medicine, Institute on Aging, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.

    Previous studies demonstrated that alpha-synuclein (alpha-syn) fibrillization is inhibited by dopamine, and studies to understand the molecular basis of this process were conducted (Conway, K. A., Rochet, J. C., Bieganski, R. M., and Lansbury, P. T., Jr. (2001) Science 294, 1346-1349). Dopamine inhibition of alpha-syn fibrillization generated exclusively spherical oligomers that depended on dopamine autoxidation but not alpha-syn oxidation, because mutagenesis of Met, His, and Tyr residues in alpha-syn did not abrogate this inhibition. However, truncation of alpha-syn at residue 125 restored the ability of alpha-syn to fibrillize in the presence of dopamine. Mutagenesis and competition studies with specific synthetic peptides identified alpha-syn residues 125-129 (i.e. YEMPS) as an important region in the dopamine-induced inhibition of alpha-syn fibrillization. Significantly, the dopamine oxidation product dopaminochrome was identified as a specific inhibitor of alpha-syn fibrillization. Dopaminochrome promotes the formation of spherical oligomers by inducing conformational changes, as these oligomers regained the ability to fibrillize by simple denaturation/renaturation. Taken together, these data indicate that dopamine inhibits alpha-syn fibrillization by inducing structural changes in alpha-syn that can occur through the interaction of dopaminochrome with the 125YEMPS129 motif of alpha-syn. These results suggest that the dopamine autoxidation can prevent alpha-syn fibrillization in dopaminergic neurons through a novel mechanism. Thus, decreased dopamine levels in substantia nigra neurons might promote alpha-syn aggregation in Parkinson's disease.

    Funded by: NIA NIH HHS: AG09215; NINDS NIH HHS: NS044233

    The Journal of biological chemistry 2005;280;22;21212-9

  • Alpha-synuclein overexpression in oligodendrocytic cells results in impaired adhesion to fibronectin and cell death.

    Tsuboi K, Grzesiak JJ, Bouvet M, Hashimoto M, Masliah E and Shults CW

    Department of Neurosciences, University of California, San Diego, La Jolla, CA 92093, USA. ktsuboi@vapop.ucsd.edu

    The cardinal pathological features of multiple system atrophy (MSA) are the presence of glial cytoplasmic inclusions (GCIs) in oligodendrocytes and loss of oligodendrocytes. To understand the mechanisms underlying MSA, we examined the effects of overexpression of human alpha-synuclein (halpha-syn) in CG-4 oligodendrocytic progenitor cells. CG-4 cells overexpressing halpha-syn (halpha-syn CG-4) demonstrated severely impaired adhesion and increased cell death when plated on fibronectin compared to laminin. The expression of the alphav integrin subunit in whole cell lysates was also significantly downregulated in halpha-syn CG-4. These results demonstrate a cytotoxic consequence of halpha-syn overexpression in CG-4. This cytotoxicity appears to be the result of alterations in cell-extracellular matrix interactions, where impaired adhesion to fibronectin is associated with downregulation of the alphav integrin subunit and increased cell death. It may, therefore, be one of the mechanisms underlying the loss of oligodendrocytes in MSA.

    Funded by: NIA NIH HHS: AG18440; NINDS NIH HHS: P01 NS44233

    Molecular and cellular neurosciences 2005;29;2;259-68

  • Antiapoptotic property of human alpha-synuclein in neuronal cell lines is associated with the inhibition of caspase-3 but not caspase-9 activity.

    Li W and Lee MK

    Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.

    Abnormalities of alpha-synuclein (alpha-Syn) are mechanistically linked to Parkinson's disease (PD) and other alpha-synucleinopathies. To gain additional insights into the relationships between alpha-Syn expression and cell death, we examined the effects of expressing human alpha-Syn (Hualpha-Syn) variants on the cellular vulnerability to apoptotic stimuli. We show that the expression of wild-type (WT) and A30P mutant, but not A53T mutant, Hualpha-Syn leads to the protection of neuronal cell lines from apoptosis but not necrosis. Significantly, Hualpha-Syn did not protect non-neuronal cell lines from apoptosis. We also show that A53T mutant is a loss of function in regards to the antiapoptotic property since the expression of WT Hualpha-Syn with an excess of A53T mutant Hualpha-Syn leads to protection of the cells from apoptosis. The antiapoptotic property is specific to human alpha-Syn as neither beta-Syn nor mouse alpha-Syn protected cells from apoptosis, and the carboxy-terminal 20 amino acids are required for the antiapoptotic property. Analyses of capase-3 and caspase-9 activation reveal that the antiapoptotic property of Hualpha-Syn in neuronal cell lines is associated with the attenuation of caspase-3 activity without affecting the caspase-9 activity or the levels of cleaved, active caspase-3. We conclude that Hualpha-Syn modulates the activity of cleaved caspase-3 product in neuronal cell lines.

    Funded by: NINDS NIH HHS: NS38065, NS38377

    Journal of neurochemistry 2005;93;6;1542-50

  • DJ-1 is present in a large molecular complex in human brain tissue and interacts with alpha-synuclein.

    Meulener MC, Graves CL, Sampathu DM, Armstrong-Gold CE, Bonini NM and Giasson BI

    Department of Biology and Laboratory of Medicine of University of Pennsylvania, Philadelphia 19104-6084, USA.

    DJ-1 is a ubiquitously expressed protein involved in various cellular processes including cell proliferation, RNA-binding, and oxidative stress. Mutations that result in loss of DJ-1 function lead to early onset parkinsonism in humans, and DJ-1 protein is present in pathological lesions of several tauopathies and synucleinopathies. In order to further investigate the role of DJ-1 in human neurodegenerative disease, we have generated novel polyclonal and monoclonal antibodies to human DJ-1 protein. We have characterized these antibodies and confirmed the pathological co-localization of DJ-1 with other neurodegenerative disease-associated proteins, as well as the decrease in DJ-1 solubility in disease tissue. In addition, we report the presence of DJ-1 in a large molecular complex (> 2000 kDa), and provide evidence for an interaction between endogenous DJ-1 and alpha-synuclein in normal and diseased tissue. These findings provide new avenues towards the study of DJ-1 function and how loss of its activity may lead to parkinsonism. Furthermore, our results provide further evidence for the interplay between neurodegenerative disease-associated proteins.

    Journal of neurochemistry 2005;93;6;1524-32

  • Alpha-synuclein and parkin contribute to the assembly of ubiquitin lysine 63-linked multiubiquitin chains.

    Doss-Pepe EW, Chen L and Madura K

    Department of Biochemistry, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854, USA. dosspeew@umdnj.edu

    Mutations in alpha-synuclein, Parkin, and UCH-L1 cause heritable forms of Parkinson disease. Unlike alpha-synuclein, for which no precise biochemical function has been elucidated, Parkin functions as a ubiquitin E3 ligase, and UCH-L1 is a deubiquitinating enzyme. The E3 ligase activity of Parkin in Parkinson disease is poorly understood and is further obscured by the fact that multiubiquitin chains can be formed through distinct types of linkages that regulate diverse cellular processes. For instance, ubiquitin lysine 48-linked multiubiquitin chains target substrates to the proteasome, whereas ubiquitin lysine 63-linked chains control ribosome function, protein sorting and trafficking, and endocytosis of membrane proteins. It is notable in this regard that ubiquitin lysine 63-linked chains promote the degradation of membrane proteins by the lysosome. Because both Parkin and alpha-synuclein can regulate the activity of the dopamine transporter, we investigated whether they influenced ubiquitin lysine 63-linked chain assembly. These studies revealed novel biochemical activities for both Parkin and alpha-synuclein. We determined that Parkin functions with UbcH13/Uev1a, a dimeric ubiquitin-conjugating enzyme, to assemble ubiquitin lysine 63-linked chains. Our results and the results of others indicate that Parkin can promote both lysine 48- and lysine 63-linked ubiquitin chains. alpha-Synuclein also stimulated the assembly of lysine 63-linked ubiquitin chains. Because UCH-L1, a ubiquitin hydrolase, was recently reported to form lysine 63-linked conjugates, it is evident that three proteins that are genetically linked to Parkinson disease can contribute to lysine 63 multiubiquitin chain formation.

    Funded by: NCI NIH HHS: CA83875; NIA NIH HHS: AG01047; NINDS NIH HHS: NS044081

    The Journal of biological chemistry 2005;280;17;16619-24

  • Heat shock protein 70 inhibits alpha-synuclein fibril formation via preferential binding to prefibrillar species.

    Dedmon MM, Christodoulou J, Wilson MR and Dobson CM

    Department of Chemistry, University of Cambridge, Cambridge, CB2 1EW, United Kingdom.

    Parkinson's disease (PD) is a neurodegenerative disorder affecting an estimated 4 million people worldwide. Intracellular proteinaceous inclusions called Lewy bodies are the histological hallmarks of PD and are primarily composed of aggregated alpha-synuclein (alphaSyn). Although the detailed mechanisms remain unclear, mounting evidence suggests that the misfolding of alphaSyn into prefibrillar and fibrillar species is the driving force responsible for cellular toxicity. We show here that the molecular chaperone heat shock protein (Hsp) 70 strongly inhibits alphaSyn fibril formation via preferential binding to prefibrillar species. Moreover, our studies reveal that Hsp70 alters the characteristics of toxic alphaSyn aggregates and indicate that cellular toxicity arises from the prefibrillar forms of alphaSyn. This work therefore elucidates a specific role of Hsp70 in the pathogenesis of PD and supports the general concept that chaperone action is a crucial aspect in protecting against the otherwise damaging consequences of protein misfolding.

    The Journal of biological chemistry 2005;280;15;14733-40

  • Lysosomal pathology associated with alpha-synuclein accumulation in transgenic models using an eGFP fusion protein.

    Rockenstein E, Schwach G, Ingolic E, Adame A, Crews L, Mante M, Pfragner R, Schreiner E, Windisch M and Masliah E

    Department of Neurosciences, University of California San Diego, School of Medicine, La Jolla, California 92093-0624, USA.

    Disorders with Lewy body (LB) formation, such as Parkinson's disease (PD) and dementia with Lewy bodies (DLB), are characterized by alpha-synuclein accumulation in the neuronal cell body. Recent studies have suggested that in addition to LBs, alpha-synuclein might accumulate more widely throughout the neurons and their processes, leading to neurodegeneration and functional impairment. The precise patterns of alpha-synuclein accumulation in vivo, however, and its relationship with subcellular neuronal alterations such as lysosomal pathology are not completely clear. To this end, we developed transgenic (tg) in vivo and in vitro models expressing a stable enhanced green fluorescent protein (eGFP) tagged in the C-terminal site of a human (h)alpha-synuclein construct under the regulatory control of the platelet-derived growth factor-beta (PDGFbeta) promoter and carried out confocal, ultrastructural, and biochemical studies. In tg mice, confocal studies demonstrated a wide distribution of halpha-synuclein-eGFP in the neuronal cell bodies, axons, and presynaptic terminals. In several neuronal cell bodies and their neurites, halpha-synuclein-eGFP was found not only as inclusions but also as discrete granular structures that in double-labeling studies colocalized with antibodies against halpha-synuclein and the lysosomal marker cathepsin D. Consistent with these findings, ultrastructural analysis showed that halpha-synuclein-eGFP overexpression resulted in the accumulation of electrodense inclusions and laminated bodies suggestive of lysosomal pathology, and that the halpha-synuclein-eGFP protein was more abundant in the lysosomal fractions of the tg animals. Taken together, these findings support the notion that enhanced visualization of alpha-synuclein utilizing a hybrid eGFP molecule reveals a more widespread accumulation of this molecule in several neuronal compartments, promoting lysosomal dysfunction. Furthermore, the PDGFbeta-halpha-synuclein-eGFP tg model might be a valuable tool in testing new treatments for LBD in a fast and reliable manner.

    Funded by: NIA NIH HHS: AG022074, AG18440, AG5131

    Journal of neuroscience research 2005;80;2;247-59

  • Joint analysis of the NACP-REP1 marker within the alpha synuclein gene concludes association with alcohol dependence.

    Bönsch D, Lederer T, Reulbach U, Hothorn T, Kornhuber J and Bleich S

    Department of Psychiatry and Psychotherapy, Friedrich-Alexander-University of Erlangen-Nuremburg, 91054 Erlangen, Germany.

    Various studies have linked alcohol dependence phenotypes to chromosome 4. One candidate gene is NACP (non-amyloid component of plaques), coding for alpha synuclein. Recently, it has been shown that alpha synuclein mRNA is increased in alcohol-dependent patients within withdrawal state. This increase is significantly associated with craving, especially obsessive craving. On the basis of these observations, the present study analysed two polymorphic repeats within the NACP gene. We found highly significant longer alleles of NACP-REP1 in alcohol-dependent patients compared with healthy controls (Kruskal-Wallis test, chi(2)=99.5; df=3, P<0.001). In addition, these lengths significantly correlate with levels of expressed alpha synuclein mRNA (chi(2)=8.83; df=2, P=0.012). The present results point to a novel approach for a genetic determination of craving, a key factor in the genesis and maintenance not only of alcoholism but also of addiction in general.

    Human molecular genetics 2005;14;7;967-71

  • Aggregated alpha-synuclein activates microglia: a process leading to disease progression in Parkinson's disease.

    Zhang W, Wang T, Pei Z, Miller DS, Wu X, Block ML, Wilson B, Zhang W, Zhou Y, Hong JS and Zhang J

    Neuropharmacology Section, Laboratory of Pharmacology and Chemistry, National Institute of Environmental Health Sciences/National Institutes of Health, Research Triangle Park, North Carolina, USA.

    A growing body of evidence indicates that an inflammatory process in the substantia nigra, characterized by activation of resident microglia, likely either initiates or aggravates nigral neurodegeneration in Parkinson's disease (PD). To study the mechanisms by which nigral microglia are activated in PD, the potential role of alpha-synuclein (a major component of Lewy bodies that can cause neurodegeneration when aggregated) in microglial activation was investigated. The results demonstrated that in a primary mesencephalic neuron-glia culture system, extracellular aggregated human alpha-synuclein indeed activated microglia; microglial activation enhanced dopaminergic neurodegeneration induced by aggregated alpha-synuclein. Furthermore, microglial enhancement of alpha-synuclein-mediated neurotoxicity depended on phagocytosis of alpha-synuclein and activation of NADPH oxidase with production of reactive oxygen species. These results suggest that nigral neuronal damage, regardless of etiology, may release aggregated alpha-synuclein into substantia nigra, which activates microglia with production of proinflammatory mediators, thereby leading to persistent and progressive nigral neurodegeneration in PD. Finally, NADPH oxidase could be an ideal target for potential pharmaceutical intervention, given that it plays a critical role in alpha-synuclein-mediated microglial activation and associated neurotoxicity.

    FASEB journal : official publication of the Federation of American Societies for Experimental Biology 2005;19;6;533-42

  • Mitochondrial associated metabolic proteins are selectively oxidized in A30P alpha-synuclein transgenic mice--a model of familial Parkinson's disease.

    Poon HF, Frasier M, Shreve N, Calabrese V, Wolozin B and Butterfield DA

    Department of Chemistry, University of Kentucky, Lexington, KY 40506-0055, USA.

    Parkinson's disease (PD) is the most common neurodegenerative movement disorder and is characterized by the loss of dopaminergic neurons in the substantia nigra compacta. alpha-Synuclein is strongly implicated in the pathophysiology of PD because aggregated alpha-synuclein accumulates in the brains of subjects with PD, mutations in alpha-synuclein cause familial PD, and overexpressing mutant human alpha-synuclein (A30P or A53T) causes degenerative disease in mice or drosophila. The pathophysiology of PD is poorly understood, but increasing evidence implicates mitochondrial dysfunction and oxidative stress. To understand how mutations in alpha-synuclein contribute to the pathophysiology of PD, we undertook a proteomic analysis of transgenic mice overexpressing A30P alpha-synuclein to investigate which proteins are oxidized. We observed more than twofold selective increases in specific carbonyl levels of three metabolic proteins in brains of symptomatic A30P alpha-synuclein mice: carbonic anhydrase 2 (Car2), alpha-enolase (Eno1), and lactate dehydrogenase 2 (Ldh2). Analysis of the activities of these proteins demonstrates decreased functions of these oxidatively modified proteins in brains from the A30P compared to control mice. Our findings suggest that proteins associated with impaired energy metabolism and mitochondria are particularly prone to oxidative stress associated with A30P-mutant alpha-synuclein.

    Funded by: NIA NIH HHS: AG-105119, AG-10836; NINDS NIH HHS: NS41786

    Neurobiology of disease 2005;18;3;492-8

  • Structural characterization of copper(II) binding to alpha-synuclein: Insights into the bioinorganic chemistry of Parkinson's disease.

    Rasia RM, Bertoncini CW, Marsh D, Hoyer W, Cherny D, Zweckstetter M, Griesinger C, Jovin TM and Fernández CO

    Department of Molecular Biology, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Göttingen, Germany.

    The aggregation of alpha-synuclein (AS) is characteristic of Parkinson's disease and other neurodegenerative synucleinopathies. We demonstrate here that Cu(II) ions are effective in accelerating AS aggregation at physiologically relevant concentrations without altering the resultant fibrillar structures. By using numerous spectroscopic techniques (absorption, CD, EPR, and NMR), we have located the primary binding for Cu(II) to a specific site in the N terminus, involving His-50 as the anchoring residue and other nitrogen/oxygen donor atoms in a square planar or distorted tetragonal geometry. The carboxylate-rich C terminus, originally thought to drive copper binding, is able to coordinate a second Cu(II) equivalent, albeit with a 300-fold reduced affinity. The NMR analysis of AS-Cu(II) complexes reveals the existence of conformational restrictions in the native state of the protein. The metallobiology of Cu(II) in Parkinson's disease is discussed by a comparative analysis with other Cu(II)-binding proteins involved in neurodegenerative disorders.

    Proceedings of the National Academy of Sciences of the United States of America 2005;102;12;4294-9

  • Metal-catalyzed oxidation of alpha-synuclein: helping to define the relationship between oligomers, protofibrils, and filaments.

    Cole NB, Murphy DD, Lebowitz J, Di Noto L, Levine RL and Nussbaum RL

    Genetic Diseases Research Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland 20892, USA. ncole@mail.nih.gov

    Oxidative stress is implicated in a number of neuro-degenerative diseases and is associated with the selective loss of dopaminergic neurons of the substantia nigra in Parkinson's disease. The role of alpha-synuclein as a potential target of intracellular oxidants has been demonstrated by the identification of posttranslational modifications of synuclein within intracellular aggregates that accumulate in Parkinson's disease brains, as well as the ability of a number of oxidative insults to induce synuclein oligomerization. The relationship between these relatively small soluble oligomers, potentially neurotoxic synuclein protofibrils, and synuclein filaments remains unclear. We have found that metal-catalyzed oxidation of alpha-synuclein inhibited formation of synuclein filaments with a concomitant accumulation of beta sheet-rich oligomers that may represent synuclein protofibrils. Similar results with a number of oxidative and enzymatic treatments suggest that the covalent association of synuclein into higher molecular mass oligomers/protofibrils represents an alternate pathway from filament formation and renders synuclein less prone to proteasomal degradation.

    The Journal of biological chemistry 2005;280;10;9678-90

  • Structure and dynamics of micelle-bound human alpha-synuclein.

    Ulmer TS, Bax A, Cole NB and Nussbaum RL

    Laboratory of Chemical Physics, NIDDK, National Institutes of Health, Bethesda, Maryland 20892 , USA. tobias.ulmer@nih.gov

    Misfolding of the protein alpha-synuclein (aS), which associates with presynaptic vesicles, has been implicated in the molecular chain of events leading to Parkinson's disease. Here, the structure and dynamics of micelle-bound aS are reported. Val3-Val37 and Lys45-Thr92 form curved alpha-helices, connected by a well ordered, extended linker in an unexpected anti-parallel arrangement, followed by another short extended region (Gly93-Lys97), overlapping the recently identified chaperone-mediated autophagy recognition motif and a highly mobile tail (Asp98-Ala140). Helix curvature is significantly less than predicted based on the native micelle shape, indicating a deformation of the micelle by aS. Structural and dynamic parameters show a reduced helical content for Ala30-Val37. A dynamic variation in interhelical distance on the microsecond timescale is complemented by enhanced sub-nanosecond timescale dynamics, particularly in the remarkably glycine-rich segments of the helices. These unusually rich dynamics may serve to mitigate the effect of aS binding on membrane fluidity. The well ordered conformation of the helix-helix connector indicates a defined interaction with lipidic surfaces, suggesting that, when bound to larger diameter synaptic vesicles, it can act as a switch between this structure and a previously proposed uninterrupted helix.

    The Journal of biological chemistry 2005;280;10;9595-603

  • The E46K mutation in alpha-synuclein increases amyloid fibril formation.

    Greenbaum EA, Graves CL, Mishizen-Eberz AJ, Lupoli MA, Lynch DR, Englander SW, Axelsen PH and Giasson BI

    Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA.

    The identification of a novel mutation (E46K) in one of the KTKEGV-type repeats in the amino-terminal region of alpha-synuclein suggests that this region and, more specifically, Glu residues in the repeats may be important in regulating the ability of alpha-synuclein to polymerize into amyloid fibrils. It was demonstrated that the E46K mutation increased the propensity of alpha-synuclein to fibrillize, but this effect was less than that of the A53T mutation. The substitution of Glu(46) for an Ala also increased the assembly of alpha-synuclein, but the polymers formed can have different ultrastructures, further indicating that this amino acid position has a significant effect on the assembly process. The effect of residue Glu(83) in the sixth repeat of alpha-synuclein, which lies closest to the amino acid stretch critical for filament assembly, was also studied. Mutation of Glu(83) to a Lys or Ala increased polymerization but perturbed some of the properties of mature amyloid. These results demonstrated that some of the Glu residues within the repeats can have significant effects on modulating the assembly of alpha-synuclein to form amyloid fibrils. The greater effect of the A53T mutation, even when compared with what may be predicted to be a more dramatic mutation such as E46K, underscores the importance of protein microenvironment in affecting protein structure. Moreover, the relative effects of the A53T and E46K mutations are consistent with the age of onset of disease. These findings support the notion that aberrant alpha-synuclein polymerization resulting in the formation of pathological inclusions can lead to disease.

    Funded by: NIA NIH HHS: AG20238; NINDS NIH HHS: NS45986

    The Journal of biological chemistry 2005;280;9;7800-7

  • beta-Synuclein reduces proteasomal inhibition by alpha-synuclein but not gamma-synuclein.

    Snyder H, Mensah K, Hsu C, Hashimoto M, Surgucheva IG, Festoff B, Surguchov A, Masliah E, Matouschek A and Wolozin B

    Department of Pharmacology, Loyola University Medical Center, Maywood, Illinois 60153, USA.

    The accumulation of aggregated alpha-synuclein is thought to contribute to the pathogenesis of Parkinson's disease. Recent studies indicate that aggregated alpha-synuclein binds to S6', a component of the 19 S subunit in the 26 S proteasome and inhibits 26 S proteasomal degradation, both ubiquitin-independent and ubiquitin-dependent. The IC(50) of aggregated alpha-synuclein for inhibition of the 26 S ubiquitin-independent proteasomal activity is approximately 1 nm. alpha-Synuclein has two close homologues, termed beta-synuclein and gamma-synuclein. In the present study we compared the effects of the three synuclein homologues on proteasomal activity. The proteasome exists as a 26 S and a 20 S species, with the 26 S proteasome containing the 20 S core and 19 S cap. Monomeric alpha- and beta-synucleins inhibited the 20 S and 26 S proteasomal activities only weakly, but monomeric gamma-synuclein strongly inhibited ubiquitin-independent proteolysis. The IC(50) of monomeric gamma-synuclein for the 20 S proteolysis was 400 nm. In monomeric form, none of the three synuclein proteins inhibited 26 S ubiquitin-dependent proteasomal activity. Although beta-synuclein had no direct effect on proteasomal activity, co-incubating monomeric beta-synuclein with aggregated alpha-synuclein antagonized the inhibition of the 26 S ubiquitin-independent proteasome by aggregated alpha-synuclein when added before the aggregated alpha-synuclein. Co-incubating beta-synuclein with gamma-synuclein had no effect on the inhibition of the 20 S proteasome by monomeric gamma-synuclein. Immunoprecipitation and pull-down experiments suggested that antagonism by beta-synuclein resulted from binding to alpha-synuclein rather than binding to S6'. Pull-down experiments demonstrated that recombinant monomeric beta-synuclein does not interact with the proteasomal subunit S6', unlike alpha-synuclein, but beta-synuclein does bind alpha-synuclein and competes with S6' for binding to alpha-synuclein. Based on these data, we hypothesize that the alpha- and gamma-synucleins regulate proteasomal function and that beta-synuclein acts as a negative regulator of alpha-synuclein.

    Funded by: NINDS NIH HHS: NS41786

    The Journal of biological chemistry 2005;280;9;7562-9

  • Interaction of alpha-synuclein and tau genotypes in Parkinson's disease.

    Mamah CE, Lesnick TG, Lincoln SJ, Strain KJ, de Andrade M, Bower JH, Ahlskog JE, Rocca WA, Farrer MJ and Maraganore DM

    Department of Neurology, Mayo Clinic College of Medicine, 200 First Street SW, Rochester, MN 55905, USA.

    To determine whether the microtubule-associated protein tau (MAPT) and alpha-synuclein (SNCA) genes interact to confer Parkinson's disease (PD) susceptibility, we conducted a study of 557 case-control pairs. There was an increased risk of PD for persons with either SNCA 261/261 or MAPT H1/H1 genotypes as compared with persons with neither (odds ratio, 1.96; 95% confidence interval, 1.34-2.86; p = 0.0003). However, the combined effect of the two genotypes was the same as for either of the genotypes alone (separate and equal). These findings are consistent with in vitro experiments that revealed tau-mediated fibrillization of alpha-synuclein protein at low concentrations (dose threshold effect).

    Funded by: NIEHS NIH HHS: R01 ES 10751; NINDS NIH HHS: P01 NS 40256, R01 NS 33978

    Annals of neurology 2005;57;3;439-43

  • Regulation of alpha-synuclein expression by poly (ADP ribose) polymerase-1 (PARP-1) binding to the NACP-Rep1 polymorphic site upstream of the SNCA gene.

    Chiba-Falek O, Kowalak JA, Smulson ME and Nussbaum RL

    Genetic Disease Research Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892, USA.

    Alleles at NACP-Rep1, the polymorphic microsatellite repeat located approximately 10 kb upstream of the alpha -synuclein gene (SNCA), are associated, in some reports, with differing risks of sporadic Parkinson disease (PD). We showed previously that NACP-Rep1 acts as a negative modulator of SNCA transcription, with an effect that varied threefold among different NACP-Rep1 alleles. Given that duplications and triplications of SNCA have been implicated in familial Parkinson disease (PD), even a 1.5-2-fold increase in alpha -synuclein expression may, over many decades, contribute to PD. Thus, the association of different NACP-Rep1 alleles with PD may be a consequence of polymorphic differences in transcriptional regulation of SNCA. Here we aimed to identify the factor(s) that bind to NACP-Rep1 and potentially contribute to SNCA transcriptional modulation, by pulling down proteins that bind to NACP-Rep1 and identifying them by mass spectrometry. One of these proteins was poly-(ADP-ribose) transferase/polymerase-1 (PARP-1), a DNA-binding protein and transcriptional regulator. Electrophoresis mobility shift and chromatin immunoprecipitation assays showed specific binding of PARP-1 to NACP-Rep1. Inhibition of PARP-1's catalytic domain increased the endogenous SNCA mRNA levels in cultured SH-SY5Y cells. Furthermore, PARP-1 binding to NACP-Rep1 specifically reduced the transcriptional activity of the SNCA promoter/enhancer in luciferase reporter assays. This down-regulation effect of PARP-1 depended on NACP-Rep1 being present in the construct and was abrogated by inhibiting PARP-1's catalytic activity with 3-aminobenzamide. The association of different NACP-Rep1 alleles with PD may be mediated, in part, by the effect of PARP-1, as well as other factors, on SNCA expression.

    Funded by: NCI NIH HHS: P01CA-74175

    American journal of human genetics 2005;76;3;478-92

  • Australian data and meta-analysis lend support for alpha-synuclein (NACP-Rep1) as a risk factor for Parkinson's disease.

    Mellick GD, Maraganore DM and Silburn PA

    Department of Neurology, School of Medicine, University of Queensland, Princess Alexandra Hospital, Brisbane, Australia. gmellick@soms.uq.edu.au

    It remains unclear whether genetic variants in SNCA (the alpha-synuclein gene) alter risk for sporadic Parkinson's disease (PD). The polymorphic mixed sequence repeat (NACP-Rep1) in the promoter region of SNCA has been previously examined as a potential susceptibility factor for PD with conflicting results. We report genotype and allele distributions at this locus from 369 PD cases and 370 control subjects of European Australian ancestry, with alleles designated as -1, 0, +1, +2, and +3 as previously described. Allele frequencies designated (0) were less common in Australian cases compared to controls (OR=0.80, 95% CI 0.62-1.03). Combined analysis including all previously published ancestral European Rep1 data yielded a highly significant association between the 0 allele and a reduced risk for PD (OR=0.79, 95% CI 0.70-0.89, p=0.0001). Further study must now proceed to examine in detail this interesting and biologically plausible genetic association.

    Neuroscience letters 2005;375;2;112-6

  • p25alpha Stimulates alpha-synuclein aggregation and is co-localized with aggregated alpha-synuclein in alpha-synucleinopathies.

    Lindersson E, Lundvig D, Petersen C, Madsen P, Nyengaard JR, Højrup P, Moos T, Otzen D, Gai WP, Blumbergs PC and Jensen PH

    Institute of Medical Biochemistry and Stereological Research and Electron Microscopy Laboratory, University of Aarhus, Aarhus, DK-8000, Denmark.

    Aggregation of the nerve cell protein alpha-synuclein is a characteristic of the common neurodegenerative alpha-synucleinopathies like Parkinson's disease and Lewy body dementia, and it plays a direct pathogenic role as demonstrated by early onset diseases caused by mis-sense mutations and multiplication of the alpha-synuclein gene. We investigated the existence of alpha-synuclein pro-aggregatory brain proteins whose dysregulation may contribute to disease progression, and we identified the brain-specific p25alpha as a candidate that preferentially binds to alpha-synuclein in its aggregated state. Functionally, purified recombinant human p25alpha strongly stimulates the aggregation of alpha-synuclein in vitro as demonstrated by thioflavin-T fluorescence and quantitative electron microscopy. p25alpha is normally only expressed in oligodendrocytes in contrast to alpha-synuclein, which is normally only expressed in neurons. This expression pattern is changed in alpha-synucleinopathies. In multiple systems atrophy, degenerating oligodendrocytes displayed accumulation of p25alpha and dystopically expressed alpha-synuclein in the glial cytoplasmic inclusions. In Parkinson's disease and Lewy body dementia, p25alpha was detectable in the neuronal Lewy body inclusions along with alpha-synuclein. The localization in alpha-synuclein-containing inclusions was verified biochemically by immunological detection in Lewy body inclusions purified from Lewy body dementia tissue and glial cytoplasmic inclusions purified from tissue from multiple systems atrophy. We suggest that p25alpha plays a pro-aggregatory role in the common neurodegenerative disorders hall-marked by alpha-synuclein aggregates.

    The Journal of biological chemistry 2005;280;7;5703-15

  • Aggregation promoting C-terminal truncation of alpha-synuclein is a normal cellular process and is enhanced by the familial Parkinson's disease-linked mutations.

    Li W, West N, Colla E, Pletnikova O, Troncoso JC, Marsh L, Dawson TM, Jäkälä P, Hartmann T, Price DL and Lee MK

    Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.

    Abnormal biology of alpha-synuclein (alpha-Syn) is directly implicated in the pathogenesis of Parkinson's disease and other alpha-synucleinopathies. Herein, we demonstrate that C-terminally truncated alpha-Syn (alpha-SynDeltaC), enriched in the pathological alpha-Syn aggregates, is normally generated from full-length alpha-Syn independent of alpha-Syn aggregation in brains and in cultured cells. The accumulation of alpha-SynDeltaC is enhanced in neuronal cells as compared with nonneuronal cells. Significantly, the expression of familial Parkinson's disease-linked mutant alpha-Syn is associated with the enhanced cellular accumulation of alpha-SynDeltaC. Moreover, substoichiometric amounts of alpha-SynDeltaC enhance the in vitro aggregation of the more abundant full-length alpha-Syn. Finally, cases of alpha-synucleinopathy exhibit increases in the total soluble alpha-Syn and a higher proportion of soluble alpha-SynDeltaC, a condition favoring the aggregation of alpha-Syn. Collectively, our results indicate that the biology behind the generation and accumulation of alpha-SynDeltaC is likely to have relevance for the initiation and the progression of alpha-Syn aggregation in vivo.

    Funded by: NINDS NIH HHS: NS38065, NS38377

    Proceedings of the National Academy of Sciences of the United States of America 2005;102;6;2162-7

  • DNA hypermethylation of the alpha synuclein promoter in patients with alcoholism.

    Bönsch D, Lenz B, Kornhuber J and Bleich S

    Department of Psychiatry and Psychotherapy, Friedrich-Alexander-University of Erlangen-Nuremberg, Schwabachanlage 6-10, D-91054 Erlangen, Germany.

    The aim of this study was to investigate whether the DNA methylation pattern within the alpha synuclein promoter region is altered in intoxicated and early abstinence patients with alcoholism undergoing alcohol withdrawal. We observed a significant increase of the alpha synuclein promoter DNA methylation in patients with alcoholism which was significantly associated with their elevated homocysteine levels. No significant differences of the promoter DNA methylation within a control gene (presenilin-1) in alcoholics and controls were found. The present results hint to a gene specific DNA promoter hypermethylation within the alpha synuclein gene. Since hypermethylation of DNA is an important epigenetic factor in the down regulation of gene expression and since alpha synuclein has been linked to craving these findings may explain the reduced value of craving under alcohol drinking conditions.

    Neuroreport 2005;16;2;167-70

  • Release of long-range tertiary interactions potentiates aggregation of natively unstructured alpha-synuclein.

    Bertoncini CW, Jung YS, Fernandez CO, Hoyer W, Griesinger C, Jovin TM and Zweckstetter M

    Department of Molecular Biology, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, D-37077 Göttingen, Germany.

    In idiopathic Parkinson's disease, intracytoplasmic neuronal inclusions (Lewy bodies) containing aggregates of the protein alpha-synuclein (alphaS) are deposited in the pigmented nuclei of the brainstem. The mechanisms underlying the structural transition of innocuous, presumably natively unfolded, alphaS to neurotoxic forms are largely unknown. Using paramagnetic relaxation enhancement and NMR dipolar couplings, we show that monomeric alphaS assumes conformations that are stabilized by long-range interactions and act to inhibit oligomerization and aggregation. The autoinhibitory conformations fluctuate in the range of nanoseconds to micro-seconds corresponding to the time scale of secondary structure formation during folding. Polyamine binding and/or temperature increase, conditions that induce aggregation in vitro, release this inherent tertiary structure, leading to a completely unfolded conformation that associates readily. Stabilization of the native, autoinhibitory structure of alphaS constitutes a potential strategy for reducing or inhibiting oligomerization and aggregation in Parkinson's disease.

    Proceedings of the National Academy of Sciences of the United States of America 2005;102;5;1430-5

  • Structural evidence for alpha-synuclein fibrils using in situ atomic force microscopy.

    Zhang F, Ji LN, Tang L, Hu J, Hu HY, Xu HJ and He JH

    Shanghai Institute of Applied Physics, Chinese Academy of Sciences, Shanghai 201800, China.

    Human alpha-synuclein is a presynaptic terminal protein and can form insoluble fibrils that are believed to play an important role in the pathogenesis of several neurodegenerative diseases such as Parkinson's disease, dementia with Lewy bodies and Lewy body variant of Alzheimer's disease. In this paper, in situ atomic force microscopy has been used to study the structural properties of alpha-synuclein fibrils in solution using two different atomic force microscopy imaging modes: tapping mode and contact mode. In the in situ contact mode atomic force microscopy experiments alpha-synuclein fibrils quickly broke into fragments, and a similar phenomenon was found using tapping mode atomic force microscopy in which alpha-synuclein fibrils were incubated with guanidine hydrochloride (0.6 M). The alpha-synuclein fibrils kept their original filamentous topography for over 1 h in the in situ tapping mode atomic force microscopy experiments. The present results provide indirect evidence on how beta-sheets assemble into alpha-synuclein fibrils on a nanometer scale.

    Acta biochimica et biophysica Sinica 2005;37;2;113-8

  • Fluorescence studies suggest a role for alpha-synuclein in the phosphatidylinositol lipid signaling pathway.

    Narayanan V, Guo Y and Scarlata S

    Department of Physiology and Biophysics, State University of New York, Stony Brook, New York 11794-8661, USA.

    Alpha-synuclein plays a key role in the pathogenesis of many neurodegenerative diseases. To date, its cellular role has yet to be determined, although it has been proposed to be connected to calcium and G protein-mediated dopamine signaling. Alpha-synuclein is known to bind strongly to model membrane surfaces where it may interact with other membrane-associated proteins. Here, we find that the membrane association of alpha-synuclein is enhanced by the presence of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P(2)] and Ca(2+). We also find that alpha-synuclein interacts with high affinity with the G protein-regulated enzyme phospholipase Cbeta(2) (PLCbeta(2)), which catalyzes the hydrolysis of PI(4,5)P(2). Binding of alpha-synuclein to PLCbeta(2) reduces its catalytic activity by 50%, but causes its level of activation by Gbetagamma subunits to increase from 4- to 24-fold. This effect is greatly reduced for A53T alpha-synuclein, which is a mutant associated with familial Parkinson's disease. PI(4,5)P(2) hydrolysis by PLCbeta(2) results in an increase in the intracellular Ca(2+) concentration, and we find that in cultured cells the presence of alpha-synuclein results in a 6-fold enhancement in the release of Ca(2+) from intracellular stores in response to agents that release Gbetagamma subunits relative to controls. Alpha-synuclein also enhances the increase in the level of inositol phosphates seen upon G protein stimulation, suggesting that it also may interact with PLCbeta(2) in cells. Given that Ca(2+) and dopamine regulation are mediated through PLCbeta and G protein signals, our results suggest that alpha-synuclein may play a role in inositol phospholipid signaling.

    Funded by: NIGMS NIH HHS: GM53132

    Biochemistry 2005;44;2;462-70

  • Characterization of the Alzheimer's disease-associated CLAC protein and identification of an amyloid beta-peptide-binding site.

    Söderberg L, Kakuyama H, Möller A, Ito A, Winblad B, Tjernberg LO and Näslund J

    Karolinska Institutet and Sumitomo Pharmaceuticals Alzheimer Center, Neurotec, Novum, SE-141 57 Huddinge, Sweden. linda.soderberg@neurotec.ki.se

    Amyloid beta-peptide (Abeta) deposition into amyloid plaques is one of the invariant neuropathological features of Alzheimer's disease. Other proteins co-deposit with Abeta in plaques, and one recently identified amyloid-associated protein is the collagen-like Alzheimer amyloid plaque component CLAC. It is not known how CLAC deposition affects Abeta plaque genesis and the progress of the disease. Here, we studied the in vitro properties of CLAC purified from a mammalian expression system. CLAC displays features characteristic of a collagen protein, e.g. it forms a partly protease-resistant triple-helical structure, exhibits an intermediate affinity for heparin, and is glycosylated. Purified CLAC was also used to investigate the interaction between CLAC and Abeta. Using a solid-phase binding assay, we show that CLAC bound with a similar affinity to aggregates formed by Abeta-(1-40) and Abeta-(1-42) and that the interaction was impaired by increasing salt concentrations. An 8-residue-long sequence located in non-collagenous domain 2 of CLAC was found to be crucial for the interaction with Abeta. These findings may be useful for future therapeutic interventions aimed at finding compounds that modulate the binding of CLAC to Abeta deposits.

    The Journal of biological chemistry 2005;280;2;1007-15

  • A topological model of the interaction between alpha-synuclein and sodium dodecyl sulfate micelles.

    Bisaglia M, Tessari I, Pinato L, Bellanda M, Giraudo S, Fasano M, Bergantino E, Bubacco L and Mammi S

    Department of Chemical Sciences, University of Padova, Via F. Marzolo 1, 35131 Padova, Italy.

    Human alpha-synuclein is a 140-amino acid protein of unknown function abundantly expressed in the brain and found in Lewy bodies, a characteristic feature of Parkinson's disease. Alpha-synuclein is random in water under physiological conditions, but the first approximately 100 residues interact with SDS micelles or acidic phospholipid small unilamellar vesicles and adopt an ordered conformation. The rest of the molecule remains disordered in the bulk of the solution. The conformation of the N-terminal portion of the molecule in lipids was described as an extended helix [Ramakrishnan, M., Jensen, P. H., and Marsh, D. (2003) Biochemistry 42, 12919-12926], as two distinct alpha-helices interrupted by a two-residue break [Chandra, S., Chen, X., Rizo, J., Jahn, R., and Sudhof, T. C. (2003) J. Biol. Chem. 278, 15313-15318], or as a noncanonical conformation, the alpha11/3 helix [Bussell, R., Jr., and Eliezer, D. (2003) J. Mol. Biol. 329, 763-778]. We characterized the topology of the different regions of alpha-synuclein relative to the surface of SDS micelles using spin probe-induced broadening of NMR signals, (15)N relaxation measurements, and fluorescence spectroscopy. Our results support the presence of two N-terminal helices, positioned on the surface of the micelle and separated by a flexible stretch. The region of residues 61-95 of the protein also adopts a helical conformation, but it is partially embedded in the micelle. These results could shed some light on the role of the membrane on the aggregation process of alpha-synuclein.

    Biochemistry 2005;44;1;329-39

  • Ubiquitination of alpha-synuclein.

    Nonaka T, Iwatsubo T and Hasegawa M

    Department of Molecular Neurobiology, Tokyo Institute of Psychiatry, Tokyo Metropolitan Organization for Medical Research, 2-1-8 Kamikitazawa, Setagaya-ku, Tokyo 156-8585, Japan.

    Filamentous alpha-synuclein depositions are the defining hallmarks of a subset of neurodegenerative diseases including Parkinson's disease (PD), dementia with Lewy bodies, and multiple system atrophy. We previously reported that alpha-synuclein in those brains are extensively phosphorylated at Ser129 [Fujiwara et al. (2002) Nat. Cell Biol. 4, 160-164] and also partially ubiquitinated [Hasegawa et al. (2002) J. Biol. Chem. 277, 49071-49076]. Here, we investigate ubiquitination of alpha-synuclein in vitro and in vivo and report the ubiquitination sites and the effects of familial PD-linked mutations, phosphorylation, and fibril formation on ubiquitination. Protein-sequence analysis revealed that Lys21, Lys23, Lys32, and Lys34 within the repeats in the amino-terminal half are liable to ubiquitination in vitro. A site-directed mutagensis study confirmed that these are the major ubiquitination sites. A53T and A30P mutations had no significant effect on ubiquitination. Similarly, phosphorylation of alpha-synuclein at Ser129 did not affect ubiquitination. Notably, we show that assembled, filamentous alpha-synuclein is less ubiquitinated than the soluble form and that the major ubiquitination sites are localized to Lys6, Lys10, and Lys12 at the amino-terminal region of filamentous alpha-synuclein. Furthermore, we successfully detected ubiquitination of alpha-synuclein in 293T cells by cotransfection with alpha-synuclein and ubiquitin. The in vivo ubiquitination sites were found to be identical to those in filamentous alpha-synuclein. PD-linked mutations and phosphorylation at Ser129 had no effects on ubiquitination of alpha-synuclein in vivo. These data may have implications for the mechanisms of the formation of alpha-synuclein deposits in alpha-synucleinopathy brains.

    Biochemistry 2005;44;1;361-8

  • Lentiviral vector delivery of parkin prevents dopaminergic degeneration in an alpha-synuclein rat model of Parkinson's disease.

    Lo Bianco C, Schneider BL, Bauer M, Sajadi A, Brice A, Iwatsubo T and Aebischer P

    Institute of Neuroscience, Swiss Federal Institute of Technology Lausanne, Ecole Polytechnique Fédérale de Lausanne, CH-1015 Lausanne, Switzerland.

    Parkinson's disease (PD) is characterized by a progressive loss of midbrain dopamine neurons and the presence of cytoplasmic inclusions called Lewy bodies. Mutations in several genes including alpha-synuclein and parkin have been linked to familial PD. The loss of parkin's E3-ligase activity leads to dopaminergic neuronal degeneration in early-onset autosomal recessive juvenile parkinsonism, suggesting a key role of parkin for dopamine neuron survival. To evaluate the potential neuroprotective role of parkin in the pathogenesis of PD, we tested whether overexpression of wild-type rat parkin could protect against the toxicity of mutated human A30P alpha-synuclein in a rat lentiviral model of PD. Animals overexpressing parkin showed significant reductions in alpha-synuclein-induced neuropathology, including preservation of tyrosine hydroxylase-positive cell bodies in the substantia nigra and sparing of tyrosine hydroxylase-positive nerve terminals in the striatum. The parkin-mediated neuroprotection was associated with an increase in hyperphosphorylated alpha-synuclein inclusions, suggesting a key role for parkin in the genesis of Lewy bodies. These results indicate that parkin gene therapy may represent a promising candidate treatment for PD.

    Proceedings of the National Academy of Sciences of the United States of America 2004;101;50;17510-5

  • Role of alpha-synuclein in presynaptic dopamine recruitment.

    Yavich L, Tanila H, Vepsäläinen S and Jäkälä P

    Department of Pharmacology and Toxicology, Clinical Research Centre/Mediteknia, University of Kuopio, Finland. leonid.yavich@uku.fi

    Real-time monitoring of stimulated dopamine release in mice with different alpha-synuclein expression was used to study the role of alpha-synuclein in presynaptic dopamine recruitment. Repeated electrical stimulations of ascending dopaminergic pathways decreased the capacity of the readily releasable pool (RRP) and temporarily increased its refilling rate, significantly slowing the rate of dopamine decline in mice with normally expressed alpha-synuclein. Mice with alpha-synuclein null mutation demonstrated a permanent increase of the refilling rate. This increase maintained stable dopamine release during stimulation (which induced dopamine decline in other animals) and served as an adaptation to altered dopamine compartmentalization. Mice without alpha-synuclein and with overexpression of human A30P mutated alpha-synuclein had a lower capacity of the dopamine storage pool than other animals. Reducing capacity of the storage pool in transgenic A30P mice led to paradoxical effects of l-dopa, which elevated dopamine release in response to single stimulation but decreased the refilling rate of the RRP.

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2004;24;49;11165-70

  • A single amino acid substitution differentiates Hsp70-dependent effects on alpha-synuclein degradation and toxicity.

    Klucken J, Shin Y, Hyman BT and McLean PJ

    MassGeneral Institute for Neurodegenerative disease, Alzheimer Disease Research Unit, Massachusetts General Hospital, 114 16th Street, Charlestown, MA 02129, USA.

    alpha-Synuclein aggregation and toxicity play a major role in Parkinson's disease and dementia with Lewy bodies. Hsp70 is a multipurpose stress response chaperone protein that mediates both refolding and degradation of misfolded proteins. We have shown that Hsp70 is able to block both alpha-synuclein toxicity and aggregation. Here we introduce a mutation into the ATPase domain of Hsp70 (K71S) and demonstrate that this abolishes Hsp70 refolding activity. Nonetheless, Hsp70K71S continues to mediate alpha-synuclein degradation and blocks aggregate formation. In contrast to wild type Hsp70, the ATPase domain mutant mediates alpha-synuclein degradation through a non-proteasome inhibitor sensitive pathway. Although Hsp70K71S can diminish levels of alpha-synuclein to an even greater extent than Hsp70, HSP70K71S does not protect against alpha-synuclein toxicity. The Hsp70K71S mutant appears to dissociate the formation of aggregates, which it blocks, and toxicity, which it does not block. These data suggest that the ability of Hsp70 to prevent toxicity is distinct from degradation of alpha-synuclein and is dependent on its ATPase domain.

    Funded by: NIA NIH HHS: AG18440; NINDS NIH HHS: NS38372

    Biochemical and biophysical research communications 2004;325;1;367-73

  • Double-stranded DNA stimulates the fibrillation of alpha-synuclein in vitro and is associated with the mature fibrils: an electron microscopy study.

    Cherny D, Hoyer W, Subramaniam V and Jovin TM

    Department of Molecular Biology, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, D-37077 Goettingen, Germany.

    Filamentous aggregates formed by alpha-synuclein are a prominent and presumably key etiological factor in Parkinson's and other neurodegenerative diseases characterized by motor disorders. Numerous studies have demonstrated that various environmental and intracellular factors affect the fibrillation properties of alpha-synuclein, e.g. by accelerating the process of assembly. Histones, the major component and constituent of chromatin, interact specifically with alpha-synuclein and enhance its fibrillation significantly. Here, we report that another component of chromatin, double-stranded DNA (dsDNA), either linear or supercoiled, also interacts with wild-type alpha-synuclein, leading to a significant stimulation of alpha-synuclein assembly into mature fibrils characterized by a reduced lag phase. In general, the morphology of the fibrils remains unchanged in the presence of linear dsDNA. Electron microscopy reveals that DNA forms various types of complexes upon association with the fibrils at their surface without distortion of the double-helical structure. The existence of these complexes was confirmed by the electrophoresis, which also demonstrated that a fraction of the associated DNA was resistant to digestion by restriction endonucleases. Fibrils assembled from the alpha-synuclein mutants A30P and A53T and the C-terminally truncated variants (encoding amino acid residues 1-108 or 1-124) also form complexes with linear dsDNA. Possible mechanisms and implications of dsDNA-alpha-synuclein interactions are discussed.

    Journal of molecular biology 2004;344;4;929-38

  • Differential expression of alpha-synuclein isoforms in dementia with Lewy bodies.

    Beyer K, Lao JI, Carrato C, Mate JL, López D, Ferrer I and Ariza A

    Department of Pathology, Hospital Universitari Germans Trias i Pujol, Autonomous University of Barcelona, Barcelona, Spain.

    Dementia with Lewy bodies (DLB) is characterized by the widespread presence of Lewy bodies (LBs) in the brain. alpha-Synuclein, the main component of LBs, is expressed as two main isoforms (112 and 140), but little is known about their differential expression in the brain. We compared alpha-synuclein 112 and alpha-synuclein 140 expression levels in the prefrontal cortices of six DLB patients, eight Alzheimer disease (AD) patients, and six control subjects. Relative alpha-synuclein 112 and alpha-synuclein 140 expression levels were determined by real-time polymerase chain reaction with competimer technology using a LightCycler System. Whereas total alpha-synuclein levels were just marginally elevated in DLB in comparison with the other groups, alpha-synuclein 112 was seen to be markedly increased in DLB compared with AD cases and controls. In contrast, alpha-synuclein 140 levels were significantly diminished in both neurodegenerative disorders in comparison with controls. These results show differential overexpression of alpha-synuclein 112 in DLB, a finding that could be of importance in DLB pathogenesis.

    Neuropathology and applied neurobiology 2004;30;6;601-7

  • Lewy bodies in the amygdala: increase of alpha-synuclein aggregates in neurodegenerative diseases with tau-based inclusions.

    Popescu A, Lippa CF, Lee VM and Trojanowski JQ

    Department of Neurology, Drexel University College of Medicine, 3300 Henry Avenue, Philadelphia, PA 19129, USA.

    Background: Increased attention has been given to alpha-synuclein aggregation in nonsynucleinopathies because alpha-synuclein-containing Lewy bodies (LBs) influence symptoms. However, the spectrum of disorders in which secondary inclusions are likely to occur has not been defined. Amygdala neurons commonly develop large numbers of secondary LBs, making it a practical region for studying this phenomenon.

    Objective: To characterize the spectrum of diseases associated with LB formation in the amygdala of neurodegenerative disease and control cases.

    Design: An autopsy series of 101 neurodegenerative disease and 34 aged control cases. Using immunohistochemistry studies, we examined the amygdala for alpha-synuclein aggregates.

    Results: Lewy bodies were often abundant in classic Pick disease, argyrophilic grain disease, Alzheimer disease, and dementia with LBs but not in cases with amygdala degeneration lacking tau-based inclusions, control cases, preclinical disease carriers, or degenerative diseases lacking pathologic involvement of the amygdala. The exposed alpha-synuclein epitopes were similar in all cases containing LBs.

    Conclusions: Abnormal alpha-synuclein aggregation in the amygdala is disease selective, but not restricted to disorders of alpha-synuclein and beta-amyloid. Our data are compatible with the notion that tau aggregates predispose neurons to develop secondary LBs.

    Archives of neurology 2004;61;12;1915-9

  • Functional consequences of alpha-synuclein tyrosine nitration: diminished binding to lipid vesicles and increased fibril formation.

    Hodara R, Norris EH, Giasson BI, Mishizen-Eberz AJ, Lynch DR, Lee VM and Ischiropoulos H

    Stokes Research Institute and Department of Biochemistry and Biophysics, Children's Hospital of Philadelphia and the University of Pennsylvania, 19104, USA.

    Previous studies have shown the presence of nitrated alpha-synuclein (alpha-syn) in human Lewy bodies and other alpha-syn inclusions. Herein, the effects of tyrosine nitration on alpha-syn fibril formation, lipid binding, chaperone-like function, and proteolytic degradation were systematically examined by employing chromatographically isolated nitrated monomeric, dimeric, and oligomeric alpha-syn. Nitrated alpha-syn monomers and dimers but not oligomers accelerated the rate of fibril formation of unmodified alpha-syn when present at low concentrations. Immunoelectron microscopy revealed that nitrated monomers and dimers are incorporated into the fibrils. However, the purified nitrated alpha-syn monomer by itself was unable to form fibrils. Nitration of the tyrosine residue at position 39 was largely responsible for decreased binding of nitrated monomeric alpha-syn to synthetic vesicles, which correlated with an impairment of the nitrated protein to adopt alpha-helical conformation in the presence of liposomes. The chaperone-like activity of alpha-syn was not inhibited by nitration or oxidation. Furthermore, the 20 S proteasome and calpain I degraded nitrated monomeric alpha-syn, although at a slower rate compared with control alpha-syn. Collectively, these data suggest that post-translational modification of alpha-syn by nitration can promote the formation of intracytoplasmic inclusions that constitute the hallmark of Parkinson disease and other synucleinopathies.

    The Journal of biological chemistry 2004;279;46;47746-53

  • DJ-1 is a redox-dependent molecular chaperone that inhibits alpha-synuclein aggregate formation.

    Shendelman S, Jonason A, Martinat C, Leete T and Abeliovich A

    Department of Pathology, Center for Neurobiology and Behavior, and Taub Institute, Columbia University, College of Physicians and Surgeons, New York, New York, USA.

    Parkinson's disease (PD) pathology is characterized by the degeneration of midbrain dopamine neurons (DNs) ultimately leading to a progressive movement disorder in patients. The etiology of DN loss in sporadic PD is unknown, although it is hypothesized that aberrant protein aggregation and cellular oxidative stress may promote DN degeneration. Homozygous mutations in DJ-1 were recently described in two families with autosomal recessive inherited PD (Bonifati et al. 2003). In a companion article (Martinat et al. 2004), we show that mutations in DJ-1 alter the cellular response to oxidative stress and proteasomal inhibition. Here we show that DJ-1 functions as a redox-sensitive molecular chaperone that is activated in an oxidative cytoplasmic environment. We further demonstrate that DJ-1 chaperone activity in vivo extends to alpha-synuclein, a protein implicated in PD pathogenesis.

    PLoS biology 2004;2;11;e362

  • Peroxidative aggregation of alpha-synuclein requires tyrosines.

    Olteanu A and Pielak GJ

    Department of Chemistry, University of North Carolina, Chapel Hill, NC 27599, USA.

    Alpha-synuclein is the main component of the intracellular protein aggregates in neurons of patients with Parkinson's disease. The occurrence of the disease is associated with oxidative damage. Although it is known that peroxidative chemistry leads to the aggregation of alpha-synuclein in vitro, the specific amino acid types of alpha-synuclein involved in this type of aggregation have not been identified. We show, using human cytochrome c plus H(2)O(2) as the source oxidative stress, that the tyrosines of alpha-synuclein are required for aggregation. The studies reveal the chemical basis for a crucial step in the aggregation process.

    Funded by: NIEHS NIH HHS: R21 ES 10774

    Protein science : a publication of the Protein Society 2004;13;11;2852-6

  • Rifampicin inhibits alpha-synuclein fibrillation and disaggregates fibrils.

    Li J, Zhu M, Rajamani S, Uversky VN and Fink AL

    Department of Chemistry and Biochemistry, University of California, Santa Cruz, Santa Cruz, CA 95064, USA.

    The aggregation of alpha-synuclein in dopaminergic neurons of the substantia nigra is a critical step in the pathogenesis of Parkinson's disease. We show that the antibiotic rifampicin inhibited alpha-synuclein fibrillation and disaggregated existing fibrils in a concentration-dependent manner. Size-exclusion chromatography data indicated that rifampicin stabilized alpha-synuclein as both a monomer and soluble oligomers comprised of partially folded alpha-synuclein. Experiments using aged samples of rifampicin indicated that the most active species in inhibiting fibrillation and disaggregating fibrils is an oxidation product of rifampicin, which was confirmed in experiments under anaerobic conditions. These results indicate that rifampicin-mediated inhibition of alpha-synuclein fibrillation and disaggregation of fibrils involves preferential stabilization of monomeric and soluble oligomeric forms, and that rifampicin potentially may have therapeutic application for Parkinson's disease.

    Funded by: NINDS NIH HHS: NS 43778

    Chemistry & biology 2004;11;11;1513-21

  • Sensitivity to oxidative stress in DJ-1-deficient dopamine neurons: an ES- derived cell model of primary Parkinsonism.

    Martinat C, Shendelman S, Jonason A, Leete T, Beal MF, Yang L, Floss T and Abeliovich A

    Department of Pathology, Center for Neurobiology and Behavior, and Taub Institute, Columbia University, New York, New York, USA.

    The hallmark of Parkinson's disease (PD) is the selective loss of dopamine neurons in the ventral midbrain. Although the cause of neurodegeneration in PD is unknown, a Mendelian inheritance pattern is observed in rare cases, indicating a genetic factor. Furthermore, pathological analyses of PD substantia nigra have correlated cellular oxidative stress and altered proteasomal function with PD. Homozygous mutations in DJ-1 were recently described in two families with autosomal recessive Parkinsonism, one of which is a large deletion that is likely to lead to loss of function. Here we show that embryonic stem cells deficient in DJ-1 display increased sensitivity to oxidative stress and proteasomal inhibition. The accumulation of reactive oxygen species in toxin-treated DJ-1-deficient cells initially appears normal, but these cells are unable to cope with the consequent damage that ultimately leads to apoptotic death. Furthermore, we find that dopamine neurons derived from in vitro-differentiated DJ-1-deficient embryonic stem cells display decreased survival and increased sensitivity to oxidative stress. These data are consistent with a protective role for DJ-1, and demonstrate the utility of genetically modified embryonic stem cell-derived neurons as cellular models of neuronal disorders.

    PLoS biology 2004;2;11;e327

  • Mutation E46K increases phospholipid binding and assembly into filaments of human alpha-synuclein.

    Choi W, Zibaee S, Jakes R, Serpell LC, Davletov B, Crowther RA and Goedert M

    MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK.

    Missense mutations (A30P and A53T) in alpha-synuclein and the overproduction of the wild-type protein cause familial forms of Parkinson's disease and dementia with Lewy bodies. Alpha-synuclein is the major component of the filamentous Lewy bodies and Lewy neurites that define these diseases at a neuropathological level. Recently, a third missense mutation (E46K) in alpha-synuclein was described in an inherited form of dementia with Lewy bodies. Here, we have investigated the functional effects of this novel mutation on phospholipid binding and filament assembly of alpha-synuclein. When compared to the wild-type protein, the E46K mutation caused a significantly increased ability of alpha-synuclein to bind to negatively charged liposomes, unlike the previously described mutations. The E46K mutation increased the rate of filament assembly to the same extent as the A53T mutation. Filaments formed from E46K alpha-synuclein often had a twisted morphology with a cross-over spacing of 43 nm. The observed effects on lipid binding and filament assembly may explain the pathogenic nature of the E46K mutation in alpha-synuclein.

    FEBS letters 2004;576;3;363-8

  • Early and progressive sensorimotor anomalies in mice overexpressing wild-type human alpha-synuclein.

    Fleming SM, Salcedo J, Fernagut PO, Rockenstein E, Masliah E, Levine MS and Chesselet MF

    Department of Neurology, The Mental Retardation Research Center, The David Geffen School of Medicine at University of California Los Angeles, Los Angeles, California 90095-1769, USA.

    Accumulation of alpha-synuclein in brain is a hallmark of synucleinopathies, neurodegenerative diseases that include Parkinson's disease. Mice overexpressing alpha-synuclein under the Thy-1 promoter (ASO) show abnormal accumulation of alpha-synuclein in cortical and subcortical regions of the brain, including the substantia nigra. We examined the motor deficits in ASO mice with a battery of sensorimotor tests that are sensitive to alterations in the nigrostriatal dopaminergic system. Male wild-type and ASO mice were tested every 2 months for 8 months for motor performance and coordination on a challenging beam, inverted grid, and pole, sensorimotor deficits in an adhesive removal test, spontaneous activity in a cylinder, and gait. Fine motor skills were assessed by the ability to grasp cotton from a bin. ASO mice displayed significant impairments in motor performance and coordination and a reduction in spontaneous activity as early as 2 months of age. Motor performance and coordination impairments became progressively worse with age and sensorimotor deficits appeared at 6 months. Fine motor skills were altered at 4 months and worsened at 8 months. These data indicate that overexpression of alpha-synuclein induced an early and progressive behavioral phenotype that can be detected in multiple tests of sensorimotor function. These behavioral deficits provide a useful way to assess novel drug therapy in genetic models of synucleinopathies.

    Funded by: NIA NIH HHS: AG022074, AG18440; NIEHS NIH HHS: U54ES12078; NINDS NIH HHS: P50NS38367, T32 NS07449-05

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2004;24;42;9434-40

  • The status, quality, and expansion of the NIH full-length cDNA project: the Mammalian Gene Collection (MGC).

    Gerhard DS, Wagner L, Feingold EA, Shenmen CM, Grouse LH, Schuler G, Klein SL, Old S, Rasooly R, Good P, Guyer M, Peck AM, Derge JG, Lipman D, Collins FS, Jang W, Sherry S, Feolo M, Misquitta L, Lee E, Rotmistrovsky K, Greenhut SF, Schaefer CF, Buetow K, Bonner TI, Haussler D, Kent J, Kiekhaus M, Furey T, Brent M, Prange C, Schreiber K, Shapiro N, Bhat NK, Hopkins RF, Hsie F, Driscoll T, Soares MB, Casavant TL, Scheetz TE, Brown-stein MJ, Usdin TB, Toshiyuki S, Carninci P, Piao Y, Dudekula DB, Ko MS, Kawakami K, Suzuki Y, Sugano S, Gruber CE, Smith MR, Simmons B, Moore T, Waterman R, Johnson SL, Ruan Y, Wei CL, Mathavan S, Gunaratne PH, Wu J, Garcia AM, Hulyk SW, Fuh E, Yuan Y, Sneed A, Kowis C, Hodgson A, Muzny DM, McPherson J, Gibbs RA, Fahey J, Helton E, Ketteman M, Madan A, Rodrigues S, Sanchez A, Whiting M, Madari A, Young AC, Wetherby KD, Granite SJ, Kwong PN, Brinkley CP, Pearson RL, Bouffard GG, Blakesly RW, Green ED, Dickson MC, Rodriguez AC, Grimwood J, Schmutz J, Myers RM, Butterfield YS, Griffith M, Griffith OL, Krzywinski MI, Liao N, Morin R, Morrin R, Palmquist D, Petrescu AS, Skalska U, Smailus DE, Stott JM, Schnerch A, Schein JE, Jones SJ, Holt RA, Baross A, Marra MA, Clifton S, Makowski KA, Bosak S, Malek J and MGC Project Team

    The National Institutes of Health's Mammalian Gene Collection (MGC) project was designed to generate and sequence a publicly accessible cDNA resource containing a complete open reading frame (ORF) for every human and mouse gene. The project initially used a random strategy to select clones from a large number of cDNA libraries from diverse tissues. Candidate clones were chosen based on 5'-EST sequences, and then fully sequenced to high accuracy and analyzed by algorithms developed for this project. Currently, more than 11,000 human and 10,000 mouse genes are represented in MGC by at least one clone with a full ORF. The random selection approach is now reaching a saturation point, and a transition to protocols targeted at the missing transcripts is now required to complete the mouse and human collections. Comparison of the sequence of the MGC clones to reference genome sequences reveals that most cDNA clones are of very high sequence quality, although it is likely that some cDNAs may carry missense variants as a consequence of experimental artifact, such as PCR, cloning, or reverse transcriptase errors. Recently, a rat cDNA component was added to the project, and ongoing frog (Xenopus) and zebrafish (Danio) cDNA projects were expanded to take advantage of the high-throughput MGC pipeline.

    Funded by: PHS HHS: N01-C0-12400

    Genome research 2004;14;10B;2121-7

  • Alpha-synuclein induces apoptosis by altered expression in human peripheral lymphocyte in Parkinson's disease.

    Kim S, Jeon BS, Heo C, Im PS, Ahn TB, Seo JH, Kim HS, Park CH, Choi SH, Cho SH, Lee WJ and Suh YH

    Department of Pharmacology, College of Medicine, National Creative Research Initiative Center for Alzheimer's Dementia and Neuroscience Research Institute, MRC, Seoul National University, Republic of Korea.

    Though the etiology of Parkinson's disease (PD) remains unclear, alpha-synuclein (alpha-SN) is regarded as a major causative agent of PD. Several lines of evidence indicate that immunological abnormalities are associated with PD for unknown reasons. The present study was performed to assess whether peripheral blood mononuclear cells (PBMCs) show altered alpha-SN expression in PD patients and to identify its functions, which may be related to peripheral immune abnormalities in PD. alpha-SN was found to be expressed more in 151 idiopathic PD (IPD) patients than in 101 healthy controls, who nevertheless showed as age-dependent increases. By in vitro transfection, alpha-SN expression was shown to be correlated with glucocorticoid sensitive apoptosis, possibly caused by the enhanced expression of glucocorticoid receptor (GR), caspase activations (caspase-8, caspase-9), CD95 up-regulation, and reactive oxygen species (ROS) production. An understanding of the correlation between alpha-SN levels and apoptosis in the presence of the coordinated involvement of multiple processes would provide an insight into the molecular basis of the disease. The present study provides a clue that the alpha-SN may be one of the primary causes of the immune abnormalities observed in PD and offers new targets for pharmacotherapeutic intervention.

    FASEB journal : official publication of the Federation of American Societies for Experimental Biology 2004;18;13;1615-7

  • alpha-Synuclein promoter confers susceptibility to Parkinson's disease.

    Pals P, Lincoln S, Manning J, Heckman M, Skipper L, Hulihan M, Van den Broeck M, De Pooter T, Cras P, Crook J, Van Broeckhoven C and Farrer MJ

    Department of Molecular Genetics VIB8, Flanders Interuniversity Institute for Biotechnology, University of Antwerp, Belgium.

    Familial Parkinson's disease (PD) has been linked to missense and genomic multiplication mutations of the alpha-synuclein gene (SNCA). Genetic variability within SNCA has been implicated in idiopathic PD in many populations. We now confirm and extend these findings, within a Belgian sample, using a high-resolution map of genetic markers across the SNCA locus. Our study implicates the SNCA promoter in susceptibility to PD, and more specifically defines a minimum promoter haplotype, spanning approximately 15.3kb of sequence, which is overrepresented in patients. Our findings represent a biomarker for PD and may have implications for patient diagnosis, longitudinal evaluation, and treatment.

    Annals of neurology 2004;56;4;591-5

  • Alpha-synuclein: stable compact and extended monomeric structures and pH dependence of dimer formation.

    Bernstein SL, Liu D, Wyttenbach T, Bowers MT, Lee JC, Gray HB and Winkler JR

    Department of Chemistry and Biochemistry, University of California at Santa Barbara, USA.

    The protein alpha-synuclein, implicated in Parkinson's disease, was studied by combining nano-electrospray ionization (N-ESI) mass spectrometry and ion mobility. It was found that both the charge-state distribution in the mass spectra and the average protein shape deduced from ion mobility data, depend on the pH of the spray solution. Negative-ion N-ESI of pH 7 solutions yielded a broad charge-state distribution from -6 to -16, centered at -11, and ion mobility data consistent with extended protein structures. Data obtained for pH 2.5 solutions, on the other hand, showed a narrow charge-state distribution from -6 to -11, centered at -8, and ion mobilities in agreement with compact alpha-synuclein structures. The data indicated that there are two distinct families of structures: one consisting of relatively compact proteins with eight or less negative charges and one consisting of relatively extended structures with nine or more charges. The average cross section of a-synuclein at pH 2.5 is 33% smaller than for the extended protein sprayed from pH 7 solution. Significant dimer formation was observed when sprayed from pH 7 solution but no dimers were observed from the low pH solution. A plausible mechanism for aggregate formation in solution is proposed.

    Journal of the American Society for Mass Spectrometry 2004;15;10;1435-43

  • G209A mutant alpha synuclein expression specifically enhances dopamine induced oxidative damage.

    Orth M, Tabrizi SJ, Tomlinson C, Messmer K, Korlipara LV, Schapira AH and Cooper JM

    University Department of Clinical Neurosciences, Royal Free and University College Medical School, Rowland Hill Street, London NW3 2PF, UK.

    Alpha synuclein protein may play an important role in familial and sporadic Parkinson's disease pathology. We have induced G209A mutant or wild-type alpha-synuclein expression in stable HEK293 cell models to determine if this influences markers of oxidative stress and damage under normal conditions or in the presence of dopamine or paraquat. Induced wild-type or mutant alpha-synuclein expression alone had no effect upon levels of oxidative stress or damage, as measured by glutathione levels or aconitase activity. Both wild-type and mutant alpha-synuclein expression decreased the oxidative damage induced by paraquat, although the protection was less marked with mutant alpha-synuclein expression. This suggests that alpha-synuclein expression may either have anti-oxidant properties or may upregulate cellular antioxidant levels, a function that was diminished by the G209A mutation. However, mutant but not wild-type alpha-synuclein expression specifically enhanced dopamine associated oxidative damage. Non-expressing cells treated with reserpine to inhibit the vesicular monoamine compartmentalisation produced similar results. However, consistent with the hypothesis that mutant alpha-synuclein disrupts vesicular dopamine compartmentalization, this effect was diminished in cells expressing mutant alpha-synuclein. This may result in increased dopamine metabolism and cause selective oxidative damage to dopaminergic cells.

    Neurochemistry international 2004;45;5;669-76

  • Study of association between alpha-synuclein gene polymorphism and methamphetamine psychosis/dependence.

    Kobayashi H, Ide S, Hasegawa J, Ujike H, Sekine Y, Ozaki N, Inada T, Harano M, Komiyama T, Yamada M, Iyo M, Shen HW, Ikeda K and Sora I

    Department of Molecular Psychiatry, Tokyo Institute of Psychiatry, Japan.

    Methamphetamine (MAP) dissipates proton gradients across the membranes of synaptic vesicles, enhances cytoplasmic dopamine (DA) concentrations, and causes calcium-independent, nonvesicular DA release into synapses. MAP is taken into the cytosol by the dopamine transporter (DAT) on the synaptic terminals of DA neurons, and endogenous DA is concurrently released through the transporter by carrier exchange mechanisms, resulting in a robust increase in DA concentration in the synaptic clefts. The enhanced DA release through DAT by MAP is the main mechanism for the reinforcing effects of MAP. The complexes of alpha-synuclein and DAT facilitate membrane clustering of the DAT, thereby accelerating DA uptake in vitro. alpha-Synuclein has been shown to be overexpressed in the midbrain DA neurons of chronic cocaine abusers. The present study was performed to study the association between the alpha-synuclein gene polymorphisms and MAP psychosis/dependence in Japanese population. Since the T10A7 polymorphic site at the 5' end of the noncoding exon 1' in the alpha-synuclein gene is highly polymorphic, we analyzed the noncoding exon 1' and intron 1, including this polymorphic site by sequencing. We confirmed four single nucleotide polymorphisms (SNPs) within 1.38 kbp of the T10A7 polymorphic site. No significant difference was found in genotype or allele frequencies in the T10A7 polymorphic site between MAP psychotic/dependent and control subjects. We found significant association between three SNPs in the vicinity of this polymorphic site in intron 1 and MAP psychosis/dependence in female subjects, but not in males. These results suggest an association of the alpha-synuclein gene polymorphisms with MAP psychosis/dependence in our female subjects. Further analyses are necessary to clarify the gender difference, by using a larger sample size and/or different ethnic groups, as well as functional variations in the alpha-synuclein gene.

    Annals of the New York Academy of Sciences 2004;1025;325-34

  • Beta-synuclein gene alterations in dementia with Lewy bodies.

    Ohtake H, Limprasert P, Fan Y, Onodera O, Kakita A, Takahashi H, Bonner LT, Tsuang DW, Murray IV, Lee VM, Trojanowski JQ, Ishikawa A, Idezuka J, Murata M, Toda T, Bird TD, Leverenz JB, Tsuji S and La Spada AR

    Department of Neurology, Brain Research Institute, Niigata University, Niigata, Japan.

    Objective: To determine whether mutations in the genes for alpha-synuclein or beta-synuclein are responsible for dementia with Lewy bodies (DLB), a disorder closely related to Parkinson disease (PD).

    Methods: The authors ascertained 33 sporadic cases of DLB and 10 kindreds segregating DLB. DNA samples from the 43 index cases were screened for alterations in the genes for alpha-synuclein and beta-synuclein, as alpha-synuclein alterations cause PD and beta-synuclein may modulate alpha-synuclein aggregation and neurotoxicity.

    Results: Two amino acid alterations were identified in unrelated DLB index cases: a valine to methionine substitution at codon 70 (V70M) and a proline to histidine substitution at codon 123 (P123H), both in the beta-synuclein gene. These amino acid substitutions occur at conserved residues in highly conserved regions of the beta-synuclein protein. Screening of at least 660 chromosomes from control subjects matched to the patients' population groups failed to identify another V70M or P123H allele. Cosegregation analysis of an extended pedigree segregating the P123H beta-synuclein alteration suggested that it is a dominant trait with reduced penetrance or a risk factor polymorphism. Histopathology and immunohistochemistry analysis of index case brain sections revealed widespread Lewy body pathology and alpha-synuclein aggregation without evidence of beta-synuclein aggregation.

    Conclusion: Mutations in the beta-synuclein gene may predispose to DLB.

    Funded by: NIA NIH HHS: AG-09215; NINDS NIH HHS: P01 NS-40256, R01 NS048595-03

    Neurology 2004;63;5;805-11

  • Impaired degradation of mutant alpha-synuclein by chaperone-mediated autophagy.

    Cuervo AM, Stefanis L, Fredenburg R, Lansbury PT and Sulzer D

    Department of Anatomy and Structural Biology, Marion Bessin Liver Research Center, Albert Einstein College of Medicine, Bronx, NY 10461, USA. amcuervo@aecom.yu.edu

    Aberrant alpha-synuclein degradation is implicated in Parkinson's disease pathogenesis because the protein accumulates in the Lewy inclusion bodies associated with the disease. Little is known, however, about the pathways by which wild-type alpha-synuclein is normally degraded. We found that wild-type alpha-synuclein was selectively translocated into lysosomes for degradation by the chaperone-mediated autophagy pathway. The pathogenic A53T and A30P alpha-synuclein mutants bound to the receptor for this pathway on the lysosomal membrane, but appeared to act as uptake blockers, inhibiting both their own degradation and that of other substrates. These findings may underlie the toxic gain-of-function by the mutants.

    Funded by: NIA NIH HHS: AG021904

    Science (New York, N.Y.) 2004;305;5688;1292-5

  • Stabilization of alpha-synuclein protein with aging and familial parkinson's disease-linked A53T mutation.

    Li W, Lesuisse C, Xu Y, Troncoso JC, Price DL and Lee MK

    Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205-2196, USA.

    We examined the potential relationship between aging and alpha-synuclein (alpha-Syn) metabolism, both of which are implicated in the pathogenesis of Parkinson's disease (PD) and other alpha-synucleinopathies. During aging,alpha-Syn and beta-Syn mRNA expression in brain decreases, but the protein levels are maintained at high levels. Significantly, the alpha-Syn protein level increases with aging in human substantia nigra. Pulse-chase analyses of alpha-Syn half-lives in neurons and neuronal cell lines indicate that, in mature neurons, the expression of alpha-Syn is regulated by the post-translational stabilization of alpha-Syn protein. Moreover, A53T mutant human alpha-Syn exhibits increased stability in neuronal cell lines, leading to higher levels of the mutant protein in cells and transgenic mice. Inhibitor studies suggest that the proteasomal and lysosomal systems may not be responsible for the differential stabilization or metabolism of alpha-Syn protein in neuronal cells. Because increased stabilization of alpha-Syn protein is associated with increased protein levels and accumulation of pathogenic protein modifications, such as oxidative damage, the stabilization of alpha-Syn with aging may be a significant factor in the pathogenesis of alpha-synucleinopathies.

    Funded by: NINDS NIH HHS: NS38065, NS38377

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2004;24;33;7400-9

  • SNCA multiplication is not a common cause of Parkinson disease or dementia with Lewy bodies.

    Johnson J, Hague SM, Hanson M, Gibson A, Wilson KE, Evans EW, Singleton AA, McInerney-Leo A, Nussbaum RL, Hernandez DG, Gallardo M, McKeith IG, Burn DJ, Ryu M, Hellstrom O, Ravina B, Eerola J, Perry RH, Jaros E, Tienari P, Weiser R, Gwinn-Hardy K, Morris CM, Hardy J and Singleton AB

    Laboratory of Neurogenetics, National Institute on Aging, NIH, Bethesda, MD 20892, USA.

    The authors recently have shown that triplication of the alpha-synuclein gene (SNCA) can cause Parkinson disease (PD) and diffuse Lewy body disease within the same kindred. The authors assessed 101 familial PD probands, 325 sporadic PD cases, 65 patients with dementia with Lewy bodies, and 366 neurologically normal control subjects for SNCA multiplication. The authors did not identify any subjects with multiplication of SNCA and conclude this mutation is a rare cause of disease.

    Neurology 2004;63;3;554-6

  • [The alpha-synuclein gene microsatellite polymorphism and late-onset sporadic Parkinson's disease susceptibility].

    Zhao XP, Zheng HM, Xie HJ, Ding SJ and Ren DM

    Department of Neurology, Changhai Hospital, the Second Military Medical University, Shanghai, 200433 PR China. xiaopingzhao1@263.net

    Objective: To explore the association of the microsatellite polymorphisms in the promoter region of alpha-synuclein gene with the late-onset sporadic Parkinson's disease (PD) susceptibility.

    Methods: The microsatellite polymorphism of alpha-synuclein gene was analyzed with amplified fragment length polymorphism (Amp-FLP) and semiautomatic fluorescent labeled genotyping technique. Association analysis was performed in 135 unrelated late-onset sporadic PD patients and 170 age-matched healthy controls.

    Results: The distribution of the alleles of the dinucleotide repeats variants of alpha-synuclein gene promoter region in PD cases was significantly different from that in the healthy controls. The most frequent allele in PD patients was allele 269 bp, but in controls it was the 271 bp allele. Alleles of <or=267 bp showed positive correlation with PD risk (OR=5.228, 95%CI: 1.248-27.202, chi-square=6.416, P=0.011), while the 273 bp allele was negatively correlated to PD (OR=0.638, 95%CI: 0.440-0.926, chi-square=5.644, P=0.018). Furthermore, no difference of genotype polymorphism distribution was shown between the two groups (chi-square=16.368, df=12, P=0.175). But the genotypes containing <or=267 bp allele may increase the susceptibility to PD (OR=4.594, 95%CI: 0.94-22.49, chi-square=4.224, P=0.04). Heterozygosity was 40% in PD patients, and 50% in controls.

    Conclusion: alpha-synuclein microsatellite polymorphism might be a genetic susceptibility factor for late-onset sporadic PD.

    Zhonghua yi xue yi chuan xue za zhi = Zhonghua yixue yichuanxue zazhi = Chinese journal of medical genetics 2004;21;4;339-41

  • A strategy for designing inhibitors of alpha-synuclein aggregation and toxicity as a novel treatment for Parkinson's disease and related disorders.

    El-Agnaf OM, Paleologou KE, Greer B, Abogrein AM, King JE, Salem SA, Fullwood NJ, Benson FE, Hewitt R, Ford KJ, Martin FL, Harriott P, Cookson MR and Allsop D

    Department of Biological Sciences, Lancaster University, Lancaster, UK. o.el-agnaf@lancaster.ac.uk

    Convergent biochemical and genetic evidence suggests that the formation of alpha-synuclein (alpha-syn) protein deposits is an important and, probably, seminal step in the development of Parkinson's disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA). It has been reported that transgenic animals overexpressing human alpha-syn develop lesions similar to those found in the brain in PD, together with a progressive loss of dopaminergic cells and associated abnormalities of motor function. Inhibiting and/or reversing alpha-syn self-aggregation could, therefore, provide a novel approach to treating the underlying cause of these diseases. We synthesized a library of overlapping 7-mer peptides spanning the entire alpha-syn sequence, and identified amino acid residues 64-100 of alpha-syn as the binding region responsible for its self-association. Modified short peptides containing alpha-syn amino acid sequences from part of this binding region (residues 69-72), named alpha-syn inhibitors (ASI), were found to interact with full-length alpha-syn and block its assembly into both early oligomers and mature amyloid-like fibrils. We also developed a cell-permeable inhibitor of alpha-syn aggregation (ASID), using the polyarginine peptide delivery system. This ASID peptide was able to inhibit the DNA damage induced by Fe(II) in neuronal cells transfected with alpha-syn(A53T), a familial PD-associated mutation. ASI peptides without this delivery system did not reverse levels of Fe(II)-induced DNA damage. Furthermore, the ASID peptide increased (P<0.0005) the number of cells stained positive for Bcl-2, while significantly (P<0.05) decreasing the percentage of cells stained positive for BAX. These short peptides could serve as lead compounds for the design of peptidomimetic drugs to treat PD and related disorders.

    FASEB journal : official publication of the Federation of American Societies for Experimental Biology 2004;18;11;1315-7

  • alpha-Synuclein-synaptosomal membrane interactions: implications for fibrillogenesis.

    Jo E, Darabie AA, Han K, Tandon A, Fraser PE and McLaurin J

    Centre for Research in Neurodegenerative Diseases, University of Toronto, Ontario, Canada.

    alpha-Synuclein exists in two different compartments in vivo-- correspondingly existing as two different forms: a membrane-bound form that is predominantly alpha-helical and a cytosolic form that is randomly structured. It has been suggested that these environmental and structural differences may play a role in aggregation propensity and development of pathological lesions observed in Parkinson's disease (PD). Such effects may be accentuated by mutations observed in familial PD kindreds. In order to test this hypothesis, wild-type and A53T mutant alpha-synuclein interactions with rat brain synaptosomal membranes were examined. Previous data has demonstrated that the A30P mutant has defective lipid binding and therefore was not examined in this study. Electron microscopy demonstrated that wild-type alpha-synuclein fibrillogenesis is accelerated in the presence of synaptosomal membranes whereas the A53T alpha-synuclein fibrillogenesis is inhibited under the same conditions. These results suggested that subtle sequence changes in alpha-synuclein could significantly alter interaction with membrane bilayers. Fluorescence and absorption spectroscopy using environment sensitive probes demonstrated variations in the inherent lipid properties in the presence and absence of alpha-synuclein. Addition of wild-type alpha-synuclein to synaptosomes did not significantly alter the membrane fluidity at either the fatty acyl chains or headgroup space, suggesting that synaptosomes have a high capacity for alpha-synuclein binding. In contrast, synaptosomal membrane fluidity was decreased by A53T alpha-synuclein binding with concomitant packing of the lipid headgroups. These results suggest that alterations in alpha-synuclein-lipid interactions may contribute to physiological changes detected in early onset PD.

    European journal of biochemistry 2004;271;15;3180-9

  • Neurobiology of alpha-synuclein.

    Vekrellis K, Rideout HJ and Stefanis L

    Neurobiology Laboratory, Institute of Biomedical Research of the Academy of Athens, Greece, Columbia University, New York, NY.

    Alpha-synuclein is an abundant neuronal protein that has been linked both to normal synaptic function and to neurodegeneration. Most significantly, mutations in the gene encoding for alpha-synuclein are responsible for Parkinson's disease (PD) in rare familial cases, and the aggregated protein is a major component of Lewy bodies found in sporadic PD. Here we review recent data regarding the structure, the regulation at the transcriptional and posttranslational level, and the physiologic and aberrant functions of alpha-synuclein. We focus in particular on the fibrilization potential of alpha-synuclein and on its link with defects in protein degradation.

    Molecular neurobiology 2004;30;1;1-21

  • Lipid rafts mediate the synaptic localization of alpha-synuclein.

    Fortin DL, Troyer MD, Nakamura K, Kubo S, Anthony MD and Edwards RH

    Department of Neurology, Graduate Programs in Biomedical Sciences, Cell Biology and Neuroscience, University of California San Francisco School of Medicine, San Francisco, California 94143-2140, USA.

    Alpha-synuclein contributes to the pathogenesis of Parkinson's disease (PD), but its precise role in the disorder and its normal function remain poorly understood. Consistent with a presumed role in neurotransmitter release and its prominent deposition in the dystrophic neurites of PD, alpha-synuclein localizes almost exclusively to the nerve terminal. In brain extracts, however, alpha-synuclein behaves as a soluble, monomeric protein. Using a binding assay to characterize the association of alpha-synuclein with cell membranes, we find that alpha-synuclein binds saturably and with high affinity to characteristic intracellular structures that double label for components of lipid rafts. Biochemical analysis demonstrates the interaction of alpha-synuclein with detergent-resistant membranes and reveals a shift in electrophoretic mobility of the raft-associated protein. In addition, the A30P mutation associated with PD disrupts the interaction of alpha-synuclein with lipid rafts. Furthermore, we find that both the A30P mutation and raft disruption redistribute alpha-synuclein away from synapses, indicating an important role for raft association in the normal function of alpha-synuclein and its role in the pathogenesis of PD.

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2004;24;30;6715-23

  • Interaction of the molecular chaperone alphaB-crystallin with alpha-synuclein: effects on amyloid fibril formation and chaperone activity.

    Rekas A, Adda CG, Andrew Aquilina J, Barnham KJ, Sunde M, Galatis D, Williamson NA, Masters CL, Anders RF, Robinson CV, Cappai R and Carver JA

    Department of Chemistry, University of Wollongong, Northfields Avenue, Wollongong, NSW 2522, Australia.

    alpha-Synuclein is a pre-synaptic protein, the function of which is not completely understood, but its pathological form is involved in neurodegenerative diseases. In vitro, alpha-synuclein spontaneously forms amyloid fibrils. Here, we report that alphaB-crystallin, a molecular chaperone found in Lewy bodies that are characteristic of Parkinson's disease (PD), is a potent in vitro inhibitor of alpha-synuclein fibrillization, both of wild-type and the two mutant forms (A30P and A53T) that cause familial, early onset PD. In doing so, large irregular aggregates of alpha-synuclein and alphaB-crystallin are formed implying that alphaB-crystallin redirects alpha-synuclein from a fibril-formation pathway towards an amorphous aggregation pathway, thus reducing the amount of physiologically stable amyloid deposits in favor of easily degradable amorphous aggregates. alpha-Synuclein acts as a molecular chaperone to prevent the stress-induced, amorphous aggregation of target proteins. Compared to wild-type alpha-synuclein, both mutant forms have decreased chaperone activity in vitro against the aggregation of reduced insulin at 37 degrees C and the thermally induced aggregation of betaL-crystallin at 60 degrees C. Wild-type alpha-synuclein abrogates the chaperone activity of alphaB-crystallin to prevent the precipitation of reduced insulin. Interaction between these two chaperones and formation of a complex are also indicated by NMR spectroscopy, size-exclusion chromatography and mass spectrometry. In summary, alpha-synuclein and alphaB-crystallin interact readily with each other and affect each other's properties, in particular alpha-synuclein fibril formation and alphaB-crystallin chaperone action.

    Journal of molecular biology 2004;340;5;1167-83

  • Cross-linking of ubiquitin, HSP27, parkin, and alpha-synuclein by gamma-glutamyl-epsilon-lysine bonds in Alzheimer's neurofibrillary tangles.

    Nemes Z, Devreese B, Steinert PM, Van Beeumen J and Fésüs L

    Department of Psychiatry, Hungarian Academy of Sciences, Research Center for Molecular Medicine, University of Debrecen, Debrecen, Hungary. znemes@dote.hu

    The accumulation of misfolded proteins in intracellular inclusions is a generic feature of neurodegenerative disorders. Although heavily ubiquitylated, the aggregated proteins are not degraded by the proteasomes. A possible reason for this phenomenon may be a modification of deposited proteins by transglutaminases forming gamma-glutamyl-epsilon-lysine (GGEL) cross-links between distinct proteins. Here, we show that the frequency of GGEL cross-links is an order of magnitude higher in Alzheimer's brain cortex than in age-matched or younger controls. This difference is due to the accumulation of GGEL cross-links in ubiquitin-immunopositive protein particles present in both Alzheimer's brains and those from aged individuals. The highly cross-linked protein aggregates show immunoreactivity to antibodies against tau and neurofilament proteins, and partially also to alpha-synuclein, indicating that these structures are inherent in Alzheimer's neurofibrillary tangles and Lewy bodies. Using mass sequence analysis, we identified the same six pairs of peptide sequences cross-linked in both senile and Alzheimer's specimens: Gln31 and Gln190 of HSP27 protein are cross-linked with Lys29 and Lys48 of ubiquitin and HSP27 therefore may cross-link two (poly)ubiquitin chains. One lysine residue of parkin and one of alpha-synuclein were also found to be cross-linked. The data suggest that cross-linking of (poly)ubiquitin moieties via HSP27 may have a role in the stabilization of the intraneuronal protein aggregates by interference with the proteasomal elimination of unfolded proteins.

    FASEB journal : official publication of the Federation of American Societies for Experimental Biology 2004;18;10;1135-7

  • Structure of membrane-bound alpha-synuclein studied by site-directed spin labeling.

    Jao CC, Der-Sarkissian A, Chen J and Langen R

    Department of Biochemistry and Molecular Biology, School of Pharmacy, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA.

    Many of the proposed physiological functions of alpha-synuclein, a protein involved in the pathogenesis of Parkinson's disease, are related to its ability to interact with phospholipids. To better understand the conformational changes that occur upon membrane binding of monomeric alpha-synuclein, we performed EPR analysis of 47 singly labeled alpha-synuclein derivatives. We show that membrane interaction is mediated by major conformational changes within seven N-terminal 11-aa repeats, which reorganize from a highly dynamic structure into an elongated helical structure devoid of significant tertiary packing. Furthermore, we find that analogous positions from different repeats are in equivalent locations with respect to membrane proximity. These and other findings suggest a curved membrane-dependent alpha-helical structure, wherein each 11-aa repeat takes up three helical turns. Similar helical structures could also apply to apolipoproteins and other lipid-interacting proteins with related 11-aa repeats.

    Funded by: NIA NIH HHS: P50 AG05142

    Proceedings of the National Academy of Sciences of the United States of America 2004;101;22;8331-6

  • Abnormal alpha-synuclein interactions with rab3a and rabphilin in diffuse Lewy body disease.

    Dalfó E, Barrachina M, Rosa JL, Ambrosio S and Ferrer I

    Unitat de Bioquímica, Departament de Ciències Fisiològiques II, Campus de Bellvitge, Universitat de Barcelona, Hospitalet de Llobregat, Barcelona, Spain.

    The present study examines alpha-synuclein interactions with rab3a and rabphilin by antibody arrays, immunoprecipitation and pull-down methods in the entorhinal cortex of control cases and in diffuse Lewy body disease (LBD) cases. Alpha-synuclein immunoprecipitation revealed alpha-synuclein binding to rabphilin in control but not in LB cases. Immunoprecipitation with rab3a disclosed rab3a binding to rabphilin in control but not in LB cases. Moreover, rab3a interacted with high molecular weight (66 kDa) alpha-synuclein only in LB cases, in agreement with parallel studies using antibody arrays. Results were compared with pull-down assays using His(6)/Flag-tagged rab3, rab5 and rab8, and anti-Flag immunoblotting. Weak bands of 17 kDa, corresponding to alpha-synuclein, were obtained in LB and, less intensely, in control cases. In addition, alpha-synuclein-immunoreactive bands of high molecular weight (36 kDa) were seen only in LB cases after pull-down assays with rab3a, rab5 or rab8. These findings corroborate previous observations showing rab3a-rabphilin interactions in control brains, and add substantial information regarding decreased binding of rab3a to rabphilin and increased binding of rab3a to alpha-synuclein aggregates in LB cases. Since, alpha-synuclein, rab3a and rabphilin participate in the docking and fusion of synaptic vesicles, it can be suggested that exocytosis of neurotransmitters may be impaired in LB diseases.

    Neurobiology of disease 2004;16;1;92-7

  • Alpha-synuclein in blood and brain from familial Parkinson disease with SNCA locus triplication.

    Miller DW, Hague SM, Clarimon J, Baptista M, Gwinn-Hardy K, Cookson MR and Singleton AB

    Laboratory of Neurogenetics, National Institute of Neurological Disorders and Stroke, NIH, Bethesda, MD 20892, USA.

    The authors recently demonstrated that genetic triplication of the SNCA locus causes Parkinson disease. Here it is shown that SNCA triplication results in a doubling in the amount of alpha-synuclein protein in blood. Examination of brain tissue showed a doubling in the level of SNCA message. However, at the protein level in brain, there was a greater effect on deposition of aggregated forms into insoluble fractions than on net expression of soluble alpha-synuclein.

    Neurology 2004;62;10;1835-8

  • Alpha-synuclein has a high affinity for packing defects in a bilayer membrane: a thermodynamics study.

    Nuscher B, Kamp F, Mehnert T, Odoy S, Haass C, Kahle PJ and Beyer K

    Department of Biochemistry, Ludwig Maximilian University, 80336 Munich, Germany.

    A number of neurodegenerative disorders, including Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy, are characterized by the intracellular deposition of fibrillar aggregates that contain a high proportion of alpha-synuclein (alphaS). The interaction with the membrane-water interface strongly modulates folding and aggregation of the protein. The present study investigates the lipid binding and the coil-helix transition of alphaS, using titration calorimetry, differential scanning calorimetry, and circular dichroism spectroscopy. Titration of the protein with small unilamellar vesicles composed of zwitterionic phospholipids below the chain melting temperature of the lipids yielded exceptionally large exothermic heat values. The sigmoidal titration curves were evaluated in terms of a simple model that assumes saturable binding sites at the vesicle surface. The cumulative heat release and the ellipticity were linearly correlated as a result of simultaneous binding and helix folding. There was no heat release and folding of alphaS in the presence of large unilamellar vesicles, indicating that a small radius of curvature is necessary for the alphaS-membrane interaction. The heat release and the negative heat capacity of the protein-vesicle interaction could not be attributed to the coil-helix transition of the protein alone. We speculate that binding and helix folding of alphaS depends on the presence of defect structures in the membrane-water interface, which in turn results in lipid ordering in the highly curved vesicular membranes. This will be discussed with regard to a possible role of the protein for the stabilization of synaptic vesicle membranes.

    The Journal of biological chemistry 2004;279;21;21966-75

  • NMR of alpha-synuclein-polyamine complexes elucidates the mechanism and kinetics of induced aggregation.

    Fernández CO, Hoyer W, Zweckstetter M, Jares-Erijman EA, Subramaniam V, Griesinger C and Jovin TM

    LANAIS RMN 300, Laboratorio Nacional de Resonancia Magnética, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Buenos Aires, Argentina. cfernand@ffyb.uba.ar <cfernand@ffyb.uba.ar&gt;

    The aggregation of alpha-synuclein is characteristic of Parkinson's disease (PD) and other neurodegenerative synucleinopathies. The 140-aa protein is natively unstructured; thus, ligands binding to the monomeric form are of therapeutic interest. Biogenic polyamines promote the aggregation of alpha-synuclein and may constitute endogenous agents modulating the pathogenesis of PD. We characterized the complexes of natural and synthetic polyamines with alpha-synuclein by NMR and assigned the binding site to C-terminal residues 109-140. Dissociation constants were derived from chemical shift perturbations. Greater polyamine charge (+2 --> +5) correlated with increased affinity and enhancement of fibrillation, for which we propose a simple kinetic mechanism involving a dimeric nucleation center. According to the analysis, polyamines increase the extent of nucleation by approximately 10(4) and the rate of monomer addition approximately 40-fold. Significant secondary structure is not induced in monomeric alpha-synuclein by polyamines at 15 degrees C. Instead, NMR reveals changes in a region (aa 22-93) far removed from the polyamine binding site and presumed to adopt the beta-sheet conformation characteristic of fibrillar alpha-synuclein. We conclude that the C-terminal domain acts as a regulator of alpha-synuclein aggregation.

    The EMBO journal 2004;23;10;2039-46

  • alpha-Synuclein regulation of the dopaminergic transporter: a possible role in the pathogenesis of Parkinson's disease.

    Sidhu A, Wersinger C and Vernier P

    Laboratory of Molecular Neurochemistry, Department of Pediatrics, Georgetown University, The Research Building, Room W222, 3970 Reservoir Road, NW, Washington, DC 20007, USA. sidhua@georgetown.edu

    Parkinson's disease (PD) is a slow progressive neurodegenerative disorder. Recent evidence suggests a central role for alpha-synuclein, a protein of unknown function, in the genesis of PD. The phenomenon of selective degeneration of dopaminergic neurons in PD may be linked to the potential toxicity of dopamine itself and aberrations in the processes which regulate dopamine content may underlie the pathogenesis of this disease. Here, we review a vital role of alpha-synuclein in the modulation of dopamine transporter (DAT) function, and describe how disruption of this modulatory process permits increased re-uptake of high levels of intracellular dopamine by DAT, causing profound neurotoxicity.

    Funded by: NINDS NIH HHS: NS-34914, NS-45326

    FEBS letters 2004;565;1-3;1-5

  • Alpha-synuclein, Parkinson's disease, and Alzheimer's disease.

    Kim S, Seo JH and Suh YH

    Department of Pharmacology, College of Medicine, Neuroscience Research Institute, MRC, National Creative Research Initiative Center for Alzheimer's Dementia, Seoul National University, 28 Yongon-dong, Chongno-gu, Seoul 110-799, South Korea.

    Alpha synuclein (alpha-SN) is a ubiquitous protein that is especially abundant in the brain and has been postulated to play a central role in the pathogenesis of Parkinson's disease (PD), Alzheimer's disease, and other neurodegenerative disorders. Here, we show that alpha-SN plays dual role of neuroprotection and neurotoxicity depending on its concentration or level of expression. In addition, our study shows that alpha-synuclein is differentially expressed in human peripheral blood mononuclear cells. PD patients expressed more alpha-synuclein than healthy controls. Thus, the alpha-synuclein expression in the peripheral immune system might be one of the primary causes of immune abnormalities in PD patients.

    Parkinsonism & related disorders 2004;10 Suppl 1;S9-13

  • Effects of Parkinson's disease-linked mutations on the structure of lipid-associated alpha-synuclein.

    Bussell R and Eliezer D

    Department of Physiology, Biophysics and Molecular Medicine, Weill Medical College of Cornell University, 1300 York Avenue, New York, New York 10021, USA.

    Alpha-synuclein (alphaS) is a lipid-binding synaptic protein of unknown function that is found in an aggregated amyloid fibril form in the intraneuronal Lewy body deposits that are a defining characteristic of Parkinson's disease (PD). Although intrinsically unstructured when free in solution, alphaS adopts a highly helical conformation in association with lipid membranes or membrane mimetic detergent micelles. Two mutations in the alphaS gene have been linked to early onset autosomal dominant hereditary forms of PD, and have been shown to affect the aggregation kinetics of the protein in vitro. We have used high-resolution NMR spectroscopy, circular dichroism, and limited proteolysis to investigate the effects of these PD-linked mutations on the helical structure adopted by alphaS in the lipid or detergent micelle-bound form. We show that neither the A53T nor the A30P mutation has a significant effect on the structure of the folded protein, although the A30P mutation may cause a minor perturbation in the helical structure around the site of the mutation. The A30P, but not the A53T, mutation also appears to decrease the affinity of the protein for lipid surfaces, possibly by perturbing the nascent helical structure of the free protein. The potential implications of these results for the role of alphaS in PD are discussed.

    Funded by: NIA NIH HHS: AG19391, R01 AG019391, R01 AG019391-04, R37 AG019391

    Biochemistry 2004;43;16;4810-8

  • Role of individual methionines in the fibrillation of methionine-oxidized alpha-synuclein.

    Hokenson MJ, Uversky VN, Goers J, Yamin G, Munishkina LA and Fink AL

    Department of Chemistry and Biochemistry, University of California, Santa Cruz, California 95064, USA.

    The aggregation of normally soluble alpha-synuclein in the dopaminergic neurons of the substantia nigra is a crucial step in the pathogenesis of Parkinson's disease. Oxidative stress is believed to be a contributing factor in this disorder. We have previously established that oxidation of all four methionine residues in alpha-synuclein (to the sulfoxide, MetO) inhibits fibrillation of this protein in vitro and that the MetO protein also inhibits fibrillation of unmodified alpha-synuclein. Here we show that the degree of inhibition of fibrillation by MetO alpha-synuclein is proportional to the number of oxidized methionines. This was accomplished be selectively converting Met residues into Leu, prior to Met oxidation. The results showed that with one oxidized Met the kinetics of fibrillation were comparable to those for the control (nonoxidized), and with increasing numbers of methionine sulfoxides the kinetics of fibrillation became progressively slower. Electron microscope images showed that the fibril morphology was similar for all species examined, although fewer fibrils were observed with the oxidized forms. The presence of zinc was shown to overcome the Met oxidation-induced inhibition. Interestingly, substitution of Met by Leu led to increased propensity for aggregation (soluble oligomers) but slower formation of fibrils.

    Funded by: NINDS NIH HHS: NS39985

    Biochemistry 2004;43;15;4621-33

  • Alpha-synuclein up-regulation and aggregation during MPP+-induced apoptosis in neuroblastoma cells: intermediacy of transferrin receptor iron and hydrogen peroxide.

    Kalivendi SV, Cunningham S, Kotamraju S, Joseph J, Hillard CJ and Kalyanaraman B

    Department of Biophysics and Free Radical Research Center, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA.

    1-Methyl-4-phenylpyridinium (MPP(+)) is a neurotoxin that causes Parkinson's disease in experimental animals and humans. Despite the fact that intracellular iron was shown to be crucial for MPP(+)-induced apoptotic cell death, the molecular mechanisms for the iron requirement remain unclear. We investigated the role of transferrin receptor (TfR) and iron in modulating the expression of alpha-synuclein (alpha-syn) in MPP(+)-induced oxidative stress and apoptosis. Results show that MPP(+) inhibits mitochondrial complex-1 and aconitase activities leading to enhanced H(2)O(2) generation, TfR expression and alpha-syn expression/aggregation. Pretreatment with cell-permeable iron chelators, TfR antibody (that inhibits TfR-mediated iron uptake), or transfection with glutathione peroxidase (GPx1) enzyme inhibits intracellular oxidant generation, alpha-syn expression/aggregation, and apoptotic signaling as measured by caspase-3 activation. Cells overexpressing alpha-syn exacerbated MPP(+) toxicity, whereas antisense alpha-syn treatment totally abrogated MPP(+)-induced apoptosis in neuroblastoma cells without affecting oxidant generation. The increased cytotoxic effects of alpha-syn in MPP(+)-treated cells were attributed to inhibition of mitogen-activated protein kinase and proteasomal function. We conclude that MPP(+)-induced iron signaling is responsible for intracellular oxidant generation, alpha-syn expression, proteasomal dysfunction, and apoptosis. Relevance to Parkinson's disease is discussed.

    Funded by: NINDS NIH HHS: NS39958

    The Journal of biological chemistry 2004;279;15;15240-7

  • Decreases in soluble alpha-synuclein in frontal cortex correlate with cognitive decline in the elderly.

    Wang DS, Bennett DA, Mufson E, Cochran E and Dickson DW

    Department of Pathology (Neuropathology), Mayo Clinic, Jacksonville, FL 32224, USA.

    Alpha-synuclein (ASN) is a presynaptic protein and major component of Lewy bodies. It is considered important in the pathogenesis of Alzheimer's disease (AD), but its relevance to progressive cognitive decline in aging is largely unknown. To address this issue, ASN immunoreactivity was measured in frontal cortex of elderly individuals with no cognitive impairment (NCI), mild cognitive impairment (MCI) and early AD using a Western blot technique and a polyclonal antibody to ASN. ASN immunoreactivity was significantly lower in AD than in MCI and NCI, but there was no difference between MCI and NCI. The ASN immunoreactivity correlated with CERAD diagnosis, as well as Mini-Mental State Exam (MMSE) score, global neuropsychologic z-score and some, but not all, frontal neuropsychology measures. Clinical correlations were stronger for ASN than synaptophysin immunoreactivity assessed in a similar manner. The correlation with MMSE was robust when NCI cases were considered separately, suggesting that decreases in soluble ASN may be an early feature of cognitive decline in aging and AD.

    Funded by: NIA NIH HHS: P01-AG14449, P30-AG10161

    Neuroscience letters 2004;359;1-2;104-8

  • Genetics of Parkinson disease.

    Pankratz N and Foroud T

    Department of Medical and Molecular Genetics, Indiana University School of Medicine, Indianapolis, Indiana 46202-525, USA. nathan@pankratz.com

    Parkinson disease (PD) is the second most common neurodegenerative disorder. Recent studies have consistently demonstrated that in some families, disease is attributable to a mutation in a single gene. To date, genetic analyses have detected linkage to six chromosomal regions and have identified three causative genes: PARK1 (alpha-synuclein), PARK2 (parkin), and PARK7 (DJ-1). In addition, mutations in several other genes have been implicated in familial PD. Identification of the mutations in these genes has led to the recognition that the ubiquitin-proteasome system is an important pathway that may be disrupted in PD. Studies are ongoing to identify additional genes that may contribute to PD susceptibility, particularly in late-onset families without a clear pattern of disease inheritance. With the identification of mutations in particular genes and the likely role of additional genes that are important in PD risk-susceptibility, appropriate protocols must be developed so that accurate and informative genetic counseling can be offered to families in which one or more members has PD. Further diagnostic testing should be delayed until more is learned about the frequency, penetrance, and risk assessment of certain gene mutations. Important lessons can be learned from the implementation of counseling protocols for other neurodegenerative disorders, such as Huntington disease and Alzheimer disease.

    Funded by: NINDS NIH HHS: R01 NS37167

    NeuroRx : the journal of the American Society for Experimental NeuroTherapeutics 2004;1;2;235-42

  • Proteasomal inhibition by alpha-synuclein filaments and oligomers.

    Lindersson E, Beedholm R, Højrup P, Moos T, Gai W, Hendil KB and Jensen PH

    Department of Medical Biochemistry, University of Aarhus, 8000 Aarhus-C, Denmark.

    A unifying feature of many neurodegenerative disorders is the accumulation of polyubiquitinated protein inclusions in dystrophic neurons, e.g. containing alpha-synuclein, which is suggestive of an insufficient proteasomal activity. We demonstrate that alpha-synuclein and 20 S proteasome components co-localize in Lewy bodies and show that subunits from 20 S proteasome particles, in contrast to subunits of the 19 S regulatory complex, bind efficiently to aggregated filamentous but not monomeric alpha-synuclein. Proteasome binding to insoluble alpha-synuclein filaments and soluble alpha-synuclein oligomers results in marked inhibition of its chymotrypsin-like hydrolytic activity through a non-competitive mechanism that is mimicked by model amyloid-Abeta peptide aggregates. Endogenous ligands of aggregated alpha-synuclein like heat shock protein 70 and glyceraldehyde-6-phosphate dehydrogenase bind filaments and inhibit their anti-proteasomal activity. The inhibitory effect of amyloid aggregates may thus be amenable to modulation by endogenous chaperones and possibly accessible for therapeutic intervention.

    The Journal of biological chemistry 2004;279;13;12924-34

  • Tyrosine-to-cysteine modification of human alpha-synuclein enhances protein aggregation and cellular toxicity.