G2Cdb::Gene report

Gene id
Gene symbol
Homo sapiens
G00001247 (Mus musculus)

Databases (8)

ENSG00000197386 (Ensembl human gene)
3064 (Entrez Gene)
261 (G2Cdb plasticity & disease)
HD (GeneCards)
143100 (OMIM)
Marker Symbol
HGNC:4851 (HGNC)
Protein Expression
2756 (human protein atlas)
Protein Sequence
P42858 (UniProt)

Synonyms (1)

  • IT15

Literature (274)

Pubmed - other

  • A double blind evaluation of cognitive decline in a Norwegian cohort of asymptomatic carriers of Huntington's disease.

    van Walsem MR, Sundet K, Retterstøl L and Sundseth Ø

    Oslo University Hospital, Rikshospitalet, Centre for Rare Disorders, Oslo, Norway.

    Previous studies investigating subclinical signs of cognitive decline in presymptomatic carriers of Huntington's disease (HD) have shown conflicting results. The current study examines cognition in 105 at-risk individuals, using a broad neuropsychological test battery and adopting strict inclusion criteria for attaining a homogeneous sample. Results obtained by analyses of variance and effect size calculations indicate no clinical evidence of significant cognitive decline in asymptomatic HD carriers very far from onset of illness compared to noncarriers. Closeness to disease onset amongst gene carriers influenced cognition negatively whereas cytosine-adenine-guanine (CAG) repeat size did not. The findings call for longitudinal follow-up studies using a combination of clinical instruments and experimental paradigms to pinpoint when subtle cognitive deficits occur and within which of the cognitive domains.

    Journal of clinical and experimental neuropsychology 2010;32;6;590-8

  • Protein folding: sticky N17 speeds huntingtin pile-up.

    Liebman SW and Meredith SC

    Funded by: NIGMS NIH HHS: R01 GM056350

    Nature chemical biology 2010;6;1;7-8

  • Expression of mutant huntingtin in mouse brain astrocytes causes age-dependent neurological symptoms.

    Bradford J, Shin JY, Roberts M, Wang CE, Li XJ and Li S

    Department of Human Genetics, Emory University School of Medicine, 615 Michael Street, Atlanta, GA 30322, USA.

    Huntington disease (HD) is an inherited neurological disorder caused by a polyglutamine expansion in the protein huntingtin and is characterized by selective neurodegeneration that preferentially occurs in striatal medium spiny neurons. Because the medium spiny neurons are innervated abundantly by glutamatergic axons from cortical neurons, the preferential degeneration in the striatal neurons supports the glutamate excitotoxicity theory for HD pathogenesis. Thus, glutamate uptake by glia may be particularly important for preventing glutamate excitotoxicity in HD. Although mutant huntingtin is expressed ubiquitously in various types of cells, it accumulates and forms aggregates in fewer glial cells than in neuronal cells. It remains largely unknown whether and how mutant huntingtin in glia can contribute to the neurological symptoms of HD. We generated transgenic mice that express N-terminal mutant huntingtin in astrocytes, a major type of glial cell that remove extracellular glutamate in the brain. Although transgenic mutant huntingtin in astrocytes is expressed below the endogenous level, it can cause age-dependent neurological phenotypes in transgenic mice. Mice expressing mutant huntingtin show body weight loss, have motor function deficits, and die earlier than wild-type or control transgenic mice. We also found that mutant huntingtin in astrocytes decreases the expression of glutamate transporter by increasing its binding to Sp1 and reducing the association of Sp1 with the promoter of glutamate transporter. These results imply an important role for glial mutant huntingtin in HD pathology and suggest possibilities for treatment.

    Funded by: NIA NIH HHS: AG031153, AG19206, R01 AG019206, R01 AG031153; NIGMS NIH HHS: T32 GM008490; NINDS NIH HHS: NS045016, NS36232, R01 NS036232, R01 NS041669, R01 NS045016

    Proceedings of the National Academy of Sciences of the United States of America 2009;106;52;22480-5

  • Serines 13 and 16 are critical determinants of full-length human mutant huntingtin induced disease pathogenesis in HD mice.

    Gu X, Greiner ER, Mishra R, Kodali R, Osmand A, Finkbeiner S, Steffan JS, Thompson LM, Wetzel R and Yang XW

    Center for Neurobehavioral Genetics, The Jane and Terry Semel Institute for Neuroscience and Human Behavior, David Geffen School of Medicine at University of California, Los Angeles, Los Angeles, CA 90095, USA.

    The N-terminal 17 amino acids of huntingtin (NT17) can be phosphorylated on serines 13 and 16; however, the significance of these modifications in Huntington's disease pathogenesis remains unknown. In this study, we developed BAC transgenic mice expressing full-length mutant huntingtin (fl-mhtt) with serines 13 and 16 mutated to either aspartate (phosphomimetic or SD) or alanine (phosphoresistant or SA). Both mutant proteins preserve the essential function of huntingtin in rescuing knockout mouse phenotypes. However, fl-mhtt-induced disease pathogenesis, including motor and psychiatric-like behavioral deficits, mhtt aggregation, and selective neurodegeneration are abolished in SD but preserved in SA mice. Moreover, modification of these serines in expanded repeat huntingtin peptides modulates aggregation and amyloid fibril formation in vitro. Together, our findings demonstrate that serines 13 and 16 are critical determinants of fl-mhtt-induced disease pathogenesis in vivo, supporting the targeting of huntingtin NT17 domain and its modifications in HD therapy.

    Funded by: NIA NIH HHS: 2P01AG022074, P01 AG022074, R01 AG019322, R01AG019322; NINDS NIH HHS: 2R01NS39074, 2R01NS45491, 3R01NS049501-05S1, NS045283, NS52789, R01 NS039074, R01 NS045283, R01 NS049501, R01 NS049501-01, R01 NS049501-02, R01 NS049501-03, R01 NS049501-04, R01 NS049501-05, R01 NS049501-05S1, R01 NS052789, R01NS049501, R24 NS045491

    Neuron 2009;64;6;828-40

  • Dopamine D2 receptor stimulation potentiates PolyQ-Huntingtin-induced mouse striatal neuron dysfunctions via Rho/ROCK-II activation.

    Deyts C, Galan-Rodriguez B, Martin E, Bouveyron N, Roze E, Charvin D, Caboche J and Bétuing S

    CNRS UMR 7102, Université Pierre et Marie Curie-Paris 6, Paris, France.

    Background: Huntington's disease (HD) is a polyglutamine-expanded related neurodegenerative disease. Despite the ubiquitous expression of expanded, polyQ-Huntingtin (ExpHtt) in the brain, striatal neurons present a higher susceptibility to the mutation. A commonly admitted hypothesis is that Dopaminergic inputs participate to this vulnerability. We previously showed that D2 receptor stimulation increased aggregate formation and neuronal death induced by ExpHtt in primary striatal neurons in culture, and chronic D2 antagonist treatment protects striatal dysfunctions induced by ExpHtt in a lentiviral-induced model system in vivo. The present work was designed to elucidate the signalling pathways involved, downstream D2 receptor (D2R) stimulation, in striatal vulnerability to ExpHtt.

    Using primary striatal neurons in culture, transfected with a tagged-GFP version of human exon 1 ExpHtt, and siRNAs against D2R or D1R, we confirm that DA potentiates neuronal dysfunctions via D2R but not D1R stimulation. We demonstrate that D2 agonist treatment induces neuritic retraction and growth cone collapse in Htt- and ExpHtt expressing neurons. We then tested a possible involvement of the Rho/ROCK signalling pathway, which plays a key role in the dynamic of the cytoskeleton, in these processes. The pharmacological inhibitors of ROCK (Y27632 and Hydroxyfasudil), as well as siRNAs against ROCK-II, reversed D2-related effects on neuritic retraction and growth cone collapse. We show a coupling between D2 receptor stimulation and Rho activation, as well as hyperphosphorylation of Cofilin, a downstream effector of ROCK-II pathway. Importantly, D2 agonist-mediated potentiation of aggregate formation and neuronal death induced by ExpHtt, was totally reversed by Y27632 and Hydroxyfasudil and ROCK-II siRNAs.

    Our data provide the first demonstration that D2R-induced vulnerability in HD is critically linked to the activation of the Rho/ROCK signalling pathway. The inclusion of Rho/ROCK inhibitors could be an interesting therapeutic option aimed at forestalling the onset of the disease.

    PloS one 2009;4;12;e8287

  • The chaperonin TRiC blocks a huntingtin sequence element that promotes the conformational switch to aggregation.

    Tam S, Spiess C, Auyeung W, Joachimiak L, Chen B, Poirier MA and Frydman J

    Department of Biology, BioX Program Stanford University, Stanford, California, USA.

    Aggregation of proteins containing polyglutamine (polyQ) expansions characterizes many neurodegenerative disorders, including Huntington's disease. Molecular chaperones modulate the aggregation and toxicity of the huntingtin (Htt) protein by an ill-defined mechanism. Here we determine how the chaperonin TRiC suppresses Htt aggregation. Unexpectedly, TRiC does not physically block the polyQ tract itself, but rather sequesters a short Htt sequence element, N-terminal to the polyQ tract, that promotes the amyloidogenic conformation. The residues of this element essential for rapid Htt aggregation are directly bound by TRiC. Our findings illustrate how molecular chaperones, which recognize hydrophobic determinants, can prevent aggregation of polar polyQ tracts associated with neurodegenerative diseases. The observation that short endogenous sequence elements can accelerate the switch of polyQ tracts to an amyloidogenic conformation provides a novel target for therapeutic strategies.

    Funded by: NEI NIH HHS: PN2 EY016525, PN2 EY016525-04, PN2 EY016525-04S1, PN2 EY016525-05, PN2 EY016525-05S1, PN2 EY016525-06; NIGMS NIH HHS: GM74074, R01 GM074074, R01 GM074074-01S1, R01 GM074074-02, R01 GM074074-03, R01 GM074074-04

    Nature structural & molecular biology 2009;16;12;1279-85

  • Mutant huntingtin impairs vesicle formation from recycling endosomes by interfering with Rab11 activity.

    Li X, Standley C, Sapp E, Valencia A, Qin ZH, Kegel KB, Yoder J, Comer-Tierney LA, Esteves M, Chase K, Alexander J, Masso N, Sobin L, Bellve K, Tuft R, Lifshitz L, Fogarty K, Aronin N and DiFiglia M

    Department of Neurology, Massachusetts General Hospital and Harvard Medical School, Charlestown, Massachusetts 02129, USA.

    Huntingtin (Htt) localizes to endosomes, but its role in the endocytic pathway is not established. Recently, we found that Htt is important for the activation of Rab11, a GTPase involved in endosomal recycling. Here we studied fibroblasts of healthy individuals and patients with Huntington's disease (HD), which is a movement disorder caused by polyglutamine expansion in Htt. The formation of endocytic vesicles containing transferrin at plasma membranes was the same in control and HD patient fibroblasts. However, HD fibroblasts were delayed in recycling biotin-transferrin back to the plasma membrane. Membranes of HD fibroblasts supported less nucleotide exchange on Rab11 than did control membranes. Rab11-positive vesicular and tubular structures in HD fibroblasts were abnormally large, suggesting that they were impaired in forming vesicles. We used total internal reflection fluorescence imaging of living fibroblasts to monitor fluorescence-labeled transferrin-carrying transport intermediates that emerged from recycling endosomes. HD fibroblasts had fewer small vesicles and more large vesicles and long tubules than did control fibroblasts. Dominant active Rab11 expressed in HD fibroblasts normalized the recycling of biotin-transferrin. We propose a novel mechanism for cellular dysfunction by the HD mutation arising from the inhibition of Rab11 activity and a deficit in vesicle formation at recycling endosomes.

    Funded by: NIDDK NIH HHS: DK P30-32520; NINDS NIH HHS: NS R01-38194

    Molecular and cellular biology 2009;29;22;6106-16

  • Phosphorylation of threonine 3: implications for Huntingtin aggregation and neurotoxicity.

    Aiken CT, Steffan JS, Guerrero CM, Khashwji H, Lukacsovich T, Simmons D, Purcell JM, Menhaji K, Zhu YZ, Green K, Laferla F, Huang L, Thompson LM and Marsh JL

    Department of Developmental and Cell Biology, University of California, Irvine, California 92697, USA.

    Huntingtin (Htt) is a widely expressed protein that causes tissue-specific degeneration when mutated to contain an expanded polyglutamine (poly(Q)) domain. Although Htt is large, 350 kDa, the appearance of amino-terminal fragments of Htt in extracts of postmortem brain tissue from patients with Huntington disease (HD), and the fact that an amino-terminal fragment, Htt exon 1 protein (Httex1p), is sufficient to cause disease in models of HD, points to the importance of the amino-terminal region of Htt in the disease process. The first exon of Htt encodes 17 amino acids followed by a poly(Q) repeat of variable length and culminating with a proline-rich domain of 50 amino acids. Because modifications to this fragment have the potential to directly affect pathogenesis in several ways, we have surveyed this fragment for potential post-translational modifications that might affect Htt behavior and detected several modifications of Httex1p. Here we report that the most prevalent modifications of Httex1p are NH(2)-terminal acetylation and phosphorylation of threonine 3 (pThr-3). We demonstrate that pThr-3 occurs on full-length Htt in vivo, and that this modification affects the aggregation and pathogenic properties of Htt. Thus, therapeutic strategies that modulate these events could in turn affect Htt pathogenesis.

    Funded by: NCI NIH HHS: CA-62203, P30 CA062203; NICHD NIH HHS: HD36081, R01 HD036081; NIGMS NIH HHS: GM-74830, R01 GM074830, T32GM0731130; NINDS NIH HHS: NS045283, NS52789, R01 NS045283, R01 NS052789

    The Journal of biological chemistry 2009;284;43;29427-36

  • Normal and mutant HTT interact to affect clinical severity and progression in Huntington disease.

    Aziz NA, Jurgens CK, Landwehrmeyer GB, EHDN Registry Study Group, van Roon-Mom WM, van Ommen GJ, Stijnen T and Roos RA

    Leiden University Medical Center, Department of Neurology, Leiden, The Netherlands. N.A.Aziz@lumc.nl

    Objective: Huntington disease (HD) is an autosomal dominant neurodegenerative disorder caused by a CAG repeat expansion in the HD gene (HTT). We aimed to assess whether interaction between CAG repeat sizes in the mutant and normal allele could affect disease severity and progression.

    Methods: Using linear regression and mixed-effects models, the influence of mutant and normal CAG repeat sizes interaction was assessed on 1) age at onset in 921 patients with HD, 2) clinical severity and progression in 512 of these patients with follow-up data available, and 3) basal ganglia volume on magnetic resonance images in 16 premanifest HD mutation carriers.

    Results: Normal and mutant CAG repeat sizes interacted to influence 1) age at onset (p = 0.001), 2) severity or progression of motor, cognitive, and functional, but not behavioral, symptoms in patients with HD (all p < 0.05), and 3) in premanifest subjects, basal ganglia volumes (p < 0.05). In subjects with mutant CAG expansions in the low range, increasing size of the normal repeat correlated with more severe symptoms and pathology, whereas for those subjects with expansions in the high range, increasing size of the normal repeat correlated with less severe symptoms and pathology.

    Conclusions: Increasing CAG repeat size in normal HTT diminishes the association between mutant CAG repeat size and disease severity and progression in Huntington disease. The underlying mechanism may involve interaction of the polyglutamine domains of normal and mutant huntingtin (fragments) and needs further elucidation. These findings may have predictive value and are essential for the design and interpretation of future therapeutic trials.

    Neurology 2009;73;16;1280-5

  • Mutant huntingtin interacts with {beta}-tubulin and disrupts vesicular transport and insulin secretion.

    Smith R, Bacos K, Fedele V, Soulet D, Walz HA, Obermüller S, Lindqvist A, Björkqvist M, Klein P, Onnerfjord P, Brundin P, Mulder H and Li JY

    Department of Experimental Medical Science, Lund University, Sweden.

    Huntington's disease is a severe progressive neurodegenerative disorder caused by a CAG expansion in the IT15 gene, which encodes huntingtin. The disease primarily affects the neostriatum and cerebral cortex and also associates with increased incidence of diabetes. Here, we show that mutant huntingtin disrupts intracellular transport and insulin secretion by direct interference with microtubular beta-tubulin. We demonstrate that mutant huntingtin impairs glucose-stimulated insulin secretion in insulin-producing beta-cells, without altering stored levels of insulin. Using VSVG-YFP, we show that mutant huntingtin retards post-Golgi transport. Moreover, we demonstrate that the speed of insulin vesicle trafficking is reduced. Using immunoprecipitation of mutant and wild-type huntingtin in combination with mass spectrometry, we reveal an enhanced and aberrant interaction between mutant huntingtin and beta-tubulin, implying the underlying mechanism of impaired intracellular transport. Thus, our findings have revealed a novel pathogenetic process by which mutant huntingtin may disrupt hormone exocytosis from beta-cells and possibly impair vesicular transport in any cell that expresses the pathogenic protein.

    Human molecular genetics 2009;18;20;3942-54

  • Secondary structure of Huntingtin amino-terminal region.

    Kim MW, Chelliah Y, Kim SW, Otwinowski Z and Bezprozvanny I

    Department of Biochemistry, University of Texas Southwestern Medical Center at Dallas, Dallas, TX 75390, USA. MeeWhi.Kim@UTSouthwestern.edu

    Huntington's disease is a genetic neurodegenerative disorder resulting from polyglutamine (polyQ) expansion (>36Q) within the first exon of Huntingtin (Htt) protein. We applied X-ray crystallography to determine the secondary structure of the first exon (EX1) of Htt17Q. The structure of Htt17Q-EX1 consists of an amino-terminal alpha helix, poly17Q region, and polyproline helix formed by the proline-rich region. The poly17Q region adopts multiple conformations in the structure, including alpha helix, random coil, and extended loop. The conformation of the poly17Q region is influenced by the conformation of neighboring protein regions, demonstrating the importance of the native protein context. We propose that the conformational flexibility of the polyQ region observed in our structure is a common characteristic of many amyloidogenic proteins. We further propose that the pathogenic polyQ expansion in the Htt protein increases the length of the random coil, which promotes aggregation and facilitates abnormal interactions with other proteins in cells.

    Funded by: Howard Hughes Medical Institute; NIGMS NIH HHS: GM053163, R01 GM053163, R01 GM053163-07; NINDS NIH HHS: NS056224, R01 NS056224, R01 NS056224-02

    Structure (London, England : 1993) 2009;17;9;1205-12

  • Interaction of postsynaptic density protein-95 with NMDA receptors influences excitotoxicity in the yeast artificial chromosome mouse model of Huntington's disease.

    Fan J, Cowan CM, Zhang LY, Hayden MR and Raymond LA

    Graduate Program in Neuroscience, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada.

    Evidence suggests that NMDA-type glutamate receptors contribute to degeneration of striatal medium-sized spiny neurons (MSNs) in Huntington's disease (HD). Previously, we demonstrated that NMDA receptor (NMDAR)-mediated current and/or toxicity is increased in MSNs from the yeast artificial chromosome (YAC) transgenic mouse model expressing polyglutamine (polyQ)-expanded (mutant) full-length human huntingtin (htt). Others have shown that membrane-associated guanylate kinases (MAGUKs), such as PSD-95 and SAP102, modulate NMDAR surface expression and excitotoxicity in hippocampal and cortical neurons and that htt interacts with PSD-95. Here, we tested the hypothesis that an altered association between MAGUKs and NMDARs in mutant huntingtin-expressing cells contributes to increased susceptibility to excitotoxicity. We show that htt coimmunoprecipitated with SAP102 in HEK293T cells and striatal tissue from wild-type and YAC transgenic mice; however, the association of SAP102 with htt or the NMDAR NR2B subunit was unaffected by htt polyQ length, whereas association of PSD-95 with NR2B in striatal tissue was enhanced by increased htt polyQ length. Treatment of cultured MSNs with Tat-NR2B9c peptide blocked binding of NR2B with SAP102 and PSD-95 and reduced NMDAR surface expression by 20% in both YAC transgenic and wild-type MSNs, and also restored susceptibility to NMDAR excitoxicity in YAC HD MSNs to levels observed in wild-type MSNs; a similar effect on excitotoxicity was observed after knockdown of PSD-95 by small interfering RNA. Unlike previous findings in cortical and hippocampal neurons, rescue of NMDA toxicity by Tat-NR2B9c occurred independently of any effect on neuronal nitric oxide synthase activity. Our results elucidate further the mechanisms underlying enhanced excitotoxicity in HD.

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2009;29;35;10928-38

  • Polyglutamine expansion in huntingtin alters its interaction with phospholipids.

    Kegel KB, Sapp E, Alexander J, Valencia A, Reeves P, Li X, Masso N, Sobin L, Aronin N and DiFiglia M

    Laboratory of Cellular Neurobiology, Department of Neurology, Massachusetts General Hospital, 11416th Street, Room 2150, Charlestown, MA 02129, USA. kkegel@partners.org

    Huntingtin has an expanded polyglutamine tract in patients with Huntington's disease. Huntingtin localizes to intracellular and plasma membranes but the function of huntingtin at membranes is unknown. Previously we reported that exogenously expressed huntingtin bound pure phospholipids using protein-lipid overlays. Here we show that endogenous huntingtin from normal (Hdh(7Q/7Q)) mouse brain and mutant huntingtin from Huntington's disease (Hdh(140Q/140Q)) mouse brain bound to large unilamellar vesicles containing phosphoinositol (PI) PI 3,4-bisphosphate, PI 3,5-bisphosphate, and PI 3,4,5-triphosphate [PI(3,4,5)P3]. Huntingtin interactions with multivalent phospholipids were similar to those of dynamin. Mutant huntingtin associated more with phosphatidylethanolamine and PI(3,4,5)P3 than did wild-type huntingtin, and associated with other phospholipids not recognized by wild-type huntingtin. Wild-type and mutant huntingtin also bound to large unilamellar vesicles containing cardiolipin, a phospholipid specific to mitochondrial membranes. Maximal huntingtin-phospholipid association required inclusion of huntingtin amino acids 171-287. Endogenous huntingtin recruited to the plasma membrane in cells that incorporated exogenous PI 3,4-bisphosphate and PI(3,4,5)P3 or were stimulated by platelet-derived growth factor or insulin growth factor 1, which both activate PI 3-kinase. These data suggest that huntingtin interacts with membranes through specific phospholipid associations and that mutant huntingtin may disrupt membrane trafficking and signaling at membranes.

    Journal of neurochemistry 2009;110;5;1585-97

  • HSP40 ameliorates impairment of insulin secretion by inhibiting huntingtin aggregation in a HD pancreatic beta cell model.

    Ye CF and Li H

    Division of Histology and Embryology, Department of Anatomy, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, PR China.

    Diabetes frequently develops in Huntington's disease patients. Here, we found that mutant huntingtin forms aggregates in the cytoplasm and reduces insulin secretion from huntingtin transfected pancreatic beta cell lines, NIT-1 cells. Activity of the pro-survival factor, Akt, is enhanced in these cells, which might improve the maintenance of insulin content. Overexpression of heat shock protein 40 (HSP40) inhibits aggregation, reverses impaired insulin release, and blocks the enhancement of Akt activity. These results suggest that impairment of beta cells is mostly linked with the aggregate formation of mutant huntingtin, and that HSP40 ameliorates the malfunction of pancreatic beta cells by inhibiting aggregation.

    Bioscience, biotechnology, and biochemistry 2009;73;8;1787-92

  • Progress and challenges in RNA interference therapy for Huntington disease.

    Harper SQ

    Department of Pediatrics, The Ohio State University Medical Center, and Center for Gene Therapy, The Research Institute at Nationwide Children's Hospital, 700 Children's Dr, Room WA2015, Columbus, OH 43205, USA. harper.104@osu.edu

    Huntington disease is an incurable, dominant neurodegenerative disorder caused by polyglutamine repeat expansion in the huntingtin protein. Reducing mutant huntingtin expression may offer a treatment for Huntington disease. RNA interference has emerged as a powerful method to silence dominant disease genes. As such, it is being developed as a prospective Huntington disease therapy. Here I discuss the current progress and important remaining challenges of RNA interference therapy for Huntington disease.

    Archives of neurology 2009;66;8;933-8

  • Pathogenic huntingtin inhibits fast axonal transport by activating JNK3 and phosphorylating kinesin.

    Morfini GA, You YM, Pollema SL, Kaminska A, Liu K, Yoshioka K, Björkblom B, Coffey ET, Bagnato C, Han D, Huang CF, Banker G, Pigino G and Brady ST

    Department of Anatomy and Cell Biology, University of Illinois at Chicago, USA. gmorfini@uic.edu

    Selected vulnerability of neurons in Huntington's disease suggests that alterations occur in a cellular process that is particularly critical for neuronal function. Supporting this idea, pathogenic Htt (polyQ-Htt) inhibits fast axonal transport (FAT) in various cellular and animal models of Huntington's disease (mouse and squid), but the molecular basis of this effect remains unknown. We found that polyQ-Htt inhibited FAT through a mechanism involving activation of axonal cJun N-terminal kinase (JNK). Accordingly, we observed increased activation of JNK in vivo in cellular and mouse models of Huntington's disease. Additional experiments indicated that the effects of polyQ-Htt on FAT were mediated by neuron-specific JNK3 and not by ubiquitously expressed JNK1, providing a molecular basis for neuron-specific pathology in Huntington's disease. Mass spectrometry identified a residue in the kinesin-1 motor domain that was phosphorylated by JNK3 and this modification reduced kinesin-1 binding to microtubules. These data identify JNK3 as a critical mediator of polyQ-Htt toxicity and provide a molecular basis for polyQ-Htt-induced inhibition of FAT.

    Funded by: NIMH NIH HHS: MH066179, R01 MH066179; NINDS NIH HHS: NS23320, NS23868, NS41170, R01 NS023320, R01 NS023868, R01 NS023868-22, R01 NS041170, R01 NS041170-06, R56 NS023868

    Nature neuroscience 2009;12;7;864-71

  • Wild-type but not mutant huntingtin modulates the transcriptional activity of liver X receptors.

    Futter M, Diekmann H, Schoenmakers E, Sadiq O, Chatterjee K and Rubinsztein DC

    CIMR, Medical Genetics, Wellcome Trust/MRC Building, Addenbrooke's Hospital, Cambridge, UK.

    Background: Huntington's disease is caused by expansion of a polyglutamine tract found in the amino-terminal of the ubiquitously expressed protein huntingtin. Well studied in its mutant form, huntingtin has a wide variety of normal functions, loss of which may also contribute to disease progression. Widespread transcriptional dysfunction occurs in brains of Huntington's disease patients and in transgenic mouse and cell models of Huntington's disease.

    Methods: To identify new transcriptional pathways altered by the normal and/or abnormal function of huntingtin, we probed several nuclear receptors, normally expressed in the brain, for binding to huntingtin in its mutant and wild-type forms.

    Results: Wild-type huntingtin could bind to a number of nuclear receptors; LXRalpha, PPARgamma, VDR and TRalpha1. Over-expression of huntingtin activated, while knockout of huntingtin decreased, LXR mediated transcription of a reporter gene. Loss of huntingtin also decreased expression of the LXR target gene, ABCA1. In vivo, huntingtin deficient zebrafish had a severe phenotype and reduced expression of LXR regulated genes. An LXR agonist was able to partially rescue the phenotype and the expression of LXR target genes in huntingtin deficient zebrafish during early development.

    Conclusion: Our data suggest a novel function for wild-type huntingtin as a co-factor of LXR. However, this activity is lost by mutant huntingtin that only interacts weakly with LXR.

    Funded by: Medical Research Council: G0400066, G0600194, G0600194(77639); Wellcome Trust: 064354

    Journal of medical genetics 2009;46;7;438-46

  • Rhes, a striatal specific protein, mediates mutant-huntingtin cytotoxicity.

    Subramaniam S, Sixt KM, Barrow R and Snyder SH

    Solomon H. Snyder Department of Neuroscience, Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205, USA.

    Huntington's disease (HD) is caused by a polyglutamine repeat in the protein huntingtin (Htt) with mutant Htt (mHtt) expressed throughout the body and similarly in all brain regions. Yet, HD neuropathology is largely restricted to the corpus striatum. We report that the small guanine nucleotide-binding protein Rhes, which is localized very selectively to the striatum, binds physiologically to mHtt. Using cultured cells, we found Rhes induces sumoylation of mHtt, which leads to cytotoxicity. Thus, Rhes-mHtt interactions can account for the localized neuropathology of HD.

    Funded by: NIDA NIH HHS: DA00074, K05 DA000074; NIMH NIH HHS: MH18501, R01 MH018501, R37 MH018501, R37 MH018501-40

    Science (New York, N.Y.) 2009;324;5932;1327-30

  • IKKalpha and IKKbeta regulation of DNA damage-induced cleavage of huntingtin.

    Khoshnan A, Ko J, Tescu S, Brundin P and Patterson PH

    Biology Division 216-76, California Institute of Technology, Pasadena, California, USA. khoshnan@caltech.edu

    Background: Proteolysis of huntingtin (Htt) plays a key role in the pathogenesis of Huntington's disease (HD). However, the environmental cues and signaling pathways that regulate Htt proteolysis are poorly understood. One stimulus may be the DNA damage that accumulates in neurons over time, and the subsequent activation of signaling pathways such as those regulated by IkappaB kinase (IKK), which can influence neurodegeneration in HD.

    We asked whether DNA damage induces the proteolysis of Htt and if activation of IKK plays a role. We report that treatment of neurons with the DNA damaging agent etoposide or gamma-irradiation promotes cleavage of wild type (WT) and mutant Htt, generating N-terminal fragments of 80-90 kDa. This event requires IKKbeta and is suppressed by IKKalpha. Elevated levels of IKKalpha, or inhibition of IKKbeta expression by a specific small hairpin RNA (shRNA) or its activity by sodium salicylate, prevents Htt proteolysis and increases neuronal resistance to DNA damage. Moreover, IKKbeta phosphorylates the anti-apoptotic protein Bcl-xL, a modification known to reduce Bcl-xL levels, and activates caspases that can cleave Htt. When IKKbeta expression is blocked, etoposide treatment does not decrease Bcl-xL and activation of caspases is diminished. Similar to silencing of IKKbeta, increasing the level of Bcl-xL in neurons prevents etoposide-induced caspase activation and Htt proteolysis.

    These results indicate that DNA damage triggers cleavage of Htt and identify IKKbeta as a prominent regulator. Moreover, IKKbeta-dependent reduction of Bcl-xL is important in this process. Thus, inhibition of IKKbeta may promote neuronal survival in HD as well as other DNA damage-induced neurodegenerative disorders.

    Funded by: NINDS NIH HHS: 5R01NS55298, R01 NS055298

    PloS one 2009;4;6;e5768

  • A majority of Huntington's disease patients may be treatable by individualized allele-specific RNA interference.

    Lombardi MS, Jaspers L, Spronkmans C, Gellera C, Taroni F, Di Maria E, Donato SD and Kaemmerer WF

    Medtronic Bakken Research Center, Corporate Science and Technology, 6229 GW Maastricht, The Netherlands.

    Use of RNA interference to reduce huntingtin protein (htt) expression in affected brain regions may provide an effective treatment for Huntington disease (HD), but it remains uncertain whether suppression of both wild-type and mutant alleles in a heterozygous patient will provide more benefit than harm. Previous research has shown suppression of just the mutant allele is achievable using siRNA targeted to regions of HD mRNA containing single nucleotide polymorphisms (SNPs). To determine whether more than a minority of patients may be eligible for an allele-specific therapy, we genotyped DNA from 327 unrelated European Caucasian HD patients at 26 SNP sites in the HD gene. Over 86% of the patients were found to be heterozygous for at least one SNP among those tested. Because the sites are genetically linked, one cannot use the heterozygosity rates of the individual SNPs to predict how many sites (and corresponding allele-specific siRNA) would be needed to provide at least one treatment possibility for this percentage of patients. By computing all combinations, we found that a repertoire of allele-specific siRNA corresponding to seven sites can provide at least one allele-specific siRNA treatment option for 85.6% of our sample. Moreover, we provide evidence that allele-specific siRNA targeting these sites are readily identifiable using a high throughput screening method, and that allele-specific siRNA identified using this method indeed show selective suppression of endogenous mutant htt protein in fibroblast cells from HD patients. Therefore, allele-specific siRNA are not so rare as to be impractical to find and use therapeutically.

    Experimental neurology 2009;217;2;312-9

  • Conformational targeting of fibrillar polyglutamine proteins in live cells escalates aggregation and cytotoxicity.

    Kvam E, Nannenga BL, Wang MS, Jia Z, Sierks MR and Messer A

    Wadsworth Center, New York State Department of Health, Albany, New York, United States of America.

    Background: Misfolding- and aggregation-prone proteins underlying Parkinson's, Huntington's and Machado-Joseph diseases, namely alpha-synuclein, huntingtin, and ataxin-3 respectively, adopt numerous intracellular conformations during pathogenesis, including globular intermediates and insoluble amyloid-like fibrils. Such conformational diversity has complicated research into amyloid-associated intracellular dysfunction and neurodegeneration. To this end, recombinant single-chain Fv antibodies (scFvs) are compelling molecular tools that can be selected against specific protein conformations, and expressed inside cells as intrabodies, for investigative and therapeutic purposes.

    Using atomic force microscopy (AFM) and live-cell fluorescence microscopy, we report that a human scFv selected against the fibrillar form of alpha-synuclein targets isomorphic conformations of misfolded polyglutamine proteins. When expressed in the cytoplasm of striatal cells, this conformation-specific intrabody co-localizes with intracellular aggregates of misfolded ataxin-3 and a pathological fragment of huntingtin, and enhances the aggregation propensity of both disease-linked polyglutamine proteins. Using this intrabody as a tool for modulating the kinetics of amyloidogenesis, we show that escalating aggregate formation of a pathologic huntingtin fragment is not cytoprotective in striatal cells, but rather heightens oxidative stress and cell death as detected by flow cytometry. Instead, cellular protection is achieved by suppressing aggregation using a previously described intrabody that binds to the amyloidogenic N-terminus of huntingtin. Analogous cytotoxic results are observed following conformational targeting of normal or polyglutamine-expanded human ataxin-3, which partially aggregate through non-polyglutamine domains.

    These findings validate that the rate of aggregation modulates polyglutamine-mediated intracellular dysfunction, and caution that molecules designed to specifically hasten aggregation may be detrimental as therapies for polyglutamine disorders. Moreover, our findings introduce a novel antibody-based tool that, as a consequence of its general specificity for fibrillar conformations and its ability to function intracellularly, offers broad research potential for a variety of human amyloid diseases.

    Funded by: NINDS NIH HHS: NS053912, NS061257, R01 NS053912, R01 NS053912-04, R21 NS061257

    PloS one 2009;4;5;e5727

  • The predicted structure of the headpiece of the Huntingtin protein and its implications on Huntingtin aggregation.

    Kelley NW, Huang X, Tam S, Spiess C, Frydman J and Pande VS

    Biophysics Program, Stanford University, Stanford, CA 94305, USA.

    We have performed simulated tempering molecular dynamics simulations to study the thermodynamics of the headpiece of the Huntingtin (Htt) protein (N17(Htt)). With converged sampling, we found this peptide is highly helical, as previously proposed. Interestingly, this peptide is also found to adopt two different and seemingly stable states. The region from residue 4 (L) to residue 9 (K) has a strong helicity from our simulations, which is supported by experimental studies. However, contrary to what was initially proposed, we have found that simulations predict the most populated state as a two-helix bundle rather than a single straight helix, although a significant percentage of structures do still adopt a single linear helix. The fact that Htt aggregation is nucleation dependent infers the importance of a critical transition. It has been shown that N17(Htt) is involved in this rate-limiting step. In this study, we propose two possible mechanisms for this nucleating event stemming from the transition between two-helix bundle state and single-helix state for N17(Htt) and the experimentally observed interactions between the N17(Htt) and polyQ domains. More strikingly, an extensive hydrophobic surface area is found to be exposed to solvent in the dominant monomeric state of N17(Htt). We propose the most fundamental role played by N17(Htt) would be initializing the dimerization and pulling the polyQ chains into adequate spatial proximity for the nucleation event to proceed.

    Funded by: NEI NIH HHS: PN1 EY016525, PN1 EY016525-02, PN2 EY016525, PN2 EY016525-05; NIGMS NIH HHS: R01 GM062868, R01 GM062868-07, R01-GM062868, U54 GM072970

    Journal of molecular biology 2009;388;5;919-27

  • Regulator of calcineurin (RCAN1-1L) is deficient in Huntington disease and protective against mutant huntingtin toxicity in vitro.

    Ermak G, Hench KJ, Chang KT, Sachdev S and Davies KJ

    Ethel Percy Andrus Gerontology Center and Division of Molecular and Computational Biology, University of Southern California, Los Angeles, California 90089-0191, USA.

    Our work suggests an important new link between the RCAN1 gene and Huntington disease. Huntington disease is caused by expansion of glutamine repeats in the huntingtin protein. How the huntingtin protein with expanded polyglutamines (mutant huntingtin) causes the disease is still unclear, but phosphorylation of huntingtin appears to be protective. Increased huntingtin phosphorylation can be produced either by inhibition of the phosphatase calcineurin or by activation of the Akt kinase. The RCAN1 gene encodes regulators of calcineurin, and we now demonstrate, for the first time, that RCAN1-1L is depressed in Huntington disease. We also show that RCAN1-1L overexpression can protect against mutant huntingtin toxicity in an ST14A cell culture model of Huntington disease and that increased phosphorylation of huntingtin via calcineurin inhibition, rather than via Akt induction or activation, is the likely mechanism by which RCAN1-1L may be protective against mutant huntingtin. These findings suggest that RCAN1-1L "deficiency" may actually play a role in the etiology of Huntington disease. In addition, our results allow for the possibility that controlled overexpression of RCAN1-1L in the striatal region of the brain might be a viable avenue for therapeutic intervention in Huntington disease patients (and perhaps other polyglutamine expansion disorders).

    The Journal of biological chemistry 2009;284;18;11845-53

  • Live axonal transport disruption by mutant huntingtin fragments in Drosophila motor neuron axons.

    Sinadinos C, Burbidge-King T, Soh D, Thompson LM, Marsh JL, Wyttenbach A and Mudher AK

    University of Southampton, Bassett Crescent East, UK.

    Huntington's Disease is a neurodegenerative condition caused by a polyglutamine expansion in the huntingtin (Htt) protein, which aggregates and also causes neuronal dysfunction. Pathogenic N-terminal htt fragments perturb axonal transport in vitro. To determine whether this occurs in vivo and to elucidate how transport is affected, we expressed htt exon 1 with either pathogenic (HttEx1Q93) or non-pathogenic (HttEx1Q20) polyglutamine tracts in Drosophila. We found that HttEx1Q93 expression causes axonal accumulation of GFP-tagged fast axonal transport vesicles in vivo and leads to aggregates within larval motor neuron axons. Time-lapse video microscopy, shows that vesicle velocity is unchanged in HttEx1Q93-axons compared to HttEx1Q20-axons, but vesicle stalling occurs to a greater extent. Whilst HttEx1Q93 expression did not affect locomotor behaviour, external heat stress unveiled a locomotion deficit in HttEx1Q93 larvae. Therefore vesicle transport abnormalities amidst axonal htt aggregation places a cumulative burden upon normal neuronal function under stressful conditions.

    Funded by: Biotechnology and Biological Sciences Research Council; Medical Research Council: G120/881

    Neurobiology of disease 2009;34;2;389-95

  • SCAMP5 links endoplasmic reticulum stress to the accumulation of expanded polyglutamine protein aggregates via endocytosis inhibition.

    Noh JY, Lee H, Song S, Kim NS, Im W, Kim M, Seo H, Chung CW, Chang JW, Ferrante RJ, Yoo YJ, Ryu H and Jung YK

    Department of Life Science, Gwangju Institute of Science and Technology, Gwangju 500-712, Korea.

    Accumulation of expanded polyglutamine proteins is considered to be a major pathogenic biomarker of Huntington disease. We isolated SCAMP5 as a novel regulator of cellular accumulation of expanded polyglutamine track protein using cell-based aggregation assays. Ectopic expression of SCAMP5 augments the formation of ubiquitin-positive and detergent-resistant aggregates of mutant huntingtin (mtHTT). Expression of SCAMP5 is markedly increased in the striatum of Huntington disease patients and is induced in cultured striatal neurons by endoplasmic reticulum (ER) stress or by mtHTT. The increase of SCAMP5 impairs endocytosis, which in turn enhances mtHTT aggregation. On the contrary, down-regulation of SCAMP5 alleviates ER stress-induced mtHTT aggregation and endocytosis inhibition. Moreover, stereotactic injection into the striatum and intraperitoneal injection of tunicamycin significantly increase mtHTT aggregation in the striatum of R6/2 mice and in the cortex of N171-82Q mice, respectively. Taken together, these results suggest that exposure to ER stress increases SCAMP5 in the striatum, which positively regulates mtHTT aggregation via the endocytosis pathway.

    The Journal of biological chemistry 2009;284;17;11318-25

  • Cross-seeding fibrillation of Q/N-rich proteins offers new pathomechanism of polyglutamine diseases.

    Furukawa Y, Kaneko K, Matsumoto G, Kurosawa M and Nukina N

    Laboratory for Structural Neuropathology, Brain Science Institute, RIKEN, Wako, Saitama 351-0198, Japan.

    A pathological hallmark of the Huntington's disease (HD) is intracellular inclusions containing a huntingtin (Htt) protein with an elongated polyglutamine tract. Aggregation of mutant Htt causes abnormal protein-protein interactions, and the functional dysregulation of aggregate-interacting proteins (AIPs) has been proposed as a pathomechanism of HD. Despite this, a molecular mechanism remains unknown how Htt aggregates sequester AIPs. We note an RNA-binding protein, TIA-1, as a model of AIPs containing a Q/N-rich sequence and suggest that in vitro and in vivo Htt fibrillar aggregates function as a structural template for inducing insoluble fibrillation of TIA-1. It is also plausible that such a cross-seeding activity of Htt aggregates represses the physiological function of TIA-1. We thus propose that Htt aggregates act as an intracellular hub for the cross-seeded fibrillation of Q/N-rich AIPs and that a cross-seeding reaction is a molecular origin to cause diverse pathologies in a polyglutamine disease.

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2009;29;16;5153-62

  • Mutant huntingtin N-terminal fragments of specific size mediate aggregation and toxicity in neuronal cells.

    Ratovitski T, Gucek M, Jiang H, Chighladze E, Waldron E, D'Ambola J, Hou Z, Liang Y, Poirier MA, Hirschhorn RR, Graham R, Hayden MR, Cole RN and Ross CA

    Division of Neurobiology, Department of Psychiatry, Mass Spectrometry and Proteomics Facility, Johns Hopkins University School of Medicine, Baltimore, Maryland 21287, USA. tratovi1@jhmi.edu

    Huntingtin proteolysis is implicated in Huntington disease pathogenesis, yet, the nature of huntingtin toxic fragments remains unclear. Huntingtin undergoes proteolysis by calpains and caspases within an N-terminal region between amino acids 460 and 600. We have focused on proteolytic steps producing shorter N-terminal fragments, which we term cp-1 and cp-2 (distinct from previously described cp-A/cp-B). We used HEK293 cells to express the first 511 residues of huntingtin and further define the cp-1 and cp-2 cleavage sites. Based on epitope mapping with huntingtin-specific antibodies, we found that cp-1 cleavage occurs between residues 81 and 129 of huntingtin. Affinity and size exclusion chromatography were used to further purify huntingtin cleavage products and enrich for the cp-1/cp-2 fragments. Using mass spectrometry, we found that the cp-2 fragment is generated by cleavage of huntingtin at position Arg(167). This site was confirmed by deletion analysis and specific detection with a custom-generated cp-2 site neo-epitope antibody. Furthermore, alterations of this cleavage site resulted in a decrease in toxicity and an increase in aggregation of huntingtin in neuronal cells. These data suggest that cleavage of huntingtin at residue Arg(167) may mediate mutant huntingtin toxicity in Huntington disease.

    Funded by: NINDS NIH HHS: NS038144-08

    The Journal of biological chemistry 2009;284;16;10855-67

  • Polyglutamine disruption of the huntingtin exon 1 N terminus triggers a complex aggregation mechanism.

    Thakur AK, Jayaraman M, Mishra R, Thakur M, Chellgren VM, Byeon IJ, Anjum DH, Kodali R, Creamer TP, Conway JF, Gronenborn AM and Wetzel R

    Department of Structural Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15260, USA.

    Simple polyglutamine (polyQ) peptides aggregate in vitro via a nucleated growth pathway directly yielding amyloid-like aggregates. We show here that the 17-amino-acid flanking sequence (HTT(NT)) N-terminal to the polyQ in the toxic huntingtin exon 1 fragment imparts onto this peptide a complex alternative aggregation mechanism. In isolation, the HTT(NT) peptide is a compact coil that resists aggregation. When polyQ is fused to this sequence, it induces in HTT(NT), in a repeat-length dependent fashion, a more extended conformation that greatly enhances its aggregation into globular oligomers with HTT(NT) cores and exposed polyQ. In a second step, a new, amyloid-like aggregate is formed with a core composed of both HTT(NT) and polyQ. The results indicate unprecedented complexity in how primary sequence controls aggregation within a substantially disordered peptide and have implications for the molecular mechanism of Huntington's disease.

    Funded by: NIA NIH HHS: R01 AG019322, R01 AG019322-08

    Nature structural & molecular biology 2009;16;4;380-9

  • 4p16.3 haplotype modifying age at onset of Huntington disease.

    Nørremølle A, Budtz-Jørgensen E, Fenger K, Nielsen JE, Sørensen SA and Hasholt L

    Department of Cellular and Molecular Medicine, University of Copenhagen, Denmark. annenoe@sund.ku.dk

    Huntington disease (HD) is caused by an expanded CAG repeat sequence in the HD gene. Although the age at onset is correlated to the CAG repeat length, this correlation only explains approximately half of the variation in onset age. Less variation between siblings indicates that the variation is, in part, explained by genetic modifiers. We analyzed polymorphic loci within or close to the HD gene on the HD chromosome in Danish HD patients. We found one specific haplotype segregating with later age at onset, compared with patients with similar CAG repeat length and another haplotype. The nine Danish families in the study carrying this haplotype most likely have a common founder. Several of the polymorphic loci displayed alleles that may be specific to the late-onset haplotype, implicating that from this study we cannot determine which of the loci tested (or other polymorphic loci in this chromosomal area) do in fact contain genetic modifiers of age at onset.

    Clinical genetics 2009;75;3;244-50

  • CAG expansion in the Huntington disease gene is associated with a specific and targetable predisposing haplogroup.

    Warby SC, Montpetit A, Hayden AR, Carroll JB, Butland SL, Visscher H, Collins JA, Semaka A, Hudson TJ and Hayden MR

    Centre for Molecular Medicine and Therapeutics, Child and Family Research Institute, University of British Columbia, BC, Canada.

    Huntington disease (HD) is an autosomal-dominant disorder that results from >or=36 CAG repeats in the HD gene (HTT). Approximately 10% of patients inherit a chromosome that underwent CAG expansion from an unaffected parent with <36 CAG repeats. This study is a comprehensive analysis of genetic diversity in HTT and reveals that HD patients of European origin (n = 65) have a significant enrichment (95%) of a specific set of 22 tagging single nucleotide polymorphisms (SNPs) that constitute a single haplogroup. The disease association of many SNPs is much stronger than any previously reported polymorphism and was confirmed in a replication cohort (n = 203). Importantly, the same haplogroup is also significantly enriched (83%) in individuals with 27-35 CAG repeats (intermediate alleles, n = 66), who are unaffected by the disease, but have increased CAG tract sizes relative to the general population (n = 116). These data support a stepwise model for CAG expansion into the affected range (>or=36 CAG) and identifies specific haplogroup variants in the general population associated with this instability. The specific variants at risk for CAG expansion are not present in the general population in China, Japan, and Nigeria where the prevalence of HD is much lower. The current data argue that cis-elements have a major predisposing influence on CAG instability in HTT. The strong association between specific SNP alleles and CAG expansion also provides an opportunity of personalized therapeutics in HD where the clinical development of only a small number of allele-specific targets may be sufficient to treat up to 88% of the HD patient population.

    American journal of human genetics 2009;84;3;351-66

  • CAG repeat lengths > or =335 attenuate the phenotype in the R6/2 Huntington's disease transgenic mouse.

    Dragatsis I, Goldowitz D, Del Mar N, Deng YP, Meade CA, Liu L, Sun Z, Dietrich P, Yue J and Reiner A

    Department of Physiology, University of Tennessee Health Science Center, Memphis, TN 38163, USA.

    With spontaneous elongation of the CAG repeat in the R6/2 transgene to > or =335, resulting in a transgene protein too large for passive entry into nuclei via the nuclear pore, we observed an abrupt increase in lifespan to >20 weeks, compared to the 12 weeks common in R6/2 mice with 150 repeats. In the > or =335 CAG mice, large ubiquitinated aggregates of mutant protein were common in neuronal dendrites and perikaryal cytoplasm, but intranuclear aggregates were small and infrequent. Message and protein for the > or =335 CAG transgene were reduced to one-third that in 150 CAG R6/2 mice. Neurological and neurochemical abnormalities were delayed in onset and less severe than in 150 CAG R6/2 mice. These findings suggest that polyQ length and pathogenicity in Huntington's disease may not be linearly related, and pathogenicity may be less severe with extreme repeats. Both diminished mutant protein and reduced nuclear entry may contribute to phenotype attenuation.

    Funded by: NINDS NIH HHS: NS19620, NS28721, R01 NS019620, R01 NS028721, R56 NS028721

    Neurobiology of disease 2009;33;3;315-30

  • Paradoxical delay in the onset of disease caused by super-long CAG repeat expansions in R6/2 mice.

    Morton AJ, Glynn D, Leavens W, Zheng Z, Faull RL, Skepper JN and Wight JM

    Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge CB2 1PD, UK. ajm41@cam.ac.uk

    Huntington's disease (HD) is caused by an expanded CAG repeat in the HD gene. The pathological threshold for expansion in HD is around 36 CAG repeats, although 'super-long' expansions are found in brains of HD patients. We examined the effect of varying the CAG repeat length (from 170 to 450) on behavior and neuropathology of R6/2 mice. Unexpectedly, we found that increasing the repeat length delayed onset of disease and prolonged survival, from around 4 months to over 18 months in mice with the longest repeats. The delay in onset correlated with a delayed appearance of neuronal intranuclear inclusions (NIIs). However, super-long CAG repeats are not neuroprotective. Mice carrying 2 copies of the mutant transgene die earlier than those carrying a single copy. Furthermore, neurodegeneration is present in super-long repeat length mice at mid-stage disease, whereas little neurodegeneration is seen in mice with shorter CAG repeats until end stage. Expanding the CAG repeat beyond the range where NII formation is the dominant pathology has unmasked a slowly progressing neurological phenotype in R6/2 mice with brain pathology, including the identification of a novel form of inclusion, that more closely resembles that seen in adult onset cases of HD. This mouse may represent a better model for adult-onset HD than R6/2 mice with shorter repeats.

    Neurobiology of disease 2009;33;3;331-41

  • DNA breakage and induction of DNA damage response proteins precede the appearance of visible mutant huntingtin aggregates.

    Illuzzi J, Yerkes S, Parekh-Olmedo H and Kmiec EB

    Department of Biological Sciences, University of Delaware, Delaware Biotechnology Institute, Newark, Delaware 19711, USA.

    Huntington's disease (HD) is a neurodegenerative disorder that follows an autosomal-dominant inheritance pattern. The pathogenesis of the disease depends on the degree of expansion of triplet (CAG) repeats located in the first exon on the gene. An expanded polyglutamine tract within the protein huntingtin (Htt) enables a gain-of-function phenotype that is often exhibited by a dysfunctional oligomerization process and the formation of protein aggregates. How this process leads to neurodegeneration remains undefined. We report that expression of a Htt-fragment containing an expanded glutamine tract induces DNA damage and activates the DNA damage response pathway. Both single-strand and double-strand breaks are observed as the mutant protein accumulates in the cell; these breaks precede the appearance of detectable protein aggregates containing mutant Htt. We also observe activation of H2AX, ATM, and p53 in cells expressing mutant Htt, a predictable response in cells containing chromosomal breakage. Expression of wild-type Htt does not affect the integrity of DNA, nor does it activate the same pathway. Furthermore, DNA damage and activated H2AX are present in HD transgenic mice before the formation of mutant Htt aggregates and HD pathogenesis. Taken together, our data suggest that the expression of mutant Htt causes an accumulation of DNA breaks that activates the DNA damage response pathway, a process that can disable cell function. Because these events can lead to apoptosis, it is possible that the DNA damage response pathway activated by single- and double-strand breaks that we found contributes to neurodegeneration.

    Journal of neuroscience research 2009;87;3;733-47

  • Effects of overexpression of huntingtin proteins on mitochondrial integrity.

    Wang H, Lim PJ, Karbowski M and Monteiro MJ

    Institute for Neurodegenerative Diseases, Medical Biotechnology Center, University of Maryland Biotechnology Institute, Baltimore, MD 21201, USA.

    Huntington's disease (HD) is caused by an expansion of a CAG trinucleotide sequence that encodes a polyglutamine tract in the huntingtin (Htt) protein. Expansion of the polyglutamine tract above 35 repeats causes disease, with the age of onset inversely related to the degree of expansion above this number. Growing evidence suggests that mitochondrial function is compromised during HD pathogenesis, but how this occurs is not understood. We examined mitochondrial properties of HeLa cells that expressed green fluorescent protein (GFP)- or FLAG-tagged N-terminal portions of the Htt protein containing either, 17, 28, 74 or 138 polyglutamine repeats. Immunofluorescence staining of cells using antibodies against Tom20, a mitochondrion localized protein, revealed that cells expressing Htt proteins with 74 or 138 polyglutamine repeats were more sensitized to oxidative stress-induced mitochondria fragmentation and had reduced ATP levels compared with cells expressing Htt proteins with 17 or 28 polyglutamine repeats. By measuring changes in fluorescence of a photoactivated GFP protein targeted to mitochondria, we found that cells expressing red fluorescent protein (RFP)-tagged Htt protein containing 74 polyglutamine repeats had mitochondria that displayed reduced movement and fusion than cells expressing RFP-Htt protein with 28 polyglutamine repeats. Overexpression of Drp-1(K38A), a dominant-negative mitochondria-fission mutant, or Mfn2, a protein that promotes mitochondria fusion, suppressed polyglutamine-induced mitochondria fragmentation, the reduction of ATP levels and cell death. In a Caenorhabditis elegans model of HD, we found that reduction of Drp-1 expression by RNA interference rescued the motility defect associated with the expression of Htt proteins with polyglutamine repeats. These results suggest that the increase in cytotoxicity induced by Htt proteins containing expanded polyglutamine tracts is likely mediated, at least in part, by an alteration in normal mitochondrial dynamics, which results in increased mitochondrial fragmentation. Furthermore, our results suggest that it might be possible to reverse polyglutamine-induced cytotoxicity by preventing mitochondrial fragmentation.

    Funded by: NIA NIH HHS: AG016839; NIGMS NIH HHS: GM 066287

    Human molecular genetics 2009;18;4;737-52

  • Huntington disease in subjects from an Israeli Karaite community carrying alleles of intermediate and expanded CAG repeats in the HTT gene: Huntington disease or phenocopy?

    Herishanu YO, Parvari R, Pollack Y, Shelef I, Marom B, Martino T, Cannella M and Squitieri F

    Department of Neurology, Soroka University Medical Center, Beer-Sheva, Israel. yuvalh@bgu.ac.il

    We report a cluster of patients from a Karaite Jew community with a movement disorder suggestive of Huntington disease (HD), in some cases associated with repeat lengths below the edge of 36 CAG repeats. The study describes the clinical and genetic features of four patients who were followed over several years. Patients belonged to an inbred family in whom progressive chorea, manifesting predominantly with dystonia and cerebellar features, developed during middle age. Although severe psychiatric symptoms ultimately developed in two of the four patients, cognitive function remained reasonably well preserved in all of them even after several disease years. Moderate cognitive deficits were limited to the visuomotor organization and abstract thinking subtests in three of the four patients. Qualitative brain imaging showed atrophy of brain predominantly involving cortex and cerebellum. Genetic testing revealed a variable mutation penetrance among family members, some affected members showing an upper allele size ranging from 34 to 49, whereas others remained unaffected despite the presence of the full mutation beyond 40 CAG repeats. Co-morbidity with recessive hereditary inclusion body myopathy was found in two subjects from one family. Although the main diagnosis of HD remains to be confirmed by further neuropathological studies, these cases may suggest that HD could manifest with as few as 34 CAG repeats, in some geographic areas, the disease phenotype most probably being influenced by additional, as yet unidentified, genes.

    Funded by: Telethon: GGP06181

    Journal of the neurological sciences 2009;277;1-2;143-6

  • Phosphorylation of huntingtin reduces the accumulation of its nuclear fragments.

    Warby SC, Doty CN, Graham RK, Shively J, Singaraja RR and Hayden MR

    Department of Medical Genetics, University of British Columbia, Vancouver, British Columbia V5Z4H4, Canada.

    Huntingtin is phosphorylated on serine-421 (S421) by the pro-survival signaling protein kinases Akt and SGK. Phosphorylation of huntingtin at S421 is variable in different regions of the brain with the lowest levels observed in the striatum, which is further reduced by the mutation for Huntington disease (HD). Cleavage of huntingtin by caspase-6 at amino acid 586 is a crucial event in the pathogenesis of HD. Nuclear localization of huntingtin is also an important marker of HD and preventing or delaying its nuclear accumulation is protective in disease models. Phosphorylation influences proteolysis and clearance of many protein substrates. We therefore sought to investigate the influence of huntingtin phosphorylation at S421 on the accumulation of huntingtin-caspase-6 fragments because these fragments are generated in the nucleus and are crucial for the disease phenotype. Using phospho-huntingtin mutants and a cleavage site-specific neo-epitope huntingtin antibody, we demonstrate that phosphorylation at S421 reduces the nuclear accumulation of huntingtin-caspase-6 fragments by reducing huntingtin cleavage by caspase-6, the levels of full-length huntingtin, and its nuclear localization.

    Molecular and cellular neurosciences 2009;40;2;121-7

  • Relationship between CAG repeat length and brain volume in premanifest and early Huntington's disease.

    Henley SM, Wild EJ, Hobbs NZ, Scahill RI, Ridgway GR, Macmanus DG, Barker RA, Fox NC and Tabrizi SJ

    Dementia Research Centre, Institute of Neurology, University College, London, UK. shenley@drc.ion.ucl.ac.uk

    Huntington's disease (HD) is caused by an expanded CAG repeat on the gene encoding for the protein huntingtin. There are conflicting findings about the extent to which repeat length predicts signs of the disease or severity of disease progression in adults. This study examined the relationship between CAG repeat length and brain volume in a large cohort of pre- and post-motor onset HD gene carriers, using voxel-based morphometry (VBM), an approach which allowed us to investigate the whole brain without defining a priori regions of interest. We also used VBM to examine group differences between 20 controls, 21 premanifest, and 40 early HD subjects. In the 61 mutation-positive subjects higher CAG repeat length was significantly associated with reduced volume of the body of the caudate nucleus bilaterally, left putamen, right insula, right parahippocampal gyrus, right anterior cingulate, and right occipital lobe, after correcting for age. The group contrasts showed significant reduction in grey matter volume in the early HD group relative to controls in widespread cortical as well as subcortical areas but there was no evidence of difference between controls and premanifest subjects. Overall we have demonstrated that increased CAG repeat length is associated with atrophy in extra-striatal as well as striatal regions, which has implications for the monitoring of disease-modifying therapies in the condition.

    Funded by: Department of Health; Medical Research Council: G0601846, G90/86

    Journal of neurology 2009;256;2;203-12

  • The gene coding for PGC-1alpha modifies age at onset in Huntington's Disease.

    Weydt P, Soyal SM, Gellera C, Didonato S, Weidinger C, Oberkofler H, Landwehrmeyer GB and Patsch W

    Department of Neurology, University of Ulm (P,W,; G,B,L,), Ulm, Germany. patrick.weydt@uni-ulm.de.

    Huntington's disease (HD) is one of the most common autosomal dominant inherited, neurodegenerative disorders. It is characterized by progressive motor, emotional and cognitive dysfunction. In addition metabolic abnormalities such as wasting and altered energy expenditure are increasingly recognized as clinical hallmarks of the disease. HD is caused by an unstable CAG repeat expansion in the HD gene (HTT), localized on chromosome 4p16.3. The number of CAG repeats in the HD gene is the main predictor of disease-onset, but the remaining variation is strongly heritable. Transcriptional dysregulation, mitochondrial dysfunction and enhanced oxidative stress have been implicated in the pathogenesis. Recent studies suggest that PGC-1alpha, a transcriptional master regulator of mitochondrial biogenesis and metabolism, is defective in HD. A genome wide search for modifier genes of HD age-of-onset had suggested linkage at chromosomal region 4p16-4p15, near the locus of PPARGC1A, the gene coding for PGC-1alpha. We now present data of 2-loci PPARGC1A block 2 haplotypes, showing an effect upon age-at-onset in 447 unrelated HD patients after statistical consideration of CAG repeat lengths in both HTT alleles. Block 1 haplotypes were not associated with the age-at-onset. Homozygosity for the 'protective' block 2 haplotype was associated with a significant delay in disease onset. To our knowledge this is the first study to show clinically relevant effects of the PGC-1alpha system on the course of Huntington's disease in humans.

    Funded by: Austrian Science Fund FWF: P 19893

    Molecular neurodegeneration 2009;4;3

  • Common variations in 4p locus are related to male completed suicide.

    Must A, Kõks S, Vasar E, Tasa G, Lang A, Maron E and Väli M

    Institute of Physiology, Tartu University, Ravila 19, Tartu, 50411, Estonia. anne.must@ut.ee

    Suicidal behavior is a multifactorial phenomenon, with a significant genetic predisposition. To assess the contribution of genes in the 4p region to suicide risk, we genotyped 36 single nucleotide polymorphisms from a 49Mb region on the chromosome arm 4p11-16 in a total of 288 male suicide victims and 327 healthy male volunteers. The nonsynonymous variants rs1383180 in EVC gene, rs6811863 in TBC1D1 gene, rs362272 in HTT gene, and rs734312 in WFS1 gene were associated to the male completed suicide. However, only EVC polymorphism remained significant after correcting for multiple comparisons (P < .05 after 10 K permutations). The function of these genes is not clear yet. WFS1 and HTT are related to the unfolded protein response and endoplasmic reticulum stress, and TBC1D1 is a GTPase activator. EVC is a protein with transmembrane and leucine zipper domains, its function has not been elucidated yet. Further studies are required in order to reveal the role of these four polymorphisms in the pathoetiology of suicide.

    Neuromolecular medicine 2009;11;1;13-9

  • Phosphorylation of mutant huntingtin at S421 restores anterograde and retrograde transport in neurons.

    Zala D, Colin E, Rangone H, Liot G, Humbert S and Saudou F

    Institut Curie, Unité Mixte de Recherche 146,F-91405 Orsay, France.

    Huntingtin (htt), the protein mutated in Huntington's disease, is a positive regulatory factor for vesicular transport whose function is lost in disease. Here, we demonstrate that phosphorylation of htt at serine 421 (S421) restores its function in axonal transport. Using a strategy involving RNA (ribonucleic acid) interference and re-expression of various constructs, we show that polyQ (polyglutamine)-htt is unable to promote transport of brain-derived neurotrophic factor (BDNF)-containing vesicles, but polyQ-htt constitutively phosphorylated at S421 is as effective as the wild-type (wt) as concerns transport of these vesicles. The S421 phosphorylated polyQ-htt displays the wt function of inducing BDNF release. Phosphorylation restores the interaction between htt and the p150(Glued) subunit of dynactin and their association with microtubules in vitro and in cells. We also show that the IGF-1 (insulin growth factor type I)/Akt pathway by promoting htt phosphorylation compensates for the transport defect. This is the first description of a mechanism that restores the htt function altered in disease.

    Human molecular genetics 2008;17;24;3837-46

  • Huntingtin regulates RE1-silencing transcription factor/neuron-restrictive silencer factor (REST/NRSF) nuclear trafficking indirectly through a complex with REST/NRSF-interacting LIM domain protein (RILP) and dynactin p150 Glued.

    Shimojo M

    Department of Molecular and Cellular Biochemistry, University of Kentucky College of Medicine, Lexington, Kentucky 40536-0509, USA. mshim1@uky.edu

    Huntingtin has been reported to regulate the nuclear translocation of the transcriptional repressor RE1-silencing transcription factor/neuron-restrictive silencer factor (REST/NRSF). The REST/NRSF-interacting LIM domain protein (RILP) has also been shown to regulate REST/NRSF nuclear translocation. Therefore, we were prompted to address the question of how two distinct proteins could have the same function. We initially used a yeast two-hybrid screen to look for an interaction between huntingtin and RILP. This screen identified dynactin p150(Glued) as an interacting protein. Coimmunoprecipitation of proteins in vitro expressed in a reticulocyte lysate system showed an interaction between REST/NRSF and RILP as well as between RILP and dynactin p150(Glued). Coimmunoprecipitation analysis further showed a complex containing RILP, dynactin p150(Glued), and huntingtin. Huntingtin did not interact directly with either REST/NRSF or RILP, but did interact with dynactin p150(Glued). The N-terminal fragment of wild-type huntingtin did not affect the interaction between dynactin p150(Glued) and RILP; however, mutant huntingtin weakened this interaction. We further show that HAP1 (huntingtin-associated protein-1) prevents this complex from translocating REST/NRSF to the nucleus. Thus, this study suggests that REST/NRSF, dynactin p150(Glued), huntingtin, HAP1, and RILP form a complex involved in the translocation of REST/NRSF into the nucleus and that HAP1 controls REST/NRSF cellular localization in neurons.

    Funded by: NCRR NIH HHS: P20RR020171; NIMH NIH HHS: K01MH067123

    The Journal of biological chemistry 2008;283;50;34880-6

  • Colocalization of transactivation-responsive DNA-binding protein 43 and huntingtin in inclusions of Huntington disease.

    Schwab C, Arai T, Hasegawa M, Yu S and McGeer PL

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

    Transactivation-responsive DNA-binding protein 43 (TDP-43) is a component of pathological inclusions in amyotrophic lateral sclerosis and several forms of sporadic and familial frontotemporal lobar degeneration. Transactivation-responsive DNA-binding protein 43-immunostained inclusions have also been found in other neurodegenerative disorders including Alzheimer disease, dementia with Lewy bodies, and parkinsonism dementia complex of Guam. Here, we analyzed the occurrence of TDP-43 immunostaining in Huntington disease, which is characterized by inclusions containing mutated huntingtin. In all Huntington disease cases studied, TDP-43 was frequently colocalized with huntingtin in dystrophic neurites and various intracellular inclusions, but not in intranuclear inclusions; the latter were only stained with huntingtin and anti-ubiquitin antibodies. Two phosphorylation-dependent TDP-43 antibodies proved to be superior for detecting pathological inclusions because they did not stain nonphosphorylated TDP-43 in normal nuclei; staining of normal nuclei with phosphorylation-independent antibodies obscured the inclusions. Our results further add to the hypothesis that TDP-43 may be involved in the pathology of a variety of neurodegenerative disorders.

    Journal of neuropathology and experimental neurology 2008;67;12;1159-65

  • Protective role of Engrailed in a Drosophila model of Huntington's disease.

    Mugat B, Parmentier ML, Bonneaud N, Chan HY and Maschat F

    Institute of Human Genetics, UPR1142, CNRS 141, Montpellier Cedex 05, France.

    Huntington's disease (HD) is caused by the expansion of the polyglutamine (polyQ) tract in the human Huntingtin (hHtt) protein (polyQ-hHtt). Although this mutation behaves dominantly, htt loss of function may also contribute to HD pathogenesis. Using a Drosophila model of HD, we found that Engrailed (EN), a transcriptional activator of endogenous Drosophila htt (dhtt), is able to prevent aggregation of polyQ-hHtt. To interpret these findings, we tested and identified a protective role of N-terminal fragments of both Drosophila and Human wild-type Htt onto polyQ-hHtt-induced cellular defects. In addition, N-terminal parts of normal hHtt were also able to rescue eye degeneration due to the loss of Drosophila endogenous dhtt function. Thus, our data indicate that Drosophila and Human Htt share biological properties, and confirm a model whereby EN activates endogenous dhtt, which in turn prevents polyQ-hHtt-induced phenotypes. The protective role of wild-type hHtt N-terminal parts, specifically onto polyQ-hHtt-induced cellular toxicity suggests that the HD may be considered as a dominant negative disease rather than solely dominant.

    Human molecular genetics 2008;17;22;3601-16

  • Weight loss in Huntington disease increases with higher CAG repeat number.

    Aziz NA, van der Burg JM, Landwehrmeyer GB, Brundin P, Stijnen T, EHDI Study Group and Roos RA

    Leiden University Medical Center, Department of Neurology, Leiden, The Netherlands. N.A.Aziz@lumc.nl

    Objective: Huntington disease (HD) is a hereditary neurodegenerative disorder caused by an expanded number of CAG repeats in the huntingtin gene. A hallmark of HD is unintended weight loss, the cause of which is unknown. In order to elucidate the underlying mechanisms of weight loss in HD, we studied its relation to other disease characteristics including motor, cognitive, and behavioral disturbances and CAG repeat number.

    Methods: In 517 patients with early stage HD, we applied mixed-effects model analyses to correlate weight changes over 3 years to CAG repeat number and various components of the Unified Huntington's Disease Rating Scale (UHDRS). We also assessed the relation between CAG repeat number and body weight and caloric intake in the R6/2 mouse model of HD.

    Results: In patients with HD, mean body mass index decreased with -0.15 units per year (p < 0.001). However, no single UHDRS component, including motor, cognitive, and behavioral scores, was independently associated with the rate of weight loss. Patients with HD with a higher CAG repeat number had a faster rate of weight loss. Similarly, R6/2 mice with a larger CAG repeat length had a lower body weight, whereas caloric intake increased with larger CAG repeat length.

    Conclusions: Weight loss in Huntington disease (HD) is directly linked to CAG repeat length and is likely to result from a hypermetabolic state. Other signs and symptoms of HD are unlikely to contribute to weight loss in early disease stages. Elucidation of the responsible mechanisms could lead to effective energy-based therapeutics.

    Neurology 2008;71;19;1506-13

  • Cross-sectional study on prevalences of psychiatric disorders in mutation carriers of Huntington's disease compared with mutation-negative first-degree relatives.

    van Duijn E, Kingma EM, Timman R, Zitman FG, Tibben A, Roos RA and van der Mast RC

    Department of Psychiatry, Leiden University Medical Center, B1-P, P.O. Box 9600, 2300 RC Leiden, The Netherlands. e.van_duijn@lumc.nl

    Objective: To investigate the prevalences of formal DSM-IV diagnoses in pre-motor- symptomatic and motor-symptomatic mutation carriers at different stages of Huntington's disease compared to a control group of first-degree noncarrier relatives and the general population.

    Method: Between May 2004 and August 2006, 154 verified mutation carriers and 56 verified noncarriers were recruited from the outpatient clinics of the Neurology and Clinical Genetics departments of Leiden University Medical Center and from a regional nursing home. To assess the 12-month prevalences of DSM-IV diagnoses, the sections for depression, mania, anxiety, obsessive-compulsive disorder, and psychosis/schizophrenia of the Composite International Diagnostic Interview were used. Prevalences in the Dutch general population were extracted from the Netherlands Mental Health Survey and Incidence Study (NEMESIS).

    Results: Both presymptomatic and symptomatic mutation carriers portrayed significantly more major depressive disorder (p = .001 and p < .001, respectively) and obsessive-compulsive disorder (p = .003 and p = .01, respectively) than the general population. Symptomatic mutation carriers also showed an increased prevalence (p = .01) of nonaffective psychosis. Psychiatric disorders were more prevalent, although not significantly (p = .06), in mutation carriers compared to first-degree relatives who were noncarriers. Noncarriers did not differ from the general population.

    Conclusion: Psychiatric disorders occur frequently in Huntington's disease, often before motor symptoms appear. In addition, first-degree noncarrier relatives do not show more psychiatric disorders compared to the general population, although they grew up in comparable, potentially stressful circumstances. Taking these findings together, psychopathology in Huntington's disease seems predominantly due to cerebral degeneration rather than to shared environmental risk factors.

    The Journal of clinical psychiatry 2008;69;11;1804-10

  • Compensatory changes in the ubiquitin-proteasome system, brain-derived neurotrophic factor and mitochondrial complex II/III in YAC72 and R6/2 transgenic mice partially model Huntington's disease patients.

    Seo H, Kim W and Isacson O

    Neuroregeneration Laboratories, Center for Neuroregeneration Research, McLean Hospital, Harvard MedicalSchool, 115 Mill Street, Belmont, MA 02478, USA.

    Intraneuronal protein aggregates of the mutated huntingtin in Huntington's disease (HD) brains suggest an overload and/or dysfunction of the ubiquitin-proteasome system (UPS). There is a general inhibition of the UPS in many brain regions (cerebellum, cortex, substantia nigra and caudate-putamen) and skin fibroblasts from HD patients. In the current experiment, the widely used mutant huntingtin-exon 1 CAG repeat HD transgenic mice model (R6/2) (with 144 CAG repeat and exon 1) during late-stage pathology, had increases in proteasome activity in the striatum. However, this discrepancy with HD patient tissue was not apparent in the mutant CAG repeat huntingtin full-length HD (YAC72) transgenic mouse model during post-symptomatic and late-stage pathology, which then also showed UPS inhibition similar to HD patients' brains. In both types of HD model mice, we determined biochemical changes, including expression of brain-derived neurotrophic factor (BDNF) and mitochondrial complex II/III (MCII/III) activities related to HD pathology. We found increases of both BDNF expression, and MCII/III activities in YAC72 transgenic mice, and no change of BDNF expression in R6/2 mice. Our data show that extreme CAG repeat lengths in R6/2 mice is paradoxically associated with increased proteasome activity, probably as a cellular compensatory biochemical change in response to the underlying mutation. Changes in HD patients for UPS function, BDNF expression and MCII/III activity are only partially modeled in R6/2 and YAC72 mice, with the latter at 16 months of age being most congruent with the human disease.

    Funded by: NINDS NIH HHS: NS-30064

    Human molecular genetics 2008;17;20;3144-53

  • Neocortical expression of mutant huntingtin is not required for alterations in striatal gene expression or motor dysfunction in a transgenic mouse.

    Brown TB, Bogush AI and Ehrlich ME

    Present address: Departments of Neurology and Pediatrics, Mt Sinai School of Medicine, New York, NY 10029, USA.

    Huntington's disease (HD) is an autosomal-dominant neurodegenerative disease caused by an expanded polyglutamine tract in the ubiquitously expressed huntingtin protein. Clinically, HD is characterized by motor, cognitive and psychiatric deficits. Striking degeneration of the striatum is observed in HD with the medium spiny neurons (MSNs) being the most severely affected neuronal subtype. Dysfunction of MSNs is marked by characteristic changes in gene expression which precede neuronal death. The ubiquitous expression of the huntingtin protein raises the question as to whether the selective vulnerability of the MSN is cell-autonomous, non-cell-autonomous, or a combination thereof. In particular, growing evidence suggests that abnormalities of the cortex and corticostriatal projections may be primary causes of striatal vulnerability. To examine this issue, we developed transgenic mice that, within the forebrain, selectively express a pathogenic huntingtin species in the MSNs, specifically excluding the neocortex. These mice develop a number of abnormalities characteristic of pan-cellular HD mouse models, including intranuclear inclusion bodies, motor impairment, and changes in striatal gene expression. As this phenotype develops in the presence of normal levels of brain-derived neurotrophic factor and its major striatal receptor, tropomyosin-related kinase B, these data represent the first demonstration of in vivo cell-autonomous transcriptional dysregulation in an HD mouse model. Furthermore, our findings suggest that therapies targeted directly to the striatum may be efficacious at reversing some of the molecular abnormalities present in HD.

    Funded by: NINDS NIH HHS: NS0045942, NS052452

    Human molecular genetics 2008;17;20;3095-104

  • Accumulation of N-terminal mutant huntingtin in mouse and monkey models implicated as a pathogenic mechanism in Huntington's disease.

    Wang CE, Tydlacka S, Orr AL, Yang SH, Graham RK, Hayden MR, Li S, Chan AW and Li XJ

    Department of Human Genetics, Emory University School ofMedicine, Atlanta, GA 30322, USA.

    A number of mouse models expressing mutant huntingtin (htt) with an expanded polyglutamine (polyQ) domain are useful for studying the pathogenesis of Huntington's disease (HD) and identifying appropriate therapies. However, these models exhibit neurological phenotypes that differ in their severity and nature. Understanding how transgenic htt leads to variable neuropathology in animal models would shed light on the pathogenesis of HD and help us to choose HD models for investigation. By comparing the expression of mutant htt at the transcriptional and protein levels in transgenic mice expressing N-terminal or full-length mutant htt, we found that the accumulation and aggregation of mutant htt in the brain is determined by htt context. HD mouse models demonstrating more severe phenotypes show earlier accumulation of N-terminal mutant htt fragments, which leads to the formation of htt aggregates that are primarily present in neuronal nuclei and processes, as well as glial cells. Similarly, transgenic monkeys expressing exon-1 htt with a 147-glutamine repeat (147Q) died early and showed abundant neuropil aggregates in swelling neuronal processes. Fractionation of HD150Q knock-in mice brains revealed an age-dependent accumulation of N-terminal mutant htt fragments in the nucleus and synaptosomes, and this accumulation was most pronounced in the striatum due to decreased proteasomal activity. Our findings suggest that the neuropathological phenotypes of HD stem largely from the accumulation of N-terminal mutant htt fragments and that this accumulation is determined by htt context and cell-type-dependent clearance of mutant htt.

    Funded by: NCRR NIH HHS: R24RR018827; NIA NIH HHS: AG019206, R01 AG019206, R01 AG031153; NIH HHS: R24 OD010930; NINDS NIH HHS: NS041669, NS045016, R01 NS036232, R01 NS041669, R01 NS045016

    Human molecular genetics 2008;17;17;2738-51

  • A common motif targets huntingtin and the androgen receptor to the proteasome.

    Chandra S, Shao J, Li JX, Li M, Longo FM and Diamond MI

    Departments of Neurology and Cellular and Molecular Pharmacology, University of California-San Francisco, 600 16th Street, San Francisco, CA 94143, USA.

    Huntington disease derives from a critically expanded polyglutamine tract in the huntingtin (Htt) protein; a similar polyglutamine expansion in the androgen receptor (AR) causes spinobulbar muscular atrophy. AR activity also plays an essential role in prostate cancer. Molecular mechanisms that regulate Htt and AR degradation are not well understood but could have important therapeutic implications. We find that a pentapeptide motif (FQKLL) within the Htt protein regulates its degradation and subcellular localization to cytoplasm puncta. Disruption of the motif by alanine substitution at the hydrophobic residues increases the steady state level of the protein. Pulsechase analyses indicate that the motif regulates degradation. A similar motif (FQNLF) has corresponding activities in the AR protein. Transfer of the Htt motif with five flanking amino acids on either side to YFP reduces the steady state YFP level by rendering it susceptible to proteasome degradation. This work defines a novel proteasome-targeting motif that is necessary and sufficient to regulate the degradation of two disease-associated proteins.

    Funded by: NCI NIH HHS: 1R01 CA 131226-01; NINDS NIH HHS: 1R01 NS 50284-01A1

    The Journal of biological chemistry 2008;283;35;23950-5

  • Huntington CAG repeat size does not modify onset age in familial Parkinson's disease: the GenePD study.

    McNicoll CF, Latourelle JC, MacDonald ME, Lew MF, Suchowersky O, Klein C, Golbe LI, Mark MH, Growdon JH, Wooten GF, Watts RL, Guttman M, Racette BA, Perlmutter JS, Ahmed A, Shill HA, Singer C, Saint-Hilaire MH, Massood T, Huskey KW, DeStefano AL, Gillis T, Mysore J, Goldwurm S, Pezzoli G, Baker KB, Itin I, Litvan I, Nicholson G, Corbett A, Nance M, Drasby E, Isaacson S, Burn DJ, Chinnery PF, Pramstaller PP, Al-Hinti J, Moller AT, Ostergaard K, Sherman SJ, Roxburgh R, Snow B, Slevin JT, Cambi F, Gusella JF and Myers RH

    Department of Neurology, Boston University School of Medicine, Boston University, Boston, Massachusetts 02118, USA.

    The ATP/ADP ratio reflects mitochondrial function and has been reported to be influenced by the size of the Huntington disease gene (HD) repeat. Impaired mitochondrial function has long been implicated in the pathogenesis of Parkinson's disease (PD), and therefore, we evaluated the relationship of the HD CAG repeat size to PD onset age in a large sample of familial PD cases. PD affected siblings (n = 495), with known onset ages from 248 families, were genotyped for the HD CAG repeat. Genotyping failed in 11 cases leaving 484 for analysis, including 35 LRRK2 carriers. All cases had HD CAG repeats (range, 15-34) below the clinical range for HD, although 5.2% of the sample (n = 25) had repeats in the intermediate range (the intermediate range lower limit = 27; upper limit = 35 repeats), suggesting that the prevalence of intermediate allele carriers in the general population is significant. No relation between the HD CAG repeat size and the age at onset for PD was found in this sample of familial PD.

    Funded by: NINDS NIH HHS: P50 NS016367, P50 NS016367-280014, P50 NS016367-280016, P50 NS16367-27, R01 NS032765, R01 NS032765-13, R01 NS036711, R01 NS036711-09, R01 NS36711-09; Telethon: GTB07001

    Movement disorders : official journal of the Movement Disorder Society 2008;23;11;1596-601

  • Activated caspase-6 and caspase-6-cleaved fragments of huntingtin specifically colocalize in the nucleus.

    Warby SC, Doty CN, Graham RK, Carroll JB, Yang YZ, Singaraja RR, Overall CM and Hayden MR

    Centre for Molecular Medicine and Therapeutics, University of British Columbia, 980 West 28th Avenue,Vancouver, British Columbia, Canada.

    Proteolysis of mutant huntingtin is crucial to the development of Huntington disease (HD). Specifically preventing proteolysis at the capase-6 (C6) consensus sequence at amino acid 586 of mutant huntingtin prevents the development of behavioural, motor and neuropathological features in a mouse model of HD. However, the mechanism underlying the selective toxicity of the 586 amino acid cleavage event is currently unknown. We have examined the subcellular localization of different caspase proteolytic fragments of huntingtin using neo-epitope antibodies. Our data suggest that the nucleus is the primary site of htt cleavage at amino acid 586. Endogenously cleaved 586 amino acid fragments are enriched in the nucleus of immortalized striatal cells and primary striatal neurons where they co-localize with active C6. Cell stress induced by staurosporine results in the nuclear translocation and activation of C6 and an increase in 586 amino acid fragments of huntingtin in the nucleus. In comparison, endogenous caspase-2/3-generated huntingtin 552 amino acid fragments localize to the perinuclear region. The different cellular itineraries of endogenously generated caspase products of huntingtin may provide an explanation for the selective toxicity of huntingtin fragments cleaved at amino acid 586.

    Human molecular genetics 2008;17;15;2390-404

  • Suppression of neuropil aggregates and neurological symptoms by an intracellular antibody implicates the cytoplasmic toxicity of mutant huntingtin.

    Wang CE, Zhou H, McGuire JR, Cerullo V, Lee B, Li SH and Li XJ

    Department of Human Genetics, Emory University School of Medicine, Atlanta, GA 30322, USA.

    Mutant huntingtin accumulates in the neuronal nuclei and processes, which suggests that its subcellular localization is critical for the pathology of Huntington's disease (HD). However, the contribution of cytoplasmic mutant huntingtin and its aggregates in neuronal processes (neuropil aggregates) has not been rigorously explored. We generated an intracellular antibody (intrabody) whose binding to a unique epitope of human huntingtin is enhanced by polyglutamine expansion. This intrabody decreases the cytotoxicity of mutant huntingtin and its distribution in neuronal processes. When expressed in the striatum of HD mice via adenoviral infection, the intrabody reduces neuropil aggregate formation and ameliorates neurological symptoms. Interaction of the intrabody with mutant huntingtin increases the ubiquitination of cytoplasmic huntingtin and its degradation. These findings suggest that the intrabody reduces the specific neurotoxicity of cytoplasmic mutant huntingtin and its associated neurological symptoms by preventing the accumulation of mutant huntingtin in neuronal processes and promoting its clearance in the cytoplasm.

    Funded by: NIA NIH HHS: AG19206, R01 AG019206, R01 AG031153; NINDS NIH HHS: NS045016, NS052806, NS36232, NS41669, R01 NS036232, R01 NS041669, R01 NS045016, R21 NS052806

    The Journal of cell biology 2008;181;5;803-16

  • Effect of CAG repeat length on psychiatric disorders in Huntington's disease.

    Vassos E, Panas M, Kladi A and Vassilopoulos D

    Neurogenetics Unit, Department of Neurology, Eginition Hospital, University of Athens, 74 Vas. Sofias Ave., Athens 11528, Greece. evangelos.vassos@iop.kcl.ac.uk

    There is strong evidence that the length of CAG repeats, in patients with Huntington's disease (HD), govern the age of onset and the rate of clinical progression of neurological symptoms. However, psychiatric manifestations of the disease have not been examined as comprehensively. Seventy two Greek patients with Huntington's disease had DNA testing and were clinically assessed by means of a semi-structured interview (SCID) and four self-rated questionnaires. Genotype-phenotype correlations were examined. The CAG repeat length had a significant negative association with the age of onset of psychiatric disorders, the total level of functioning and the MMSE. However, the probability of developing a psychiatric disorder and the severity of psychiatric symptoms were not determined by the trinucleotide expansion, after controlling for the duration of illness, sex, and age of the subjects. The factors that determine the development of psychiatric symptoms in HD patients seem not to be limited to a dose related toxicity of the expanded Huntington. It is hypothesized that alternative genetic or environmental factors underlie the pathogenesis of the psychiatric phenotype.

    Journal of psychiatric research 2008;42;7;544-9

  • Dopamine determines the vulnerability of striatal neurons to the N-terminal fragment of mutant huntingtin through the regulation of mitochondrial complex II.

    Benchoua A, Trioulier Y, Diguet E, Malgorn C, Gaillard MC, Dufour N, Elalouf JM, Krajewski S, Hantraye P, Déglon N and Brouillet E

    Unité de Recherche Associée, Commissariat à l'Energie Atomique (CEA)-Centre Nationale de la Recherche Scientifique (CNRS) 2210, Service Hospitalier Frédéric Joliot, Orsay Cedex, France.

    In neurodegenerative disorders associated with primary or secondary mitochondrial defects such as Huntington's disease (HD), cells of the striatum are particularly vulnerable to cell death, although the mechanisms by which this cell death is induced are unclear. Dopamine, found in high concentrations in the striatum, may play a role in striatal cell death. We show that in primary striatal cultures, dopamine increases the toxicity of an N-terminal fragment of mutated huntingtin (Htt-171-82Q). Mitochondrial complex II protein (mCII) levels are reduced in HD striatum, indicating that this protein may be important for dopamine-mediated striatal cell death. We found that dopamine enhances the toxicity of the selective mCII inhibitor, 3-nitropropionic acid. We also demonstrated that dopamine doses that are insufficient to produce cell loss regulate mCII expression at the mRNA, protein and catalytic activity level. We also show that dopamine-induced down-regulation of mCII levels can be blocked by several dopamine D2 receptor antagonists. Sustained overexpression of mCII subunits using lentiviral vectors abrogated the effects of dopamine, both by high dopamine concentrations alone and neuronal death induced by low dopamine concentrations together with Htt-171-82Q. This novel pathway links dopamine signaling and regulation of mCII activity and could play a key role in oxidative energy metabolism and explain the vulnerability of the striatum in neurodegenerative diseases.

    Funded by: NINDS NIH HHS: NS 036821

    Human molecular genetics 2008;17;10;1446-56

  • Searching for mutation in the JPH3, ATN1 and TBP genes in Polish patients suspected of Huntington's disease and without mutation in the IT15 gene.

    Sułek-Piatkowska A, Krysa W, Zdzienicka E, Szirkowiec W, Hoffman-Zacharska D, Rajkiewicz M, Fidziańska E, Kowalska G and Zaremba J

    Instytut Psychiatrii i Neurologii, Zak(3)ad Genetyki, Al. Sobieskiego 9, 02-957 Warszawa. suleka@ipin.edu.pl

    The aim of this study was to perform DNA analysis in patients with clinical diagnosis of Huntington's disease (HD) after molecular exclusion of HD and further molecular examinations for other neurodegenerative diseases such as Huntington's disease-like 2 (HDL-2; gene JPH3), dentatorubral pallidoluysian atrophy (DRPLA; gene ATN1) and spinocerebellar ataxia type 17 (SCA17; gene TBP).

    The material comprised 224 DNA samples isolated from peripheral blood from patients suspected of HD and 100 DNA samples from unaffected controls. The control group was used to determine the normal range of the number of CAG/CTG repeats in genes JPH3, ATN1 and TBP in the Polish population. Molecular analysis was carried out by PCR reaction, embracing microsatellite repeats in genes JPH3, ATN1 and TBP with specific, fluorescently labelled primers. PCR products were separated in polyacrylamide gels. The normal ranges of the number of repeats established for the control group in genes JPH3, ATN1 and TBP were 7-19, 9-27 and 29-45, respectively.

    Results: Molecular analysis of DNA from 224 individuals suspected of HD (117 women and 107 men) revealed one case of dynamic mutation - 55 CAG repeats - in the TBP locus (SCA17). No cases of DRPLA or HDL-2 were detected. The range of CAG/CTG repeats for the JPH3 gene in the patient group was 11-19, with the most common alleles containing 14 and 16 repeats. For the ATN1 gene in patients the range of 8-27 repeats was established and the most frequent allele with 16 triplets was present.

    Conclusions: The study on 244 patients referred with the clinical diagnosis of HD and without mutation of the IT15 gene revealed one case of SCA17 but did not disclose the presence of two other diseases with a similar clinical manifestation: DRPLA and HDL2.

    Neurologia i neurochirurgia polska 2008;42;3;203-9

  • Altered histone monoubiquitylation mediated by mutant huntingtin induces transcriptional dysregulation.

    Kim MO, Chawla P, Overland RP, Xia E, Sadri-Vakili G and Cha JH

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

    Although transcriptional dysregulation is a critical pathogenic mechanism in Huntington's disease (HD), it is still not known how mutant huntingtin causes it. Here we show that alteration of histone monoubiquitylation is a key mechanism. Disrupted interaction of huntingtin with Bmi-1, a component of the hPRC1L E3 ubiquitin ligase complex, increases monoubiquityl histone H2A (uH2A) levels in a cell culture model of HD. Genes with expression that is repressed in transgenic R6/2 mouse brain have increased uH2A and decreased uH2B at their promoters, whereas actively transcribed genes show the opposite pattern. Reduction in uH2A reverses transcriptional repression and inhibits methylation of histone H3 at lysine 9 in cell culture. In contrast, reduction in uH2B induces transcriptional repression and inhibits methylation of histone H3 at lysine 4. This is the first report to demonstrate hPRC1L as a huntingtin-interacting histone modifying complex and a crucial role for histone monoubiquitylation in mammalian brain gene expression, which broadens our understanding of histone code. These findings also provide a rationale for targeting histone monoubiquitylation for therapy in HD.

    Funded by: NINDS NIH HHS: NS38106, NS45242, P01 NS045242, P01 NS045242-030004, R01 NS038106, R01 NS038106-06

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2008;28;15;3947-57

  • Wheel running from a juvenile age delays onset of specific motor deficits but does not alter protein aggregate density in a mouse model of Huntington's disease.

    van Dellen A, Cordery PM, Spires TL, Blakemore C and Hannan AJ

    Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, OX1 3PT, UK. anthony.hannan@florey.edu.au

    Background: Huntington's disease (HD) is a neurodegenerative disorder predominantly affecting the cerebral cortex and striatum. Transgenic mice (R6/1 line), expressing a CAG repeat encoding an expanded polyglutamine tract in the N-terminus of the huntingtin protein, closely model HD. We have previously shown that environmental enrichment of these HD mice delays the onset of motor deficits. Furthermore, wheel running initiated in adulthood ameliorates the rear-paw clasping motor sign, but not an accelerating rotarod deficit.

    Results: We have now examined the effects of enhanced physical activity via wheel running, commenced at a juvenile age (4 weeks), with respect to the onset of various behavioral deficits and their neuropathological correlates in R6/1 HD mice. HD mice housed post-weaning with running wheels only, to enhance voluntary physical exercise, have delayed onset of a motor co-ordination deficit on the static horizontal rod, as well as rear-paw clasping, although the accelerating rotarod deficit remains unaffected. Both wheel running and environmental enrichment rescued HD-induced abnormal habituation of locomotor activity and exploratory behavior in the open field. We have found that neither environment enrichment nor wheel running ameliorates the shrinkage of the striatum and anterior cingulate cortex (ACC) in HD mice, nor the overall decrease in brain weight, measured at 9 months of age. At this age, the density of ubiquitinated protein aggregates in the striatum and ACC is also not significantly ameliorated by environmental enrichment or wheel running.

    Conclusion: These results indicate that enhanced voluntary physical activity, commenced at an early presymptomatic stage, contributes to the positive effects of environmental enrichment. However, sensory and cognitive stimulation, as well as motor stimulation not associated with running, may constitute major components of the therapeutic benefits associated with enrichment. Comparison of different environmental manipulations, performed in specific time windows, can identify critical periods for the induction of neuroprotective 'brain reserve' in animal models of HD and related neurodegenerative diseases.

    Funded by: Medical Research Council

    BMC neuroscience 2008;9;34

  • Mitogen- and stress-activated protein kinase-1 deficiency is involved in expanded-huntingtin-induced transcriptional dysregulation and striatal death.

    Roze E, Betuing S, Deyts C, Marcon E, Brami-Cherrier K, Pagès C, Humbert S, Mérienne K and Caboche J

    Université Pierre et Marie Curie-Paris 6, CNRS, UMR 7102, 9 quai St. Bernard, 75005, Paris, France.

    Huntington's disease (HD) is a neurodegenerative disorder due to an abnormal polyglutamine expansion in the N-terminal region of huntingtin protein (Exp-Htt). This expansion causes protein aggregation and neuronal dysfunction and death. Transcriptional dysregulation due to Exp-Htt participates in neuronal death in HD. Here, using the R6/2 transgenic mouse model of HD, we identified a new molecular alteration that could account for gene dysregulation in these mice. Despite a nuclear activation of the mitogen-activated protein kinase/extracellular regulated kinase (ERK) along with Elk-1 and cAMP responsive element binding, two transcription factors involved in c-Fos transcription, we failed to detect any histone H3 phosphorylation, which is expected after nuclear ERK activation. Accordingly, we found in the striatum of these mice a deficiency of mitogen- and stress-activated kinase-1 (MSK-1), a kinase downstream ERK, critically involved in H3 phosphorylation and c-Fos induction. We extended this observation to Exp-Htt-expressing striatal neurons and postmortem brains of HD patients. In vitro, knocking out MSK-1 expression potentiated Exp-Htt-induced striatal death. Its overexpression induced H3 phosphorylation and c-Fos expression and totally protected against striatal neurodegeneration induced by Exp-Htt. We propose that MSK-1 deficiency is involved in transcriptional dysregulation and striatal degeneration. Restoration of its expression and activity may be a new therapeutic target in HD.

    FASEB journal : official publication of the Federation of American Societies for Experimental Biology 2008;22;4;1083-93

  • p21-activated kinase 1 promotes soluble mutant huntingtin self-interaction and enhances toxicity.

    Luo S, Mizuta H and Rubinsztein DC

    Department of Medical Genetics, Cambridge Institute for Medical Research, Wellcome/MRC Building, Addenbrooke's Hospital, Hills Road, Cambridge CB2 2XY, UK.

    Huntington's disease (HD) is caused by a polyglutamine (polyQ) expansion in the huntingtin (htt) protein. While aggregation is a pathological hallmark of HD and related polyQ expansion diseases, the role of aggregates has been disputed. Here we report that p21-activated kinase 1 (Pak1) binds to htt in vivo and in vitro. Pak1 colocalized with mutant htt (muhtt) aggregates in cell models and in human HD brains. Pak1 overexpression enhanced the aggregation of muhtt. Furthermore, we observed SDS-soluble wild-type htt (wthtt)-wthtt, wthtt-muhtt and muhtt-muhtt interactions, which were enhanced by the presence of Pak1. We show that Pak1 overexpression enhanced htt toxicity in cell models and neurons in parallel with its ability to promote aggregation, while Pak1 knockdown suppressed both aggregation and toxicity. Overexpression of either kinase-dead or wild-type Pak enhanced both aggregation and toxicity. Our data reveal a novel mechanism regulating muhtt oligomerization and toxicity and suggest that pathology may be at least partly dependent on soluble muhtt-muhtt interactions.

    Funded by: Medical Research Council: G0600194, G0600194(77639); Wellcome Trust: 064354

    Human molecular genetics 2008;17;6;895-905

  • N-terminal mutant huntingtin associates with mitochondria and impairs mitochondrial trafficking.

    Orr AL, Li S, Wang CE, Li H, Wang J, Rong J, Xu X, Mastroberardino PG, Greenamyre JT and Li XJ

    Department of Human Genetics, Emory University School of Medicine, Atlanta, Georgia 30322, USA.

    Huntington's disease (HD) is caused by polyglutamine (polyQ) expansion in huntingtin (htt), a large (350 kDa) protein that localizes predominantly to the cytoplasm. Proteolytic cleavage of mutant htt yields polyQ-containing N-terminal fragments that are prone to misfolding and aggregation. Disease progression in HD transgenic models correlates with age-related accumulation of soluble and aggregated forms of N-terminal mutant htt fragments, suggesting that multiple forms of mutant htt are involved in the selective neurodegeneration in HD. Although mitochondrial dysfunction is implicated in the pathogenesis of HD, it remains unclear which forms of cytoplasmic mutant htt associate with mitochondria to affect their function. Here we demonstrate that specific N-terminal mutant htt fragments associate with mitochondria in Hdh(CAG)150 knock-in mouse brain and that this association increases with age. The interaction between soluble N-terminal mutant htt and mitochondria interferes with the in vitro association of microtubule-based transport proteins with mitochondria. Mutant htt reduces the distribution and transport rate of mitochondria in the processes of cultured neuronal cells. Reduced ATP level was also found in the synaptosomal fraction isolated from Hdh(CAG)150 knock-in mouse brain. These findings suggest that specific N-terminal mutant htt fragments, before the formation of aggregates, can impair mitochondrial function directly and that this interaction may be a novel target for therapeutic strategies in HD.

    Funded by: NIA NIH HHS: AG19206, R01 AG019206, R01 AG019206-07, R01 AG031153; NINDS NIH HHS: NS41669, R01 NS041669, R01 NS045016

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2008;28;11;2783-92

  • Inhibition of endoplasmic reticulum stress counteracts neuronal cell death and protein aggregation caused by N-terminal mutant huntingtin proteins.

    Reijonen S, Putkonen N, Nørremølle A, Lindholm D and Korhonen L

    Medical Research Institute Minerva, Biomedicum Helsinki, Haartmaninkatu 8, Helsinki, Finland.

    Accumulation of abnormal proteins occurs in many neurodegenerative diseases including Huntington's disease (HD). However, the precise role of protein aggregation in neuronal cell death remains unclear. We show here that the expression of N-terminal huntingtin proteins with expanded polyglutamine (polyQ) repeats causes cell death in neuronal PC6.3 cell that involves endoplasmic reticulum (ER) stress. These mutant huntingtin fragment proteins elevated Bip, an ER chaperone, and increased Chop and the phosphorylation of c-Jun-N-terminal kinase (JNK) that are involved in cell death regulation. Caspase-12, residing in the ER, was cleaved in mutant huntingtin expressing cells, as was caspase-3 mediating cell death. In contrast, cytochrome-c or apoptosis inducing factor (AIF) was not released from mitochondria after the expression of these proteins. Treatment with salubrinal that inhibits ER stress counteracted cell death and reduced protein aggregations in the PC6.3 cells caused by the mutant huntingtin fragment proteins. Salubrinal upregulated Bip, reduced cleavage of caspase-12 and increased the phosphorylation of eukaryotic translation initiation factor-2 subunit-alpha (eIF2alpha) that are neuroprotective. These results show that N-terminal mutant huntingtin proteins activate cellular pathways linked to ER stress, and that inhibition of ER stress by salubrinal increases cell survival. The data suggests that compounds targeting ER stress may be considered in designing novel approaches for treatment of HD and possibly other polyQ diseases.

    Experimental cell research 2008;314;5;950-60

  • Disruption of striatal glutamatergic transmission induced by mutant huntingtin involves remodeling of both postsynaptic density and NMDA receptor signaling.

    Torres-Peraza JF, Giralt A, García-Martínez JM, Pedrosa E, Canals JM and Alberch J

    Departament de Biologia Cellular i Anatomia Patològica, Facultat de Medicina, Universitat de Barcelona, Barcelona, Spain.

    We study the striatal susceptibility to NMDA receptor (NMDAR)-mediated injury of two Huntington's disease (HD) transgenic mice: R6/1 and R6/1:BDNF(+/-). We found that R6/1:BDNF(+/-) mice--which express reduced levels of BDNF--were more resistant than R6/1 mice to intrastriatal injection of quinolinate. This increased resistance is related to a differential reduction in expression of NMDAR scaffolding proteins, MAGUKs (PSD-95, PSD-93, SAP-102 and SAP-97) but not to altered levels or synaptic location of NMDAR. A robust reorganization of postsynaptic density (PSD) was detected in HD transgenic mice, shown by a switch of PSD-93 by PSD-95 in PSD. Furthermore, NMDAR signaling pathways were affected by different BDNF levels in HD mice; we found a reduction of synaptic alpha CaMKII (but not of nNOS) in R6/1:BDNF(+/-) compared to R6/1 mice. The specific regulation of MAGUKs and alpha CaMKII in striatal neurons may reflect a protective mechanism against expression of mutant huntingtin exon-1.

    Neurobiology of disease 2008;29;3;409-21

  • Mutant huntingtin can paradoxically protect neurons from death.

    Zuchner T and Brundin P

    Neuronal Survival Unit, Department of Experimental Medical Science, Wallenberg Neuroscience Center, Lund University, BMC A10, Lund 22184, Sweden. Thole.Zuechner@bbz.uni-leipzig.de

    Huntington's disease (HD) is a progressive neurodegenerative disorder caused by a mutation in the gene huntingtin and characterized by motor, cognitive and psychiatric symptoms. Huntingtin contains a CAG repeat in exon 1. An expansion of this CAG repeat above 35 results in misfolding of Huntingtin, giving rise to protein aggregates and neuronal cell death. There are several transgenic HD mouse models that reproduce most of the features of the human disorder, for example protein inclusions, some neurodegeneration as well as motor and cognitive symptoms. At the same time, a subgroup of the HD transgenic mouse models exhibit dramatically reduced susceptibility to excitotoxicity. The mechanism behind this is unknown. Here, we review the literature regarding this phenomenon, attempt to explain what protein domains are crucial for this phenomenon and point toward a putative mechanism. We suggest, that the C-terminal domain of exon 1 Huntingtin, namely the proline rich domain, is responsible for mediating a neuroprotective effect against excitotoxicity. Furthermore, we point out the possible importance of this mechanism for future therapies in neurological disorders that have been suggested to be associated with excitotoxicity, for example Alzheimer's disease, Parkinson's disease and amyotrophic lateral sclerosis.

    Cell death and differentiation 2008;15;3;435-42

  • 14-3-3zeta is indispensable for aggregate formation of polyglutamine-expanded huntingtin protein.

    Omi K, Hachiya NS, Tanaka M, Tokunaga K and Kaneko K

    Department of Neurophysiology, Tokyo Medical University, Shinjuku-ku, Tokyo 160-8402, Japan.

    Huntington's disease (HD) is an autosomal dominant progressive neurodegenerative disorder caused by polyglutamine (polyQ) expansions in the huntingtin (Htt) protein. A hallmark of HD is the presence of aggregates-predominantly composed of NH(2)-terminal fragments of polyQ-expanded Htt-in the nucleus and cytoplasm of affected neurons. We previously proposed that 14-3-3zeta might act as a sweeper of misfolded proteins by facilitating the formation of aggregates possibly for neuroprotection; these aggregates are referred to as inclusion bodies. However, evidence available in this regard is indirect and circumstantial. In this study, analysis of the aggregation-prone protein Htt encoded by HD gene exon 1 containing polyglutamine expansions (Htt86Q) revealed that 17 residues in the NH(2)-terminal of this protein are indispensable for its aggregate formation. Immunoprecipitation assays revealed that 14-3-3beta, gamma, eta, and zeta interact with Htt86Q transfected in N2a cells. Interestingly, the small interfering ribonucleic acid (siRNA) suppression of 14-3-3zeta exclusively abolished Htt86Q aggregate formation, whereas 14-3-3beta or eta siRNA suppression did not. This indicates that 14-3-3zeta participates in aggregate formation under nonnative conditions. Our data support a novel role for 14-3-3zeta in the aggregate formation of nonnative, aggregation-prone proteins.

    Neuroscience letters 2008;431;1;45-50

  • HspB8 chaperone activity toward poly(Q)-containing proteins depends on its association with Bag3, a stimulator of macroautophagy.

    Carra S, Seguin SJ, Lambert H and Landry J

    Centre de Recherche en Cancérologie and Département de Médecine, Université Laval, Québec, Canada.

    Mutations in HspB8, a member of the B group of heat shock proteins (Hsp), have been associated with human neuromuscular disorders. However, the exact function of HspB8 is not yet clear. We previously demonstrated that overexpression of HspB8 in cultured cells prevents the accumulation of aggregation-prone proteins such as the polyglutamine protein Htt43Q. Here we report that HspB8 forms a stable complex with Bag3 in cells and that the formation of this complex is essential for the activity of HspB8. Bag3 overexpression resulted in the accelerated degradation of Htt43Q, whereas Bag3 knockdown prevented HspB8-induced Htt43Q degradation. Additionally, depleting Bag3 caused a reduction in the endogenous levels of LC3-II, a key molecule involved in macroautophagy, whereas overexpressing Bag3 or HspB8 stimulated the formation LC3-II. These results suggested that the HspB8-Bag3 complex might stimulate the degradation of Htt43Q by macroautophagy. This was confirmed by the observation that treatments with macroautophagy inhibitors significantly decreased HspB8- and Bag3-induced degradation of Htt43Q. We conclude that the HspB8 activity is intrinsically dependent on Bag3, a protein that may facilitate the disposal of doomed proteins by stimulating macroautophagy.

    The Journal of biological chemistry 2008;283;3;1437-44

  • HYPK, a Huntingtin interacting protein, reduces aggregates and apoptosis induced by N-terminal Huntingtin with 40 glutamines in Neuro2a cells and exhibits chaperone-like activity.

    Raychaudhuri S, Sinha M, Mukhopadhyay D and Bhattacharyya NP

    Structural Genomics Section, Saha Institute of Nuclear Physics, 1/AF Bidhan Nagar, Kolkata 700 064, India.

    Expansion of polymorphic glutamine (Q) numbers present at the protein Huntingtin (Htt) beyond 36Q results in its misfolding and aggregation, and the aggregates recruit several other proteins. Here we show that HYPK, initially identified as an Htt-interacting partner by yeast two-hybrid assay, physically interacts with N-terminal Htt in Neuro2A cells and alters the numbers and distribution of aggregates formed by N-terminal Htt with 40Q. HYPK also alters the kinetics of mutated N-terminal Htt-mediated aggregate formation. Fluorescence recovery after photobleaching studies reveal that over-expression of HYPK results in the appearance of Htt poly Q aggregates, which upon bleaching recovers approximately 80% of initial fluorescence intensity within 6 min. Fluorescence loss in photobleaching studies indicate loss off fluorescence intensity of the aggregates with time in presence of HYPK. Over-expression of this protein reduces poly Q-mediated caspase-2, caspase-3 and caspase-8 activations, whereas gamma ray-induced activations of these enzymes are not affected. In vitro and in vivo studies demonstrate that HYPK possesses a novel chaperone-like activity. We conclude that HYPK, without having any sequence similarity with known chaperones, plays an effective role in protecting neuronal cells against apoptosis induced by mutated N-terminal Htt by modulating the aggregate formation.

    Human molecular genetics 2008;17;2;240-55

  • Analysis of CCG repeats in Huntingtin gene among HD patients and normal populations in Japan.

    Morovvati S, Nakagawa M, Osame M and Karami A

    Research Center of Molecular Biology, Baqiyatallah Medical Sciences University, Tehran, Iran. morovvati@hotmail.com

    Background: Huntington's disease (HD) is a hereditary autosomal dominant neurodegenerative disease characterized by motor, cognitive, and psychiatric symptoms. The molecular basis of the disease is the expansion of the trinucleotide CAG in the first exon of a gene on chromosome four (4p 16.3). There is another triplet sequence, a CCG repeat, immediately 3' adjacent to the CAG repeat in Huntingtin. This triplet sequence is also polymorphic, alleles of 7 or 10 repeats are predominant in populations, and strong linkage disequilibrium between the CCG (7) allele and HD has been shown in western HD chromosomes, whereas Japanese HD chromosomes strongly associate with an allele of (CCG)10.

    Methods: Distribution of CAG and the CCG repeats in Huntingtin in 15 patients with HD living in southern Japan were selected to evaluate the regional difference in the CCG repeat number in Japan.

    Results: Among our 15 HD patients, only 4 patients had the (CCG)7 allele, and the (CCG)10 alleles were found in the remaining 11 patients.

    Conclusions: In this study, a linkage disequilibrium was found between Japanese HD chromosomes and (CCG)10, whereas western HD chromosomes are strongly associated with (CCG)7. These data suggest that (CCG)10 allele is dominant in southern Japan.

    Archives of medical research 2008;39;1;131-3

  • Huntington disease mutation in Venezuela: age of onset, haplotype analyses and geographic aggregation.

    Paradisi I, Hernández A and Arias S

    Laboratorio de Genética Humana, Centro de Medicina Experimental, Instituto Venezolano de Investigaciones Científicas, Apartado 21827, Caracas 1020A, Venezuela.

    The aggregation of patients with Huntington's disease (HD) around Lake Maracaibo, Zulia State, Venezuela, is widely recognized, but the epidemiology of HD in the whole country is relatively unstudied. We have examined 279 individuals from 60 unrelated affected families residing in various areas of Venezuela for the presence of CAG repeats and other features associated with HD. The number of expanded repeats in 139 carriers varied from 35 to 112. Based on our examination of 71 symptomatic individuals, we developed a log-transformed regression equation, y= -0.0238x + 2.6616, to enable the prediction of age of onset in asymptomatic carriers. Intragenic haplotypes were constructed with two VNTRs (variable number of tandem repeats) and two SNPs (single nucleotide polymorphisms) in the promoter region as well as CCG repeat and Delta2642 polymorphisms to assess kinship between families. In 43 of 45 tested families, the haplotype on the mutated chromosome was 1;G;C;7;(A). The other haplotypes observed, 1;G;C;7;(B) and 4;G;C;7;(A), were of Peruvian and French origins, respectively. The geographic source of the first affected ancestor was assessed in 54 families from 15 different states. Residents of the states of Miranda, Lara and Táchira, excluding those of Zulia, had a mutated allele prevalence five- to ninefold higher than that of other areas. A low (approx. 1/200,000) prevalence, a wide-spread distribution with aggregation in some states and a likely remote European Caucasoid origin are defining epidemiologic features of HD in Venezuela.

    Journal of human genetics 2008;53;2;127-35

  • Neuropsychological deficits in Huntington's disease gene carriers and correlates of early "conversion".

    Brandt J, Inscore AB, Ward J, Shpritz B, Rosenblatt A, Margolis RL and Ross CA

    Department of Psychiatry and Behavioral Sciences, The Johns Hopkins University School of Medicine, Baltimore, MD, USA. jbrandt@jhmi.edu

    The authors examined whether the baseline cognitive functioning of 21 clinically normal huntingtin mutation carriers who developed manifest Huntington's disease on follow-up differed from that of 49 mutation carriers who remain asymptomatic over the same period in a longitudinal study. One hundred thirty-four gene-negative offspring of Huntington's disease patients were studied as well. Overall, there were no differences in cognitive test performance among the three groups. However, "converters" who developed signs of Huntington's disease within 8.6 years demonstrated poorer performance on the Wisconsin Card Sorting Test at baseline. People with the Huntington's disease mutation who are carefully examined neurologically and found to be asymptomatic have, at most, very minimal problem-solving impairment, and only if they are within a few years of clinical onset.

    Funded by: NINDS NIH HHS: NS16375, P01 NS016375, P01 NS016375-250012

    The Journal of neuropsychiatry and clinical neurosciences 2008;20;4;466-72

  • dAtaxin-2 mediates expanded Ataxin-1-induced neurodegeneration in a Drosophila model of SCA1.

    Al-Ramahi I, Pérez AM, Lim J, Zhang M, Sorensen R, de Haro M, Branco J, Pulst SM, Zoghbi HY and Botas J

    Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas, United States of America.

    Spinocerebellar ataxias (SCAs) are a genetically heterogeneous group of neurodegenerative disorders sharing atrophy of the cerebellum as a common feature. SCA1 and SCA2 are two ataxias caused by expansion of polyglutamine tracts in Ataxin-1 (ATXN1) and Ataxin-2 (ATXN2), respectively, two proteins that are otherwise unrelated. Here, we use a Drosophila model of SCA1 to unveil molecular mechanisms linking Ataxin-1 with Ataxin-2 during SCA1 pathogenesis. We show that wild-type Drosophila Ataxin-2 (dAtx2) is a major genetic modifier of human expanded Ataxin-1 (Ataxin-1[82Q]) toxicity. Increased dAtx2 levels enhance, and more importantly, decreased dAtx2 levels suppress Ataxin-1[82Q]-induced neurodegeneration, thereby ruling out a pathogenic mechanism by depletion of dAtx2. Although Ataxin-2 is normally cytoplasmic and Ataxin-1 nuclear, we show that both dAtx2 and hAtaxin-2 physically interact with Ataxin-1. Furthermore, we show that expanded Ataxin-1 induces intranuclear accumulation of dAtx2/hAtaxin-2 in both Drosophila and SCA1 postmortem neurons. These observations suggest that nuclear accumulation of Ataxin-2 contributes to expanded Ataxin-1-induced toxicity. We tested this hypothesis engineering dAtx2 transgenes with nuclear localization signal (NLS) and nuclear export signal (NES). We find that NLS-dAtx2, but not NES-dAtx2, mimics the neurodegenerative phenotypes caused by Ataxin-1[82Q], including repression of the proneural factor Senseless. Altogether, these findings reveal a previously unknown functional link between neurodegenerative disorders with common clinical features but different etiology.

    Funded by: NINDS NIH HHS: NS42179, R01 NS042179, R56 NS042179

    PLoS genetics 2007;3;12;e234

  • R6/2 neurons with intranuclear inclusions survive for prolonged periods in the brains of chimeric mice.

    Reiner A, Del Mar N, Deng YP, Meade CA, Sun Z and Goldowitz D

    Department of Anatomy & Neurobiology, College of Medicine, University of Tennessee Health Science Center, Memphis, Tennessee 38163, USA. areiner@utmem.edu

    The R6/2 mouse possesses mutant exon 1 of human Hdh, and R6/2 mice with 150 CAG repeats show neurological abnormalities by 10 weeks and die by 15 weeks. Few brain abnormalities, however, are evident at death, other than widespread ubiquitinated neuronal intranuclear inclusions (NIIs). We constructed R6/2t+/t- <--> wildtype (WT) chimeric mice to prolong survival of R6/2 cells and determine if neuronal death and/or neuronal injury become evident with longer survival. ROSA26 mice (which bear a lacZ transgene) were used as WT to distinguish between R6/2 and WT neurons. Chimeric mice consisting partly of R6/2 cells lived longer than pure R6/2 mice (up to 10 months), with the survival proportional to the R6/2 contribution. Genotypically R6/2 cells formed NIIs in the chimeras, and these NIIs grew only slightly larger than in 12-week pure R6/2 mice, even after 10 months. Additionally, neuropil aggregates formed near R6/2 neurons in chimeric mice older than 15 weeks. Thus, R6/2 neurons could survive well beyond 15 weeks in chimeras. Moreover, little neuronal degeneration was evident in either cortex or striatum by routine histological stains. Nonetheless, striatal shrinkage and ventricular enlargement occurred, and striatal projection neuron markers characteristically reduced in Huntington's disease were diminished. Consistent with such abnormalities, cortex and striatum in chimeras showed increased astrocytic glial fibrillary acidic protein. These results suggest that while cortical and striatal neurons can survive nearly a year with nuclear and extranuclear aggregates of mutant huntingtin, such lengthy survival does reveal cortical and striatal abnormality brought on by the truncated mutant protein.

    Funded by: NINDS NIH HHS: NS19620, NS28721, R01 NS028721

    The Journal of comparative neurology 2007;505;6;603-29

  • Are cognitive changes progressive in prediagnostic HD?

    Stout JC, Weaver M, Solomon AC, Queller S, Hui S, Johnson SA, Gray J, Beristain X, Wojcieszek J and Foroud T

    Department of Psychological and Brain Sciences, Indiana University, Bloomington, IN, USA.

    Objective: To characterize neurocognitive signs of disease progression in prediagnosis and early Huntington disease (HD) and compare the sensitivity of 2 disease staging classification schemes for detecting these signs.

    Methods: Three hundred and six individuals at-risk for or recently diagnosed with HD completed the Unified Huntington's Disease Rating Scale, genetic testing, and a neurocognitive battery. Two schemes were used to estimate latency to onset of disease. One was based on genetic information (CAG repeat length) and the other was based on the extent of motor signs. Effect sizes were compared to assess the relative sensitivity of the 2 schemes for detecting signs of disease progression.

    Results: CAG-expanded participants far from estimated diagnosis performed similarly to controls, whereas those near to estimated diagnosis were impaired relative to controls. Overall, the method employing genetic information yielded larger effect sizes than the motor scheme, particularly for strategic and executive function measures; the motor scheme resulted in a larger effect size for a measure of motor/psychomotor function.

    Conclusions: Neurocognitive function is not uniformly affected in prediagnosis and early HD; individuals near to their estimated age of diagnosis have cognitive signs similar to HD, whereas individuals far from estimated diagnosis appear cognitively normal. Classification schemes that incorporate both genetic and phenotypic information may be more sensitive for tracking neurocognitive signs of disease progression.

    Funded by: NCRR NIH HHS: M01RR-00750; NINDS NIH HHS: N01-NS-3-2357, R01 NS042659

    Cognitive and behavioral neurology : official journal of the Society for Behavioral and Cognitive Neurology 2007;20;4;212-8

  • Cognitive changes in asymptomatic carriers of the Huntington disease mutation gene.

    Verny C, Allain P, Prudean A, Malinge MC, Gohier B, Scherer C, Bonneau D, Dubas F and Le Gall D

    Centre National de Référence pour les Maladies Neurogénétiques et Mitochondriales de l'Adulte, Centre Hospitalier Universitaire, Angers, France. chverny@chu-angers.fr

    Huntington disease (HD) is a neurodegenerative disorder due to an excessive number of CAG repeats in the IT15 gene on chromosome 4. Studies of cognitive function in asymptomatic gene carriers have yielded contradictory results. This study compared cognitive performance in 44 subjects with the HD mutation (group of carriers) who had no clinical signs of HD and 39 at-risk individuals without HD mutation (group of non-carriers). Neuropsychological evaluation focused on global cognitive efficiency, psychomotor speed, attentional, executive and memory functions. Significant differences, with lower performances in the group of gene carriers, were detected for some measures of psychomotor speed, attention and executive functioning (all P < 0.01). More differences between groups were observed for memory measures, in particular on the California Verbal Memory Test. Complementing these observations, cognitive scores were correlated with age in the group of gene carriers, but not in the group of non-carriers. This suggests that the cognitive changes precede the appearance of the motor and psychiatric symptoms in HD and that tests proved to be sensitive to early HD deficiencies are better suited than global cognitive efficiency scales to observe them.

    European journal of neurology 2007;14;12;1344-50

  • Increased caspase-2, calpain activations and decreased mitochondrial complex II activity in cells expressing exogenous huntingtin exon 1 containing CAG repeat in the pathogenic range.

    Majumder P, Raychaudhuri S, Chattopadhyay B and Bhattacharyya NP

    Saha Institute of Nuclear Physics, 1/AF Bidhan Nagar, Kolkata, 700064, India.

    (1) Huntington's disease (HD) is an autosomal dominant neurodegenerative disease caused by the expansion of polymorphic CAG repeats beyond 36 at exon 1 of huntingtin gene (htt). To study cellular effects by expressing N-terminal domain of Huntingtin (Htt) in specific cell lines, we expressed exon 1 of htt that codes for 40 glutamines (40Q) and 16Q in Neuro2A and HeLa cells. (2) Aggregates and various apoptotic markers were detected at various time points after transfection. In addition, we checked the alterations of expressions of few apoptotic genes by RT-PCR. (3) Cells expressing exon 1 of htt coding 40Q at a stretch exhibited nuclear and cytoplasmic aggregates, increased caspase-1, caspase-2, caspase-8, caspase-9/6, and calpain activations, release of cytochrome c and AIF from mitochondria in a time-dependent manner. Truncation of Bid was increased, while the activity of mitochondrial complex II was decreased in such cells. These changes were significantly higher in cells expressing N-terminal Htt with 40Q than that obtained in cells expressing N-terminal Htt with 16Q. Expressions of caspase-1, caspase-2, caspase-3, caspase-7, and caspase-8 were increased while expression of Bcl-2 was decreased in cells expressing mutated Htt-exon 1. (4) Results presented in this communication showed that expression of mutated Htt-exon 1 could mimic the cellular phenotypes observed in Huntington's disease and this cell model can be used for screening the agents that would interfere with the apoptotic pathway and aggregate formation.

    Cellular and molecular neurobiology 2007;27;8;1127-45

  • Factors associated with HD CAG repeat instability in Huntington disease.

    Wheeler VC, Persichetti F, McNeil SM, Mysore JS, Mysore SS, MacDonald ME, Myers RH, Gusella JF, Wexler NS and US-Venezuela Collaborative Research Group

    Molecular Neurogenetics Unit, Center for Human Genetic Research, Massachusetts General Hospital, Boston, MA 02114, USA. wheeler@helix.mgh.harvard.edu

    Background: The Huntington disease (HD) CAG repeat exhibits dramatic instability when transmitted to subsequent generations. The instability of the HD disease allele in male intergenerational transmissions is reflected in the variability of the CAG repeat in DNA from the sperm of male carriers of the HD gene.

    Results: In this study, we used a collection of 112 sperm DNAs from male HD gene-positive members of a large Venezuelan cohort to investigate the factors associated with repeat instability. We confirm previous observations that CAG repeat length is the strongest predictor of repeat-length variability in sperm, but we did not find any correlation between CAG repeat instability and either age at the time of sperm donation or affectedness status. We also investigated transmission instability for 184 father-offspring and 311 mother-offspring pairs in this Venezuelan pedigree. Repeat-length changes were dependent upon the sex of the transmitting parent and parental CAG repeat length but not parental age or birth order. Unexpectedly, in maternal transmissions, repeat-length changes were also dependent upon the sex of the offspring, with a tendency for expansion in male offspring and contraction in female offspring.

    Conclusion: Significant sibling-sibling correlation for repeat instability suggests that genetic factors play a role in intergenerational CAG repeat instability.

    Funded by: NINDS NIH HHS: NS049206, P50 NS016367, P50 NS016367-28, R01 NS049206, R01 NS049206-04

    Journal of medical genetics 2007;44;11;695-701

  • Huntington's disease and mitochondrial DNA deletions: event or regular mechanism for mutant huntingtin protein and CAG repeats expansion?!

    Banoei MM, Houshmand M, Panahi MS, Shariati P, Rostami M, Manshadi MD and Majidizadeh T

    Department of Medical Genetics, National Institute of Genetic Engineering and Biotechnology, PO Box 14155-6343, Tehran, Iran.

    The mitochondrial DNA (mtDNA) may play an essential role in the pathogenesis of the respiratory chain complex activities in neurodegenerative disorders such as Huntington's disease (HD). Research studies were conducted to determine the possible levels of mitochondrial defect (deletion) in HD patients and consideration of interaction between the expanded Huntingtin gene as a nuclear gene and mitochondria as a cytoplasmic organelle. To determine mtDNA damage, we investigated deletions based in four areas of mitochondrial DNA, in a group of 60 Iranian patients clinically diagnosed with HD and 70 healthy controls. A total of 41 patients out of 60 had CAG expansion (group A). About 19 patients did not show expansion but had the clinical symptoms of HD (group B). MtDNA deletions were classified into four groups according to size; 9 kb, 7.5 kb, 7 kb, and 5 kb. We found one of the four-mtDNA deletions in at least 90% of samples. Multiple deletions have also been observed in 63% of HD patients. None of the normal control (group C) showed mtDNA deletions. The sizes or locations of the deletions did not show a clear correlation with expanded CAG repeat and age in our samples. The study presented evidence that HD patients had higher frequencies of mtDNA deletions in lymphocytes in comparison to the controls. It is thus proposed that CAG repeats instability and mutant Htt are causative factor in mtDNA damage.

    Cellular and molecular neurobiology 2007;27;7;867-75

  • Nucleocytoplasmic trafficking and transcription effects of huntingtin in Huntington's disease.

    Truant R, Atwal RS and Burtnik A

    McMaster University, Department of Biochemistry and Biomedical Sciences, HSC4H24A, 1200 Main Street West, Hamilton, Ontario, Canada L8N3Z5. truantr@mcmaster.ca

    There are nine genetic neurodegenerative diseases caused by a similar genetic defect, a CAG DNA triplet-repeat expansion in the disease gene's open reading frame resulting in a polyglutamine expansion in the disease proteins. Despite the commonality of polyglutamine expansion, each of the polyglutamine diseases manifest as unique diseases, with some similarities, but important differences. These differences suggest that the context of the polyglutamine expansion is important to the mechanism of pathology of the disease proteins. Therefore, it is becoming increasingly paramount to understand the normal functions of these polyglutamine disease proteins, which include huntingtin, the polyglutamine-expanded protein in Huntington's disease (HD). Transcriptional dysregulation is seen in HD. Here we discuss the role of normal huntingtin in transcriptional regulation and misregulation in Huntington's disease in relation to potentially analogous model systems, and to other polyglutamine disease proteins. Huntingtin has functional roles in both the cytoplasm and the nucleus. One commonality of activity of polyglutamine disease proteins is at the level of protein dynamics and ability to import and export to and from the nucleus. Knowing the temporal location of huntingtin protein in response to signaling and neuronal communication could lead to valuable insights into an important trigger of HD pathology.

    Progress in neurobiology 2007;83;4;211-27

  • Sequestration of glyceraldehyde-3-phosphate dehydrogenase to aggregates formed by mutant huntingtin.

    Wu J, Lin F and Qin Z

    Department of Pharmacology and Laboratory of Aging and Nervous Diseases, Soochow University School of Medicine, Suzhou 215123, China.

    Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has been reported to interact with proteins containing the polyglutamine (polyQ) domain. The present study was undertaken to evaluate the potential contributions of the polyQ and polyproline (polyP) domains to the co-localization of mutant huntingtin (htt) and GAPDH. Overexpression of N-terminal htt (1-969 amino acids) with 100Q and 46Q (htt1-969-100Q and httl-969-46Q, mutant htt) in human mammary gland carcinoma MCF-7 cells formed more htt aggregates than that of htt1-969-18Q (wild-type htt). The co-localization of GAPDH with htt aggregates was found in the cells expressing mutant but not wild-type htt. Deletion of the polyP region in the N-terminal htt had no effect on the co-localization of GAPDH and mutant htt aggregates. These results suggest that the polyQ domain, but not the polyP domain, plays a role in the sequestration of GAPDH to aggregates by mutant htt. This effect might contribute to the dysfunction of neurons caused by mutant htt in Huntington's disease.

    Acta biochimica et biophysica Sinica 2007;39;11;885-90

  • Mutant huntingtin's effects on striatal gene expression in mice recapitulate changes observed in human Huntington's disease brain and do not differ with mutant huntingtin length or wild-type huntingtin dosage.

    Kuhn A, Goldstein DR, Hodges A, Strand AD, Sengstag T, Kooperberg C, Becanovic K, Pouladi MA, Sathasivam K, Cha JH, Hannan AJ, Hayden MR, Leavitt BR, Dunnett SB, Ferrante RJ, Albin R, Shelbourne P, Delorenzi M, Augood SJ, Faull RL, Olson JM, Bates GP, Jones L and Luthi-Carter R

    Ecole Polytechnique Fédérale de Lausanne (EPFL), 1015 Lausanne, Switzerland.

    To test the hypotheses that mutant huntingtin protein length and wild-type huntingtin dosage have important effects on disease-related transcriptional dysfunction, we compared the changes in mRNA in seven genetic mouse models of Huntington's disease (HD) and postmortem human HD caudate. Transgenic models expressing short N-terminal fragments of mutant huntingtin (R6/1 and R6/2 mice) exhibited the most rapid effects on gene expression, consistent with previous studies. Although changes in the brains of knock-in and full-length transgenic models of HD took longer to appear, 15- and 22-month CHL2(Q150/Q150), 18-month Hdh(Q92/Q92) and 2-year-old YAC128 animals also exhibited significant HD-like mRNA signatures. Whereas it was expected that the expression of full-length huntingtin transprotein might result in unique gene expression changes compared with those caused by the expression of an N-terminal huntingtin fragment, no discernable differences between full-length and fragment models were detected. In addition, very high correlations between the signatures of mice expressing normal levels of wild-type huntingtin and mice in which the wild-type protein is absent suggest a limited effect of the wild-type protein to change basal gene expression or to influence the qualitative disease-related effect of mutant huntingtin. The combined analysis of mouse and human HD transcriptomes provides important temporal and mechanistic insights into the process by which mutant huntingtin kills striatal neurons. In addition, the discovery that several available lines of HD mice faithfully recapitulate the gene expression signature of the human disorder provides a novel aspect of validation with respect to their use in preclinical therapeutic trials.

    Funded by: Medical Research Council: G0500794, G9810900

    Human molecular genetics 2007;16;15;1845-61

  • Unbiased gene expression analysis implicates the huntingtin polyglutamine tract in extra-mitochondrial energy metabolism.

    Lee JM, Ivanova EV, Seong IS, Cashorali T, Kohane I, Gusella JF and MacDonald ME

    Center for Human Genetic Research, Massachusetts General Hospital, Boston, Massachusetts, United States of America.

    The Huntington's disease (HD) CAG repeat, encoding a polymorphic glutamine tract in huntingtin, is inversely correlated with cellular energy level, with alleles over approximately 37 repeats leading to the loss of striatal neurons. This early HD neuronal specificity can be modeled by respiratory chain inhibitor 3-nitropropionic acid (3-NP) and, like 3-NP, mutant huntingtin has been proposed to directly influence the mitochondrion, via interaction or decreased PGC-1alpha expression. We have tested this hypothesis by comparing the gene expression changes due to mutant huntingtin accurately expressed in STHdh(Q111/Q111) cells with the changes produced by 3-NP treatment of wild-type striatal cells. In general, the HD mutation did not mimic 3-NP, although both produced a state of energy collapse that was mildly alleviated by the PGC-1alpha-coregulated nuclear respiratory factor 1 (Nrf-1). Moreover, unlike 3-NP, the HD CAG repeat did not significantly alter mitochondrial pathways in STHdh(Q111/Q111) cells, despite decreased Ppargc1a expression. Instead, the HD mutation enriched for processes linked to huntingtin normal function and Nf-kappaB signaling. Thus, rather than a direct impact on the mitochondrion, the polyglutamine tract may modulate some aspect of huntingtin's activity in extra-mitochondrial energy metabolism. Elucidation of this HD CAG-dependent pathway would spur efforts to achieve energy-based therapeutics in HD.

    Funded by: NINDS NIH HHS: NS16367, NS32765, P50 NS016367, R01 NS032765; NLM NIH HHS: LM008748, U54 LM008748

    PLoS genetics 2007;3;8;e135

  • Phosphorylation of huntingtin by cyclin-dependent kinase 5 is induced by DNA damage and regulates wild-type and mutant huntingtin toxicity in neurons.

    Anne SL, Saudou F and Humbert S

    Institut Curie, F-91405 Orsay, France.

    Huntingtin is an antiapoptotic protein that becomes toxic when its polyglutamine stretch is expanded, resulting in Huntington's disease (HD). Protein context and posttranslational modifications regulate huntingtin toxicity. Identifying signaling pathways that act on huntingtin is, therefore, key to understanding huntingtin function in normal and pathological conditions. We show here that huntingtin is phosphorylated by the cyclin-dependent kinase 5 (Cdk5) at serines 1181 and 1201. Phosphorylation can be induced by DNA damage in vitro and in vivo. The state of huntingtin phosphorylation is a crucial regulator of neuronal cell death. Absence of phosphorylation of huntingtin at serines 1181 and 1201 confers toxic properties to wild-type huntingtin in a p53-dependent manner in striatal neurons and accelerates neuronal death induced by DNA damage. In contrast, phosphorylation at serines 1181 and 1201 protects against polyQ-induced toxicity. Finally, we show in late stages of HD a sustained DNA damage that is associated with a decrease in Cdk5/p35 levels. We propose that wild-type huntingtin is a component of the DNA damage response signal in neurons and that the Cdk5/DNA damage pathway is dysregulated in HD.

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2007;27;27;7318-28

  • Intergeneration CAG expansion in a Wuhan juvenile-onset Huntington disease family.

    Liu Y, Shen Y, Li H, Wang H, Yang ZR, Chen Y and Tang YP

    Department of Medical Biology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.

    Objective: To make early diagnosis of IT15 gene mutation in a Wuhan juvenile-onset Huntington disease (HD) family, for providing them with genetic counseling, and making preparation for the further research on pathogenesis and experimental therapy of HD.

    Methods: According to the principle of informed consent, we extracted genomic DNA from peripheral blood samples and carried genetic diagnosis of pathogenic exon 1 of IT15 gene by modified touchdown PCR and DNA sequencing methods.

    Results: Eight of twenty-five family members carried abnormal allele: III(10), III(12), III(14), IV(3), and V(2) carried (CAG) (48), IV(11) and IV(12) carried (CAG) (67), and IV(14) carried (CAG) (63), in contrast with the 8-25 CAG trinucleotides in the members of control group. IV(14) carried 15 more CAG trinucleotides than her father III(10).

    Conclusion: The results definitely confirm the diagnosis of HD and indicate the CAG trinucleotide repeat expansion of IT15 gene in this HD family. In addition, CAG expansion results in juvenile-onset and anticipation (characterized by earlier age of onset and increasing severity) of the patient IV(12).

    Neuroscience bulletin 2007;23;4;198-202

  • Type 2 transglutaminase differentially modulates striatal cell death in the presence of wild type or mutant huntingtin.

    Ruan Q, Quintanilla RA and Johnson GV

    Department of Psychiatry, University of Alabama at Birmingham, Birmingham, Alabama 35294-0017, USA.

    Huntington's disease (HD), which is caused by an expanded polyglutamine tract in huntingtin (htt), is characterized by extensive loss of striatal neurons. The dysregulation of type 2 transglutaminase (TG2) has been proposed to contribute to the pathogenesis in HD as TG2 is up-regulated in HD brain and knocking out TG2 in mouse models of HD ameliorates the disease process. To understand the role of TG2 in the pathogenesis of HD, immortalized striatal cells established from mice in which mutant htt with a polyglutamine stretch of 111 Gln had been knocked-in and wild type (WT) littermates, were stably transfected with human TG2 in a tetracycline inducible vector. Overexpression of TG2 in the WT striatal cells resulted in significantly greater cell death under basal conditions as well as in response to thapsigargin treatment, which causes increased intracellular calcium concentrations. Furthermore, in WT striatal cells TG2 overexpression potentiated mitochondrial membrane depolarization, intracellular reactive oxygen species production, and apoptotic cell death in response to thapsigargin. In contrast, in mutant striatal cells, TG2 overexpression did not increase cell death, nor did it potentiate thapsigargin-induced mitochondrial membrane depolarization or intracellular reactive oxygen species production. Instead, TG2 overexpression in mutant striatal cells attenuated the thapsigargin-activated apoptosis. When in situ transglutaminase activity was quantitatively analyzed in these cell lines, we found that in response to thapsigargin treatment TG2 was activated in WT, but not mutant striatal cells. These data suggest that mutant htt alters the activation of TG2 in response to certain stimuli and therefore differentially modulates how TG2 contributes to cell death processes.

    Funded by: NIA NIH HHS: AG012396

    Journal of neurochemistry 2007;102;1;25-36

  • Huntingtin facilitates dynein/dynactin-mediated vesicle transport.

    Caviston JP, Ross JL, Antony SM, Tokito M and Holzbaur EL

    Department of Physiology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA.

    Cytoplasmic dynein is a multisubunit microtubule motor complex that, together with its activator, dynactin, drives vesicular cargo toward the minus ends of microtubules. Huntingtin (Htt) is a vesicle-associated protein found in both neuronal and nonneuronal cells that is thought to be involved in vesicular transport. In this study, we demonstrate through yeast two-hybrid and affinity chromatography assays that Htt and dynein intermediate chain interact directly; endogenous Htt and dynein co-immunoprecipitate from mouse brain cytosol. Htt RNAi in HeLa cells results in Golgi disruption, similar to the effects of compromising dynein/dynactin function. In vitro studies reveal that Htt and dynein are both present on vesicles purified from mouse brain. Antibodies to Htt inhibited vesicular transport along microtubules, suggesting that Htt facilitates dynein-mediated vesicle motility. In vivo inhibition of dynein function results in a significant redistribution of Htt to the cell periphery, suggesting that dynein transports Htt-associated vesicles toward the cell center. Together these findings indicate that Htt binds to dynein and acts in a complex along with dynactin and Htt-associated protein-1 to facilitate vesicular transport.

    Funded by: NIGMS NIH HHS: GM 48661, R01 GM048661

    Proceedings of the National Academy of Sciences of the United States of America 2007;104;24;10045-50

  • Testicular degeneration in Huntington disease.

    Van Raamsdonk JM, Murphy Z, Selva DM, Hamidizadeh R, Pearson J, Petersén A, Björkqvist M, Muir C, Mackenzie IR, Hammond GL, Vogl AW, Hayden MR and Leavitt BR

    Department of Medical Genetics, Centre for Molecular Medicine and Therapeutics, Child and Family Research Institute, University of British Columbia, 950 West 28th Ave., Vancouver, BC, Canada V5Z 4H4.

    Huntington disease (HD) is an adult onset, neurodegenerative disorder that results from CAG expansion in the HD gene. Recent work has demonstrated testicular degeneration in mouse models of HD and alterations in the hypothalamic-pituitary-gonadal (HPG) axis in HD patients. Here, we show that HD patients have specific testicular pathology with reduced numbers of germ cells and abnormal seminiferous tubule morphology. In the YAC128 mouse model, testicular degeneration develops prior to 12 months of age, but at 12 months, there is no evidence for decreased testosterone levels or loss of GnRH neurons in the hypothalamus. This suggests that testicular pathology results from a direct toxic effect of mutant huntingtin in the testis and is supported by the fact that huntingtin is highly expressed in the affected cell populations in the testis. Understanding the pathogenesis of HD in the testis may reveal common critical pathways which lead to degeneration in both the brain and testis.

    Neurobiology of disease 2007;26;3;512-20

  • Triplet repeat mutation length gains correlate with cell-type specific vulnerability in Huntington disease brain.

    Shelbourne PF, Keller-McGandy C, Bi WL, Yoon SR, Dubeau L, Veitch NJ, Vonsattel JP, Wexler NS, US-Venezuela Collaborative Research Group, Arnheim N and Augood SJ

    Huntington disease is caused by the expansion of a CAG repeat encoding an extended glutamine tract in a protein called huntingtin. Here, we provide evidence supporting the hypothesis that somatic increases of mutation length play a role in the progressive nature and cell-selective aspects of HD pathogenesis. Results from micro-dissected tissue and individual laser-dissected cells obtained from human HD cases and knock-in HD mice indicate that the CAG repeat is unstable in all cell types tested although neurons tend to have longer mutation length gains than glia. Mutation length gains occur early in the disease process and continue to accumulate as the disease progresses. In keeping with observed patterns of cell loss, neuronal mutation length gains tend to be more prominent in the striatum than in the cortex of low-grade human HD cases, less so in more advanced cases. Interestingly, neuronal sub-populations of HD mice appear to have different propensities for mutation length gains; in particular, smaller mutation length gains occur in nitric oxide synthase-positive striatal interneurons (a relatively spared cell type in HD) compared with the pan-striatal neuronal population. More generally, the data demonstrate that neuronal changes in HD repeat length can be at least as great, if not greater, than those observed in the germline. The fact that significant CAG repeat length gains occur in non-replicating cells also argues that processes such as inappropriate mismatch repair rather than DNA replication are involved in generating mutation instability in HD brain tissue.

    Funded by: NIGMS NIH HHS: GM36745, R01 GM036745, R01 GM036745-21

    Human molecular genetics 2007;16;10;1133-42

  • Huntingtin interacting proteins are genetic modifiers of neurodegeneration.

    Kaltenbach LS, Romero E, Becklin RR, Chettier R, Bell R, Phansalkar A, Strand A, Torcassi C, Savage J, Hurlburt A, Cha GH, Ukani L, Chepanoske CL, Zhen Y, Sahasrabudhe S, Olson J, Kurschner C, Ellerby LM, Peltier JM, Botas J and Hughes RE

    Prolexys Pharmaceuticals, Salt Lake City, Utah, United States of America.

    Huntington's disease (HD) is a fatal neurodegenerative condition caused by expansion of the polyglutamine tract in the huntingtin (Htt) protein. Neuronal toxicity in HD is thought to be, at least in part, a consequence of protein interactions involving mutant Htt. We therefore hypothesized that genetic modifiers of HD neurodegeneration should be enriched among Htt protein interactors. To test this idea, we identified a comprehensive set of Htt interactors using two complementary approaches: high-throughput yeast two-hybrid screening and affinity pull down followed by mass spectrometry. This effort led to the identification of 234 high-confidence Htt-associated proteins, 104 of which were found with the yeast method and 130 with the pull downs. We then tested an arbitrary set of 60 genes encoding interacting proteins for their ability to behave as genetic modifiers of neurodegeneration in a Drosophila model of HD. This high-content validation assay showed that 27 of 60 orthologs tested were high-confidence genetic modifiers, as modification was observed with more than one allele. The 45% hit rate for genetic modifiers seen among the interactors is an order of magnitude higher than the 1%-4% typically observed in unbiased genetic screens. Genetic modifiers were similarly represented among proteins discovered using yeast two-hybrid and pull-down/mass spectrometry methods, supporting the notion that these complementary technologies are equally useful in identifying biologically relevant proteins. Interacting proteins confirmed as modifiers of the neurodegeneration phenotype represent a diverse array of biological functions, including synaptic transmission, cytoskeletal organization, signal transduction, and transcription. Among the modifiers were 17 loss-of-function suppressors of neurodegeneration, which can be considered potential targets for therapeutic intervention. Finally, we show that seven interacting proteins from among 11 tested were able to co-immunoprecipitate with full-length Htt from mouse brain. These studies demonstrate that high-throughput screening for protein interactions combined with genetic validation in a model organism is a powerful approach for identifying novel candidate modifiers of polyglutamine toxicity.

    Funded by: NINDS NIH HHS: NS42179, R01 NS040251, R01 NS042179, R01 NS40251, R56 NS042179

    PLoS genetics 2007;3;5;e82

  • Cellular and subcellular localization of Huntingtin [corrected] aggregates in the brain of a rat transgenic for Huntington disease.

    Petrasch-Parwez E, Nguyen HP, Löbbecke-Schumacher M, Habbes HW, Wieczorek S, Riess O, Andres KH, Dermietzel R and Von Hörsten S

    Department of Neuroanatomy and Molecular Brain Research, Ruhr-University Bochum, 44801 Bochum, Germany.

    Huntington disease (HD) is a progressive neurodegenerative disorder characterized by emotional, cognitive, and motor dysfunctions. Aggregation of huntingtin is a hallmark of HD and, therefore, a crucial parameter for the evaluation of HD animal models. We investigated here the regional, cellular, and subcellular distribution of N-terminal huntingtin aggregates and associated neuropathological changes in the forebrain of a rat transgenic for HD (tgHD). The tgHD rat brain showed enormously enlarged lateral ventricles and a similar atrophy of cortical and subcortical areas as known in HD patients. Huntingtin aggregates of varying size and forms were regionally identified in neuronal nuclei, cytoplasm, dendrites, dendritic spines, axons, and synaptic terminals, closely resembling the results described earlier for human HD brains and in established HD mouse models. Huntingtin aggregates in mitochondria support mitochondrial dysfunction as contributing to the disease pathogenesis. Dark cell degeneration was reminiscent of results in HD individuals and HD mouse models. Interestingly, huntingtin aggregates were especially well accumulated in two interacting limbic forebrain systems, the ventral striatopallidum and the extended amygdala, which may contribute to the early onset of emotional changes observed in the tgHD rat. In conclusion, the tgHD rat model reflects to a remarkable extent the cellular and subcellular neuropathological key features as observed in human HD and HD mouse brains and hints of changes in limbic forebrain systems, which may elucidate the emotional dysfunction in the tgHD rat and affective disturbances in HD patients.

    The Journal of comparative neurology 2007;501;5;716-30

  • Ubiquitin-interacting motifs inhibit aggregation of polyQ-expanded huntingtin.

    Miller SL, Scappini EL and O'Bryan J

    Laboratory of Signal Transduction, NIEHS, National Institutes of Health, Department of Health and Human Services, Research Triangle Park, North Carolina 27709, USA.

    Expansion of polyglutamine (polyQ) tracts within proteins underlies a number of neurodegenerative diseases, such as Huntington disease, Kennedy disease, and spinocerebellar ataxias. The resulting mutant proteins are unstable, forming insoluble aggregates that are associated with components of the ubiquitin system, including ubiquitin, ubiquitin-like proteins, and proteins that bind to ubiquitin. Given the presence of these ubiquitin-binding proteins in the insoluble aggregates, we examined whether heterologous expression of short motifs that bind ubiquitin, termed ubiquitin-interacting motifs (UIMs), altered the aggregation of polyQ-expanded huntingtin (Htt), the protein product of the Huntington disease gene. We found that a subset of UIMs associated with mutant Htt. The ability to interact with ubiquitin was necessary, but not sufficient, for interaction with mutant Htt. Furthermore, we found that expression of single, isolated UIMs inhibited aggregation of mutant Htt. These data suggest that isolated UIMs might serve as potential inhibitors of polyQ-aggregation in vivo.

    Funded by: Intramural NIH HHS

    The Journal of biological chemistry 2007;282;13;10096-103

  • Aggregate-centered redistribution of proteins by mutant huntingtin.

    Swayne LA and Braun JE

    Hotchkiss Brain Institute, Department of Physiology and Biophysics, University of Calgary, Calgary, Alta., Canada T2N 4N1.

    Huntingtin is a widely expressed 350-kDa cytosolic multidomain of unknown function. Aberrant expansion of the polyglutamine tract located in the N-terminal region of huntingtin results in Huntington's disease. The presence of insoluble huntingtin inclusions in the brains of patients is one of the hallmarks of Huntington's disease. Experimentally, both full-length huntingtin and N-terminal fragments of huntingtin with expanded polyglutamine tracts trigger aggregate formation. Here, we report that upon the formation of huntingtin aggregates; endogenous cytosolic huntingtin, Hsc70/Hsp70 (heat shock protein and cognate protein of 70kDa) and syntaxin 1A become aggregate-centered. This redistribution suggests that these proteins are eventually depleted and become unavailable for normal cellular function. These results indicate that the cellular targeting of several key proteins are altered in the presence of mutant huntingtin and suggest that aggregate depletion of these proteins may underlie, in part, the sequence of disease progression.

    Biochemical and biophysical research communications 2007;354;1;39-44

  • Reduced penetrance alleles for Huntington's disease: a multi-centre direct observational study.

    Quarrell OW, Rigby AS, Barron L, Crow Y, Dalton A, Dennis N, Fryer AE, Heydon F, Kinning E, Lashwood A, Losekoot M, Margerison L, McDonnell S, Morrison PJ, Norman A, Peterson M, Raymond FL, Simpson S, Thompson E and Warner J

    Objective: To obtain penetrance data for Huntington's disease when DNA results are in the range of 36-39 CAG repeats and assess the consistency of reporting the upper allele from two reference centres.

    Method: Data were collected anonymously on age of onset or age last known to be unaffected from a cohort of individuals with results in this range. DNA samples were re-analysed in two reference centres. Kaplan-Meier analysis was used to construct an age of onset curve and penetrance figures.

    Results: Clinical data and concordant DNA results from both reference centres were available for 176 samples; penetrance figures (and 95% confidence intervals) for this cohort, at age 65 and 75 years, were 63.9% (55.5% to 73.2%) and 74.2% (64.2% to 84.2%), respectively. Inclusion of 28 additional subjects for whom repeat DNA results were unavailable, obtained from only one reference centre, or discrepant by one repeat within this range, gave penetrance data (including 95% confidence intervals) at ages 65 and 75 years of 62.4% (54.4% to 70.4%) and 72.7.% (63.3% to 82.1%), respectively. 238 duplicate results were available from the reference centres; 10 (4.2%) differed by one CAG repeat in the reporting of the upper allele and in two (0.84%) of these cases the discrepancy was between 39 and 40 repeats.

    Conclusion: When DNA results are in this range, a conservative approach is to say that there is at least a 40% chance the person will be asymptomatic at age 65 years and at least a 30% chance the person will be asymptomatic at age 75 years.

    Journal of medical genetics 2007;44;3;e68

  • Wild-type huntingtin participates in protein trafficking between the Golgi and the extracellular space.

    Strehlow AN, Li JZ and Myers RM

    Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305-5120, USA.

    Huntington disease (HD) is an autosomal dominant neurodegenerative disease caused by an expanded CAG trinucleotide repeat in the first exon of the HD gene, which results in a toxic polyglutamine stretch within huntingtin, the protein it encodes. Understanding the normal function of this essential protein is vital to understanding the root of the disease, yet despite more than a decade of investigation, its role in the cell remains elusive. Identifying the subcellular localization of huntingtin and understanding its effects on global gene expression are critical to this endeavor. While most reports agree that huntingtin is predominantly a cytoplasmic protein, conflicting distribution patterns have been demonstrated at the subcellular level. Here, we examine wild-type huntingtin's localization in cultured cells by expressing the full-length human protein tagged with enhanced green fluorescent protein (EGFP) within its unspliced genomic context. In fibrosarcoma and neuroblastoma cells, huntingtin shows discrete punctate, perinuclear localization overlapping largely with the trans-Golgi and cytoplasmic clathrin-coated vesicles, implicating huntingtin in vesicle trafficking. To determine whether huntingtin is involved in trafficking a specific subset of proteins, we measured changes in global transcription levels in embryonic stem cells and neurons lacking huntingtin. Huntingtin null neurons exhibit a significant reduction in transcripts encoding proteins destined for the extracellular space, many of which are components of the extracellular matrix or involved in cellular adhesion, receptor binding and hormone activity. Together, these findings support a role for huntingtin in the intracellular trafficking of proteins required for the construction of the extracellular matrix.

    Funded by: NIGMS NIH HHS: T32 GM 07790-25

    Human molecular genetics 2007;16;4;391-409

  • Ubiquitin ligase Hrd1 enhances the degradation and suppresses the toxicity of polyglutamine-expanded huntingtin.

    Yang H, Zhong X, Ballar P, Luo S, Shen Y, Rubinsztein DC, Monteiro MJ and Fang S

    Medical Biotechnology Center, University of Maryland Biotechnology Institute, 725 W. Lombard Street, Baltimore, MD 21201, USA.

    E3 ubiquitin ligases catalyze the conjugation of ubiquitin onto proteins, which acts as a signal for targeting proteins for degradation by the proteasome. Hrd1 is an endoplasmic reticulum (ER) membrane-spanning E3 with its catalytic active RING finger facing the cytosol. We speculated that this topology might allow Hrd1 to ubiquitinate misfolded proteins in the cytosol. We tested this idea by using polyglutamine (polyQ)-containing huntingtin (htt) protein as a model substrate. We found that the protein levels of Hrd1 were increased in cells overexpressing the N-terminal fragment of htt containig an expanded polyQ tract (httN). Forced expression of Hrd1 enhanced the degradation of httN in a RING finger-dependent manner, whereas silencing of endogenous Hrd1 expression by RNA interference stabilized httN. Degradation of httN was found to be p97/VCP-dependent, but independent of Ufd1 and Npl4, all of which are thought to form a complex with Hrd1 during ER-associated degradation. Consistent with its role as an E3 for httN, we demonstrate that Hrd1 interacts with and ubiquitinates httN. Subcellular fractionation and confocal microscopy revealed that Hrd1recruits HttN to the ER and co-localizes with juxtanuclear aggregates of httN in cells. Interaction of Hrd1 with httN was found to be independent of the length of the polyglutamine tract. However, httN with expanded polyglutamine tracts appeared to be a preferred substrate for Hrd1. Functionally, we found that Hrd1 protects cells against the httN-induced cell death. These results suggest that Hrd1 is a novel htt-interacting protein that can target pathogenic httN for degradation and is able to protect cells against httN-induced cell death.

    Funded by: Medical Research Council: G0600194, G0600194(77639); NIGMS NIH HHS: GM066287, R01 GM069967, R01 GM69967; Wellcome Trust: 064354

    Experimental cell research 2007;313;3;538-50

  • Cardiac dysfunction in the R6/2 mouse model of Huntington's disease.

    Mihm MJ, Amann DM, Schanbacher BL, Altschuld RA, Bauer JA and Hoyt KR

    Center for Cardiovascular Medicine, Columbus Children's Research Institute, 700 Children's Drive, Columbus, OH 43205, USA.

    Recent evidence suggests that mutant huntingtin protein-induced energetic perturbations contribute to neuronal dysfunction in Huntington's disease (HD). Given the ubiquitous expression of huntingtin, other cell types with high energetic burden may be at risk for HD-related dysfunction. Early-onset cardiovascular disease is the second leading cause of death in HD patients; a direct role for mutant huntingtin in this phenomenon remains unevaluated. Here we tested the hypothesis that expression of mutant huntingtin is sufficient to induce cardiac dysfunction, using a well-described transgenic model of HD (line R6/2). R6/2 mice developed cardiac dysfunction by 8 weeks of age, progressing to severe failure at 12 weeks, assessed by echocardiography. Limited evidence of cardiac remodeling (e.g. hypertrophy, fibrosis, apoptosis, beta(1) adrenergic receptor downregulation) was observed. Immunogold electron microscopy demonstrated significant elevations in nuclear and mitochondrial polyglutamine presence in the R6/2 myocyte. Significant alterations in mitochondrial ultrastructure were seen, consistent with metabolic stress. Increased cardiac lysine acetylation and protein nitration were observed and were each significantly associated with impairments in cardiac performance. These data demonstrate that mutant huntingtin expression has potent cardiotoxic effects; cardiac failure may be a significant complication of this important experimental model of HD. Investigation of the potential cardiotropic effects of mutant huntingtin in humans may be warranted.

    Funded by: NHLBI NIH HHS: HL59791, HL63067, R01 HL059791, R01 HL063067; NINDS NIH HHS: NS41003, R01 NS041003

    Neurobiology of disease 2007;25;2;297-308

  • The first 17 amino acids of Huntingtin modulate its sub-cellular localization, aggregation and effects on calcium homeostasis.

    Rockabrand E, Slepko N, Pantalone A, Nukala VN, Kazantsev A, Marsh JL, Sullivan PG, Steffan JS, Sensi SL and Thompson LM

    Department of Psychiatry and Human Behavior, University of California, Gillespie 2121, Irvine, CA 92697, USA.

    A truncated form of the Huntington's disease (HD) protein that contains the polyglutamine repeat, Httex1p, causes HD-like phenotypes in multiple model organisms. Molecular signatures of pathogenesis appear to involve distinct domains within this polypeptide. We studied the contribution of each domain, singly or in combination, to sub-cellular localization, aggregation and intracellular Ca2+ ([Ca2+]i) dynamics in cells. We demonstrate that sub-cellular localization is most strongly influenced by the first 17 amino acids, with this sequence critically controlling Httex1p mitochondrial localization and also promoting association with the endoplasmic reticulum (ER) and Golgi. This domain also enhances the formation of visible aggregates and together with the expanded polyQ repeat acutely disrupts [Ca2+]i levels in glutamate-challenged PC12 cells. Isolated cortical mitochondria incubated with Httex1p resulted in uncoupling and depolarization of these organelles, further supporting the idea that Httex1p-dependent mitochondrial dysfunction could be instrumental in promoting acute Ca2+ dyshomeostasis. Interestingly, neither mitochondrial nor ER associations seem to be required to promote long-term [Ca2+]i dyshomeostasis.

    Funded by: NCI NIH HHS: CA-62203; NICHD NIH HHS: HD36081; NINDS NIH HHS: NS045283, NS42157-04

    Human molecular genetics 2007;16;1;61-77

  • Autopsy-proven Huntington's disease with 29 trinucleotide repeats.

    Kenney C, Powell S and Jankovic J

    Parkinson's Disease Center and Movement Disorders Clinic, Department of Neurology, Baylor College of Medicine, Houston, Texas 77030, USA. kenney@bcm.edu

    Huntington's disease (HD) is a neurodegenerative disorder associated with expansion of CAG trinucleotide repeats in the huntingtin gene. A minimum of 36 CAG repeats is usually reported in patients with clinical features of HD; 30 to 35 repeats represent an intermediate range. Here we report a 65-year-old male with autopsy-proven HD and 29 CAG repeats.

    Movement disorders : official journal of the Movement Disorder Society 2007;22;1;127-30

  • Early cognitive deficits in Swedish gene carriers of Huntington's disease.

    Robins Wahlin TB, Lundin A and Dear K

    Karolinska Institutet, Department of Neurobiology, Caring Sciences and Society, Stockholm, Sweden. tarja-brita.robins.wahlin@ki.se

    The primary focus of this study was to examine whether there is early neuropsychological impairment in presymptomatic Huntington's disease (HD). A broad neuropsychological assessment battery was administered to 24 asymptomatic gene carriers (HD+) and 31 noncarriers (HD-). The gene carriers revealed inferior cognitive functioning as compared with the noncarriers in memory and executive functions. When the gene carriers were assigned to 2 groups based on predicted years to onset (with 15 and over being HD+ late and under 15 being HD+ near), the HD+ near group performed significantly worse than the HD+ late group in all domains but ability to shift conceptually and visuospatial memory. Results suggest that early cognitive deficits are detectable prior to motor symptoms, first in memory functions and then in executive functions and perceptual motor speed.

    Neuropsychology 2007;21;1;31-44

  • Huntingtin inhibits caspase-3 activation.

    Zhang Y, Leavitt BR, van Raamsdonk JM, Dragatsis I, Goldowitz D, MacDonald ME, Hayden MR and Friedlander RM

    Neuroapoptosis Laboratory, Department of Neurosurgery, Brigham and Women's Hospital, Harvard Medical School, 75 Francis Street, Boston, MA 02115, USA.

    Huntington's disease results from a mutation in the HD gene encoding for the protein huntingtin. The function of huntingtin, although beginning to be elucidated, remains largely unclear. To probe the prosurvival function of huntingtin, we modulate levels of wild-type huntingtin in a number of cellular and in vivo models. Huntingtin depletion resulted in caspase-3 activation, and overexpression of huntingtin resulted in caspase-3 inhibition. Additionally, we demonstrate that huntingtin physically interacts with active caspase-3. Interestingly, mutant huntingtin binds active caspase-3 with a lower affinity and lower inhibitory effect on active caspase-3 than does wild-type huntingtin. Although reduction of huntingtin levels resulted in caspase-3 activation in all conditions examined, the cellular response was cell-type specific. Depletion of huntingtin resulted in either overt cell death, or in increased vulnerability to cell death. These data demonstrate that huntingtin inhibits caspase-3 activity, suggesting a mechanism whereby caspase-mediated huntingtin depletion results in a detrimental amplification cascade leading to further caspase-3 activation, resulting in cell dysfunction and cell death.

    Funded by: NINDS NIH HHS: NS 16367, NS 32765, P50 NS016367, R01 NS032765

    The EMBO journal 2006;25;24;5896-906

  • Mutant huntingtin impairs the post-Golgi trafficking of brain-derived neurotrophic factor but not its Val66Met polymorphism.

    del Toro D, Canals JM, Ginés S, Kojima M, Egea G and Alberch J

    Departament de Biologia Cel.lular i Anatomia Patològica, Facultat de Medicina, Institut d'Investigacions Biomèdiques August Pi i Sunyer, Universitat de Barcelona, 08036 Barcelona, Spain.

    Brain-derived neurotrophic factor (BDNF) polymorphism is associated with the pathophysiology of several neurodegenerative disorders, including Huntington's disease. In view of these data and the involvement of huntingtin in intracellular trafficking, we examined the intracellular transport and release of Val66Val BDNF (Val-BDNF) and Val66Met BDNF (Met-BDNF) in transfected striatal knock-in cells expressing wild-type or mutant full-length huntingtin. Colocalization studies with specific markers for endoplasmic reticulum showed no differences between the Val-BDNF and Met-BDNF and were not modified by mutant huntingtin. However, post-Golgi trafficking was altered by mutant huntingtin dependent on the BDNF form. Thus, fluorescence recovery after photobleaching (FRAP) and inverse FRAP analysis showed retention of Met-BDNF in the Golgi apparatus with respect to Val-BDNF in wild-type cells. Strikingly, mutant huntingtin diminished post-Golgi trafficking of Val-BDNF, whereas Met-BDNF was not modified. Accordingly, a reduction in the number of transport vesicles was only observed in mutant huntingtin cells transfected with Val-BDNF but not Met-BDNF. Moreover, mutant huntingtin severely affected the KCl-evoked release of Val-BDNF, although it had little effect on Met-BDNF regulated release. The constitutive release of Val-BDNF or Met-BDNF in mutant cells was only slightly reduced. Interestingly, mutant huntingtin only perturbed post-Golgi trafficking of proteins that follow the regulated secretory pathway (epidermal growth factor receptor or atrial natriuretic factor), whereas it did not change those that follow the constitutive pathway (p75(NTR)). We conclude that mutant huntingtin differently affects intracellular transport and release of Val-BDNF and Met-BDNF. In addition, our findings reveal a new role for huntingtin in the regulation of the post-Golgi trafficking of the regulated secretory pathway.

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2006;26;49;12748-57

  • Increased apoptosis, Huntingtin inclusions and altered differentiation in muscle cell cultures from Huntington's disease subjects.

    Ciammola A, Sassone J, Alberti L, Meola G, Mancinelli E, Russo MA, Squitieri F and Silani V

    Department of Neurology and Laboratory of Neuroscience, Dino Ferrari Center, University of Milan Medical School, IRCCS Istituto Auxologico Italiano, Milan, Italy. a.ciammola@auxologico.it

    Mutated huntingtin (htt) is ubiquitously expressed in tissues of Huntington's disease (HD) patients. In the brain, the mutated protein leads to neuronal cell dysfunction and death, associated with formation of htt-positive inclusions. Given increasing evidence of abnormalities in HD skeletal muscle, we extensively analyzed primary muscle cell cultures from seven HD subjects (including two unaffected mutation carriers). Myoblasts from presymptomatic and symptomatic HD subjects showed cellular abnormalities in vitro, namely mitochondrial depolarization, cytochrome c release, increased caspase-3, -8, and -9 activities, and defective cell differentiation. Another notable feature was the formation of htt inclusions in differentiated myotubes. This study helps to advance current knowledge about the downstream effects of the htt mutation in human tissues. Further applications may include drug screening using this human cellular model.

    Cell death and differentiation 2006;13;12;2068-78

  • Structural insights into the specific binding of huntingtin proline-rich region with the SH3 and WW domains.

    Gao YG, Yan XZ, Song AX, Chang YG, Gao XC, Jiang N, Zhang Q and Hu HY

    State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China.

    The interactions of huntingtin (Htt) with the SH3 domain- or WW domain-containing proteins have been implicated in the pathogenesis of Huntington's disease (HD). We report the specific interactions of Htt proline-rich region (PRR) with the SH3GL3-SH3 domain and HYPA-WW1-2 domain pair by NMR. The results show that Htt PRR binds with the SH3 domain through nearly its entire chain, and that the binding region on the domain includes the canonical PxxP-binding site and the specificity pocket. The C terminus of PRR orients to the specificity pocket, whereas the N terminus orients to the PxxP-binding site. Htt PRR can also specifically bind to WW1-2; the N-terminal portion preferentially binds to WW1, while the C-terminal portion binds to WW2. This study provides structural insights into the specific interactions between Htt PRR and its binding partners as well as the alteration of these interactions that involve PRR, which may have implications for the understanding of HD.

    Structure (London, England : 1993) 2006;14;12;1755-65

  • Context-dependent dysregulation of transcription by mutant huntingtin.

    Cornett J, Smith L, Friedman M, Shin JY, Li XJ and Li SH

    Department of Human Genetics, Emory University School of Medicine, Atlanta, Georgia 30322, USA.

    Huntington disease (HD) is an adult-onset neurodegenerative disease caused by expansion of a polyglutamine (poly(Q) tract in the N-terminal region of huntingtin (htt). Although the precise mechanisms leading to neurodegeneration in HD have not been fully elucidated, transcriptional dysregulation has been implicated in disease pathogenesis. In HD, multiple N-terminal mutant htt fragments smaller than the first 500 amino acids have been found to accumulate in the nucleus and adversely affect gene transcription. It is unknown whether different htt fragments in the nucleus can differentially bind transcription factors and affect transcription. Here, we report that shorter N-terminal htt fragments, which are more prone to misfolding and aggregation, are more competent to bind Sp1 and inhibit its activity. These effects can be reversed by Hsp40, a molecular chaperone that reduces the misfolding of mutant htt. Our results provide insight into the beneficial effects of molecular chaperones and suggest that context dependent transcriptional dysregulation may contribute to differential toxicity of various N-terminal htt fragments.

    Funded by: NIA NIH HHS: AG19206; NINDS NIH HHS: NS045016, NS41669

    The Journal of biological chemistry 2006;281;47;36198-204

  • Mutant huntingtin expression induces mitochondrial calcium handling defects in clonal striatal cells: functional consequences.

    Milakovic T, Quintanilla RA and Johnson GV

    Department of Psychiatry, University of Alabama at Birmingham, Birmingham, Alabama 35294-0017, USA.

    Huntington disease (HD) is caused by a pathological elongation of CAG repeats in the huntingtin protein gene and is characterized by atrophy and neuronal loss primarily in the striatum. Mitochondrial dysfunction and impaired Ca2+ homeostasis in HD have been suggested previously. Here, we elucidate the effects of Ca2+ on mitochondria from the wild type (STHdhQ7/Q7) and mutant (STHdhQ111/Q111) huntingtin-expressing cells of striatal origin. When treated with increasing Ca2+ concentrations, mitochondria from mutant huntingtin-expressing cells showed enhanced sensitivity to Ca2+, as they were more sensitive to Ca2+-induced decreases in state 3 respiration and DeltaPsim, than mitochondria from wild type cells. Further, mutant huntingtin-expressing cells had a reduced mitochondrial Ca2+ uptake capacity in comparison with wild type cells. Decreases in state 3 respiration were associated with increased mitochondrial membrane permeability. The DeltaPsim defect was attenuated in the presence of ADP and the decreases in Ca2+ uptake capacity were abolished in the presence of Permeability Transition Pore (PTP) inhibitors. These findings clearly indicate that mutant huntingtin-expressing cells have mitochondrial Ca2+ handling defects that result in respiratory deficits and that the increased sensitivity to Ca2+ induced mitochondrial permeabilization maybe a contributing mechanism to the mitochondrial dysfunction in HD.

    Funded by: NINDS NIH HHS: NS041744

    The Journal of biological chemistry 2006;281;46;34785-95

  • [IT15 gene analysis in two pedigrees of Huntington's disease].

    Zhang BR, Song F, Yin XZ, Xia K, Tian J, Huang JZ and Xia JH

    Department of Neurology, Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310009, China. brzhang@zju.edu.cn

    To investigate the relationship between the clinical features and (CAG)n trinucleotide repeats in two pedigrees of Chinese Huntington's disease (HD). Clinical and neuroimaging features, the age of disease onset and pattern of transmission of the patients were studied in the two pedigrees of HD. Genomic DNA of 42 family members was used for amplification of the (CAG)n repeats of IT15 gene by PCR. The numbers of (CAG)n were determined by electrophoresis through a 6% polyacrylamide gel and direct sequence analysis. Results showed that patients in pedigree 1 were absent of the typical triad of HD symptoms or caudate atrophy. A total of 9 (5 patients and 4 asymptomatic) out of 18 family members had 40-50 (CAG)n repeats in the IT15 gene. In pedigree 2, all the patients were characterized by a triad of symptoms, including motor disturbance, cognitive impairment and psychiatric features. Three patients and two asymptomatic relatives had more than 50 (CAG)n repeats in the IT15 gene. In conclusion, the clinical symptoms are partly determined by (CAG)n repeats in the IT15 gene. The age of onset was correlated with (CAG)n repeats over 50, and the phenomenon called "anticipation" was found to have played a role.

    Yi chuan = Hereditas 2006;28;11;1345-9

  • Cytotoxicity of a mutant huntingtin fragment in yeast involves early alterations in mitochondrial OXPHOS complexes II and III.

    Solans A, Zambrano A, Rodríguez M and Barrientos A

    Department of Neurology, Dr. John T. Macdonald Foundation Center for Medical Genetics, University of Miami, Miller School of Medicine, 1600 NW 10th Avenue, Miami, FL 33136, USA.

    Mitochondrial dysfunction may play an important role in the pathogenic mechanism of Huntington's disease (HD). However, the exact mechanism by which mutated huntingtin could cause bioenergetic dysfunction is still unknown. We have constructed a stable inducible yeast model of HD by expressing a human huntingtin fragment containing a mutant polyglutamine tract of 103Q fused to green fluorescent protein (GFP), and a control expressing a wild-type 25Q domain fused to GFP in a wild-type strain. We showed that in yeast cells expressing 103Q, cell respiration was progressively reduced after 4-6 h of induction with galactose, down to 50% of the control after 10 h of induction. The cell respiration defect results from an alteration in the function and amount of mitochondrial respiratory chain complex II+III, in congruency to data obtained from postmortem brain of HD patients and from toxin models. In our model, the production of reactive oxygen species (ROS) is significantly enhanced in cells expressing 103Q. Quenching of ROS with resveratrol partially prevents the cell respiration defect. Mitochondrial morphology and distribution were also altered in cells expressing 103Q, probably resulting from the interaction of aggregates with portions of the mitochondrial web and from a progressive disruption of the actin cytoskeleton. We propose a mechanism for mitochondrial dysfunction in our yeast model of HD in which the interactions of misfolded/aggregated polyglutamine domains with the mitochondrial and actin networks lead to disturbances in mitochondrial distribution and function and to increase in ROS production. Oxidative damage could preferentially affect the stability and function of enzymes containing iron-sulfur clusters such as complexes II and III. Our yeast model represents a very useful paradigm to study mitochondrial physiology alterations in the pathogenic mechanism of HD.

    Human molecular genetics 2006;15;20;3063-81

  • Huntingtin phosphorylation sites mapped by mass spectrometry. Modulation of cleavage and toxicity.

    Schilling B, Gafni J, Torcassi C, Cong X, Row RH, LaFevre-Bernt MA, Cusack MP, Ratovitski T, Hirschhorn R, Ross CA, Gibson BW and Ellerby LM

    The Buck Institute for Age Research, Novato, California 94945, USA.

    Huntingtin (Htt) is a large protein of 3144 amino acids, whose function and regulation have not been well defined. Polyglutamine (polyQ) expansion in the N terminus of Htt causes the neurodegenerative disorder Huntington disease (HD). The cytotoxicity of mutant Htt is modulated by proteolytic cleavage with caspases and calpains generating N-terminal polyQ-containing fragments. We hypothesized that phosphorylation of Htt may modulate cleavage and cytotoxicity. In the present study, we have mapped the major phosphorylation sites of Htt using cell culture models (293T and PC12 cells) expressing full-length myc-tagged Htt constructs containing 23Q or 148Q repeats. Purified myc-tagged Htt was subjected to mass spectrometric analysis including matrix-assisted laser desorption/ionization mass spectrometry and nano-HPLC tandem mass spectrometry, used in conjunction with on-target alkaline phosphatase and protease digestions. We have identified more than six novel serine phosphorylation sites within Htt, one of which lies in the proteolytic susceptibility domain. Three of the sites have the consensus sequence for ERK1 phosphorylation, and addition of ERK1 inhibitor blocks phosphorylation at those sites. Other observed phosphorylation sites are possibly substrates for CDK5/CDC2 kinases. Mutation of amino acid Ser-536, which is located in the proteolytic susceptibility domain, to aspartic acid, inhibited calpain cleavage and reduced mutant Htt toxicity. The results presented here represent the first detailed mapping of the phosphorylation sites in full-length Htt. Dissection of phosphorylation modifications in Htt may provide clues to Huntington disease pathogenesis and targets for therapeutic development.

    Funded by: NINDS NIH HHS: F32 NS043937, NS 16375, NS 38144, NS40251A, RL1 NS062413

    The Journal of biological chemistry 2006;281;33;23686-97

  • Huntingtin interacts with the receptor sorting family protein GASP2.

    Horn SC, Lalowski M, Goehler H, Dröge A, Wanker EE and Stelzl U

    Department of Neuroproteomics, Max-Delbrück-Centrum for Molecular Medicine (MDC), Berlin, Germany.

    Protein interaction networks are useful resources for the functional annotation of proteins. Recently, we have generated a highly connected protein-protein interaction network for Huntington's disease (HD) by automated yeast two-hybrid (Y2H) screening (Goehler et al., 2004). The network included several novel direct interaction partners for the disease protein huntingtin (htt). Some of these interactions, however, have not been validated by independent methods. Here we describe the verification of the interaction between htt and GASP2 (G protein-coupled receptor associated sorting protein 2), a protein involved in membrane receptor degradation. Using membrane-based and classical coimmunoprecipitation assays we demonstrate that htt and GASP2 form a complex in cotransfected mammalian cells. Moreover, we show that the two proteins colocalize in SH-SY5Y cells, raising the possibility that htt and GASP2 interact in neurons. As the GASP protein family plays a role in G protein-coupled receptor sorting, our data suggest that htt might influence receptor trafficking via the interaction with GASP2.

    Journal of neural transmission (Vienna, Austria : 1996) 2006;113;8;1081-90

  • Cleavage at the caspase-6 site is required for neuronal dysfunction and degeneration due to mutant huntingtin.

    Graham RK, Deng Y, Slow EJ, Haigh B, Bissada N, Lu G, Pearson J, Shehadeh J, Bertram L, Murphy Z, Warby SC, Doty CN, Roy S, Wellington CL, Leavitt BR, Raymond LA, Nicholson DW and Hayden MR

    Department of Medical Genetics, Centre for Molecular Medicine and Therapeutics, University of British Columbia, Vancouver, BC V5Z 4H4, Canada.

    Cleavage of huntingtin (htt) has been characterized in vitro, and accumulation of caspase cleavage fragments represents an early pathological change in brains of Huntington's disease (HD) patients. However, the relationship between htt proteolysis and the pathogenesis of HD is unknown. To determine whether caspase cleavage of htt is a key event in the neuronal dysfunction and selective neurodegeneration in HD, we generated YAC mice expressing caspase-3- and caspase-6-resistant mutant htt. Mice expressing mutant htt, resistant to cleavage by caspase-6 but not caspase-3, maintain normal neuronal function and do not develop striatal neurodegeneration. Furthermore, caspase-6-resistant mutant htt mice are protected against neurotoxicity induced by multiple stressors including NMDA, quinolinic acid (QA), and staurosporine. These results are consistent with proteolysis of htt at the caspase-6 cleavage site being an important event in mediating neuronal dysfunction and neurodegeneration and highlight the significant role of htt proteolysis and excitotoxicity in HD.

    Cell 2006;125;6;1179-91

  • Expression and characterization of full-length human huntingtin, an elongated HEAT repeat protein.

    Li W, Serpell LC, Carter WJ, Rubinsztein DC and Huntington JA

    Department of Haematology, University of Cambridge, Cambridge Institute for Medical Research, Cambridge CB2 2XY, United Kingdom.

    Huntington disease is an inherited neurodegenerative disorder that is caused by expanded CAG trinucleotide repeats, resulting in a polyglutamine stretch of >37 on the N terminus of the protein huntingtin (htt). htt is a large (347 kDa), ubiquitously expressed protein. The precise functions of htt are not clear, but its importance is underscored by the embryonic lethal phenotype in htt knock-out mice. Despite the fact that the htt gene was cloned 13 years ago, little is known about the properties of the full-length protein. Here we report the expression and preliminary characterization of recombinant full-length wild-type human htt. Our results support a model of htt composed entirely of HEAT repeats that stack to form an elongated superhelix.

    Funded by: Wellcome Trust

    The Journal of biological chemistry 2006;281;23;15916-22

  • Regulation of intracellular accumulation of mutant Huntingtin by Beclin 1.

    Shibata M, Lu T, Furuya T, Degterev A, Mizushima N, Yoshimori T, MacDonald M, Yankner B and Yuan J

    Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115, USA.

    Intracellular accumulation of mutant Huntingtin with expanded polyglutamine provides a context-dependent cytotoxicity critical for the pathogenesis of Huntington disease (Everett, C. M., and Wood, N. W. (2004) Brain 127, 2385-2405). Here we demonstrate that the accumulation of mutant Huntingtin is highly sensitive to the expression of beclin 1, a gene essential for autophagy. Moreover, we show that the accumulated mutant Huntingtin recruits Beclin 1 and impairs the Beclin 1-mediated long lived protein turnover. Thus, sequestration of Beclin 1 in the vulnerable neuronal population of Huntington disease patients might further reduce Beclin 1 function and autophagic degradation of mutant Huntingtin. Finally, we demonstrate that the expression of beclin 1 decreases in an age-dependent fashion in human brains. Because beclin 1 gene is haploid insufficient in regulating autophagosome function (Qu, X., Yu, J., Bhagat, G., Furuya, N., Hibshoosh, H., Troxel, A., Rosen, J., Eskelinen, E. L., Mizushima, N., Ohsumi, Y., Cattoretti, G., and Levine, B. (2003) J. Clin. Invest. 112, 1809-1820; Yue, Z., Jin, S., Yang, C., Levine, A. J., and Heintz, N. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 15077-15082), we propose that the age-dependent decrease of beclin 1 expression may lead to a reduction of autophagic activity during aging, which in turn promotes the accumulation of mutant Htt and the progression of the disease.

    Funded by: NINDS NIH HHS: NS16367, NS32765; PHS HHS: R37-12859

    The Journal of biological chemistry 2006;281;20;14474-85

  • Mutant huntingtin aggregates impair mitochondrial movement and trafficking in cortical neurons.

    Chang DT, Rintoul GL, Pandipati S and Reynolds IJ

    Department of Pharmacology, University of Pittsburgh, Pittsburgh, PA 15261, USA.

    Huntington's disease (HD) is a neurodegenerative disorder caused by a polyglutamine repeat in the huntingtin gene (Htt). Mitochondrial defects and protein aggregates are characteristic of affected neurons. Recent studies suggest that these aggregates impair cellular transport mechanisms by interacting with cytoskeletal components and molecular motors. Here, we investigated whether mutant Htt alters mitochondrial trafficking and morphology in primary cortical neurons. We demonstrate that full-length mutant Htt was more effective than N-terminal mutant Htt in blocking mitochondrial movement, an effect that correlated with its heightened expression in the cytosolic compartment. Aggregates impaired the passage of mitochondria along neuronal processes, causing mitochondria to accumulate adjacent to aggregates and become immobilized. Furthermore, mitochondrial trafficking was reduced specifically at sites of aggregates while remaining unaltered in regions lacking aggregates. We conclude that in cortical neurons, an early event in HD pathophysiology is the aberrant mobility and trafficking of mitochondria caused by cytosolic Htt aggregates.

    Funded by: NINDS NIH HHS: NS049560

    Neurobiology of disease 2006;22;2;388-400

  • The association of CAG repeat length with clinical progression in Huntington disease.

    Rosenblatt A, Liang KY, Zhou H, Abbott MH, Gourley LM, Margolis RL, Brandt J and Ross CA

    Department of Psychiatry, Johns Hopkins University School of Medicine, Baltimore, MD, USA. arosenba@jhmi.edu

    Objective: To determine whether the rate of clinical progression in Huntington disease (HD) is influenced by the size of the CAG expansion.

    Methods: The dataset consisted of 3,402 examinations of 512 subjects seen through the Baltimore Huntington's Disease Center. Subjects were seen for a mean of 6.64 visits, with mean follow-up of 6.74 years. Subjects were administered the Quantified Neurological Examination, with its subsets the Motor Impairment and Chorea Scores, the Mini-Mental State Examination, and the HD Activities of Daily Living (ADL) Scale.

    Results: In an analysis based on the Random Effects Model, CAG length was significantly associated with the rate of progression of all measures except chorea and ADL. There was a significant interaction term between CAG length and disease duration for all measures except chorea. Further graphical exploration of the data supported these linear models and suggested that subjects at the low end of the expanded CAG repeat range may experience a more benign late course.

    Conclusions: CAG repeat length has a small effect on rate of progression that may be clinically important over time. Individuals with the shortest expansions appear to have the best prognosis. These effects of the CAG length may be relevant in the analysis of clinical trials.

    Neurology 2006;66;7;1016-20

  • Involvement of mitochondrial complex II defects in neuronal death produced by N-terminus fragment of mutated huntingtin.

    Benchoua A, Trioulier Y, Zala D, Gaillard MC, Lefort N, Dufour N, Saudou F, Elalouf JM, Hirsch E, Hantraye P, Déglon N and Brouillet E

    URA CEA-CNRS 2210, Service Hospitalier Frédéric Joliot, MIRCen Program, Département de Recherches Médicales, Direction des Sciences du Vivant, Commissariat à l'Energie Atomique (CEA), 91401 Orsay Cedex, France.

    Alterations of mitochondrial function may play a central role in neuronal death in Huntington's disease (HD). However, the molecular mechanisms underlying such functional deficits of mitochondria are not elucidated yet. We herein showed that the expression of two important constituents of mitochondrial complex II, the 30-kDa iron-sulfur (Ip) subunit and the 70-kDa FAD (Fp) subunit, was preferentially decreased in the striatum of HD patients compared with controls. We also examined several mitochondrial proteins in striatal neurons that were infected with lentiviral vectors coding for the N-terminus part of huntingtin (Htt) with either a pathological (Htt171-82Q) or physiological (Htt171-19Q) polyglutamine tract. Compared with Htt171-19Q, expression of Htt171-82Q preferentially decreased the levels of Ip and Fp subunits and affected the dehydrogenase activity of the complex. The Htt171-82Q-induced preferential loss of complex II was not associated with a decrease in mRNA levels, suggesting the involvement of a posttranscriptional mechanism. Importantly, the overexpression of either Ip or Fp subunit restored complex II levels and blocked mitochondrial dysfunction and striatal cell death induced by Htt171-82Q in striatal neurons. The present results strongly suggest that complex II defects in HD may be instrumental in striatal cell death.

    Molecular biology of the cell 2006;17;4;1652-63

  • Wild-type huntingtin protects neurons from excitotoxicity.

    Leavitt BR, van Raamsdonk JM, Shehadeh J, Fernandes H, Murphy Z, Graham RK, Wellington CL, Raymond LA and Hayden MR

    Centre for Molecular Medicine and Therapeutics, British Colombia Research Institute for Children's and Women's Health, Department of Medical Genetics, University of British Columbia, Vancouver, British Columbia, Canada. bleavitt@cmmt.ubc.ca

    Huntingtin is a caspase substrate, and loss of normal huntingtin function resulting from caspase-mediated proteolysis may play a role in the pathogenesis of Huntington disease. Here we tested the hypothesis that increasing huntingtin levels protect striatal neurons from NMDA receptor-mediated excitotoxicity. Cultured striatal neurons from yeast artificial chromosome (YAC)18 transgenic mice over-expressing full-length wild-type huntingtin were dramatically protected from apoptosis and caspase-3 activation compared with cultured striatal neurons from non-transgenic FVB/N littermates and YAC72 mice expressing mutant human huntingtin. NMDA receptor activation induced by intrastriatal injection of quinolinic acid initiated a form of apoptotic neurodegeneration within the striatum of mice that was associated with caspase-3 cleavage of huntingtin in neurons and astrocytes, decreased levels of full-length huntingtin, and the generation of a specific N-terminal caspase cleavage product of huntingtin. In vivo, over-expression of wild-type huntingtin in YAC18 transgenic mice conferred significant protection against NMDA receptor-mediated apoptotic neurodegeneration. These data provide in vitro and in vivo evidence that huntingtin may regulate the balance between neuronal survival and death following acute excitotoxic stress, and that the levels of huntingtin may modulate neuronal sensitivity to excitotoxic neurodegeneration. We suggest that further study of huntingtin's anti-apoptotic function will contribute to our understanding of the pathogenesis of Huntingdon's disease and provide insights into the selective vulnerability of striatal neurons to excitotoxic cell death.

    Journal of neurochemistry 2006;96;4;1121-9

  • p53 tumor suppressor protein regulates the levels of huntingtin gene expression.

    Feng Z, Jin S, Zupnick A, Hoh J, de Stanchina E, Lowe S, Prives C and Levine AJ

    Cancer Institute of New Jersey, University of Medicine and Dentistry of New Jersey, New Brunswick, NJ, USA.

    The p53 protein is a transcription factor that integrates various cellular stress signals. The accumulation of the mutant huntingtin protein with an expanded polyglutamine tract plays a central role in the pathology of human Huntington's disease. We found that the huntingtin gene contains multiple putative p53-responsive elements and p53 binds to these elements both in vivo and in vitro. p53 activation in cultured human cells, either by a temperature-sensitive mutant p53 protein or by gamma-irradiation (gamma-irradiation), increases huntingtin mRNA and protein expression. Similarly, murine huntingtin also contains multiple putative p53-responsive elements and its expression is induced by p53 activation in cultured cells. Moreover, gamma-irradiation, which activates p53, increases huntingtin gene expression in the striatum and cortex of mouse brain, the major pathological sites for Huntington's disease, in p53+/+ but not the isogenic p53-/- mice. These results demonstrate that p53 protein can regulate huntingtin expression at transcriptional level, and suggest that a p53 stress response could be a modulator of the process of Huntington's disease.

    Oncogene 2006;25;1;1-7

  • Expression of mutant huntingtin in glial cells contributes to neuronal excitotoxicity.

    Shin JY, Fang ZH, Yu ZX, Wang CE, Li SH and Li XJ

    Department of Human Genetics, Emory University School of Medicine, Atlanta, GA 30322, USA.

    Huntington disease (HD) is characterized by the preferential loss of striatal medium-sized spiny neurons (MSNs) in the brain. Because MSNs receive abundant glutamatergic input, their vulnerability to excitotoxicity may be largely influenced by the capacity of glial cells to remove extracellular glutamate. However, little is known about the role of glia in HD neuropathology. Here, we report that mutant huntingtin accumulates in glial nuclei in HD brains and decreases the expression of glutamate transporters. As a result, mutant huntingtin (htt) reduces glutamate uptake in cultured astrocytes and HD mouse brains. In a neuron-glia coculture system, wild-type glial cells protected neurons against mutant htt-mediated neurotoxicity, whereas glial cells expressing mutant htt increased neuronal vulnerability. Mutant htt in cultured astrocytes decreased their protection of neurons against glutamate excitotoxicity. These findings suggest that decreased glutamate uptake caused by glial mutant htt may critically contribute to neuronal excitotoxicity in HD.

    Funded by: NIA NIH HHS: AG19206, R01 AG019206; NINDS NIH HHS: NS36232, R01 NS036232

    The Journal of cell biology 2005;171;6;1001-12

  • HDAC6 and microtubules are required for autophagic degradation of aggregated huntingtin.

    Iwata A, Riley BE, Johnston JA and Kopito RR

    Department of Biological Sciences, BIO-X Program, Stanford University, Stanford, California 94305-5430, USA.

    CNS neurons are endowed with the ability to recover from cytotoxic insults associated with the accumulation of proteinaceous polyglutamine aggregates via a process that appears to involve capture and degradation of aggregates by autophagy. The ubiquitin-proteasome system protects cells against proteotoxicity by degrading soluble monomeric misfolded aggregation-prone proteins but is ineffective against, and impaired by, non-native protein oligomers. Here we show that autophagy is induced in response to impaired ubiquitin proteasome system activity. We show that ATG proteins, molecular determinants of autophagic vacuole formation, and lysosomes are recruited to pericentriolar cytoplasmic inclusion bodies by a process requiring an intact microtubule cytoskeleton and the cytoplasmic deacetylase HDAC6. These data suggest that HDAC6-dependent retrograde transport on microtubules is used by cells to increase the efficiency and selectivity of autophagic degradation.

    Funded by: NINDS NIH HHS: NS-042842

    The Journal of biological chemistry 2005;280;48;40282-92

  • Progressive and selective striatal degeneration in primary neuronal cultures using lentiviral vector coding for a mutant huntingtin fragment.

    Zala D, Benchoua A, Brouillet E, Perrin V, Gaillard MC, Zurn AD, Aebischer P and Déglon N

    Institute of Neurosciences, Swiss Federal Institute of Technology Lausanne, EPFL, 1015 Lausanne, Switzerland.

    A lentiviral vector expressing a mutant huntingtin protein (htt171-82Q) was used to generate a chronic model of Huntington's disease (HD) in rat primary striatal cultures. In this model, the majority of neurons expressed the transgene so that Western blot analysis and flow cytometry measurement could complement immunohistological evaluation. Mutant huntingtin produced a slowly progressing pathology characterized after 1 month by the appearance of neuritic aggregates followed by intranuclear inclusions, morphological anomalies of neurites, loss of neurofilament 160, increased expression in stress response protein Hsp70, and later loss of neuronal markers such as NeuN and MAP-2. At 2 months post-infection, a significant increase in TUNEL-positive cells confirmed actual striatal cell loss. Interestingly, cortical cultures infected with the same vector showed no sign of neuronal dysfunction despite accumulation of numerous inclusions. We finally examined whether the trophic factors CNTF and BDNF that were found neuroprotective in acute HD models could prevent striatal degeneration in a chronic model. Results demonstrated that both agents were neuroprotective without modifying inclusion formation. The present study demonstrates that viral vectors coding for mutant htt provides an advantageous system for histological and biochemical analysis of HD pathogenesis in primary striatal cultures.

    Neurobiology of disease 2005;20;3;785-98

  • Reduced hippocampal neurogenesis in R6/2 transgenic Huntington's disease mice.

    Gil JM, Mohapel P, Araújo IM, Popovic N, Li JY, Brundin P and Petersén A

    Neuronal Survival Unit, Wallenberg Neuroscience Center, Department of Experimental Medical Sciences, Lund University, BMC A10, SE-221 84 Lund, Sweden.

    We investigated whether cell proliferation and neurogenesis are altered in R6/2 transgenic Huntington's disease mice. Using bromodeoxyuridine (BrdU), we found a progressive decrease in the number of proliferating cells in the dentate gyrus of R6/2 mice. This reduction was detected in pre-symptomatic mice, and by 11.5 weeks, R6/2 mice had 66% fewer newly born cells in the hippocampus. The results were confirmed by immunohistochemistry for the cell cycle markers Ki-67 and proliferating cell nuclear antigen (PCNA). We did not observe changes in cell proliferation in the R6/2 subventricular zone, indicating that the decrease in cell proliferation is specific for the hippocampus. This decrease corresponded to a reduction in actual hippocampal neurogenesis as assessed by double immunostaining for BrdU and the neuronal marker neuronal nuclei (NeuN) and by immunohistochemistry for the neuroblast marker doublecortin. Reduced hippocampal neurogenesis may be a novel neuropathological feature in R6/2 mice that could be assessed when evaluating potential therapies.

    Neurobiology of disease 2005;20;3;744-51

  • p62/SQSTM1 forms protein aggregates degraded by autophagy and has a protective effect on huntingtin-induced cell death.

    Bjørkøy G, Lamark T, Brech A, Outzen H, Perander M, Overvatn A, Stenmark H and Johansen T

    Biochemistry Department, Institute of Medical Biology, University of Tromsø, 9037 Tromsø, Norway.

    Autophagic degradation of ubiquitinated protein aggregates is important for cell survival, but it is not known how the autophagic machinery recognizes such aggregates. In this study, we report that polymerization of the polyubiquitin-binding protein p62/SQSTM1 yields protein bodies that either reside free in the cytosol and nucleus or occur within autophagosomes and lysosomal structures. Inhibition of autophagy led to an increase in the size and number of p62 bodies and p62 protein levels. The autophagic marker light chain 3 (LC3) colocalized with p62 bodies and co-immunoprecipitated with p62, suggesting that these two proteins participate in the same complexes. The depletion of p62 inhibited recruitment of LC3 to autophagosomes under starvation conditions. Strikingly, p62 and LC3 formed a shell surrounding aggregates of mutant huntingtin. Reduction of p62 protein levels or interference with p62 function significantly increased cell death that was induced by the expression of mutant huntingtin. We suggest that p62 may, via LC3, be involved in linking polyubiquitinated protein aggregates to the autophagy machinery.

    The Journal of cell biology 2005;171;4;603-14

  • Overexpression of yeast hsp104 reduces polyglutamine aggregation and prolongs survival of a transgenic mouse model of Huntington's disease.

    Vacher C, Garcia-Oroz L and Rubinsztein DC

    Department of Medical Genetics, Cambridge Institute for Medical Research, Wellcome/MRC Building, Addenbrooke's Hospital, UK

    Huntington's disease is a devastating neurodegenerative condition associated with the formation of intraneuronal aggregates by mutant huntingtin. Aggregate formation is a property shared by the nine related diseases caused by polyglutamine codon expansion mutations and also by other neurodegenerative conditions like Parkinsons's disease. The roles of aggregates and aggregation in these diseases have been a subject of heated controversy. Here, we have addressed the question in vivo by generating a new transgenic mouse overexpressing the yeast chaperone hsp104, as hsp104 overexpression reduced mutant huntingtin aggregation and toxicity in cell models. Hsp104 has no close mammalian orthologues and does not appear to have effects on mammalian cell death pathways. We crossed hsp104 transgenic mice with mice expressing the first 171 residues of mutant huntingtin. Hsp104 reduced aggregate formation and prolonged the lifespan of the HD mice by 20%. This protection may be mediated at the level of changing the conformation of a putative toxic monomer, reducing oligomerization or aggregation, reducing the levels of oligomeric species or aggregates or combinations of these non-mutually exclusive possibilities.

    Funded by: Medical Research Council: G0000872

    Human molecular genetics 2005;14;22;3425-33

  • Crosstalk between huntingtin and syntaxin 1A regulates N-type calcium channels.

    Swayne LA, Chen L, Hameed S, Barr W, Charlesworth E, Colicos MA, Zamponi GW and Braun JE

    Department of Physiology and Biophysics, Hotchkiss Brain Institute, The University of Calgary, Calgary, Alberta, Canada T2N 4N1.

    We have identified a novel interaction between huntingtin (htt) and N-type calcium channels, a channel key in coupling calcium influx with synaptic vesicle exocytosis. Htt is a widely expressed 350-kDa cytosolic protein bearing an N-terminal polyglutamine tract. Htt is proteolytically cleaved by calpains and caspases and the resultant htt N-terminal fragments have been proposed to be biologically active; however, the cellular function of htt and/or the htt fragments remains enigmatic. We show that N-terminal fragments of htt (consisting of exon1) and full-length htt associate with the synaptic protein interaction (synprint) region of the N-type calcium channel. Given that synprint has previously been shown to bind syntaxin 1A and that this association elicits inhibition of N-type calcium channels, we tested whether htt(exon1) affects the modulation of these channels. Our data indicate that htt(exon1) enhances calcium influx by blocking syntaxin 1A inhibition of N-type calcium channels and attributes a key role for htt N-terminal fragments in the fine tuning of neurotransmission.

    Molecular and cellular neurosciences 2005;30;3;339-51

  • Huntingtin is cleaved by caspases in the cytoplasm and translocated to the nucleus via perinuclear sites in Huntington's disease patient lymphoblasts.

    Sawa A, Nagata E, Sutcliffe S, Dulloor P, Cascio MB, Ozeki Y, Roy S, Ross CA and Snyder SH

    Department of Psychiatry and Behavioral Sciences, Johns Hopkins University School of Medicine, 600 North Wolfe Street, Baltimore, MD 21287, USA.

    Accumulation of mutant Huntingtin (Htt), especially the N-terminal-cleaved Htt, participates in the pathophysiology of Huntington's disease (HD). It is difficult to elucidate temporal properties of the translocation of "endogenous" Htt using autopsy HD patient brains. Thus, we examined the cell biology of "endogenous" Htt cleavage and nuclear translocation in cultured lymphoblasts of HD patients and controls. Apoptotic stimulation of lymphoblasts elicits caspase-dependent cleavage and selective nuclear translocation of N-terminal portions of Htt. Discrete clusters of the N-terminal Htt accumulate at unique perinuclear sites prior to nuclear translocation. Our findings suggest that caspase cleavage of Htt is cytoplasmic and precedes sorting to specific perinuclear sites followed by nuclear translocation in HD patient tissue.

    Funded by: NIDA NIH HHS: DA-00074; NIMH NIH HHS: MH-18501, MH-69853; NINDS NIH HHS: NS-34172/NS-16375

    Neurobiology of disease 2005;20;2;267-74

  • Huntingtin associates with acidic phospholipids at the plasma membrane.

    Kegel KB, Sapp E, Yoder J, Cuiffo B, Sobin L, Kim YJ, Qin ZH, Hayden MR, Aronin N, Scott DL, Isenberg G, Goldmann WH and DiFiglia M

    Department of Neurology, Massachusetts General Hospital, Charlestown, Massachusetts 02129, USA. kegel@helix.mgh.harvard.edu

    We have identified a domain in the N terminus of huntingtin that binds to membranes. A three-dimensional homology model of the structure of the binding domain predicts helical HEAT repeats, which emanate a positive electrostatic potential, consistent with a charge-based mechanism for membrane association. An amphipathic helix capable of inserting into pure lipid bilayers may serve to anchor huntingtin to the membrane. In cells, N-terminal huntingtin fragments targeted to regions of plasma membrane enriched in phosphatidylinositol 4,5-bisphosphate, receptor bound-transferrin, and endogenous huntingtin. N-terminal huntingtin fragments with an expanded polyglutamine tract aberrantly localized to intracellular regions instead of plasma membrane. Our data support a new model in which huntingtin directly binds membranes through electrostatic interactions with acidic phospholipids.

    Funded by: NINDS NIH HHS: NS16367, NS35711, NS38194

    The Journal of biological chemistry 2005;280;43;36464-73

  • Contribution of nuclear and extranuclear polyQ to neurological phenotypes in mouse models of Huntington's disease.

    Benn CL, Landles C, Li H, Strand AD, Woodman B, Sathasivam K, Li SH, Ghazi-Noori S, Hockly E, Faruque SM, Cha JH, Sharpe PT, Olson JM, Li XJ and Bates GP

    King's College London, Medical and Molecular Genetics, GKT School of Medicine, UK.

    In postmortem Huntington's disease brains, mutant htt is present in both nuclear and cytoplasmic compartments. To dissect the impact of nuclear and extranuclear mutant htt on the initiation and progression of disease, we generated a series of transgenic mouse lines in which nuclear localization or nuclear export signal sequences have been placed N-terminal to the htt exon 1 protein carrying 144 glutamines. Our data indicate that the exon 1 mutant protein is present in the nucleus as part of an oligomeric or aggregation complex. Increasing the concentration of the mutant transprotein in the nucleus is sufficient for and dramatically accelerates the onset and progression of behavioral phenotypes. Furthermore, nuclear exon 1 mutant protein is sufficient to induce cytoplasmic neurodegeneration and transcriptional dysregulation. However, our data suggest that cytoplasmic mutant exon 1 htt, if present, contributes to disease progression.

    Funded by: NIA NIH HHS: AG19206; NINDS NIH HHS: NS38106, NS41669, NS45242; Wellcome Trust

    Human molecular genetics 2005;14;20;3065-78

  • Ataxin-2 and huntingtin interact with endophilin-A complexes to function in plastin-associated pathways.

    Ralser M, Nonhoff U, Albrecht M, Lengauer T, Wanker EE, Lehrach H and Krobitsch S

    Max Planck Institute for Molecular Genetics, Ihnestrasse 73, 14195 Berlin, Germany.

    Spinocerebellar ataxia type 2 is an inherited neurodegenerative disorder that is caused by an expanded trinucleotide repeat in the SCA2 gene, encoding a polyglutamine stretch in the gene product ataxin-2. Although evidence has been provided that ataxin-2 is involved in RNA metabolism, the physiological function of ataxin-2 remains unclear. Here, we demonstrate that ataxin-2 interacts with two members of the endophilin family, endophilin-A1 and endophilin-A3. To elucidate the physiological implications of these interactions, we exploited yeast as a model system and discovered that expression of ataxin-2 as well as both endophilin proteins is toxic for yeast lacking the SAC6 gene product fimbrin, a protein involved in actin filament organization and endocytotic processes. Intriguingly, expression of huntingtin, another polyglutamine protein interacting with endophilin-A3, was also toxic in Deltasac6 yeast. These effects can be suppressed by simultaneous expression of one of the two human fimbrin orthologs, L- or T-plastin. Moreover, we have discovered that ataxin-2 associates with L- and T-plastin and that overexpression of ataxin-2 leads to accumulation of T-plastin in mammalian cells. Thus, our findings suggest an interplay between ataxin-2, endophilin proteins and huntingtin in plastin-associated cellular pathways.

    Human molecular genetics 2005;14;19;2893-909

  • HD CAG repeat implicates a dominant property of huntingtin in mitochondrial energy metabolism.

    Seong IS, Ivanova E, Lee JM, Choo YS, Fossale E, Anderson M, Gusella JF, Laramie JM, Myers RH, Lesort M and MacDonald ME

    Molecular Neurogenetics UNIT, Center for Human Genetic Research, Massachusetts General Hospital, Richard B. Simches Research Center, Boston, MA 02114, USA.

    The 'expanded' HD CAG repeat that causes Huntington's disease (HD) encodes a polyglutamine tract in huntingtin, which first targets the death of medium-sized spiny striatal neurons. Mitochondrial energetics, related to N-methyl-d-aspartate (NMDA) Ca2+-signaling, has long been implicated in this neuronal specificity, implying an integral role for huntingtin in mitochondrial energy metabolism. As a genetic test of this hypothesis, we have looked for a relationship between the length of the HD CAG repeat, expressed in endogenous huntingtin, and mitochondrial ATP production. In STHdhQ111 knock-in striatal cells, a juvenile onset HD CAG repeat was associated with low mitochondrial ATP and decreased mitochondrial ADP-uptake. This metabolic inhibition was associated with enhanced Ca2+-influx through NMDA receptors, which when blocked resulted in increased cellular [ATP/ADP]. We then evaluated [ATP/ADP] in 40 human lymphoblastoid cell lines, bearing non-HD CAG lengths (9-34 units) or HD-causing alleles (35-70 units). This analysis revealed an inverse association with the longer of the two allelic HD CAG repeats in both the non-HD and HD ranges. Thus, the polyglutamine tract in huntingtin appears to regulate mitochondrial ADP-phosphorylation in a Ca2+-dependent process that fulfills the genetic criteria for the HD trigger of pathogenesis, and it thereby determines a fundamental biological parameter--cellular energy status, which may contribute to the exquisite vulnerability of striatal neurons in HD. Moreover, the evidence that this polymorphism can determine energy status in the non-HD range suggests that it should be tested as a potential physiological modifier in both health and disease.

    Funded by: NINDS NIH HHS: NS16367, NS32765, NS41552, R01 NS041552, R01 NS041552-04

    Human molecular genetics 2005;14;19;2871-80

  • A human protein-protein interaction network: a resource for annotating the proteome.

    Stelzl U, Worm U, Lalowski M, Haenig C, Brembeck FH, Goehler H, Stroedicke M, Zenkner M, Schoenherr A, Koeppen S, Timm J, Mintzlaff S, Abraham C, Bock N, Kietzmann S, Goedde A, Toksöz E, Droege A, Krobitsch S, Korn B, Birchmeier W, Lehrach H and Wanker EE

    Max Delbrueck Center for Molecular Medicine, 13092 Berlin-Buch, Germany.

    Protein-protein interaction maps provide a valuable framework for a better understanding of the functional organization of the proteome. To detect interacting pairs of human proteins systematically, a protein matrix of 4456 baits and 5632 preys was screened by automated yeast two-hybrid (Y2H) interaction mating. We identified 3186 mostly novel interactions among 1705 proteins, resulting in a large, highly connected network. Independent pull-down and co-immunoprecipitation assays validated the overall quality of the Y2H interactions. Using topological and GO criteria, a scoring system was developed to define 911 high-confidence interactions among 401 proteins. Furthermore, the network was searched for interactions linking uncharacterized gene products and human disease proteins to regulatory cellular pathways. Two novel Axin-1 interactions were validated experimentally, characterizing ANP32A and CRMP1 as modulators of Wnt signaling. Systematic human protein interaction screens can lead to a more comprehensive understanding of protein function and cellular processes.

    Cell 2005;122;6;957-68

  • Mutant huntingtin represses CBP, but not p300, by binding and protein degradation.

    Cong SY, Pepers BA, Evert BO, Rubinsztein DC, Roos RA, van Ommen GJ and Dorsman JC

    CBG-Center of Human and Clinical Genetics, Leiden University Medical Center, Leiden, The Netherlands.

    Huntington's disease can be used as a model to study neurodegenerative disorders caused by aggregation-prone proteins. It has been proposed that the entrapment of transcription factors in aggregates plays an important role in pathogenesis. We now report that the transcriptional activity of CBP is already repressed in the early time points by soluble mutant huntingtin, whereas the histone acetylase activity of CBP/p300 is gradually diminished over time. Mutant huntingtin bound much stronger to CBP than normal huntingtin, possibly contributing to repression. Especially at the later time points, CBP protein level was gradually reduced via the proteasome pathway. In sharp contrast, p300 was unaffected by mutant huntingtin. This selective degradation of CBP was absent in spinocerebellar ataxia 3. Thus, mutant huntingtin specifically affects CBP and not p300 both at the early and later time points, via multiple mechanisms. In addition to the reduction of CBP, also the altered ratio of these closely related histone acetyltransferases may affect chromatin structure and transcription and thus contribute to neurodegeneration.

    Molecular and cellular neurosciences 2005;30;1;12-23

  • p53 mediates cellular dysfunction and behavioral abnormalities in Huntington's disease.

    Bae BI, Xu H, Igarashi S, Fujimuro M, Agrawal N, Taya Y, Hayward SD, Moran TH, Montell C, Ross CA, Snyder SH and Sawa A

    Department of Neuroscience, Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205, USA.

    We present evidence for a specific role of p53 in the mitochondria-associated cellular dysfunction and behavioral abnormalities of Huntington's disease (HD). Mutant huntingtin (mHtt) with expanded polyglutamine (polyQ) binds to p53 and upregulates levels of nuclear p53 as well as p53 transcriptional activity in neuronal cultures. The augmentation is specific, as it occurs with mHtt but not mutant ataxin-1 with expanded polyQ. p53 levels are also increased in the brains of mHtt transgenic (mHtt-Tg) mice and HD patients. Perturbation of p53 by pifithrin-alpha, RNA interference, or genetic deletion prevents mitochondrial membrane depolarization and cytotoxicity in HD cells, as well as the decreased respiratory complex IV activity of mHtt-Tg mice. Genetic deletion of p53 suppresses neurodegeneration in mHtt-Tg flies and neurobehavioral abnormalities of mHtt-Tg mice. Our findings suggest that p53 links nuclear and mitochondrial pathologies characteristic of HD.

    Funded by: NEI NIH HHS: EY08117; NIDA NIH HHS: DA-00074, DA-00266; NIMH NIH HHS: MH-069853

    Neuron 2005;47;1;29-41

  • Caudate nucleus atrophy in Huntington's disease and its relationship with clinical and genetic parameters.

    Roth J, Klempìi J, Jech R, Zidovská J, Uhrová T, Doubek P, Ulmanová O, Brozová H, Volfová M, Serranová T and Ruzicka E

    Department of Neurology, 1st Medical Faculty, Charles University, Prague, Czech Republic. roth@beba.cesnet.cz

    We analysed clinical data in 80 genetically confirmed Huntington?s disease (HD) patients and measured the severity of the head of the caudate nucleus (HCN) atrophy using computed tomography-guided planimetry. The results were compared with measurements obtained in 43 age-matched healthy subjects. Mean planimetric measurements of the HCN differed significantly between the HD patients and healthy controls (p<0.001). We observed a significant inverse correlation between duration of HD and HCN planimetric values (p<0.001). Physiological atrophy of the HCN with age was also present in healthy controls, but did not overlap with values obtained in HD patients (p<0.01). Furthermore, we found in our patients a statistically significant inverse correlation between the number of CAG triplet repeats and the age at onset of HD (p<0.001). Neither the number of CAG triplet repeats, nor the age at onset of HD was found to be related to the character of the initial clinical symptoms (motor vs mental). Similarly, no relationship emerged between maternal or paternal inheritance and the number of CAG triplet repeats. Moreover, the type of inheritance did not influence the age at onset of HD in our patients. Planimetric measurement of the HCN appears to be a simple and useful paraclinical tool for the diagnosis of HD.

    Functional neurology 2005;20;3;127-30

  • Phosphorylation of arfaptin 2 at Ser260 by Akt Inhibits PolyQ-huntingtin-induced toxicity by rescuing proteasome impairment.

    Rangone H, Pardo R, Colin E, Girault JA, Saudou F and Humbert S

    UMR 146 CNRS/Institut Curie, Centre Universitaire, 91405 Orsay Cedex, France.

    Huntington disease (HD) is caused by an abnormal expanded polyglutamine repeat in the huntingtin protein. Insulin-like growth factor-1 is of particular interest in HD because it strongly inhibits polyQ-huntingtin-induced neurotoxicity. This neuroprotective effect involves the phosphorylation of huntingtin at Ser(421) by the prosurvival kinase Akt (Humbert, S., Bryson, E. A., Cordelieres, F. P., Connors, N. C., Datta, S. R., Finkbeiner, S., Greenberg, M. E., and Saudou, F. (2002) Dev. Cell 2, 831-837). Here, we report that Akt inhibits polyQ-huntingtin-induced toxicity in the absence of phosphorylation of huntingtin at Ser(421), suggesting that Akt also acts on other downstream effector(s) to prevent neuronal death in HD. We show that this survival effect involves the ADP-ribosylation factor-interacting protein arfaptin 2, the levels of which are increased in HD patients. Akt phosphorylated arfaptin 2 at Ser(260). Lack of phosphorylation of arfaptin 2 at this site substantially modified its subcellular distribution and increased neuronal death and intranuclear inclusions caused by polyQ-huntingtin. In contrast, arfaptin 2 had a neuroprotective effect on striatal neurons when phosphorylated by Akt. This effect is mediated through the proteasome, as phosphorylated arfaptin 2 inhibited the blockade of the proteasome induced by polyQ-huntingtin. This study points out a new mechanism by which Akt promotes neuroprotection in HD, emphasizing the potential therapeutic interest of this pathway in the disease.

    Funded by: NIMH NIH HHS: MH.NS 31862

    The Journal of biological chemistry 2005;280;23;22021-8

  • HSJ1 is a neuronal shuttling factor for the sorting of chaperone clients to the proteasome.

    Westhoff B, Chapple JP, van der Spuy J, Höhfeld J and Cheetham ME

    Institute for Cell Biology, Rheinische Friedrich-Wilhelms University Bonn, Ulrich-Haberland-Strasse 61a, D-53121 Bonn, Germany.

    Protein degradation in eukaryotic cells usually involves the attachment of a ubiquitin chain to a substrate protein and its subsequent sorting to the proteasome. Molecular mechanisms underlying the sorting process only recently began to emerge and rely on a cooperation of chaperone machineries and ubiquitin-chain recognition factors [1-3]. Here, we identify isoforms of the cochaperone HSJ1 as neuronal shuttling factors for ubiquitylated proteins. HSJ1 combines a J-domain that stimulates substrate loading onto the Hsc70 chaperone with ubiquitin interaction motifs (UIMs) involved in binding ubiquitylated chaperone clients. HSJ1 prevents client aggregation, shields clients against chain trimming by ubiquitin hydrolases, and stimulates their sorting to the proteasome. In this way, HSJ1 isoforms participate in ER-associated degradation (ERAD) and protect neurons against cytotoxic protein aggregation.

    Funded by: Wellcome Trust

    Current biology : CB 2005;15;11;1058-64

  • Huntingtin phosphorylation on serine 421 is significantly reduced in the striatum and by polyglutamine expansion in vivo.

    Warby SC, Chan EY, Metzler M, Gan L, Singaraja RR, Crocker SF, Robertson HA and Hayden MR

    Centre for Molecular Medicine and Therapeutics (CMMT), British Columbia Children's and Women's Hospital, University of British Columbia, 980 West 28th Avenue, Vancouver, British Columbia V5Z 4H4, Canada.

    Huntington disease (HD) results from polyglutamine expansion in the huntingtin protein (htt). Despite the widespread tissue expression pattern of htt, neuronal loss is highly selective to medium spiny neurons of the striatum. Huntingtin is phosphorylated on serine-421 (S421) by the pro-survival signaling protein kinase Akt (PKB) and this has been previously shown to be protective against the toxicity of polyglutamine-expanded htt in cell culture. Using an antibody specific for htt phosphorylated on S421, we now demonstrate that htt phosphorylation is present at significant levels under normal physiological conditions in human and mouse brain. Furthermore, htt phosphorylation shows a regional distribution with the highest levels in the cerebellum, less in the cortex, and least in the striatum. In cell cultures and in YAC transgenic mice, the endogenous phosphorylation of polyglutamine-expanded htt is significantly reduced relative to wild-type htt. The presence and pattern of significant htt phosphorylation in the brain indicates that this dynamic post-translational modification is important for the regulation of htt and may contribute to the selective neurodegeneration seen in HD.

    Human molecular genetics 2005;14;11;1569-77

  • Cdk5 phosphorylation of huntingtin reduces its cleavage by caspases: implications for mutant huntingtin toxicity.

    Luo S, Vacher C, Davies JE and Rubinsztein DC

    Department of Medical Genetics, Cambridge Institute for Medical Research, Addenbrooke's Hospital, Cambridge, CB2 2XY, England, UK.

    Huntington's disease (HD) is a neurodegenerative disorder caused by an expanded polyglutamine (polyQ) tract in the huntingtin (htt) protein. Mutant htt toxicity is exposed after htt cleavage by caspases and other proteases release NH(2)-terminal fragments containing the polyQ expansion. Here, we show htt interacts and colocalizes with cdk5 in cellular membrane fractions. Cdk5 phosphorylates htt at Ser434, and this phosphorylation reduces caspase-mediated htt cleavage at residue 513. Reduced mutant htt cleavage resulting from cdk5 phosphorylation attenuated aggregate formation and toxicity in cells expressing the NH(2)-terminal 588 amino acids (htt588) of mutant htt. Cdk5 activity is reduced in the brains of HD transgenic mice compared with controls. This result can be accounted for by the polyQ-expanded htt fragments reducing the interaction between cdk5 and its activator p35. These data predict that the ability of cdk5 phosphorylation to protect against htt cleavage, aggregation, and toxicity is compromised in cells expressing toxic fragments of htt.

    Funded by: Medical Research Council: G0000872; Wellcome Trust

    The Journal of cell biology 2005;169;4;647-56

  • Adenovirus-mediated silencing of huntingtin expression by shRNA.

    Huang B and Kochanek S

    Division of Gene Therapy, University of Ulm, D-89081 Ulm, Germany.

    Huntington's disease (HD) is an inherited autosomal dominant, neurodegenerative disease that is caused by a gain of function mutation characterized by the expansion of a CAG trinucleotide repeat in exon 1 of the huntingtin (htt) gene. Since hairpin small interference RNA (shRNA) technology allows inhibition of specific gene expression in vitro and in vivo, vector-mediated expression of an shRNA directed to htt mRNA could form the basis of a new treatment modality for HD. By initial plasmid transfection of 293 cells, we identified one exon 1-targeted shRNA, which efficiently inhibited expression of an htt exon 1-GFP fusion protein and the endogenous htt gene. A replication-deficient adenovirus (Ad) vector Adie-1-1 was constructed to express this shRNA from the U6 promoter. In A549 cells expressing exon 1 of htt with an expanded CAG allele, Adie- 1-1 efficiently prevented htt exon 1 expression and htt aggregate formation. In addition, in different neuronal and nonneuronal cell lines, Adie-1-1 efficiently inhibited the expression of endogenous htt. Together, this data indicates the delineation of an shRNA strategy that may become the basis for treatment of HD.

    Human gene therapy 2005;16;5;618-26

  • Ancient origin of the CAG expansion causing Huntington disease in a Spanish population.

    García-Planells J, Burguera JA, Solís P, Millán JM, Ginestar D, Palau F and Espinós C

    Laboratory of Genetics and Medicine, Department of Genomics and Proteomics, Instituto de Biomedicina, CSIC, Valencia, Spain.

    Huntington disease (HD) is an autosomal dominant neurodegenerative disorder characterized clinically by progressive motor impairment, cognitive decline, and emotional deterioration. The disease is caused by the abnormal expansion of a CAG trinucleotide repeat in the first exon of the huntingtin gene in chromosome 4p16.3. HD is spread worldwide and it is generally accepted that few mutational events account for the origin of the pathogenic CAG expansion in most populations. We have investigated the genetic history of HD mutation in 83 family probands from the Land of Valencia, in Eastern Spain. An analysis of the HD/CCG repeat in informative families suggested that at least two main chromosomes were associated in the Valencian population, one associated with allele 7 (77 mutant chromosomes) and one associated with allele 10 (two mutant chromosomes). Haplotype A-7-A (H1) was observed in 47 out of 48 phase-known mutant chromosomes, obtained by segregation analysis, through the haplotype analysis of rs1313770-HD/CCG-rs82334, as it also was in 120 out of 166 chromosomes constructed by means of the PHASE program. The genetic history and geographical distribution of the main haplotype H1 were both studied by constructing extended haplotypes with flanking short tandem repeats (STRs) D4S106 and D4S3034. We found that we were able to determine the age of the CAG expansion associated with the haplotype H1 as being between 4,700 and 10,000 years ago. Furthermore, we observed a nonhomogenous distribution in the different regions associated with the different extended haplotypes of the ancestral haplotype H1, suggesting that local founder effects have occurred.

    Human mutation 2005;25;5;453-9

  • Optineurin links myosin VI to the Golgi complex and is involved in Golgi organization and exocytosis.

    Sahlender DA, Roberts RC, Arden SD, Spudich G, Taylor MJ, Luzio JP, Kendrick-Jones J and Buss F

    Cambridge Institute for Medical Research, University of Cambridge, England, UK.

    Myosin VI plays a role in the maintenance of Golgi morphology and in exocytosis. In a yeast 2-hybrid screen we identified optineurin as a binding partner for myosin VI at the Golgi complex and confirmed this interaction in a range of protein interaction studies. Both proteins colocalize at the Golgi complex and in vesicles at the plasma membrane. When optineurin is depleted from cells using RNA interference, myosin VI is lost from the Golgi complex, the Golgi is fragmented and exocytosis of vesicular stomatitis virus G-protein to the plasma membrane is dramatically reduced. Two further binding partners for optineurin have been identified: huntingtin and Rab8. We show that myosin VI and Rab8 colocalize around the Golgi complex and in vesicles at the plasma membrane and overexpression of constitutively active Rab8-Q67L recruits myosin VI onto Rab8-positive structures. These results show that optineurin links myosin VI to the Golgi complex and plays a central role in Golgi ribbon formation and exocytosis.

    Funded by: Wellcome Trust: 071162

    The Journal of cell biology 2005;169;2;285-95

  • cAMP-response element-binding protein contributes to suppression of the A2A adenosine receptor promoter by mutant Huntingtin with expanded polyglutamine residues.

    Chiang MC, Lee YC, Huang CL and Chern Y

    Division of Neuroscience, Institute of Biomedical Sciences, Academia Sinica and Institute of Neuroscience, National Yang Ming University, Taipei 11529, Taiwan.

    Huntington's disease is a neurodegenerative disease resulting from a CAG (glutamine) trinucleotide expansion in exon 1 of the Huntingtin (Htt) gene. The role of the striatum-enriched A2A adenosine receptor (A2A-R) in Huntington's disease has attracted much attention lately. In the present study, we found that expression of mutant Htt with expanded poly(Q) significantly reduced the transcript levels of the endogenous A2A-R in PC12 cells and primary striatal neurons. Cotransfection of various promoter constructs of the A2A-R gene and an expression construct of poly(Q)-expanded Htt revealed that the Htt mutant suppressed the core promoter activity of the A2A-R gene. Stimulation of the A2A-R using CGS21680, forskolin, and a constitutively active cAMP-response element-binding protein (CREB) mutant elevated the reduced promoter activity of the A2A-R gene by mutant Htt. Moreover, the effect of CGS was blocked by an A2A-R-selective antagonist (CSC), two inhibitors of protein kinase A, and two dominant negative mutants of (CREB). The protein kinase A/CREB pathway therefore is involved in regulating A2A-R promoter activity. Consistently, an atypical CRE site (TCCAGG) is located in the core promoter region of the A2A-R gene. Electrophoretic gel mobility shift assay and mutational inactivation further demonstrated the functional binding of CREB to the core promoter region and showed that expression of poly(Q)-expanded Htt abolished the binding of CREB to this site. Stimulation of the A2A-R restored the reduced CREB binding caused by the mutant and concurrently reduced mutant Htt aggregation. Collectively, the poly(Q)-expanded mutant Htt suppressed expression of the A2A-R by inhibiting its core promoter at least partially by preventing CREB binding.

    The Journal of biological chemistry 2005;280;14;14331-40

  • Generation and annotation of the DNA sequences of human chromosomes 2 and 4.

    Hillier LW, Graves TA, Fulton RS, Fulton LA, Pepin KH, Minx P, Wagner-McPherson C, Layman D, Wylie K, Sekhon M, Becker MC, Fewell GA, Delehaunty KD, Miner TL, Nash WE, Kremitzki C, Oddy L, Du H, Sun H, Bradshaw-Cordum H, Ali J, Carter J, Cordes M, Harris A, Isak A, van Brunt A, Nguyen C, Du F, Courtney L, Kalicki J, Ozersky P, Abbott S, Armstrong J, Belter EA, Caruso L, Cedroni M, Cotton M, Davidson T, Desai A, Elliott G, Erb T, Fronick C, Gaige T, Haakenson W, Haglund K, Holmes A, Harkins R, Kim K, Kruchowski SS, Strong CM, Grewal N, Goyea E, Hou S, Levy A, Martinka S, Mead K, McLellan MD, Meyer R, Randall-Maher J, Tomlinson C, Dauphin-Kohlberg S, Kozlowicz-Reilly A, Shah N, Swearengen-Shahid S, Snider J, Strong JT, Thompson J, Yoakum M, Leonard S, Pearman C, Trani L, Radionenko M, Waligorski JE, Wang C, Rock SM, Tin-Wollam AM, Maupin R, Latreille P, Wendl MC, Yang SP, Pohl C, Wallis JW, Spieth J, Bieri TA, Berkowicz N, Nelson JO, Osborne J, Ding L, Meyer R, Sabo A, Shotland Y, Sinha P, Wohldmann PE, Cook LL, Hickenbotham MT, Eldred J, Williams D, Jones TA, She X, Ciccarelli FD, Izaurralde E, Taylor J, Schmutz J, Myers RM, Cox DR, Huang X, McPherson JD, Mardis ER, Clifton SW, Warren WC, Chinwalla AT, Eddy SR, Marra MA, Ovcharenko I, Furey TS, Miller W, Eichler EE, Bork P, Suyama M, Torrents D, Waterston RH and Wilson RK

    Genome Sequencing Center, Washington University School of Medicine, Campus Box 8501, 4444 Forest Park Avenue, St. Louis, Missouri 63108, USA.

    Human chromosome 2 is unique to the human lineage in being the product of a head-to-head fusion of two intermediate-sized ancestral chromosomes. Chromosome 4 has received attention primarily related to the search for the Huntington's disease gene, but also for genes associated with Wolf-Hirschhorn syndrome, polycystic kidney disease and a form of muscular dystrophy. Here we present approximately 237 million base pairs of sequence for chromosome 2, and 186 million base pairs for chromosome 4, representing more than 99.6% of their euchromatic sequences. Our initial analyses have identified 1,346 protein-coding genes and 1,239 pseudogenes on chromosome 2, and 796 protein-coding genes and 778 pseudogenes on chromosome 4. Extensive analyses confirm the underlying construction of the sequence, and expand our understanding of the structure and evolution of mammalian chromosomes, including gene deserts, segmental duplications and highly variant regions.

    Nature 2005;434;7034;724-31

  • Incidence and mutation rates of Huntington's disease in Spain: experience of 9 years of direct genetic testing.

    Ramos-Arroyo MA, Moreno S and Valiente A

    Servicio de Genética Médica, Hospital Virgen del Camino, C/ Irunlarrea 4, 31008 Pamplona, Navarra, Spain. ma.ramos.arroyo@cfnavarra.es

    Background: Prior to the discovery of the Huntington's disease (HD) mutation, the prevalence, incidence, and new mutation rates for this disease were based on the presence of progressive choreic movements and a positive family history.

    Objective: To evaluate the uptake of the HD genetic analysis in Spain, and to provide additional information on the epidemiology of this disease from the experience of 9 years of direct genetic testing.

    Methods: From 1994 to 2002, CAG repeat length was determined in 317 patients with symptoms compatible with HD. In all cases, demographic, clinical, and family data were carefully reviewed.

    Results: HD diagnosis (CAG repeat length >/=36) was confirmed in 166 (52%) symptomatic cases. Of these, 76 (45.8%) reported a positive family history and in 21 cases (12.7%) family history was negative. New mutation events were genetically proven in three families and highly suspected in another, estimating that the minimum new mutation rate for HD in our population is >4%, with a potential mutation rate of 8%. More than 16% of all HD cases had late onset (>59 years) of symptoms, and in three quarters of these the family history was negative. The incidence rate for the autonomous communities of Navarra and the Basque country, based on the number of newly diagnosed cases by genetic testing, was 4.7 per million per year.

    Conclusions: Direct HD genetic testing shows that the incidence and mutation rates of the disease are 2-3 times higher than previously reported. We also demonstrated the relevance of CAG repeat length assessment in diagnosing patients with late onset of symptoms and negative family history for HD.

    Journal of neurology, neurosurgery, and psychiatry 2005;76;3;337-42

  • Polyglutamine expansion of huntingtin impairs its nuclear export.

    Cornett J, Cao F, Wang CE, Ross CA, Bates GP, Li SH and Li XJ

    Department of Human Genetics, Emory University School of Medicine, 615 Michael Street, Atlanta, Georgia 30322, USA.

    Proteins with polyglutamine (polyQ) expansions accumulate in the nucleus and affect gene expression. The mechanism by which mutant huntingtin (htt) accumulates intranuclearly is not known; wild-type htt, a 350-kDa protein of unknown function, is normally found in the cytoplasm. N-terminal fragments of mutant htt, which contain a polyQ expansion (>37 glutamines), have no conserved nuclear localization sequences or nuclear export sequences but can accumulate in the nucleus and cause neurological problems in transgenic mice. Here we report that N-terminal htt shuttles between the cytoplasm and nucleus in a Ran GTPase-independent manner. Small N-terminal htt fragments interact with the nuclear pore protein translocated promoter region (Tpr), which is involved in nuclear export. PolyQ expansion and aggregation decrease this interaction and increase the nuclear accumulation of htt. Reducing the expression of Tpr by RNA interference or deletion of ten amino acids of N-terminal htt, which are essential for the interaction of htt with Tpr, increased the nuclear accumulation of htt. These results suggest that Tpr has a role in the nuclear export of N-terminal htt and that polyQ expansion reduces this nuclear export to cause the nuclear accumulation of htt.

    Nature genetics 2005;37;2;198-204

  • Interaction of the nuclear matrix protein NAKAP with HypA and huntingtin: implications for nuclear toxicity in Huntington's disease pathogenesis.

    Sayer JA, Manczak M, Akileswaran L, Reddy PH and Coghlan VM

    Neurological Sciences Institute, Oregon Health & Science University, Beaverton, OR 97006, USA.

    Although expansion of a polyglutamine tract in the huntingtin protein is known to cause Huntington's disease (HD), there is considerable debate as to how this mutation leads to the selective neuronal loss that characterizes the disease. The observation that mutant huntingtin accumulates in neuronal nuclei has led to the hypothesis that the molecular mechanism may involve the disruption of specific nuclear activities. Recently, several nuclear interaction partners for huntingtin have been identified, including HypA, a splicing factor-like protein of unknown function. Using a yeast two-hybrid screen, we have identified the interaction of HypA with the nuclear scaffold protein NAKAP. Interaction of NAKAP with HypA is specific and occurs both in yeast and in vitro. Deletion-mapping studies indicate that binding occurs via a proline-rich domain in NAKAP with a WW domain of HypA. In cultured cells, NAKAP and HypA localize within the nucleus and copurify with the nuclear matrix. Furthermore, NAKAP associates with HypA from human brain and copurifies with huntingtin protein in brain tissue obtained from HD patients. In HD neurons, NAKAP and mutant huntingtin were colocalized to the nuclear matrix and were found to be components of nuclear aggregates. Hence, the NAKAP-HypA scaffold is a potential nuclear docking site for huntingtin protein and may contribute to the nuclear accumulation of huntingtin observed in HD.

    Funded by: NCRR NIH HHS: RR016858; NIA NIH HHS: AG22643; NIDDK NIH HHS: DK062340; NINDS NIH HHS: NS 31862

    Neuromolecular medicine 2005;7;4;297-310

  • Huntingtin-interacting protein HIP14 is a palmitoyl transferase involved in palmitoylation and trafficking of multiple neuronal proteins.

    Huang K, Yanai A, Kang R, Arstikaitis P, Singaraja RR, Metzler M, Mullard A, Haigh B, Gauthier-Campbell C, Gutekunst CA, Hayden MR and El-Husseini A

    Department of Psychiatry, Brain Research Centre, University of British Columbia, Vancouver, British Columbia, Canada.

    In neurons, posttranslational modification by palmitate regulates the trafficking and function of signaling molecules, neurotransmitter receptors, and associated synaptic scaffolding proteins. However, the enzymatic machinery involved in protein palmitoylation has remained elusive. Here, using biochemical assays, we show that huntingtin (htt) interacting protein, HIP14, is a neuronal palmitoyl transferase (PAT). HIP14 shows remarkable substrate specificity for neuronal proteins, including SNAP-25, PSD-95, GAD65, synaptotagmin I, and htt. Conversely, HIP14 is catalytically invariant toward paralemmin and synaptotagmin VII. Exogenous HIP14 enhances palmitoylation-dependent vesicular trafficking of several acylated proteins in both heterologous cells and neurons. Moreover, interference with endogenous expression of HIP14 reduces clustering of PSD-95 and GAD65 in neurons. These findings define HIP14 as a mammalian palmitoyl transferase involved in the palmitoylation and trafficking of multiple neuronal proteins.

    Neuron 2004;44;6;977-86

  • Early onset Huntington disease: a neuronal degeneration syndrome.

    Seneca S, Fagnart D, Keymolen K, Lissens W, Hasaerts D, Debulpaep S, Desprechins B, Liebaers I and De Meirleir L

    Department of Medical Genetics, AZ-VUB, Laarbeeklaan 101, 1090 Brussels, Belgium. sara.seneca@az.vub.ac.be

    Unlabelled: Huntington disease (HD) is an autosomal dominant, lethal neurodegenerative disorder of the central nervous system, caused by an uncontrolled expansion of a CAG dynamic mutation in the coding region of the IT15gene. Although a majority of patients have a midlife onset of the disease, in a small number of patients the disease manifests before 20 years of age. In adults, HD is mainly characterised by involuntary movements, personality changes and dementia. By contrast, in children a dominant picture of bradykinesia, rigidity, dystonia and epileptic seizures is noticed. The earlier onset is often associated with a paternal transmission of the disease allele to the offspring. We report here a rather unusual infantile onset of the disease in a little girl who presented with a history of seizures and psychomotor regression starting at the age of 3 years. A progressive cortical-subcortical atrophy, progressive cerebellar atrophy and lesions in the basal ganglia were found on MRI. An important expansion, of 214 triplet numbers, of the CAG repeat size associated with HD, was observed.

    Conclusion: Juvenile Huntingdon disease should be considered in children suffering from a progressive neurodegenerative disease.

    European journal of pediatrics 2004;163;12;717-21

  • Mutant huntingtin affects the rate of transcription of striatum-specific isoforms of phosphodiesterase 10A.

    Hu H, McCaw EA, Hebb AL, Gomez GT and Denovan-Wright EM

    Laboratory of Molecular Neurobiology, Department of Pharmacology, Dalhousie University, Canada B3H 1X5.

    Huntington's disease (HD) is caused by the inheritance of a copy of the gene encoding mutant huntingtin with an expanded CAG repeat. Phosphodiesterase 10A (PDE10A) mRNA decreases in transgenic HD mice expressing exon 1 of the human huntingtin gene (HD). The mouse PDE10A mRNA is expressed through alternative splicing and polyadenylation in a tissue-specific manner and that transcription of striatal PDE10A mRNA is driven by two promoters. PDE10A2 is the predominant isoform of the gene is expressed in the striatum. Using in situ hybridization and quantitative RT-PCR, we determined that decreased steady-state levels of PDE10A2 mRNA were caused by an altered transcription initiation rate rather than by post-transcriptional mRNA instability in HD mice. Transcription from three initiation sites located within a 50-bp region in the PDE10A2-specific promoter was differentially affected by the presence of the mutant huntingtin transgene. The mouse and human PDE10A2 promoters are highly conserved with respect to the relative position of cis-regulatory elements. Several transcription factors that have been shown to interact with mutant huntingtin, including Sp1, neuron restrictive silencing factor, TATA-binding protein and cAMP-response element binding protein, are unlikely to be involved in mutant huntingtin-induced PDE10A2 transcriptional dysregulation.

    The European journal of neuroscience 2004;20;12;3351-63

  • Enhanced striatal NR2B-containing N-methyl-D-aspartate receptor-mediated synaptic currents in a mouse model of Huntington disease.

    Li L, Murphy TH, Hayden MR and Raymond LA

    Kinsmen Laboratory, Department of Psychiatry, University of British Columbia, 4N3-2255 Wesbrook Mall, Vancouver, BC V6T 1Z3, Canada.

    Huntington disease (HD) is an inherited neurodegenerative disease caused by expansion of a polyglutamine tract near the N terminus of the protein huntingtin, leading to dramatic loss of striatal medium-sized spiny GABAergic projection neurons (MSNs). Evidence suggests overactivation of N-methyl-D-aspartate (NMDA)-type glutamate receptors (NMDARs) contributes to selective degeneration of MSNs in HD. Striatal MSNs are enriched in NR2B, and whole cell current and excitotoxicity mediated predominantly by the NR2B subtype of NMDARs is increased with expression of mutant huntingtin in transfected cell lines and striatal MSNs from mice models. To test whether synaptic NMDAR current is altered by mutant huntingtin expression, we recorded striatal MSN excitatory postsynaptic currents (EPSCs) evoked by stimulation of cortical afferents in corticostriatal slices from YAC72 mice and their wild-type (WT) littermates at age 21-31 days. The ratio of NMDAR- to AMPAR-mediated EPSC amplitude was significantly increased in YAC72 compared to WT mice. Furthermore, using a paired-pulse stimulation protocol as a measure of presynaptic glutamate release probability, we found no significant differences between YAC72 and WT striatal MSN responses. These data suggest selective potentiation of postsynaptic NMDAR activity at corticostriatal synapses in YAC72 mice. Measurements of EPSC decay kinetics, as well as the effects of NR2B-subtype selective antagonists and glycine concentration on EPSC amplitude, are consistent with the majority of postsynaptic NMDARs being triheteromers of NR1/NR2A/NR2B in both WT and YAC72 mice. Together with previous results, our data suggest that enhanced activity of NR2B-containing NMDARs is one of the earliest changes leading to neuronal degeneration in HD.

    Journal of neurophysiology 2004;92;5;2738-46

  • Huntington's Disease-like 2 (HDL2) in North America and Japan.

    Margolis RL, Holmes SE, Rosenblatt A, Gourley L, O'Hearn E, Ross CA, Seltzer WK, Walker RH, Ashizawa T, Rasmussen A, Hayden M, Almqvist EW, Harris J, Fahn S, MacDonald ME, Mysore J, Shimohata T, Tsuji S, Potter N, Nakaso K, Adachi Y, Nakashima K, Bird T, Krause A and Greenstein P

    Department of Psychiatry, Johns Hopkins University School of Medicine, Baltimore, MD 21287, USA. rmargoli@jhmi.edu

    Huntington's Disease-like 2 (HDL2) is a progressive, autosomal dominant, neurodegenerative disorder with marked clinical and pathological similarities to Huntington's disease (HD). The causal mutation is a CTG/CAG expansion mutation on chromosome 16q24.3, in a variably spliced exon of junctophilin-3. The frequency of HDL2 was determined in nine independent series of patients referred for HD testing or selected for the presence of an HD-like phenotype in North America or Japan. The repeat length, ancestry, and age of onset of all North American HDL2 cases were determined. The results show that HDL2 is very rare, with a frequency of 0 to 15% among patients in the nine case series with an HD-like presentation who do not have the HD mutation. HDL2 is predominantly, and perhaps exclusively, found in individuals of African ancestry. Repeat expansions ranged from 44 to 57 triplets, with length instability in maternal transmission detected in a repeat of r2=0.29, p=0.0098). The results further support the evidence that the repeat expansion at the chromosome 16q24.3 locus is the direct cause of HDL2 and provide preliminary guidelines for the genetic testing of patients with an HD-like phenotype.

    Funded by: NINDS NIH HHS: NS16367, NS16375, NS38054, NS41547

    Annals of neurology 2004;56;5;670-4

  • Biochemical, ultrastructural, and reversibility studies on huntingtin filaments isolated from mouse and human brain.

    Díaz-Hernández M, Moreno-Herrero F, Gómez-Ramos P, Morán MA, Ferrer I, Baró AM, Avila J, Hernández F and Lucas JJ

    Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas, Universidad Autónoma de Madrid, 28029 Madrid, Spain.

    Huntington's disease (HD) and eight additional inherited neurological disorders are caused by CAG triplet-repeat expansions leading to expanded polyglutamine-sequences in their respective proteins. These triplet-CAG repeat disorders have in common the formation of aberrant intraneuronal proteinaceous inclusions containing the expanded polyglutamine sequences. These aggregates have been postulated to contribute to pathogenesis caused by conformational toxicity, sequestration of other polyglutamine-containing proteins, or by interfering with certain enzymatic activities. Testing these hypotheses has been hampered by the difficulty to isolate these aggregates from brain. Here we report that polyglutamine aggregates can be isolated from the brain of the Tet/HD94 conditional mouse model of HD, by following a method based on high salt buffer homogenization, nonionic detergent extraction, and gradient fractionation. We then verified that the method can be successfully applied to postmortem HD brains. Immunoelectron microscopy, both in human and mouse samples, revealed that the stable component of the inclusions are mutant huntingtin-containing and ubiquitin-containing fibrils. Atomic-force microscopy revealed that these fibrils have a "beads on a string" morphology. Thus, they resemble the in vitro assembled filaments made of recombinant mutant-huntingtin, as well as the Abeta and alpha-synuclein amyloid protofibrils. Finally, by shutting down transgene expression in the Tet/HD94 conditional mouse model of HD, we were able to demonstrate that these filaments, although stable in vitro, are susceptible to revert in vivo, thus demonstrating that the previously reported reversal of ubiquitin-immunoreactive inclusions does not simply reflect disassembling of the inclusions into their constituent fibrils and suggesting that any associated conformational or protein-sequestration toxicity is also likely to revert.

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2004;24;42;9361-71

  • Double-stranded siRNA targeted to the huntingtin gene does not induce DNA methylation.

    Park CW, Chen Z, Kren BT and Steer CJ

    Department of Medicine, University of Minnesota Medical School, Minneapolis, MN 55455, USA.

    RNA interference is an evolutionarily conserved mechanism of post-transcriptional gene silencing. Small interfering RNAs (siRNA) of 21-23 nucleotides generated from processing double-stranded RNA (dsRNA) by ribonuclease III, Dicer, are widely used for selective sequence-specific gene silencing in a broad range of organisms. In plants, siRNA is associated with de novo RNA-directed DNA methylation (RdDM) at the homologous target genomic region. To examine RdDM in somatic cells, human glioblastoma cell lines were treated with siRNAs homologous to the human huntingtin gene responsible for Huntington's disease. Methylation of CpG dinucleotides in the plasmid vectors expressing the dsRNAs and homologous genomic region was investigated by bisulfite-mediated genomic sequencing. Target regions of the siRNA in the huntingtin gene showed no significant change in the pattern of DNA methylation, and no CpG methylation was observed on the plasmid vectors. These results indicate that siRNA is not directly linked to DNA methylation at the target huntingtin genomic locus in human cells.

    Funded by: NHLBI NIH HHS: P01 HL55552, P01 HL65578

    Biochemical and biophysical research communications 2004;323;1;275-80

  • Huntingtin and its role in neuronal degeneration.

    Li SH and Li XJ

    Department of Human Genetics, School of Medicine, Emory University, Atlanta, GA 30322, USA.

    Huntington's disease results from a polyglutamine expansion in the N-terminal region of huntingtin (htt). This abnormality causes protein aggregation and leads to neurotoxicity. Despite its widespread expression in the brain and body, mutant htt causes selective neurodegeneration in Huntington's disease patient brains. However, Huntington's disease mouse models expressing mutant htt do not have obvious neurodegeneration despite significant neurological symptoms. Most Huntington's disease mouse models display the accumulation of toxic N-terminal mutant htt fragments in both the nucleus and neuronal processes, suggesting that these subcellular sites are hotspots for the early neuropathology of Huntington's disease. Intranuclear htt affects gene expression and may cause neuronal dysfunction. Mutant htt in neuronal processes affects axonal transport and induces degeneration, and these effects may be more relevant to the selective neurodegeneration in Huntington's disease. Growing evidence has also suggested that mutant htt mediates multiple pathological pathways. This review discusses the early pathological changes identified in Huntington's disease cellular and animal models. These changes may be the causes of neurode-generation.

    The Neuroscientist : a review journal bringing neurobiology, neurology and psychiatry 2004;10;5;467-75

  • A protein interaction network links GIT1, an enhancer of huntingtin aggregation, to Huntington's disease.

    Goehler H, Lalowski M, Stelzl U, Waelter S, Stroedicke M, Worm U, Droege A, Lindenberg KS, Knoblich M, Haenig C, Herbst M, Suopanki J, Scherzinger E, Abraham C, Bauer B, Hasenbank R, Fritzsche A, Ludewig AH, Büssow K, Buessow K, Coleman SH, Gutekunst CA, Landwehrmeyer BG, Lehrach H and Wanker EE

    Max-Delbrueck-Center for Molecular Medicine, 13125 Berlin-Buch, Germany.

    Analysis of protein-protein interactions (PPIs) is a valuable approach for characterizing proteins of unknown function. Here, we have developed a strategy combining library and matrix yeast two-hybrid screens to generate a highly connected PPI network for Huntington's disease (HD). The network contains 186 PPIs among 35 bait and 51 prey proteins. It revealed 165 new potential interactions, 32 of which were confirmed by independent binding experiments. The network also permitted the functional annotation of 16 uncharacterized proteins and facilitated the discovery of GIT1, a G protein-coupled receptor kinase-interacting protein, which enhances huntingtin aggregation by recruitment of the protein into membranous vesicles. Coimmunoprecipitations and immunofluorescence studies revealed that GIT1 and huntingtin associate in mammalian cells under physiological conditions. Moreover, GIT1 localizes to neuronal inclusions, and is selectively cleaved in HD brains, indicating that its distribution and function is altered during disease pathogenesis.

    Funded by: NINDS NIH HHS: NS31862

    Molecular cell 2004;15;6;853-65

  • Mutant huntingtin impairs axonal trafficking in mammalian neurons in vivo and in vitro.

    Trushina E, Dyer RB, Badger JD, Ure D, Eide L, Tran DD, Vrieze BT, Legendre-Guillemin V, McPherson PS, Mandavilli BS, Van Houten B, Zeitlin S, McNiven M, Aebersold R, Hayden M, Parisi JE, Seeberg E, Dragatsis I, Doyle K, Bender A, Chacko C and McMurray CT

    Department of Molecular Pharmacology and Experimental Therapeutics, Mayo ClinicFoundation, Rochester, Minnesota 55905, USA.

    Recent data in invertebrates demonstrated that huntingtin (htt) is essential for fast axonal trafficking. Here, we provide direct and functional evidence that htt is involved in fast axonal trafficking in mammals. Moreover, expression of full-length mutant htt (mhtt) impairs vesicular and mitochondrial trafficking in mammalian neurons in vitro and in whole animals in vivo. Particularly, mitochondria become progressively immobilized and stop more frequently in neurons from transgenic animals. These defects occurred early in development prior to the onset of measurable neurological or mitochondrial abnormalities. Consistent with a progressive loss of function, wild-type htt, trafficking motors, and mitochondrial components were selectively sequestered by mhtt in human Huntington's disease-affected brain. Data provide a model for how loss of htt function causes toxicity; mhtt-mediated aggregation sequesters htt and components of trafficking machinery leading to loss of mitochondrial motility and eventual mitochondrial dysfunction.

    Funded by: NIDDK NIH HHS: DK 43694-01; NIMH NIH HHS: MH/NS 31862; NINDS NIH HHS: NS40738, R01 NS040738

    Molecular and cellular biology 2004;24;18;8195-209

  • Cognitive changes in patients with Huntington's disease (HD) and asymptomatic carriers of the HD mutation--a longitudinal follow-up study.

    Lemiere J, Decruyenaere M, Evers-Kiebooms G, Vandenbussche E and Dom R

    Department of Neurology, UZ Gasthuisberg, Herestraat 49, 3000, Leuven, Belgium.

    Objective: Objective information about the onset and progression of cognitive impairment in Huntington's disease (HD) is very important in the light of appropriate outcome measures when conducting clinical trials. Therefore, we evaluated the progression of cognitive functions in HD patients and asymptomatic carriers of the HD mutation (AC) over a 2.5-year period. We also sought to detect the earliest markers of cognitive impairment in AC.

    Methods: A prospective study comparing HD patients, clinically asymptomatic HD mutation-carriers (AC) and non-carriers (NC). These groups were examined three times during a period of 2.5 years. At baseline the study sample consisted of 49 subjects. Forty-two subjects (19 HD patients, 12 AC and 11 NC) completed three assessments. A battery of neuropsychological tests measuring intelligence, attention, memory, language, visuospatial perception, and executive functions was performed.

    Results: The performance of HD patients deteriorated on the following cognitive tests: Symbol Digit Modalities Test (SDMT), Stroop Colour and Word, Boston Naming Test (BNT), Object and Space Perception and Trail Making Test-B. Longitudinal comparison of AC and NC revealed that performances on SDMT, Block Span, Digit Span Backwards, Hopkins Verbal Learning Test (learning and delayed recall) and Conditional Associative Learning Test are impaired in AC.

    Conclusions: Tasks measuring mainly attention, object and space perception and executive functions adequately assess the progression of HD disease. Other cognitive functions do not significantly deteriorate. Furthermore, problems in attention, working memory, verbal learning, verbal long-term memory and learning of random associations are the earliest cognitive manifestations in AC.

    Journal of neurology 2004;251;8;935-42

  • Mutant huntingtin directly increases susceptibility of mitochondria to the calcium-induced permeability transition and cytochrome c release.

    Choo YS, Johnson GV, MacDonald M, Detloff PJ and Lesort M

    Department of Psychiatry, University of Alabama at Birmingham, 35294, USA.

    Huntington's disease (HD) is initiated by an abnormally expanded polyglutamine stretch in the huntingtin protein, conferring a novel property on the protein that leads to the loss of striatal neurons. Defects in mitochondrial function have been implicated in the pathogenesis of HD. Here, we have examined the hypothesis that the mutant huntingtin protein may directly interact with the mitochondrion and affect its function. In human neuroblastoma cells and clonal striatal cells established from HdhQ7 (wild-type) and HdhQ111 (mutant) homozygote mouse knock-in embryos, huntingtin was present in a purified mitochondrial fraction. Subfractionation of the mitochondria and limited trypsin digestion of the organelle demonstrated that huntingtin was associated with the outer mitochondrial membrane. We further demonstrated that a recombinant truncated mutant huntingtin protein, but not a wild-type, directly induced mitochondrial permeability transition (MPT) pore opening in isolated mouse liver mitochondria, an effect that was prevented completely by cyclosporin A (CSA) and ATP. Importantly, the mutant huntingtin protein significantly decreased the Ca2+ threshold necessary to trigger MPT pore opening. We found a similar increased susceptibility to the calcium-induced MPT in liver mitochondria isolated from a knock-in HD mouse model. The mutant huntingtin protein-induced MPT pore opening was accompanied by a significant release of cytochrome c, an effect completely inhibited by CSA. These findings suggest that the development of specific MPT inhibitors may be an interesting therapeutic avenue to delay the onset of HD.

    Funded by: NINDS NIH HHS: NS32765, NS41552, NS41744, R01 NS041552, R01 NS041552-04

    Human molecular genetics 2004;13;14;1407-20

  • Cellular toxicity of polyglutamine expansion proteins: mechanism of transcription factor deactivation.

    Schaffar G, Breuer P, Boteva R, Behrends C, Tzvetkov N, Strippel N, Sakahira H, Siegers K, Hayer-Hartl M and Hartl FU

    Department of Cellular Biochemistry, Max-Planck Institute of Biochemistry, Am Klopferspitz 18, D-82152 Martinsried, Germany.

    The expression of polyglutamine-expanded mutant proteins in Huntington's disease and other neurodegenerative disorders is associated with the formation of intraneural inclusions. These aggregates could potentially cause cellular toxicity by sequestering essential proteins possessing normal polyQ repeats, including the transcription factors TBP and CBP. We show, in vitro and in cells, that monomers or small soluble oligomers of huntingtin exon1 accumulate in the nucleus and inhibit the function of TBP in a polyQ-dependent manner. FRET experiments indicate that these toxic forms are generated through a conformational rearrangement in huntingtin. Interaction of toxic huntingtin with the benign polyQ repeat of TBP structurally destabilizes the transcription factor, independent of the formation of insoluble coaggregates. Hsp70/Hsp40 chaperones interfere with the conformational change in mutant huntingtin and inhibit the deactivation of TBP. These results outline a molecular mechanism of cellular toxicity in polyQ disease and can explain the beneficial effects of molecular chaperones.

    Molecular cell 2004;15;1;95-105

  • Huntington's disease: how does huntingtin, an anti-apoptotic protein, become toxic?

    Rangone H, Humbert S and Saudou F

    UMR 146 CNRS, Institut Curie, Bldg. 110, Centre Universitaire, 91405 Orsay, France.

    Huntington's disease belongs to a class of inherited neurological disorders that are caused by the presence of a polyglutamine expansion in apparently unrelated proteins. In Huntington's disease, expansion occurs in the huntingtin protein. Together with the characteristic formation of aggregates in the diseased state, several post-translational modifications affect huntingtin during the pathological process and lead to the dysfunction and eventual death of selective neurons in the brain of patients. These mechanisms are not completely described but could involve the gain of a new toxic function as well as the loss of the beneficial properties of huntingtin.

    Pathologie-biologie 2004;52;6;338-42

  • Distinct aggregation and cell death patterns among different types of primary neurons induced by mutant huntingtin protein.

    Tagawa K, Hoshino M, Okuda T, Ueda H, Hayashi H, Engemann S, Okado H, Ichikawa M, Wanker EE and Okazawa H

    Department of Molecular Therapeutics, Tokyo Metropolitan Institute for Neuroscience, Fuchu, Tokyo, Japan.

    Aggregation of disease proteins is believed to be a central event in the pathology of polyglutamine diseases, whereas the relationship between aggregation and neuronal death remains controversial. We investigated this question by expressing mutant huntingtin (htt) with a defective adenovirus in different types of neurons prepared from rat cerebral cortex, striatum or cerebellum. The distribution pattern of inclusions is not identical among different types of primary neurons. On day 2 after infection, cytoplasmic inclusions are dominant in cortical and striatal neurons, whereas at day 4 the ratio of nuclear inclusions overtakes that of cytoplasmic inclusions. Meanwhile, nuclear inclusions are always predominantly present in cerebellar neurons. The percentage of inclusion-positive cells is highest in cerebellar neurons, whereas mutant htt induces cell death most remarkably in cortical neurons. As our system uses htt exon 1 protein and thus aggregation occurs independently from cleavage of the full-length htt, our observations indicate that the aggregation process is distinct among different neurons. Most of the neurons containing intracellular (either nuclear or cytoplasmic) aggregates are viable. Our findings suggest that the process of mutant htt aggregation rather than the resulting inclusion body is critical for neuronal cell death.

    Journal of neurochemistry 2004;89;4;974-87

  • Modulating huntingtin half-life alters polyglutamine-dependent aggregate formation and cell toxicity.

    Kaytor MD, Wilkinson KD and Warren ST

    Department of Human Genetics, Emory University School of Medicine, Atlanta, Georgia, USA. kaytor001@umn.edu

    A common finding among the expanded polyglutamine disorders is intracellular protein aggregates. Although the precise role of these aggregates in the disease process is unclear, they are generally ubiquitinated, implicating the ubiquitin-proteasome pathway in neuronal degeneration. To investigate the mechanism of aggregate formation, we have developed a cell culture model to express huntingtin designed to have an altered degradation rate through the ubiquitin-dependent N-end rule pathway. We fused the first 171 amino acids of huntingtin, containing either a pathogenic or normal polyglutamine tract, to the enhanced green fluorescent protein (EGFP). The half-life of soluble huntingtin-EGFP was dependent on the degradation signal and the polyglutamine tract length. However, once huntingtin-EGFP with a pathogenic tract had aggregated, the protein was extremely stable. Huntingtin-EGFP with a pathogenic glutamine tract and a shorter half-life displayed a delayed onset of aggregate formation and was more toxic to transfected cells. These data suggest that rapid clearance through the ubiquitin-proteasome pathway slows aggregate formation, yet increases cellular toxicity. Polyglutamine-induced neurotoxicity may therefore be triggered by non-aggregated protein, and aggregate formation itself may be a cellular defense mechanism.

    Funded by: NICHD NIH HHS: HD20521

    Journal of neurochemistry 2004;89;4;962-73

  • SUMO modification of Huntingtin and Huntington's disease pathology.

    Steffan JS, Agrawal N, Pallos J, Rockabrand E, Trotman LC, Slepko N, Illes K, Lukacsovich T, Zhu YZ, Cattaneo E, Pandolfi PP, Thompson LM and Marsh JL

    Department of Psychiatry and Human Behavior, Gillespie 2121, University of California, Irvine, CA 92697, USA.

    Huntington's disease (HD) is characterized by the accumulation of a pathogenic protein, Huntingtin (Htt), that contains an abnormal polyglutamine expansion. Here, we report that a pathogenic fragment of Htt (Httex1p) can be modified either by small ubiquitin-like modifier (SUMO)-1 or by ubiquitin on identical lysine residues. In cultured cells, SUMOylation stabilizes Httex1p, reduces its ability to form aggregates, and promotes its capacity to repress transcription. In a Drosophila model of HD, SUMOylation of Httex1p exacerbates neurodegeneration, whereas ubiquitination of Httex1p abrogates neurodegeneration. Lysine mutations that prevent both SUMOylation and ubiquitination of Httex1p reduce HD pathology, indicating that the contribution of SUMOylation to HD pathology extends beyond preventing Htt ubiquitination and degradation.

    Funded by: NCI NIH HHS: CA-62203; NICHD NIH HHS: HD36049, HD36081

    Science (New York, N.Y.) 2004;304;5667;100-4

  • Huntington's disease genetics.

    Myers RH

    Department of Neurology, Boston University School of Medicine, Boston, Massachusetts 02118, USA. rmyers@bu.edu

    Huntington's disease (HD) is a dominantly transmitted neurodegenerative disorder with wide variation in onset age but with an average age at onset of 40 years. Children of HD gene carriers have a 50% chance of inheriting the disease. The characteristic symptoms of HD are involuntary choreiform movements, cognitive impairment, mood disorders, and behavioral changes which are chronic and progressive over the course of the illness. HD is a "trinucleotide repeat" disorder, which is caused by an increase in the number of CAG repeats in the HD gene. Repeats of 40 or larger are associated with disease expression, whereas repeats of 26 and smaller are normal. Intermediate numbers of repeats, between 27 and 35, are not associated with disease expression but may expand in paternal transmission, resulting in the disease in descendents. Repeats of 36-39 are associated with reduced penetrance whereby some develop HD and others do not. The identification of the genetic defect in HD permits direct genetic testing for the presence of the gene alteration responsible for the disease. Tests may be performed in three circumstances: (1) confirmation of diagnosis, (2) predictive testing of persons at genetic risk for inheriting HD, and (3) prenatal testing. Testing is widely available and much experience has been gained with protocols that assist the individual in making an informed choice about test options, and minimize the occurrence of adverse emotional outcomes.

    Funded by: NINDS NIH HHS: P50 NS016367, P50NS016367

    NeuroRx : the journal of the American Society for Experimental NeuroTherapeutics 2004;1;2;255-62

  • Specific caspase interactions and amplification are involved in selective neuronal vulnerability in Huntington's disease.

    Hermel E, Gafni J, Propp SS, Leavitt BR, Wellington CL, Young JE, Hackam AS, Logvinova AV, Peel AL, Chen SF, Hook V, Singaraja R, Krajewski S, Goldsmith PC, Ellerby HM, Hayden MR, Bredesen DE and Ellerby LM

    The Buck Institute for Age Research, Novato, CA, USA.

    Huntington's disease (HD) is an autosomal dominant progressive neurodegenerative disorder resulting in selective neuronal loss and dysfunction in the striatum and cortex. The molecular pathways leading to the selectivity of neuronal cell death in HD are poorly understood. Proteolytic processing of full-length mutant huntingtin (Htt) and subsequent events may play an important role in the selective neuronal cell death found in this disease. Despite the identification of Htt as a substrate for caspases, it is not known which caspase(s) cleaves Htt in vivo or whether regional expression of caspases contribute to selective neuronal cells loss. Here, we evaluate whether specific caspases are involved in cell death induced by mutant Htt and if this correlates with our recent finding that Htt is cleaved in vivo at the caspase consensus site 552. We find that caspase-2 cleaves Htt selectively at amino acid 552. Further, Htt recruits caspase-2 into an apoptosome-like complex. Binding of caspase-2 to Htt is polyglutamine repeat-length dependent, and therefore may serve as a critical initiation step in HD cell death. This hypothesis is supported by the requirement of caspase-2 for the death of mouse primary striatal cells derived from HD transgenic mice expressing full-length Htt (YAC72). Expression of catalytically inactive (dominant-negative) forms of caspase-2, caspase-7, and to some extent caspase-6, reduced the cell death of YAC72 primary striatal cells, while the catalytically inactive forms of caspase-3, -8, and -9 did not. Histological analysis of post-mortem human brain tissue and YAC72 mice revealed activation of caspases and enhanced caspase-2 immunoreactivity in medium spiny neurons of the striatum and the cortical projection neurons when compared to controls. Further, upregulation of caspase-2 correlates directly with decreased levels of brain-derived neurotrophic factor in the cortex and striatum of 3-month YAC72 transgenic mice and therefore suggests that these changes are early events in HD pathogenesis. These data support the involvement of caspase-2 in the selective neuronal cell death associated with HD in the striatum and cortex.

    Funded by: NINDS NIH HHS: F32 NS043937

    Cell death and differentiation 2004;11;4;424-38

  • Cytoplasmic aggregates trap polyglutamine-containing proteins and block axonal transport in a Drosophila model of Huntington's disease.

    Lee WC, Yoshihara M and Littleton JT

    Picower Center for Learning and Memory, Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.

    Huntington's disease is an autosomal dominant neurodegenerative disorder caused by expansion of a polyglutamine tract in the huntingtin protein that results in intracellular aggregate formation and neurodegeneration. Pathways leading from polyglutamine tract expansion to disease pathogenesis remain obscure. To elucidate how polyglutamine expansion causes neuronal dysfunction, we generated Drosophila transgenic strains expressing human huntingtin cDNAs encoding pathogenic (Htt-Q128) or nonpathogenic proteins (Htt-Q0). Whereas expression of Htt-Q0 has no discernible effect on behavior, lifespan, or neuronal morphology, pan-neuronal expression of Htt-Q128 leads to progressive loss of motor coordination, decreased lifespan, and time-dependent formation of huntingtin aggregates specifically in the cytoplasm and neurites. Huntingtin aggregates sequester other expanded polyglutamine proteins in the cytoplasm and lead to disruption of axonal transport and accumulation of aggregates at synapses. In contrast, Drosophila expressing an expanded polyglutamine tract alone, or an expanded polyglutamine tract in the context of the spinocerebellar ataxia type 3 protein, display only nuclear aggregates and do not disrupt axonal trafficking. Our findings indicate that nonnuclear events induced by cytoplasmic huntingtin aggregation play a central role in the progressive neurodegeneration observed in Huntington's disease.

    Funded by: NINDS NIH HHS: R01 NS040296, R01 NS040296-04

    Proceedings of the National Academy of Sciences of the United States of America 2004;101;9;3224-9

  • Calmodulin regulates transglutaminase 2 cross-linking of huntingtin.

    Zainelli GM, Ross CA, Troncoso JC, Fitzgerald JK and Muma NA

    Department of Pharmacology, Loyola University Medical Center, Maywood, Illinois 60153, USA.

    Striatal and cortical intranuclear inclusions and cytoplasmic aggregates of mutant huntingtin are prominent neuropathological hallmarks of Huntington's disease (HD). We demonstrated previously that transglutaminase 2 cross-links mutant huntingtin in cells in culture and demonstrated the presence of transglutaminase-catalyzed cross-links in the HD cortex that colocalize with transglutaminase 2 and huntingtin. Because calmodulin regulates transglutaminase activity in erythrocytes, platelets, and the gizzard, we hypothesized that calmodulin increases cross-linking of huntingtin in the HD brain. We found that calmodulin colocalizes at the confocal level with transglutaminase 2 and with huntingtin in HD intranuclear inclusions. Calmodulin coimmunoprecipitates with transglutaminase 2 and huntingtin in cells transfected with myc-tagged N-terminal huntingtin fragments containing 148 polyglutamine repeats (htt-N63-148Q-myc) and transglutaminase 2 but not in cells transfected with myc-tagged N-terminal huntingtin fragments containing 18 polyglutamine repeats. Our previous studies demonstrated that transfection with both htt-N63-148Q-myc and transglutaminase 2 resulted in cross-linking of mutant huntingtin protein fragments and the formation of insoluble high-molecular-weight aggregates of huntingtin protein fragments. Transfection with transglutaminase 2 and htt-N63-148Q-myc followed by treatment of cells with N-(6-aminohexyl)-1-naphthalenesulfonamide, a calmodulin inhibitor, resulted in a decrease in cross-linked huntingtin. Inhibiting the interaction of calmodulin with transglutaminase and huntingtin protein could decrease cross-linking and diminish huntingtin aggregate formation in the HD brain.

    Funded by: NIMH NIH HHS: MH/NS31862; NINDS NIH HHS: NS16375

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2004;24;8;1954-61

  • Adverse effects of dopamine potentiation by long-term treatment with selegiline.

    Hollán S, Vécsei L and Magyar K

    Department of Cell Biology, National Blood Transfusion Service, Budapest, Hungary. hollan@ella.hu

    A patient with triosephosphate isomerase (TPI) deficiency exhibited worsening of abnormal involuntary movements of the dystonic type and developed psychiatric symptoms while on selegiline. When selegiline was stopped after 9 years of treatment, abnormal involuntary movements improved to pretreatment level and psychiatric behaviour returned to normal. Monoamine oxidase-B platelet activity was low in this patient.

    Movement disorders : official journal of the Movement Disorder Society 2004;19;1;107-9

  • Predictive genetic test decisions for Huntington's disease: context, appraisal and new moral imperatives.

    Taylor SD

    School of Social Work and Social Policy, The University of Queensland, St. Lucia, Queensland 4072, Australia. s.taylor@social.uq.edu.au

    Predictive testing is one of the new genetic technologies which, in conjunction with developing fields such as pharmacogenomics, promises many benefits for preventive and population health. Understanding how individuals appraise and make genetic test decisions is increasingly relevant as the technology expands. "Lay" understandings of genetic risk and test decision-making, located within holistic life frameworks including family or kin relationships, may vary considerably from clinical representations of these phenomena. The predictive test for Huntington's disease (HD), whilst specific to a single-gene, serious, mature-onset but currently untreatable disorder, is regarded as a model in this context. This paper reports upon a qualitative Australian study which investigated predictive test decision-making by individuals at risk for HD, the contexts of their decisions and the appraisals which underpinned them. In-depth interviews were conducted in Australia with 16 individuals at 50% risk for HD, with variation across testing decisions, gender, age and selected characteristics. Findings suggested predictive testing was regarded as a significant life decision with important implications for self and others, while the right "not to know" genetic status was staunchly and unanimously defended. Multiple contexts of reference were identified within which test decisions were located, including intra- and inter-personal frameworks, family history and experience of HD, and temporality. Participants used two main criteria in appraising test options: perceived value of, or need for the test information, for self and/or significant others, and degree to which such information could be tolerated and managed, short and long-term, by self and/or others. Selected moral and ethical considerations involved in decision-making are examined, as well as the clinical and socio-political contexts in which predictive testing is located. The paper argues that psychosocial vulnerabilities generated by the availability of testing technologies and exacerbated by policy imperatives towards individual responsibility and self-governance should be addressed at broader societal levels.

    Social science & medicine (1982) 2004;58;1;137-49

  • Predictive testing for Huntington's disease: relationship with partners after testing.

    Decruyenaere M, Evers-Kiebooms G, Cloostermans T, Boogaerts A, Demyttenaere K, Dom R and Fryns JP

    Psychosocial Genetics Unit, University of Leuven-Herestraat, Leuven, Belgium. marleen.decruyenaere@med.kuleuven.ac.be

    This study focuses on the partner relationship of tested persons, 5 years after their predictive test result for Huntington's disease (HD). We describe changes in marital status, quality of the relationship, and perceived changes in the relationship. Twenty-six carriers, 14 of their partners, 33 non-carriers, and 17 of their partners participated in the study. Qualitative and quantitative methods were used. For the majority of tested persons (about 70%), the marital status was unchanged 5 years post test. Overall, carriers rated the quality of the relationship higher than their partners did and they perceived more positive changes. Qualitative data show that a test result leading to changed roles may induce significant marital distress. Another consequence of the test may be the changes in dynamics in asymptomatic carrier couples. A pre-test discussion of the possible impact of the test result on the relationship should result in a better preparation for and more understanding of the reactions after testing. Counselling after testing should stimulate an open communication between partners with consideration of needs and anxieties of both partners.

    Clinical genetics 2004;65;1;24-31

  • DNA haplotype analysis of CAG repeat in Taiwanese Huntington's disease patients.

    Wang CK, Wu YR, Hwu WL, Chen CM, Ro LS, Chen ST, Gwinn-Hardy K, Yang CC, Wu RM, Chen TF, Wang HC, Chao MC, Chiu MJ, Lu CJ and Lee-Chen GJ

    Department of Life Science, National Taiwan Normal University, Taipei, Taiwan, ROC.

    We studied the expanded CAG repeat and adjacent CCG repeat in 53 Huntington's disease (HD) patients and 172 unrelated normal subjects matched to the patients for ethnic origin. The range of the CAG repeat varied from 38 to 109 in the HD patients and from 10 to 29 in the control group. A significant negative correlation was found between the age at onset and the CAG expansion, with no significant influence of the adjacent CCG repeat on the age at onset by multiple regression analysis. Allelic association using CCG repeat and 2 flanking dinucleotide repeat markers within 150 kb of the HD gene revealed linkage disequilibrium for 2 of 3 markers. Haplotype analysis of 24 HD families using these markers identified 3 major haplotypes underlying 87.5% of HD chromosomes. The data suggested frequent haplotypes in the Taiwanese population on which one or more mutational events leading to the disease occurred.

    European neurology 2004;52;2;96-100

  • Expanded huntingtin activates the c-Jun terminal kinase/c-Jun pathway prior to aggregate formation in striatal neurons in culture.

    Garcia M, Charvin D and Caboche J

    Laboratoire Signalisation Neuronale et Régulations Géniques, Unité Mixte de Recherche 7102, Université Pierre et Marie Curie, 9 quai Saint Bernard, 75005 Paris, France.

    Huntington's disease (HD) is an autosomal neurodegenerative disorder, caused by expansion of a glutamine repeat in the Huntingtin protein. Pathogenesis in HD includes the cytoplasmic cleavage of Huntingtin and release of an amino-terminal fragment capable of nuclear localization, where expanded-Huntingtin (Exp-Htt) might lead to aberrant transcriptional regulation, neuronal dysfunction and degeneration. Recent evidence, from hippocampal cell lines, also implicates altered interaction of Exp-Htt with components of the c-Jun N-terminal kinase (JNK) cascade. However, there is yet no proven implication of the JNK/c-Jun module in degeneration of striatal neurons, the more vulnerable cell population, in HD. In the present study, we used primary striatal neurons in culture to analyze c-Jun activation by Exp-Htt. Green fluorescent protein (GFP)-tagged exon 1 of human Huntingtin either in its normal (25Q, normal-Htt) or expanded (103Q, Exp-Htt) version was transiently transfected in these cells. We first set out, in our conditions, the time course of striatal degeneration produced by Exp-Htt, and found it occurred rapidly. At 48 h post-transfection, 60% of striatal neurons expressing Exp-Htt had apoptotic characteristics including DNA fragmentation and neuritic retraction. Most of these neurons also showed nuclear aggregates of GFP-Exp Htt. Kinetics of c-Jun activation were tested in transfected cells using immunocytochemical detection of phospho-c-Jun. We found a significant activation and induction of c-Jun in Exp-Htt but not normal-Htt-transfected neurons. Of interest, these events occurred prior to nuclear translocation of Exp-Htt. Finally, overexpression of a dominant negative version of c-Jun, as well as pharmacological inhibition of JNK strongly protected against DNA fragmentation and neuritic retraction induced by Exp-Htt. Thus our data suggest that c-Jun activation and induction, is an early event in the pathogenesis of HD, occurring prior to formation of nuclear aggregates of Exp-Htt.

    Neuroscience 2004;127;4;859-70

  • Cysteine string protein (CSP) inhibition of N-type calcium channels is blocked by mutant huntingtin.

    Miller LC, Swayne LA, Chen L, Feng ZP, Wacker JL, Muchowski PJ, Zamponi GW and Braun JE

    Cellular and Molecular Neurobiology Research Group, Department of Physiology and Biophysics, University of Calgary, Calgary, Alberta T2N 4N1, Canada.

    Cysteine string protein (CSP), a 34-kDa molecular chaperone, is expressed on synaptic vesicles in neurons and on secretory vesicles in endocrine, neuroendocrine, and exocrine cells. CSP can be found in a complex with two other chaperones, the heat shock cognate protein Hsc70, and small glutamine-rich tetratricopeptide repeat domain protein (SGT). CSP function is vital in synaptic transmission; however, the precise nature of its role remains controversial. We have previously reported interactions of CSP with both heterotrimeric GTP-binding proteins (G proteins) and N-type calcium channels. These associations give rise to a tonic G protein inhibition of the channels. Here we have examined the effects of huntingtin fragments (exon 1) with (huntingtin(exon1/exp)) and without (huntingtin(exon1/nonexp)) expanded polyglutamine (polyQ) tracts on the CSP chaperone system. In vitro huntingtin(exon1/exp) sequestered CSP and blocked the association of CSP with G proteins. In contrast, huntingtin(exon1/nonexp) did not interact with CSP and did not alter the CSP/G protein association. Similarly, co-expression of huntingtin(exon1/exp) with CSP and N-type calcium channels eliminated CSP's tonic G protein inhibition of the channels, while coexpression of huntingtin(exon1/nonexp) did not alter the robust inhibition promoted by CSP. These results indicate that CSP's modulation of G protein inhibition of calcium channel activity is blocked in the presence of a huntingtin fragment with expanded polyglutamine tracts.

    The Journal of biological chemistry 2003;278;52;53072-81

  • Dramatic tissue-specific mutation length increases are an early molecular event in Huntington disease pathogenesis.

    Kennedy L, Evans E, Chen CM, Craven L, Detloff PJ, Ennis M and Shelbourne PF

    Division of Molecular Genetics, Faculty of Biomedical and Life Scienes, University of Glasgow, Anderson College Complex, 56 Dumbarton Road, Glasgow G11 6NU, UK.

    Huntington disease is caused by the expansion of a CAG repeat encoding an extended glutamine tract in a protein called huntingtin. Although the mutant protein is widely expressed, the earliest and most striking neuropathological changes are observed in the striatum. Here we show dramatic mutation length increases (gains of up to 1000 CAG repeats) in human striatal cells early in the disease course, most likely before the onset of pathological cell loss. Studies of knock-in HD mouse models indicate that the size of the initial CAG repeat mutation may influence both onset and tissue-specific patterns of age-dependent, expansion-biased mutation length variability. Given that CAG repeat length strongly correlates with clinical severity, we suggest that somatic increases of mutation length may play a major role in the progressive nature and cell-selective aspects of both adult-onset and juvenile-onset HD pathogenesis and we discuss the implications of this interpretation of the data presented.

    Human molecular genetics 2003;12;24;3359-67

  • Yeast genes that enhance the toxicity of a mutant huntingtin fragment or alpha-synuclein.

    Willingham S, Outeiro TF, DeVit MJ, Lindquist SL and Muchowski PJ

    Department of Pharmacology, University of Washington, Seattle, WA 98195-7280, USA.

    Genome-wide screens were performed in yeast to identify genes that enhance the toxicity of a mutant huntingtin fragment or of alpha-synuclein. Of 4850 haploid mutants containing deletions of nonessential genes, 52 were identified that were sensitive to a mutant huntingtin fragment, 86 that were sensitive to alpha-synuclein, and only one mutant that was sensitive to both. Genes that enhanced toxicity of the mutant huntingtin fragment clustered in the functionally related cellular processes of response to stress, protein folding, and ubiquitin-dependent protein catabolism, whereas genes that modified alpha-synuclein toxicity clustered in the processes of lipid metabolism and vesicle-mediated transport. Genes with human orthologs were overrepresented in our screens, suggesting that we may have discovered conserved and nonoverlapping sets of cell-autonomous genes and pathways that are relevant to Huntington's disease and Parkinson's disease.

    Science (New York, N.Y.) 2003;302;5651;1769-72

  • Differential hydrophobicity drives self-assembly in Huntington's disease.

    Burke MG, Woscholski R and Yaliraki SN

    Department of Chemistry, Imperial College London, South Kensington Campus, London SW7 2AZ, United Kingdom.

    Identifying the driving forces and the mechanism of association of huntingtin-exon1, a close marker for the progress of Huntington's disease, is an important prerequisite to finding potential drug targets and, ultimately, a cure. We introduce here a modeling framework based on a key analogy of the physicochemical properties of the exon1 fragment to block copolymers. We use a systematic mesoscale methodology, based on dissipative particle dynamics, which is capable of overcoming kinetic barriers, thus capturing the dynamics of significantly larger systems over longer times than considered before. Our results reveal that the relative hydrophobicity of the poly(glutamine) block as compared with the rest of the (proline-based) exon1 fragment, ignored to date, constitutes a major factor in the initiation of the self-assembly process. We find that the assembly is governed by both the concentration of exon1 and the length of the poly(glutamine) stretch, with a low-length threshold for association, even at the lowest volume fractions we considered. Moreover, this self-association occurs irrespective of whether the glutamine stretch is in random-coil or hairpin configuration, leading to spherical or cylindrical assemblies, respectively. We discuss the implications of these results for reinterpretation of existing research within this context, including that the routes toward aggregation of exon1 may be distinct from those of the widely studied homopolymeric poly(glutamine) peptides.

    Proceedings of the National Academy of Sciences of the United States of America 2003;100;24;13928-33

  • Early and reversible neuropathology induced by tetracycline-regulated lentiviral overexpression of mutant huntingtin in rat striatum.

    Régulier E, Trottier Y, Perrin V, Aebischer P and Déglon N

    Institute of Neuroscience, Swiss Federal Institute of Technology Lausanne, EPFL, Lausanne, Switzerland.

    The ability to overexpress full-length huntingtin or large fragments represents an important challenge to mimic Huntington's pathology and reproduce all stages of the disease in a time frame compatible with rodent life span. In the present study, tetracycline-regulated lentiviral vectors leading to high expression levels were used to accelerate the pathological process. Rats were simultaneously injected with vectors coding for the transactivator and wild type (WT) or mutated huntingtin (TRE-853-19Q/82Q) in the left and right striatum, respectively, and analyzed in the 'on' and 'off' conditions. Overexpression of TRE-853-19Q protein or residual expression of TRE-853-82Q in 'off' condition did not cause any significant neuronal pathology. Overexpressed TRE-853-82Q protein led to proteolytic release of N-terminal htt fragments, nuclear aggregation, and a striatal dysfunction as revealed by decrease of DARPP-32 staining but absence of NeuN down-regulation. The differential effect on the DARPP-32/NeuN neuronal staining was observed as early as 1 month after injection and maintained at 3 months. In contrast, expression of a shorter htt form (htt171-82Q) did not require processing prior formation of nuclear aggregates and caused decrease of both DARPP-32 and NeuN neuronal markers at one month post-injection suggesting that polyQ pathology may be dependent on protein context. Finally, the reversibility of the pathology was assessed. Huntingtin expression was turn 'on' for 1 month and then shut 'off' for 2 months. Recovery of DARPP-32 immunoreactivity and clearance of huntingtin aggregates were observed in animals treated with doxycycline. These results suggest that a tetracycline-regulated system may be particularly attractive to model Huntington's disease and induce early and reversible striatal neuropathology in vivo.

    Human molecular genetics 2003;12;21;2827-36

  • [Analysis of polymorphic loci of Huntington genes in peoples from the Volga-Ural region].

    Kutuev IA, Fatkhlislamova RI, Khidiiatova IM and Khusnutdinova EK

    Institute of Biochemistry and Genetics, Ufa Research Center, Russian Academy of Sciences, Ufa, 450054 Russia. 021gen@mail.ru

    Eleven populations of the Volga-Ural region were analyzed with respect to three intragenic polymorphisms of the Huntington disease gene (IT15), including highly polymorphic (CAG)n and moderately polymorphic (CCG)n of exon 1 and neutral del2642 of exon 58. In the case of (CAG)n, 101 genotypes were observed, with genotype number varying from 15 in Southeastern Bashkirs to 34 in Mari. Allele diversity RS ranged from 9.70 in Southeastern Bashkirs to 18.00 in Chuvash, averaging 13.79 +/- 2.12. The (CAG)n allele frequency distribution was unimodal and had a maximum at (CAG)17. In the case of (CCG)n, six alleles with 6-10 or 12 repeats were observed. RS was 4.13 +/- 0.44, ranging from 3.73 in Udmurts to 4.99 in Tatars. In the case of del2642, allele del- was detected at a frequency 0.830 in Mari to 0.932 in Udmurts. Of all Volga-Ural ethnic populations, Finno-Ugric ones proved to be most heterogeneous with respect to the three polymorphisms, whereas Turkic populations and, in particular, Bashkirs were homogeneous. Micro-differentiation of the Volga-Ural populations corresponded to the European type.

    Molekuliarnaia biologiia 2003;37;6;961-70

  • Microtubule destabilization and nuclear entry are sequential steps leading to toxicity in Huntington's disease.

    Trushina E, Heldebrant MP, Perez-Terzic CM, Bortolon R, Kovtun IV, Badger JD, Terzic A, Estévez A, Windebank AJ, Dyer RB, Yao J and McMurray CT

    Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic and Foundation, Second Street SW, Rochester, MN 55905, USA.

    There has been a longstanding debate regarding the role of proteolysis in Huntington's disease. The toxic peptide theory posits that N-terminal cleavage fragments of mutant Huntington's disease protein [mutant huntingtin (mhtt)] enter the nucleus to cause transcriptional dysfunction. However, recent data suggest a second model in which proteolysis of full-length mhtt is inhibited. Importantly, the two competing theories differ with respect to subcellular distribution of mhtt at initiation of toxicity: nuclear if cleaved and cytoplasmic in the absence of cleavage. Using quantitative single-cell analysis and time-lapse imaging, we show here that transcriptional dysfunction is "downstream" of cytoplasmic dysfunction. Primary and reversible toxic events involve destabilization of microtubules mediated by full-length mhtt before cleavage. Restoration of microtubule structure by taxol inhibits nuclear entry and increases cell survival.

    Funded by: NIDDK NIH HHS: DK 43694-01; NIMH NIH HHS: MH 56207; NINDS NIH HHS: NS 40738, R01 NS040738

    Proceedings of the National Academy of Sciences of the United States of America 2003;100;21;12171-6

  • Molecular analysis of Huntington's disease and linked polymorphisms in the Indian population.

    Saleem Q, Roy S, Murgood U, Saxena R, Verma IC, Anand A, Muthane U, Jain S and Brahmachari SK

    Functional Genomics Unit, Centre for Biochemical Technology (CSIR), Mall Road, Delhi, India.

    Objectives: To understand the population variation and haplotypes of Huntington's disease (HD) in India we have analysed CAG repeats at the HD locus together with closely linked polymorphisms in both HD patients and normal controls.

    The CAG repeat and linked polymorphisms were analysed in 30 Indian HD families together with 250 ethnically matched controls using fluorescent polymerase chain reaction (PCR) based size estimation.

    Results: CAG repeats at the HD locus in the normal population showed a mean size of 17.99 +/- 2.66 repeats (range nine to 33 repeats). The HD mutation in our families did not show any significant association with either the (CCG)7 or (CCG)10 allele while haplotype analysis suggested the over-representation of the 7-2-I (CCG-D4s127-Delta 2642 loci) haplotype in a subset of families.

    Conclusion: The distribution of CAG repeats in the normal population suggests a higher prevalence of HD, closer to that seen in Western Europe. Haplotype analysis suggests the presence of a founder mutation in a subset of families and provides evidence for multiple and geographically distinct origins for the HD mutation in India.

    Acta neurologica Scandinavica 2003;108;4;281-6

  • Huntingtin interacts with REST/NRSF to modulate the transcription of NRSE-controlled neuronal genes.

    Zuccato C, Tartari M, Crotti A, Goffredo D, Valenza M, Conti L, Cataudella T, Leavitt BR, Hayden MR, Timmusk T, Rigamonti D and Cattaneo E

    Department of Pharmacological Sciences and Center of Excellence on Neurodegenerative Diseases, University of Milano, Via Balzaretti 9, 20133 Milano, Italy.

    Huntingtin protein is mutated in Huntington disease. We previously reported that wild-type but not mutant huntingtin stimulates transcription of the gene encoding brain-derived neurotrophic factor (BDNF; ref. 2). Here we show that the neuron restrictive silencer element (NRSE) is the target of wild-type huntingtin activity on BDNF promoter II. Wild-type huntingtin inhibits the silencing activity of NRSE, increasing transcription of BDNF. We show that this effect occurs through cytoplasmic sequestering of repressor element-1 transcription factor/neuron restrictive silencer factor (REST/NRSF), the transcription factor that binds to NRSE. In contrast, aberrant accumulation of REST/NRSF in the nucleus is present in Huntington disease. We show that wild-type huntingtin coimmunoprecipitates with REST/NRSF and that less immunoprecipitated material is found in brain tissue with Huntington disease. We also report that wild-type huntingtin acts as a positive transcriptional regulator for other NRSE-containing genes involved in the maintenance of the neuronal phenotype. Consistently, loss of expression of NRSE-controlled neuronal genes is shown in cells, mice and human brain with Huntington disease. We conclude that wild-type huntingtin acts in the cytoplasm of neurons to regulate the availability of REST/NRSF to its nuclear NRSE-binding site and that this control is lost in the pathology of Huntington disease. These data identify a new mechanism by which mutation of huntingtin causes loss of transcription of neuronal genes.

    Funded by: Telethon: E.0840

    Nature genetics 2003;35;1;76-83

  • Expression of polyglutamine-expanded huntingtin induces tyrosine phosphorylation of N-methyl-D-aspartate receptors.

    Song C, Zhang Y, Parsons CG and Liu YF

    Department of Pharmacology, Boston University School of Medicine, Boston, Massachusetts 02118, USA.

    In our previous studies, we found that expression of polyglutamine-expanded huntingtin in HN33 cells induced sensitization of N-methyl-D-aspartate (NMDA) receptors (Sun, Y., Savinainen, A., and Liu, Y. F. (2001) J. Biol. Chem. 276, 24713-24718). Following this study, we investigated whether tyrosine phosphorylation of NMDA receptors might contribute to the altered property of the receptors. Expression of polyglutamine-expanded huntingtin induced elevation of phosphorylated or activated Src and increased targeting of PSD-95 (post-synaptic density 95) and activated Src to cell surface membrane. Expression of the mutated huntingtin also induced tyrosine phosphorylation of NR2B (NMDA receptor 2B) subunits, and co-expression of PSD-95 enhanced the phosphorylation. Treatment of SU6656 (a specific Src inhibitor) or co-expression of a mutated NR2B subunit with mutations of all three major tyrosine phosphorylation sites significantly attenuated neuronal toxicity induced by the mutated huntingtin. Addition of AP-5 did not further inhibit the neuronal toxicity. Taken together, our studies show that polyglutamine-expanded huntingtin increases tyrosine phosphorylation of NMDA receptors via PSD-95 and Src, and increased tyrosine phosphorylation may contribute to the sensitization of the receptors mediated by polyglutamine-expanded huntingtin.

    The Journal of biological chemistry 2003;278;35;33364-9

  • Expression of mutant huntingtin blocks exocytosis in PC12 cells by depletion of complexin II.

    Edwardson JM, Wang CT, Gong B, Wyttenbach A, Bai J, Jackson MB, Chapman ER and Morton AJ

    Department of Pharmacology, Tennis Court Road, Cambridge CB2 1PD, United Kingdom.

    Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder caused by an expanded CAG repeat in the HD gene. We reported recently that complexin II, a protein involved in neurotransmitter release, is depleted from both the brains of mice carrying the HD mutation and from the striatum of post mortem HD brains. Here we show that this loss of complexin II is recapitulated in PC12 cells expressing the HD mutation and is accompanied by a dramatic decline in Ca2+-triggered exocytosis of neurotransmitter. Overexpression of complexin II (but not complexin I) rescued exocytosis, demonstrating that the decline in neurotransmitter release is a direct consequence of complexin II depletion. Complexin II depletion in the brain may account for some of the abnormalities in neurotransmission associated with HD.

    The Journal of biological chemistry 2003;278;33;30849-53

  • Stimulation of NeuroD activity by huntingtin and huntingtin-associated proteins HAP1 and MLK2.

    Marcora E, Gowan K and Lee JE

    Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Campus Box 347, Boulder, CO 80309, USA.

    NeuroD (ND) is a basic helix-loop-helix transcription factor important for neuronal development and survival. By using a yeast two-hybrid screen, we identified two proteins that interact with ND, huntingtin-associated protein 1 (HAP1) and mixed-lineage kinase 2 (MLK2), both of which are known to interact with huntingtin (Htt). Htt is a ubiquitous protein important for neuronal transcription, development, and survival, and loss of its function has been implicated in the pathogenesis of Huntington's disease, a neurodegenerative disorder. However, the mechanism by which Htt exerts its neuron-specific function at the molecular level is unknown. Here we report that Htt interacts with ND via HAP1, and that MLK2 phosphorylates and stimulates the activity of ND. Furthermore, we show that Htt and HAP1 facilitate the activation of ND by MLK2. To our knowledge, ND is the first example of a neuron-specific transcription factor involved in neuronal development and survival whose activity is modulated by Htt. We propose that Htt, together with HAP1, may function as a scaffold for the activation of ND by MLK2.

    Proceedings of the National Academy of Sciences of the United States of America 2003;100;16;9578-83

  • Huntingtin and huntingtin-associated protein 1 influence neuronal calcium signaling mediated by inositol-(1,4,5) triphosphate receptor type 1.

    Tang TS, Tu H, Chan EY, Maximov A, Wang Z, Wellington CL, Hayden MR and Bezprozvanny I

    Department of Physiology, University of Texas Southwestern Medical Center at Dallas, Dallas, TX 75390, USA.

    Huntington's disease (HD) is caused by polyglutamine expansion (exp) in huntingtin (Htt). The type 1 inositol (1,4,5)-triphosphate receptor (InsP3R1) is an intracellular calcium (Ca2+) release channel that plays an important role in neuronal function. In a yeast two-hybrid screen with the InsP3R1 carboxy terminus, we isolated Htt-associated protein-1A (HAP1A). We show that an InsP3R1-HAP1A-Htt ternary complex is formed in vitro and in vivo. In planar lipid bilayer reconstitution experiments, InsP3R1 activation by InsP3 is sensitized by Httexp, but not by normal Htt. Transfection of full-length Httexp or caspase-resistant Httexp, but not normal Htt, into medium spiny striatal neurons faciliates Ca2+ release in response to threshold concentrations of the selective mGluR1/5 agonist 3,5-DHPG. Our findings identify a novel molecular link between Htt and InsP3R1-mediated neuronal Ca2+ signaling and provide an explanation for the derangement of cytosolic Ca2+ signaling in HD patients and mouse models.

    Funded by: NINDS NIH HHS: R01 NS038082, R01 NS038082-02, R01 NS38082

    Neuron 2003;39;2;227-39

  • Huntingtin contains a highly conserved nuclear export signal.

    Xia J, Lee DH, Taylor J, Vandelft M and Truant R

    Department of Biochemistry, McMaster University, Health Sciences Centre Rm 4H45, 1200 Main Street West, Hamilton, Ontario, Canada L8N 3Z5.

    Huntington's disease (HD), is a genetic neurodegenerative disease characterized by a DNA CAG triplet repeat expansion in the first exon of the disease gene, HD. CAG DNA expansion results in a polyglutamine tract expansion in mutant huntingtin protein. Wild-type and mutant full-length huntingtin have been detected in the nucleus, but elevated levels of mutant huntingtin and huntingtin amino-terminal proteolytic fragments are seen to accumulate in the nuclei of HD-affected neurons. The presence of huntingtin in both the nucleus and the cytoplasm suggested that huntingtin may be dynamic between these compartments. By live cell time-lapse video microscopy, we have been able to visualize polyglutamine-mediated aggregation and the transient nuclear localization of huntingtin over time in a striatal cell line. A classical nuclear localization signal could not be detected in huntingtin, but we have discovered a nuclear export signal (NES) in the carboxy-terminus of huntingtin. Leptomycin B treatment of clonal striatal cells enhanced the nuclear localization of huntingtin, and a mutant NES huntingtin displayed increased nuclear localization, indicating that huntingtin can shuttle to and from the nucleus. The huntingtin NES is strictly conserved among all huntingtin proteins from diverse species. This export signal may be important in Huntington's disease because this fragment of huntingtin is proteolytically cleaved away during HD. The huntingtin NES therefore defines a potential role for huntingtin as a member of a nucleocytoplasmic dynamic protein complex.

    Human molecular genetics 2003;12;12;1393-403

  • Interaction of normal and expanded CAG repeat sizes influences age at onset of Huntington disease.

    Djoussé L, Knowlton B, Hayden M, Almqvist EW, Brinkman R, Ross C, Margolis R, Rosenblatt A, Durr A, Dode C, Morrison PJ, Novelletto A, Frontali M, Trent RJ, McCusker E, Gómez-Tortosa E, Mayo D, Jones R, Zanko A, Nance M, Abramson R, Suchowersky O, Paulsen J, Harrison M, Yang Q, Cupples LA, Gusella JF, MacDonald ME and Myers RH

    Section of Preventive Medicine and Epidemiology, Boston University School of Medicine, Boston, Massachusetts 02118, USA.

    Huntington disease (HD) is a neurodegenerative disorder caused by the abnormal expansion of CAG repeats in the HD gene on chromosome 4p16.3. Past studies have shown that the size of expanded CAG repeat is inversely associated with age at onset (AO) of HD. It is not known whether the normal Huntington allele size influences the relation between the expanded repeat and AO of HD. Data collected from two independent cohorts were used to test the hypothesis that the unexpanded CAG repeat interacts with the expanded CAG repeat to influence AO of HD. In the New England Huntington Disease Center Without Walls (NEHD) cohort of 221 HD affected persons and in the HD-MAPS cohort of 533 HD affected persons, we found evidence supporting an interaction between the expanded and unexpanded CAG repeat sizes which influences AO of HD (P = 0.08 and 0.07, respectively). The association was statistically significant when both cohorts were combined (P = 0.012). The estimated heritability of the AO residual was 0.56 after adjustment for normal and expanded repeats and their interaction. An analysis of tertiles of repeats sizes revealed that the effect of the normal allele is seen among persons with large HD repeat sizes (47-83). These findings suggest that an increase in the size of the normal repeat may mitigate the expression of the disease among HD affected persons with large expanded CAG repeats.

    Funded by: NINDS NIH HHS: P50NS016367

    American journal of medical genetics. Part A 2003;119A;3;279-82

  • Mutant huntingtin increases nuclear corepressor function and enhances ligand-dependent nuclear hormone receptor activation.

    Yohrling GJ, Farrell LA, Hollenberg AN and Cha JH

    Department of Neurology, Center for Aging, Genetics, and Neurodegeneration, Massachusetts General Hospital, Charlestown 02129, USA.

    There is increasing evidence that transcriptional dysregulation is important in Huntington's disease pathogenesis. The transcriptional protein, nuclear corepressor (NCoR), acts to repress transcription of nuclear hormone receptors, such as the thyroid hormone receptor (TR) and retinoic acid receptor, in the absence of their appropriate ligand. NCoR has been shown to bind to the mutated huntingtin protein in a yeast two-hybrid screen. This aberrant interaction may have profound effects on both the function of the NCoR protein and on its control of nuclear hormone receptor-mediated transcription. To test this hypothesis, reporter gene assays were performed in inducible PC12 cell lines expressing exon 1 of the human huntingtin protein (Htt) with either a 25 or 103 polyglutamine (Q) repeat. Expression of mutant 103Q protein appears to enhance the ability of NCoR to repress TR-mediated transcription in the absence of ligand. Western analyses indicated that the expression of the mutant 103Q Htt protein did not change the endogenous NCoR levels in the HD103Q PC12 cells when compared to uninduced cells. Interestingly, using GST pull-down assays we found that a mutant Htt exon 1 construct with 53 polyglutamine (HD53Q) did not bind to NCoR in a polyglutamine-dependent fashion. These findings suggest that an aberrant NCoR-Htt interaction does not exist in vitro. Expression of the mutant 103Q protein was also found to enhance ligand-dependent activation of TR and retinoic acid receptor. In vitro binding data shows that TR binds to HD53Q in the presence of ligand. Taken together these data suggest that Htt may function as a transcriptional coactivator of nuclear hormone receptors.

    Funded by: NIDDK NIH HHS: R01-DK-56123; NINDS NIH HHS: R01-NS-38106

    Molecular and cellular neurosciences 2003;23;1;28-38

  • Early Huntington disease prenatal diagnosis by maternal semiquantitative fluorescent-PCR.

    González-González MC, Trujillo MJ, Rodríguez de Alba M and Ramos C

    Department of Genetics, Fundación Jiménez Díaz, Madrid, Spain.

    Neurology 2003;60;7;1214-5

  • Mutant huntingtin causes context-dependent neurodegeneration in mice with Huntington's disease.

    Yu ZX, Li SH, Evans J, Pillarisetti A, Li H and Li XJ

    Department of Human Genetics, Emory University School of Medicine, Atlanta, Georgia 30322, USA.

    Huntington's disease (HD) mouse models that express N-terminal huntingtin fragments show rapid disease progression and have been used for developing therapeutics. However, light microscopy reveals no significant neurodegeneration in these mice. It remains unclear how mutant huntingtin induces neurodegeneration. Using caspase staining, terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling, and electron microscopy, we observed that N171-82Q mice, which express the first 171 aa of mutant huntingtin, displayed more degenerated neurons than did other HD mouse models. The neurodegeneration was also evidenced by increased immunostaining for glial fibrillary acidic protein and ultrastructural features of apoptosis. R6/2 mice, which express exon 1 of mutant huntingtin, showed dark, nonapoptotic neurons and degenerated mitochondria associated with mutant huntingtin. In HD repeat knock-in mice (HdhCAG150), which express full-length mutant huntingtin, degenerated cytoplasmic organelles were found in both axons and neuronal cell bodies in association with mutant huntingtin that was not labeled by an antibody to huntingtin amino acids 342-456. Transfection of cultured cells with mutant huntingtin revealed that an N-terminal huntingtin fragment (amino acids 1-208 plus a 120 glutamine repeat) caused a greater increase in caspase activity than did exon 1 huntingtin and longer huntingtin fragments. These results suggest that context-dependent neurodegeneration in HD may be mediated by different N-terminal huntingtin fragments. In addition, this study has identified neurodegenerative markers for the evaluation of therapeutic treatments in HD mouse models.

    Funded by: NIA NIH HHS: AG19206; NINDS NIH HHS: NS41669

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2003;23;6;2193-202

  • Cdc42-interacting protein 4 binds to huntingtin: neuropathologic and biological evidence for a role in Huntington's disease.

    Holbert S, Dedeoglu A, Humbert S, Saudou F, Ferrante RJ and Néri C

    Laboratory of Genomic Biology, Institut National de la Santé et de la Recherche Médicale-Avenir Group and Fondation Jean Dausset-Centre d'Etude du Polymorphisme Humain, 75010 Paris, France.

    Huntington's disease (HD) is a neurodegenerative disease caused by polyglutamine (polyQ) expansion in the protein huntingtin (htt). Pathogenesis in HD seems to involve the formation of neuronal intranuclear inclusions and the abnormal regulation of transcription and signal transduction. To identify previously uncharacterized htt-interacting proteins in a simple model system, we used a yeast two-hybrid screen with a Caenorhabditis elegans activation domain library. We found a predicted SH3 domain protein (K08E3.3b) that interacts with N-terminal htt in two-hybrid tests. A human homolog of K08E3.3b is the Cdc42-interacting protein 4 (CIP4), a protein involved in Cdc42 and Wiskott-Aldrich syndrome protein-dependent signal transduction. CIP4 interacted in vitro with full-length htt from lymphoblastoid cells. Neuronal CIP4 immunoreactivity increased with neuropathological severity in the neostriatum of HD patients and partially colocalized to ubiquitin-positive aggregates. Marked CIP4 overexpression also was observed in Western blot from human HD brain striatum. The overexpression of CIP4 induced the death of striatal neurons. Our data suggest that CIP4 accumulation and cellular toxicity may have a role in HD pathogenesis.

    Funded by: NCCIH NIH HHS: AT00613, R01 AT000613, U01 AT000613; NIA NIH HHS: AG12992, AG13846, P01 AG012992, P30 AG013846; NIMH NIH HHS: MH/NS 31862; NINDS NIH HHS: NS35255

    Proceedings of the National Academy of Sciences of the United States of America 2003;100;5;2712-7

  • Characterization of Endophilin B1b, a brain-specific membrane-associated lysophosphatidic acid acyl transferase with properties distinct from endophilin A1.

    Modregger J, Schmidt AA, Ritter B, Huttner WB and Plomann M

    Center for Biochemistry II, Medical Faculty, Joseph-Stelzmann-Strasse 52, University of Cologne, Germany.

    We have characterized mammalian endophilin B1, a novel member of the endophilins and a representative of their B subgroup. The endophilins B show the same domain organization as the endophilins A, which contain an N-terminal domain responsible for lipid binding and lysophosphatidic acid acyl transferase activity, a central coiled-coil domain for oligomerization, a less conserved linker region, and a C-terminal Src homology 3 (SH3) domain. The endophilin B1 gene gives rise to at least three splice variants, endophilin B1a, which shows a widespread tissue distribution, and endophilins B1b and B1c, which appear to be brain-specific. Endophilin B1, like endophilins A, binds to palmitoyl-CoA, exhibits lysophosphatidic acid acyl transferase activity, and interacts with dynamin, amphiphysins 1 and 2, and huntingtin. However, in contrast to endophilins A, endophilin B1 does not bind to synaptojanin 1 and synapsin 1, and overexpression of its SH3 domain does not inhibit transferrin endocytosis. Consistent with this, immunofluorescence analysis of endophilin B1b transfected into fibroblasts shows an intracellular reticular staining, which in part overlaps with that of endogenous dynamin. Upon subcellular fractionation of brain and transfected fibroblasts, endophilin B1 is largely recovered in association with membranes. Together, our results suggest that the action of the endophilins is not confined to the formation of endocytic vesicles from the plasma membrane, with endophilin B1 being associated with, and presumably exerting a functional role at, intracellular membranes.

    The Journal of biological chemistry 2003;278;6;4160-7

  • Huntingtin in health and disease.

    Young AB

    Neurology Service, Massachusetts General Hospital, Boston, Massachusetts 02114, USA. young@helix.mgh.harvard.edu

    Funded by: NIA NIH HHS: AG13617, R01 AG013617, R37 AG013617; NINDS NIH HHS: NS38106, R01 NS038106

    The Journal of clinical investigation 2003;111;3;299-302

  • Testing the test--why pursue a better test for Huntington disease?

    Timman R, Maat-Kievit A, Brouwer-DudokdeWit C, Zoeteweij M, Breuning MH and Tibben A

    Department of Neurology, Leiden University Medical Center, Leiden, The Netherlands. timman@mpp.fgg.eur.nl

    In 1993, the gene mutation for Huntington disease (HD) was identified and testing became possible with a reliability of >99%, without the need for co-operation of relatives. In 1997, a systematic information program offered the mutation retest to individuals who had earlier received a linkage test result for HD, which has a residual uncertainty of 1-9%. The characteristics of 129 individuals tested by linkage analysis for HD are reported on, as well as the reasons for their reassessment by mutation testing. Three groups were compared: (1) people who were retested between 1993 and 1997, before this study had started, (2) people who were retested after we provided information, and (3) persons who refrained from retesting. Nearly half of the linkage-tested individuals were retested, with the exception of noncarriers with a residual risk of 1 or 2%. Of them, less than one out of five were retested. Carriers with a hopeful view on the future (BHS) and a better sense of well-being (GHQ) were more likely to have the retest. Female carriers were also more likely to have the retest before we contacted them. Noncarriers who were retested were more anxious (HADS) than noncarriers who refrained from the retest. Retestees were younger at the time of testing. No risk reversals were revealed by this study.

    American journal of medical genetics. Part B, Neuropsychiatric genetics : the official publication of the International Society of Psychiatric Genetics 2003;117B;1;79-85

  • DNA testing for Huntington disease in the Turkish population.

    Akbas F and Erginel-Unaltuna N

    Department of Genetics, Experimental Medical Research Institute, Istanbul University, Istanbul, Turkey.

    Huntington disease (HD) is an autosomal dominant inherited disease, characterized by involuntary movements, behavioral and personality changes and dementia. Although the mean age at onset is about 40 years, onset varies from 5 to 79 years. Therefore, at-risk individuals are never sure to have escaped the disease. The genetic defect is a CAG trinucleotide repeat expansion at the 5' end of the IT-15 gene on chromosome 4. In this study, we analyzed 127 patients with HD and 122 healthy controls. The numbers of CAG repeats varied from 38 to 78 (median: 42) in 127 HD patients, while in healthy controls we observed only 10-35 CAG repeats (median: 18). The length of the CAG repeat expansion in Turkish HD patients and normal controls was similar to that reported from other populations. Negative correlations (r = -0.67) were also found between age of disease onset and repeat length.

    European neurology 2003;50;1;20-4

  • An upstream open reading frame impedes translation of the huntingtin gene.

    Lee J, Park EH, Couture G, Harvey I, Garneau P and Pelletier J

    Department of Biochemistry and McGill Cancer Center, McIntyre Medical Sciences Building, McGill University, Montreal, Quebec H3G 1Y6, Canada.

    Expansion of a CAG tract within the huntingtin gene, leading to the production of a protein with an expanded polyglutamine tract, is responsible for Huntington's disease. We show here that the 5' untranslated region (UTR) of the huntingtin gene plays an important role in controlling the synthesis of huntingtin. In particular, the 5' UTR contains an upstream open reading frame (uORF) encoding a 21 amino acid peptide. We demonstrate that the presence of this uORF negatively influences expression from the huntingtin mRNA. Our results suggest a role for the uORF in limiting ribosomal access to downstream initiation sites. Mechanisms involving the post-transcriptional regulation of huntingtin are not well understood, and this may be an important way of regulating huntingtin protein levels.

    Nucleic acids research 2002;30;23;5110-9

  • Presymptomatic testing in Huntington's disease and autosomal dominant cerebellar ataxias.

    Goizet C, Lesca G, Dürr A and French Group for Presymptomatic Testing in Neurogenetic Disorders

    Service de Génétique Médicale, Hôpital Pellegrin-Enfants, Bordeaux, France.

    Objective: To report a 7-year experience of presymptomatic testing in persons at risk for Huntington disease (HD) and to compare their characteristics and outcomes with those of persons at risk for a less disabling condition, autosomal dominant cerebellar ataxias (ADCA).

    Methods: The authors collected data on presymptomatic testing for HD (n = 712) and ADCA (n = 46) in 10 French centers.

    Results: The characteristics of applicants were similar in HD and ADCA, revealing a predominance of women, a low rate of completing the presymptomatic testing program, and a high rate of follow-up. The frequency of serious events was low (2% for HD, 5% for ADCA), but such events were also found after favorable results. Family planning was a more frequent reason for seeking presymptomatic testing in ADCA than in HD. Prenatal diagnosis was performed in only half of the pregnancies in HD carriers (n = 35) but in all of those in ADCA carriers (n = 4).

    Conclusion: Counseling in multistep and multidisciplinary teams is important not only for presymptomatic testing in HD but also for less disabling diseases.

    Neurology 2002;59;9;1330-6

  • HIP14, a novel ankyrin domain-containing protein, links huntingtin to intracellular trafficking and endocytosis.

    Singaraja RR, Hadano S, Metzler M, Givan S, Wellington CL, Warby S, Yanai A, Gutekunst CA, Leavitt BR, Yi H, Fichter K, Gan L, McCutcheon K, Chopra V, Michel J, Hersch SM, Ikeda JE and Hayden MR

    Centre for Molecular Medicine and Therapeutics, Department of Medical Genetics, University of British Columbia, Vancouver, British Columbia, Canada V5Z 4H4.

    Huntington disease (HD) is caused by polyglutamine [poly(Q)] expansion in the protein huntingtin (htt). Although the exact mechanism of disease progression remains to be elucidated, altered interactions of mutant htt with its protein partners could contribute to the disease. Using the yeast two-hybrid system, we have isolated a novel htt interacting protein, HIP14. HIP14's interaction with htt is inversely correlated to the poly(Q) length in htt. mRNAs of 9 and 6 bp are transcribed from the HIP14 gene, with the 6 kb transcript being predominantly expressed in the brain. HIP14 protein is enriched in the brain, shows partial co-localization with htt in the striatum, and is found in medium spiny projection neurons, the subset of neurons affected in HD. HIP14 localizes to the Golgi, and to vesicles in the cytoplasm. The HIP14 protein has sequence similarity to Akr1p, a protein essential for endocytosis in Saccharomyces cerevisiae. Expression of human HIP14 results in rescue of the temperature-sensitive lethality in akr1 Delta yeast cells and, furthermore, restores their defect in endocytosis, demonstrating a role for HIP14 in intracellular trafficking. Our findings suggest that decreased interaction between htt and HIP14 could contribute to the neuronal dysfunction in HD by perturbing normal intracellular transport pathways in neurons.

    Funded by: NINDS NIH HHS: NS35255

    Human molecular genetics 2002;11;23;2815-28

  • Evaluation of psychological symptoms among presymptomatic HD gene carriers as measured by selected MMPI scales.

    Close Kirkwood S, Siemers E, Viken RJ, Hodes ME, Conneally PM, Christian JC and Foroud T

    Department of Medical and Molecular Genetics, Indiana University School of Medicine, 975 West Walnut Street IB 130, Indianapolis, IN 46202, USA.

    Individuals at-risk for Huntington disease (HD), both HD gene carriers and nongene carriers, were recruited to determine whether psychological changes are detectable among clinically presymptomatic individuals who carry the HD allele. Each participant underwent genotyping to determine HD gene carrier status and a clinical assessment that included a quantified neurological examination and an abbreviated Minnesota Multiphasic Personality Inventory (MMPI): the Hypochondriasis, Depression, Psychasthenia, Neuroticism, Cynical Hostility, and Irritability Scales and the Harris Subscales of Depression. The results of the MMPI were evaluated for differences between nongene carriers (NGC) (n = 363), presymptomatic gene carriers (PSGC) (n = 149), and those with manifest HD (MHD) (n = 26). The overall multiple analysis of variance was not significant, indicating that there was no overall difference among the three groups. However, when subscales of the MMPI were examined individually, participants with manifest HD scored higher on the Psychasthenia scale (MHD vs. PSGC, P = 0.005; MHD vs. NGC, P = 0.03) and the Harris Depression subscale, Brooding (MHD vs. PSGC, P=0.0005; MHD vs. NGC, P = 0.003). No significant correlation was found between the number of trinucleotide repeats on the disease-producing allele and any of the MMPI scales for the gene carriers, MHD or PSGC. These results verify the presence of psychological symptoms in the early phases of MHD but not in PSGC. Thus, further study of the behavioral and mood symptoms thought to accompany HD using measures designed specifically to detect depressive symptoms and changes in behavior specific to HD is warranted to delineate the timing of onset of the psychological symptoms.

    Funded by: NCRR NIH HHS: M01-RR-750; NIA NIH HHS: R-01-AG-08918; NICHD NIH HHS: T3HD07373; NINDS NIH HHS: N01-NS-2326, R01-NS-42659

    Journal of psychiatric research 2002;36;6;377-82

  • The predominantly HEAT-like motif structure of huntingtin and its association and coincident nuclear entry with dorsal, an NF-kB/Rel/dorsal family transcription factor.

    Takano H and Gusella JF

    Molecular Neurogenetics Unit, Massachusetts General Hospital and Department of Genetics, Harvard Medical School, MGH-East Building 149, 13th Street, Charlestown, Massachusetts 02129, USA. hiroki@bri.niigata-u.ac.jp

    Background: Huntington's disease (HD) pathogenesis is due to an expanded polyglutamine tract in huntingtin, but the specificity of neuronal loss compared with other polyglutamine disorders also implies a role for the protein's unknown inherent function. Huntingtin is moderately conserved, with 10 HEAT repeats reported in its amino-terminal half. HD orthologues are evident in vertebrates and Drosophila, but not in Saccharomyces cerevisiae, Caenorhabditis elegans or Arabidopsis thaliana, a phylogenetic profile similar to the NF-kB/Rel/dorsal family transcription factors, suggesting a potential functional relationship.

    Results: We initially tested the potential for a relationship between huntingtin and dorsal by overexpression experiments in Drosophila S2 cells. Drosophila huntingtin complexes via its carboxyl-terminal region with dorsal, and the two enter the nucleus concomitantly, partly in a lipopolysaccharide (LPS)- and Nup88-dependent manner. Similarly, in HeLa cell extracts, human huntingtin co-immunoprecipitates with NF-kB p50 but not with p105. By cross-species comparative analysis, we find that the carboxyl-terminal segment of huntingtin that mediates the association with dorsal possesses numerous HEAT-like sequences related to those in the amino-terminal segment. Thus, Drosophila and vertebrate huntingtins are composed predominantly of 28 to 36 degenerate HEAT-like repeats that span the entire protein.

    Conclusion: Like other HEAT-repeat filled proteins, huntingtin is made up largely of degenerate HEAT-like sequences, suggesting that it may play a scaffolding role in the formation of particular protein-protein complexes. While many proteins have been implicated in complexes with the amino-terminal region of huntingtin, the NF-kB/Rel/dorsal family transcription factors merit further examination as direct or indirect interactors with huntingtin's carboxyl-terminal segment.

    Funded by: NINDS NIH HHS: NS16367, P50 NS016367

    BMC neuroscience 2002;3;15

  • PACSIN 1 interacts with huntingtin and is absent from synaptic varicosities in presymptomatic Huntington's disease brains.

    Modregger J, DiProspero NA, Charles V, Tagle DA and Plomann M

    Institute for Biochemistry II, University of Cologne, D-50931 Cologne, Germany.

    Huntington's disease (HD) is caused by a pathological expansion of a CAG repeat in the first exon of the gene coding for huntingtin, resulting in an abnormally long polyglutamine stretch. Despite its widespread expression, mutant huntingtin leads to selective neuronal loss in the striatum and cortex. Here we report that the neurospecific phosphoprotein PACSIN 1, which has been implicated as playing a central role in synaptic vesicle recycling, interacts with huntingtin via its C-terminal SH3 domain. Moreover, two other isoforms, PACSIN 2 and 3, which show a wider tissue distribution including the brain, do not interact with huntingtin despite a highly conserved SH3 domain. Furthermore, this interaction is repeat-length-dependent and is enhanced with mutant huntingtin, possibly causing the sequestration of PACSIN 1. Normally, PACSIN 1 is located along neurites and within synaptic boutons, but in HD patient neurons, there is a progressive loss of PACSIN 1 immunostaining in synaptic varicosities, beginning in presymptomatic and early-stage HD. Further, PACSIN 1 immunostaining of HD patient tissue reveals a more cytoplasmic distribution of the protein, with particular concentration in the perinuclear region coincident with mutant huntingtin. Thus, the specific interaction of huntingtin with the neuronal PACSIN isoform, PACSIN 1, and its altered intracellular distribution in pathological tissue, together with the observed differences in the binding behavior, suggest a role for PACSIN 1 during early stages of the selective neuropathology of HD.

    Funded by: NIMH NIH HHS: MH/NS 31862

    Human molecular genetics 2002;11;21;2547-58

  • CAG mutation effect on rate of progression in Huntington's disease.

    Squitieri F, Cannella M and Simonelli M

    Neurogenetics Unit, IRCCS Neuromed, Pozzilli (IS), Italy.

    Huntington's disease (HD) is progressively invalidating and caused by a CAG expanded mutation. We tested the effect of the mutation length on the rate of progression in a cohort of 80 patients clinically followed-up and genetically characterized. Two patients presenting an infantile and aggressive HD form starting under 10 years had over 90 repeats; the other patients did not show any influence of the CAG expanded number on the rate of progression. In conclusion, the CAG expanded repeat affects the disease progression only at a very upper pathological range and in rare cases initiating very early in the life, while it does not seem to affect in any way the severity of the phenotype in most HD patients. Other factors affecting the motor symptom progression, other than the expanded repeats, therefore have to be investigated.

    Neurological sciences : official journal of the Italian Neurological Society and of the Italian Society of Clinical Neurophysiology 2002;23 Suppl 2;S107-8

  • Genetic background of Huntington disease in Croatia: Molecular analysis of CAG, CCG, and Delta2642 (E2642del) polymorphisms.

    Hećimović S, Klepac N, Vlasić J, Vojta A, Janko D, Skarpa-Prpić I, Canki-Klain N, Marković D, Bozikov J, Relja M and Pavelić K

    Division of Molecular Medicine, Ruder Bosković Institute, Zagreb, Croatia. silva@icarus.wustl.edu

    This study presents the first molecular data on the basis and the origin of Huntington disease in Croatia and is the first such analysis performed among a Slavic population. We analyzed three trinucleotide polymorphisms in the HD gene: CAG, CCG and GAG Delta2642 (E2642del) triplets. Analysis of the CAG repeat size among 44 Huntington patients (39-66 CAGs) and 51 normal individuals (9-34 CAGs) showed that the range of the repeats was similar to previous findings. The frequency of the CCG and Delta2642 polymorphic alleles on N and HD chromosomes was found to correlate well with earlier reports for Western European populations. We found significance for both the CCG7 allele (p=0.004) and the Delta2642 allele (p<0.001) among HD chromosomes. The CCG7 allele was overpresented among affected chromosomes (94.6%), but was also the most frequent CCG allele among normal chromosomes (66.7%). Interestingly, the Delta2642 allele was present on 40.5% HD chromosomes compared to only 9.8% of control chromosomes. Our results indicate that HD mutations in Croatia could be of the same origin as in Western populations and also support the multi-step hypothesis for generating new HD alleles. Similar frequencies and distributions of both the CCG and the Delta2642 polymorphisms in Croatia and Western European normal chromosomes indicate that the prevalence rate of HD in Croatia may be as high as in Western populations. Since we estimated a lower prevalence rate (1 : 100,000), we assume that there are still many misdiagnosed and/or unrecognized cases of Huntington disease in Croatia.

    Human mutation 2002;20;3;233

  • Inhibition of tryptophan hydroxylase activity and decreased 5-HT1A receptor binding in a mouse model of Huntington's disease.

    Yohrling IV GJ, Jiang GC, DeJohn MM, Robertson DJ, Vrana KE and Cha JH

    Department of Neurology, Massachusetts General Hospital, Charlestown, Massachusetts 02129, USA. yohrling@helix.mgh.harvard.edu

    The pathogenic mechanisms of the mutant huntingtin protein that cause Huntington's disease (HD) are unknown. Previous studies have reported significant decreases in the levels of serotonin (5-HT) and its metabolite 5-hydroxyindoleacetic acid (5-HIAA) in the brains of the R6/2 transgenic mouse model of HD. In an attempt to elucidate the cause of these neurochemical perturbations in HD, the protein levels and enzymatic activity of tryptophan hydroxylase (TPH), the rate-limiting enzyme in 5-HT biosynthesis, were determined. Enzyme activity was measured in brainstem homogenates from 4-, 8-, and 12-week-old R6/2 mice and compared with aged-matched wild-type control mice. We observed a 62% decrease in brainstem TPH activity (p = 0.009) in 4-week-old R6/2 mice, well before the onset of behavioral symptoms. In addition, significant decreases in TPH activity were also observed at 8 and 12 weeks of age (61%, p = 0.02 and 86%, p = 0.005, respectively). In the 12-week-old-mice, no change in immunoreactive TPH was observed. In vitro binding showed that TPH does not bind to exon 1 of huntingtin in a polyglutamine-dependent manner. Specifically, glutathione-S-transferase huntingtin exon 1 proteins with 20, 32 or 53 polyglutamines did not interact with radiolabeled tryptophan hydroxylase. Therefore, the inhibition of TPH activity does not appear to result from a direct huntingtin/TPH interaction. Receptor binding analyses for the 5-HT1A receptor in 12-week-old R6/2 mice revealed significant reductions in 8-OH-[3H]DPAT binding in several hippocampal and cortical regions. These results demonstrate that the serotonergic system in the R6/2 mice is severely disrupted in both presymptomatic and symptomatic mice. The presymptomatic inhibition of TPH activity in the R6/2 mice may help explain the functional consequences of HD and provide insights into new targets for pharmacotherapy.

    Funded by: NIGMS NIH HHS: R01 GM-38931; NINDS NIH HHS: R01 NS-38106

    Journal of neurochemistry 2002;82;6;1416-23

  • Impaired glutamate transport and glutamate-glutamine cycling: downstream effects of the Huntington mutation.

    Behrens PF, Franz P, Woodman B, Lindenberg KS and Landwehrmeyer GB

    Department of Neurology, Universitätsklinik Freiburg, Germany. pfbehrens@web.de

    The pathogenesis of Huntington's disease is still not completely understood. Several lines of evidence from toxic/non-transgenic animal models of Huntington's disease suggest that excitotoxic mechanisms may contribute to the pathological phenotype. Evidence from transgenic animal models of Huntington's disease, however, is sparse. To explore potential alterations in brain glutamate handling we studied transgenic mice expressing an N-terminal fragment of mutant huntingtin (R6/2). Intracerebral microdialysis in freely moving mice showed similar extracellular glutamate levels in R6/2 and littermate controls. However, partial inhibition of glutamate transport by L-trans-pyrrolidine-2,4-dicarboxylate (4 mM) disclosed an age-dependent increase in extracellular glutamate levels in R6/2 mice compared with controls, consistent with a reduction of functional glutamate transport capacity. Biochemical studies demonstrated an age-dependent downregulation of the glial glutamate transporter GLT-1 mRNA and protein, resulting in a progressive reduction of transporter function. Glutamate transporters other than GLT-1 were unchanged. In addition, increased extracellular glutamine levels and alterations to glutamine synthetase immunoreactivity suggested a perturbation of the glutamate-glutamine cycle. These findings demonstrate that the Huntington's disease mutation results in a progressively deranged glutamate handling in the brain, beginning before the onset of symptoms in mice. They also provide evidence for a contribution of excitotoxicity to the pathophysiology of Huntington's disease, and thus Huntington's disease may be added to the growing list of neurodegenerative disorders associated with compromised glutamate transport capacity.

    Brain : a journal of neurology 2002;125;Pt 8;1908-22

  • Proteases acting on mutant huntingtin generate cleaved products that differentially build up cytoplasmic and nuclear inclusions.

    Lunkes A, Lindenberg KS, Ben-Haïem L, Weber C, Devys D, Landwehrmeyer GB, Mandel JL and Trottier Y

    Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP, B.P.163, 67404 Illkirch Cédex, CU de Strasbourg, France. astrid.lunkes@embo.org

    Proteolytic processing of mutant huntingtin (mhtt) is regarded as a key event in the pathogenesis of Huntington's disease (HD). Mhtt fragments containing a polyglutamine expansion form intracellular inclusions and are more cytotoxic than full-length mhtt. Here, we report that two distinct mhtt fragments, termed cp-A and cp-B, differentially build up nuclear and cytoplasmic inclusions in HD brain and in a cellular model for HD. Cp-A is released by cleavage of htt in a 10 amino acid domain and is the major fragment that aggregates in the nucleus. Furthermore, we provide evidence that cp-A and cp-B are most likely generated by aspartic endopeptidases acting in concert with the proteasome to ensure the normal turnover of htt. These proteolytic processes are thus potential targets for therapeutic intervention in HD.

    Funded by: NIMH NIH HHS: MH/NS31862

    Molecular cell 2002;10;2;259-69

  • Sp1 and TAFII130 transcriptional activity disrupted in early Huntington's disease.

    Dunah AW, Jeong H, Griffin A, Kim YM, Standaert DG, Hersch SM, Mouradian MM, Young AB, Tanese N and Krainc D

    Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Center for Aging, Genetics and Neurodegeneration, Charlestown, MA 02129, USA.

    Huntington's disease (HD) is an inherited neurodegenerative disease caused by expansion of a polyglutamine tract in the huntingtin protein. Transcriptional dysregulation has been implicated in HD pathogenesis. Here, we report that huntingtin interacts with the transcriptional activator Sp1 and coactivator TAFII130. Coexpression of Sp1 and TAFII130 in cultured striatal cells from wild-type and HD transgenic mice reverses the transcriptional inhibition of the dopamine D2 receptor gene caused by mutant huntingtin, as well as protects neurons from huntingtin-induced cellular toxicity. Furthermore, soluble mutant huntingtin inhibits Sp1 binding to DNA in postmortem brain tissues of both presymptomatic and affected HD patients. Understanding these early molecular events in HD may provide an opportunity to interfere with the effects of mutant huntingtin before the development of disease symptoms.

    Funded by: NCCIH NIH HHS: AT00613; NIA NIH HHS: 5R37AG13617; NINDS NIH HHS: NS02174, NS34361, NS35255

    Science (New York, N.Y.) 2002;296;5576;2238-43

  • The IGF-1/Akt pathway is neuroprotective in Huntington's disease and involves Huntingtin phosphorylation by Akt.

    Humbert S, Bryson EA, Cordelières FP, Connors NC, Datta SR, Finkbeiner S, Greenberg ME and Saudou F

    UMR 146 CNRS/Institut Curie, Centre Universitaire, 91405 Orsay Cedex, France.

    In the search for neuroprotective factors in Huntington's disease, we found that insulin growth factor 1 via activation of the serine/threonine kinase Akt/PKB is able to inhibit neuronal death specifically induced by mutant huntingtin containing an expanded polyglutamine stretch. The IGF-1/Akt pathway has a dual effect on huntingtin-induced toxicity, since activation of this pathway also results in a decrease in the formation of intranuclear inclusions of mutant huntingtin. We demonstrate that huntingtin is a substrate of Akt and that phosphorylation of huntingtin by Akt is crucial to mediate the neuroprotective effects of IGF-1. Finally, we show that Akt is altered in Huntington's disease patients. Taken together, these results support a potential role of the Akt pathway in Huntington's disease.

    Funded by: NICHD NIH HHS: HD18655, P01 HD24926; NINDS NIH HHS: R01 NS39074

    Developmental cell 2002;2;6;831-7

  • Huntingtin inclusions do not deplete polyglutamine-containing transcription factors in HD mice.

    Yu ZX, Li SH, Nguyen HP and Li XJ

    Department of Human Genetics, Emory University, School of Medicine, Atlanta, GA 30322, USA.

    A pathological hallmark of polyglutamine diseases is the presence of inclusions or aggregates of the expanded polyglutamine protein. Polyglutamine inclusions are present in the neuronal nucleus in a number of inherited neurodegenerative disorders, including Huntington disease (HD). Recent studies suggest that polyglutamine inclusions may sequester polyglutamine-containing transcription factors and deplete their concentration in the nucleus, leading to altered gene expression. To test this hypothesis, we examined the expression and localization of the polyglutamine-containing or glutamine-rich transcription factors TBP, CBP and Sp1 in HD mouse models. All three transcription factors were diffusely distributed in the nucleus, despite the presence of abundant intranuclear inclusions. There were no differences in the nuclear staining of these transcription factors between HD and wild-type mouse brains. Although some CBP staining appeared as dots in the selective brain regions (e.g. hypothalamus and amygdala), double labeling showed that most CBP was not co-localized with huntingtin nuclear inclusions. Electron microscopy confirmed that CBP was diffusely distributed in the nucleus. Western blots showed that these transcription factors were not trapped in huntingtin inclusions. In the striatum of HD mice, which suffers a significant reduction in the expression of a number of genes, mutant huntingtin was present in both an aggregated and a diffuse form. These findings suggest that altered gene expression may result from the interactions of soluble mutant huntingtin with nuclear transcription factors, rather than from the depletion of transcription factors by nuclear inclusions.

    Funded by: NIA NIH HHS: AG19206; NINDS NIH HHS: NS41669

    Human molecular genetics 2002;11;8;905-14

  • Increased sensitivity to N-methyl-D-aspartate receptor-mediated excitotoxicity in a mouse model of Huntington's disease.

    Zeron MM, Hansson O, Chen N, Wellington CL, Leavitt BR, Brundin P, Hayden MR and Raymond LA

    Kinsmen Laboratory of Neurological Research, Department of Psychiatry, 221 84, Lund, Sweden.

    Previous work suggests N-methyl-D-aspartate receptor (NMDAR) activation may be involved in degeneration of medium-sized spiny striatal neurons in Huntington's disease (HD). Here we show that these neurons are more vulnerable to NMDAR-mediated death in a YAC transgenic FVB/N mouse model of HD expressing full-length mutant huntingtin, compared with wild-type FVB/N mice. Excitotoxic death of these neurons was increased after intrastriatal injection of quinolinate in vivo, and after NMDA but not AMPA exposure in culture. NMDA-induced cell death was abolished by an NR2B subtype-specific antagonist. In contrast, NMDAR-mediated death of cerebellar granule neurons was not enhanced, consistent with cell-type and NMDAR subtype specificity. Moreover, increased NMDA-evoked current amplitude and caspase-3 activity were observed in transgenic striatal neurons. Our data support a role for NR2B-subtype NMDAR activation as a trigger for selective neuronal degeneration in HD.

    Neuron 2002;33;6;849-60

  • Huntingtin is present in the nucleus, interacts with the transcriptional corepressor C-terminal binding protein, and represses transcription.

    Kegel KB, Meloni AR, Yi Y, Kim YJ, Doyle E, Cuiffo BG, Sapp E, Wang Y, Qin ZH, Chen JD, Nevins JR, Aronin N and DiFiglia M

    Department of Neurology, Massachusetts General Hospital, Boston, MA 02114, USA. kegel@helix.mgh.harvard.edu

    Huntingtin is a protein of unknown function that contains a polyglutamine tract, which is expanded in patients with Huntington's disease (HD). We investigated the localization and a potential function for huntingtin in the nucleus. In human fibroblasts from normal and HD patients, huntingtin localized diffusely in the nucleus and in subnuclear compartments identified as speckles, promyelocytic leukemia protein bodies, and nucleoli. Huntingtin-positive nuclear bodies redistributed after treatment with sodium butyrate. By Western blot, purified nuclei had low levels of full-length huntingtin compared with the cytoplasm but contained high levels of N- and C-terminal huntingtin fragments, which tightly bound the nuclear matrix. Full-length huntingtin co-immunoprecipitated with the transcriptional corepressor C-terminal binding protein, and polyglutamine expansion in huntingtin reduced this interaction. Full-length wild-type and mutant huntingtin repressed transcription when targeted to DNA. Truncated N-terminal mutant huntingtin repressed transcription, whereas the corresponding wild-type fragment did not repress transcription. We speculate that wild-type huntingtin may function in the nucleus in the assembly of nuclear matrix-bound protein complexes involved with transcriptional repression and RNA processing. Proteolysis of mutant huntingtin may alter nuclear functions by disrupting protein complexes and inappropriately repressing transcription in HD.

    Funded by: NIA NIH HHS: T32 AG 00222; NINDS NIH HHS: NS 16367, NS 35711, NS 38194

    The Journal of biological chemistry 2002;277;9;7466-76

  • Perinuclear localization of huntingtin as a consequence of its binding to microtubules through an interaction with beta-tubulin: relevance to Huntington's disease.

    Hoffner G, Kahlem P and Djian P

    CNRS --- UPR 2228, Régulation de la Transcription et Maladies Génétiques, Université René Descartes, 45 rue des Saints-Pères, 75270 Paris Cedex 06, France.

    Huntington's disease results from an expansion of a series of glutamine repeats in the protein huntingtin. We have discovered from immunopurification studies that huntingtin combines specifically with the beta subunit of tubulin. This binding explains why huntingtin can be shown on assembled microtubules by electron microscopy. Immunostaining shows that most of the huntingtin in the cytoplasm is associated with microtubules. Huntingtin is particularly abundant in the perinuclear region, where it is also associated with microtubules and in the centrosomal region, where it co-localizes with gamma-tubulin. In Huntington's disease, inclusions are often nuclear or perinuclear. Since the perinuclear concentration of huntingtin does not depend on the number of its glutamine repeats, we propose that inclusions are found in perinuclear and intranuclear locations because the beta-tubulin binding property of huntingtin brings it to the perinuclear region, from which it readily gains access to the nucleus. The mutational glutamine expansion then promotes insolubility and results in an inclusion.

    Journal of cell science 2002;115;Pt 5;941-8

  • Arfaptin 2 regulates the aggregation of mutant huntingtin protein.

    Peters PJ, Ning K, Palacios F, Boshans RL, Kazantsev A, Thompson LM, Woodman B, Bates GP and D'Souza-Schorey C

    Division of Tumor Biology, Netherlands Cancer Institute, Amsterdam, The Netherlands.

    Huntington's disease (HD) is an inherited neurodegenerative disorder. Here we demonstrate that expression of arfaptin 2/POR1 (partner of Rac1) in cultured cells induces the formation of pericentriolar and nuclear aggregates, which morphologically resemble mutant huntingtin aggregates characteristic of HD. Endogenous arfaptin 2 localizes to aggregates induced by expression of an abnormal amino-terminal fragment of huntingtin that contains polyglutamine (polyQ) expansions. A dominant inhibitory mutant of arfaptin 2 inhibits aggregation of mutant huntingtin, but not in the presence of proteasome inhibitors. Using cell-free biochemical assays, we show that arfaptin 2 inhibits proteasome activity. Finally, we show that expression of arfaptin 2 is increased at sites of neurodegeneration and the protein localizes to huntingtin aggregates in HD transgenic mouse brains. Our data suggest that arfaptin 2 is involved in regulating huntingtin protein aggregation, possibly by impairing proteasome function.

    Nature cell biology 2002;4;3;240-5

  • Interaction of Huntington disease protein with transcriptional activator Sp1.

    Li SH, Cheng AL, Zhou H, Lam S, Rao M, Li H and Li XJ

    Department of Human Genetics, Emory University School of Medicine, Atlanta, Georgia 30322, USA.

    Polyglutamine expansion causes Huntington disease (HD) and at least seven other neurodegenerative diseases. In HD, N-terminal fragments of huntingtin with an expanded glutamine tract are able to aggregate and accumulate in the nucleus. Although intranuclear huntingtin affects the expression of numerous genes, the mechanism of this nuclear effect is unknown. Here we report that huntingtin interacts with Sp1, a transcription factor that binds to GC-rich elements in certain promoters and activates transcription of the corresponding genes. In vitro binding and immunoprecipitation assays show that polyglutamine expansion enhances the interaction of N-terminal huntingtin with Sp1. In HD transgenic mice (R6/2) that express N-terminal-mutant huntingtin, Sp1 binds to the soluble form of mutant huntingtin but not to aggregated huntingtin. Mutant huntingtin inhibits the binding of nuclear Sp1 to the promoter of nerve growth factor receptor and suppresses its transcriptional activity in cultured cells. Overexpression of Sp1 reduces the cellular toxicity and neuritic extension defects caused by intranuclear mutant huntingtin. These findings suggest that the soluble form of mutant huntingtin in the nucleus may cause cellular dysfunction by binding to Sp1 and thus reducing the expression of Sp1-regulated genes.

    Funded by: NIA NIH HHS: AG19206, R01 AG019206; NINDS NIH HHS: NS41669, R01 NS041669

    Molecular and cellular biology 2002;22;5;1277-87

  • Predictive DNA-testing for Huntington's disease and reproductive decision making: a European collaborative study.

    Evers-Kiebooms G, Nys K, Harper P, Zoeteweij M, Dürr A, Jacopini G, Yapijakis C and Simpson S

    Psychosocial Genetics Unit, Center for Human Genetics, K.U. Leuven, Leuven, Belgium. Gerry.Kiebooms@med.kuleuven.ac.be

    This European collaborative study addresses the question whether a predictive test result for Huntington's disease (HD) has an effect on subsequent reproduction by comparing carriers and non-carriers of the Huntington mutation. A unique characteristic of this study is that this evaluation is done in persons at reproductive age who had a predictive test after the identification of the Huntington gene and who were counselled in one of the participating centres. Data were collected for 180 carriers and 271 non-carriers who received a predictive test result in the period 1993-1998 in Aberdeen, Athens, Cardiff, Leiden, Leuven, Paris or Rome. The mean age of the total study group was 31.5 years and for about half of the group the follow-up interval was 3 years or more, with a maximum of 7 years. The collaborative study clearly revealed an overall impact of the predictive test result on subsequent reproduction: 14% of the carriers had one or more subsequent pregnancies vs 28% of the non-carriers. In the total carrier group a prenatal test was carried out in about two thirds of the pregnancies and one child was born after preimplantation genetic diagnosis; artificial insemination by donor, egg cell donation or adoption were not reported. A more refined analysis was performed in the subgroup with a follow-up interval of at least 3 years and who reported 'family planning' as a motive to apply for predictive testing in the pretest period. The complexity of this motive is discussed. In this subgroup with a desire for children in the pretest period the effect of the predictive test result was more pronounced: 69% of the non-carriers had subsequent pregnancies while only 39% of the carriers who mentioned 'family planning' as one of the major reasons to apply for predictive testing had a subsequent pregnancy. Of the carriers with one or more subsequent pregnancies the percentage using prenatal diagnosis was slightly higher than the percentage not using it, although there were clear differences from one centre to another. The latter group's decisions may seem more intriguing but may be partially understood based on stage theories of health behaviour. Last, but not least, whatever option is chosen by a couple at increased risk of transmitting the Huntington mutation, it is of the utmost importance that professionals fully respect this decision and support the couple.

    European journal of human genetics : EJHG 2002;10;3;167-76

  • Psychiatric symptoms in neurologically asymptomatic Huntington's disease gene carriers: a comparison with gene negative at risk subjects.

    Berrios GE, Wagle AC, Marková IS, Wagle SA, Rosser A and Hodges JR

    Department of Psychiatry, University of Cambridge, Addenbrooke's Hospital, Cambridge, UK. geb11@cam.ac.uk

    Objective: Psychiatric profiles of two at-risk groups [Huntington's disease (HD) gene carriers and non-carriers] were compared by means of a computerized battery and a structured interview.

    Method: To avoid confounding, only subjects who were free from neurological and cognitive deficits (neurologically asymptomatic) were included in the study. To avoid evaluation biases, all subjects were seen before the genetic testing was undertaken.

    Results: Gene carriers had significantly worse recognition memory and scored higher in measures of irritability than controls. The groups also differed in terms of the factor structure of their psychiatric symptoms. None of the subjects qualified for a psychiatric diagnosis at the time of assessment.

    Conclusion: The groups differed with respect to their profile of psychiatric symptoms. It is hypothesized that these differences are the expression of different mechanisms, i.e. that cognitive deficits relate more to genetic factors and neurotic complaints more to being brought up in a disturbed family background. Issues concerning instrument sensitivity, selection bias and the advantage of seriatim assessments are discussed.

    Acta psychiatrica Scandinavica 2002;105;3;224-30

  • Recruitment and activation of caspase-8 by the Huntingtin-interacting protein Hip-1 and a novel partner Hippi.

    Gervais FG, Singaraja R, Xanthoudakis S, Gutekunst CA, Leavitt BR, Metzler M, Hackam AS, Tam J, Vaillancourt JP, Houtzager V, Rasper DM, Roy S, Hayden MR and Nicholson DW

    Department of Biochemistry and Molecular Biology, Merck Frosst Centre for Therapeutic Research, Pointe-Claire-Dorval, Quebec, Canada H9R 4P8.

    In Huntington disease, polyglutamine expansion of the protein huntingtin (Htt) leads to selective neurodegenerative loss of medium spiny neurons throughout the striatum by an unknown apoptotic mechanism. Binding of Hip-1, a protein normally associated with Htt, is reduced by polyglutamine expansion. Free Hip-1 binds to a hitherto unknown polypeptide, Hippi (Hip-1 protein interactor), which has partial sequence homology to Hip-1 and similar tissue and subcellular distribution. The availability of free Hip-1 is modulated by polyglutamine length within Htt, with disease-associated polyglutamine expansion favouring the formation of pro-apoptotic Hippi-Hip-1 heterodimers. This heterodimer can recruit procaspase-8 into a complex of Hippi, Hip-1 and procaspase-8, and launch apoptosis through components of the 'extrinsic' cell-death pathway. We propose that Htt polyglutamine expansion liberates Hip-1 so that it can form a caspase-8 recruitment complex with Hippi. This novel non-receptor-mediated pathway for activating caspase-8 might contribute to neuronal death in Huntington disease.

    Nature cell biology 2002;4;2;95-105

  • Effects of intracellular expression of anti-huntingtin antibodies of various specificities on mutant huntingtin aggregation and toxicity.

    Khoshnan A, Ko J and Patterson PH

    Biology Division, California Institute of Technology, Pasadena, CA 91125, USA.

    We have generated eight mAbs (MW1-8) that bind the epitopes polyglutamine (polyQ), polyproline (polyP), or the C terminus of exon 1 in huntingtin (htt) protein. In the brains of Huntington's disease (HD) mouse models, the anti-polyQ mAbs bind to various cytoplasmic compartments, whereas the anti-polyP and anti-C terminus mAbs bind nuclear inclusions containing htt. To use these mAbs as intracellular perturbation agents, we have cloned and expressed the antigen-binding domains of three of the mAbs as single-chain variable region fragment Abs (scFvs). In 293 cells cotransfected with htt exon 1 containing an expanded polyQ domain, MW1, MW2, and MW7 scFvs colocalize with htt exon 1. Moreover, these scFvs coimmunoprecipitate with htt exon 1 in cell extracts. In perturbation experiments, MW7 scFv, recognizing the polyP domains of htt, significantly inhibits aggregation as well as the cell death induced by mutant htt protein. In contrast, MW1 and MW2 scFvs, recognizing the polyQ stretch, stimulate htt aggregation and apoptosis. Therefore, these anti-htt scFvs can be used to investigate the role of the polyP and polyQ domains in HD pathogenesis, and antibody binding to the polyP domain has potential therapeutic value in HD.

    Proceedings of the National Academy of Sciences of the United States of America 2002;99;2;1002-7

  • Is the 31 CAG repeat allele of the spinocerebellar ataxia 1 (SCA1) gene locus non-specifically associated with trinucleotide expansion diseases?

    Savić D, Topisirović I, Keckarević M, Keckarević D, Major T, Culjković B, Stojković O, Rakocević-Stojanović V, Mladenović J, Todorović S, Apostolski S and Romac S

    Faculty of Biology, University of Belgrade, Belgrade, Yugoslavia.

    A number of human hereditary neuromuscular and neurodegenerative disorders are caused by the expansion of trinucleotide repeats within certain genes. The molecular mechanisms that underlie these expansions are not yet known. We have analyzed six trinucleotide repeat-containing loci [spinocerebellar ataxias (SCA1, SCA3, SCA8), dentatorubral-pallidoluysian atrophy (DRPLA), Huntington chorea (HD) and fragile X syndrome (FRAXA)] in myotonic dystrophy type 1 (DM1) patients (n = 52). As controls, we analyzed two groups of subjects: healthy control subjects (n =133), and a group of patients with non-triplet neuromuscular diseases (n = 68) caused by point mutations, deletions or duplications (spinal muscular atrophy, Charcot-Marie-Tooth disease, type 1A, hereditary neuropathy with liability to pressure palsies, and Duchenne and Becker muscular dystrophy). Allele frequency distributions for all tested loci were similar in these three groups with the exception of the SCA1 locus. In DM1 patients, the SCA1 allele with 31 CAG repeats account for 40.4% of all chromosomes tested, which is significantly higher than in two other groups (11.3% in healthy controls and 6.6% in the group of non-triplet diseased patients; P < 0.001, Fisher's exact test). This is consistent with our previous findings in HD patients. The absence of this association in non-triplet diseases as well as in healthy controls could indicate a possible role of this SCA1 allele with 31 repeats in triplet diseases. Here we discuss a possible role of the SCA1 region in pathological trinucleotide repeat expansions.

    Psychiatric genetics 2001;11;4;201-5

  • Psychological impact of news of genetic risk for Huntington disease.

    Horowitz MJ, Field NP, Zanko A, Donnelly EF, Epstein C and Longo F

    University of California, San Francisco, California 94143, USA. mardi@itsa.ucsf.edu

    A one-year longitudinal study was conducted investigating the psychological effects of the news of genetic testing for the Huntington disease (HD) gene. Participants were assessed at baseline (before obtaining news of test results) and at three, six, and 12 months after test results on stress-specific symptom measures. Among carriers of the HD gene, a considerable number (55%) showed evidence of neurological impairment at baseline, indicative of HD. Also noteworthy, these individuals had significantly higher psychological symptom scores at baseline than carriers without neurological impairment or noncarriers. Despite this, these individuals were no more aware of their carrier status at baseline than carriers without HD symptoms or noncarriers. Furthermore, the psychological symptom levels of HD carriers with neurological impairment remained elevated across the follow-up assessments. Results for noncarriers and carriers without HD neurological symptoms were consistent with the findings of previous studies indicating that news of genetic testing for the HD gene had limited detrimental impact. The clinical implications of the results are discussed.

    American journal of medical genetics 2001;103;3;188-92

  • [Results of a program of presymptomatic diagnosis of Huntington's disease: evaluation of a 6 year period].

    Solís-Pérez MP, Burguera JA, Palau F, Livianos L, Vila M and Rojo L

    Servicio de Neurología, Hospital Universitario La Fe, Valencia. msolisp@meditex.es

    Objective: To know the uptake of predictive testing of Huntington's disease, the characteristics of the applicants, as well as the consequences for them.

    Methods: Prospective observational study between January of 1994 and December of 1999 of the predictive testing applicants who entered in the protocol consisted of: informative interview, psychiatric interview, blood extraction for molecular study, as well as outcome and follow-up interviews.

    Results: There were 87 applicants with a 50% risk. The mean age of the applicants was 28 years (SD = 7). Thirty one per cent already had children in the moment of predictive testing. The application rate according to the estimate population with 50% risk for the Comunidad Valenciana is 13,4%. The rate varies depending on the access to the information of the population in risk, being of 24,7% when they have direct access and of 8,3% when they do not have it (p < 0,01). Forty per cent did not come to the post-outcome visit, the positive or negative result for the mutation not influencing over it. Only 6,8% had some adverse event in the six years of follow-up all being slight.

    Conclusions: The application rate is determined by the access to the information of the population in risk. The fulfilment of the protocol designed for presymptomatic diagnosis of Huntington's disease keeps the adverse events presentation to a minimum.

    Neurologia (Barcelona, Spain) 2001;16;8;348-52

  • High incidence rate and absent family histories in one quarter of patients newly diagnosed with Huntington disease in British Columbia.

    Almqvist EW, Elterman DS, MacLeod PM and Hayden MR

    Department of Medical Genetics, Centre for Molecular Medicine and Therapeutics, University of British Columbia, Vancouver, Canada.

    The advent of the direct mutation test for Huntington disease (HD) has made it possible to identify a previously unrecognized symptomatic population of HD, including those with an atypical presentation or patients without a family history of HD. The present study investigated the uptake of this test in the province of British Columbia (BC), Canada and assessed the incidence rate and rate of identification of new mutations for HD. All symptomatic individuals residing in BC who were referred for the genetic test for HD between 1993 and 2000 (n=205) were analyzed for CAG expansion, baseline demographics and clinical data, and a family history of HD. A total of 141 (or 68.8%) had a CAG expansion > or =36. Of these, almost one-quarter (24.1%) did not have a family history of HD. An extensive chart review revealed that 11 patients (or 7.8%) had reliable information on both parents (who lived well into old age) and therefore possibly could represent new mutations for HD. This indicates a three to four times higher new mutation rate than previously reported. Our findings also show that the yearly incidence rate for HD was 6.9 per million, which is two times higher than previous incidence studies performed prior to the identification of the HD mutation. We also identified five persons with a clinical presentation of HD but without CAG expansion (genocopies) (2.4%).

    Clinical genetics 2001;60;3;198-205

  • Double-stranded RNA-dependent protein kinase, PKR, binds preferentially to Huntington's disease (HD) transcripts and is activated in HD tissue.

    Peel AL, Rao RV, Cottrell BA, Hayden MR, Ellerby LM and Bredesen DE

    The Buck Institute, 8001 Redwood Boulevard, Novato, CA 94945, USA. apeel@buckinstitute.org

    Fourteen neurological diseases have been associated with the expansion of trinucleotide repeat regions. These diseases have been categorized into those that give rise to the translation of toxic polyglutamine proteins and those that are untranslated. Thus far, compelling evidence has not surfaced for the inclusion of a model in which a common mechanism may participate in the pathobiology of both translated and untranslated trinucleotide diseases. In these studies we show that a double-stranded RNA-binding protein, PKR, which has previously been linked to virally-induced and stress-mediated apoptosis, preferentially binds mutant huntingtin RNA transcripts immobilized on streptavidin columns that have been incubated with human brain extracts. These studies also show, by immunodetection in tissue slices, that PKR is present in its activated form in both human Huntington autopsy material and brain tissue derived from Huntington yeast artificial chromosome transgenic mice. The increased immunolocalization of the activated kinase is more pronounced in areas most affected by the disease and, coupled with the RNA binding results, suggests a role for PKR activation in the disease process.

    Funded by: NIMH NIH HHS: MH/NS 31862

    Human molecular genetics 2001;10;15;1531-8

  • Polyglutamine-expanded huntingtin promotes sensitization of N-methyl-D-aspartate receptors via post-synaptic density 95.

    Sun Y, Savanenin A, Reddy PH and Liu YF

    Department of Pharmaceutical Sciences, Northeastern University, 715 Albany St., Boston, MA 02115, USA.

    Increased glutamate-mediated excitotoxicity seems to play an important role in the pathogenesis of Huntington's disease (Tabrizi, S. J., Cleeter, M. W., Xuereb, J., Taaman, J. W., Cooper, J. M., and Schapira, A. H. (1999) Ann. Neurol. 45, 25-32). However, how polyglutamine expansion in huntingtin promotes glutamate-mediated excitotoxicity remains a mystery. In this study we provide evidence that (i) normal huntingtin is associated with N-methyl-d-aspartate (NMDA) and kainate receptors via postsynaptic density 95 (PSD-95), (ii) the SH3 domain of PSD-95 mediates its binding to huntingtin, and (iii) polyglutamine expansion interferes with the ability of huntingtin to interact with PSD-95. The expression of polyglutamine-expanded huntingtin causes sensitization of NMDA receptors and promotes neuronal apoptosis induced by glutamate. The addition of the NMDA receptor antagonist significantly attenuates neuronal toxicity induced by glutamate and polyglutamine-expanded huntingtin. The overexpression of normal huntingtin significantly inhibits neuronal toxicity mediated by NMDA or kainate receptors. Our results demonstrate that polyglutamine expansion impairs the ability of huntingtin to bind PSD-95 and inhibits glutamate-mediated excitotoxicity. These changes may be essential for the pathogenesis of Huntington's disease.

    The Journal of biological chemistry 2001;276;27;24713-8

  • Identification of the full-length huntingtin- interacting protein p231HBP/HYPB as a DNA-binding factor.

    Rega S, Stiewe T, Chang DI, Pollmeier B, Esche H, Bardenheuer W, Marquitan G and Pützer BM

    Center for Cancer Research and Cancer Therapy, Department of Internal Medicine, Institute of Molecular Biology, University of Essen, Hufelandstrasse 55, Essen, D-45122, Germany.

    Neurodegeneration in Huntington's disease (HD) is associated with an elongated glutamine tract in the widely expressed huntingtin protein. Although the pathogenic mechanisms are still unknown, the distinct physical properties of mutant huntingtin in the brain suggest that other factors including huntingtin-interacting proteins might play a specific role. We have previously identified a DNA-binding motif in the proximal E1A promoter of adenovirus serotype 12 as responsible for E1A autoregulation. Here, we identified the p231HBP protein as a DNA-binding factor, the C-terminal portion of which has recently been characterized as the huntingtin-interacting protein HYPB of unknown function. We have determined the full-length cDNA sequence, identified several domains supporting its gene regulatory functions, and mapped the HBP231 gene to chromosome 3p21.2-p21.3. Our results provide an interesting molecular link between huntingtin and a DNA-binding factor, implicating that this interaction might result in the alteration of cellular gene expression involved in HD pathogenesis.

    Molecular and cellular neurosciences 2001;18;1;68-79

  • Wild type Huntingtin reduces the cellular toxicity of mutant Huntingtin in mammalian cell models of Huntington's disease.

    Ho LW, Brown R, Maxwell M, Wyttenbach A and Rubinsztein DC

    Department of Medical Genetics, Wellcome Trust Centre for Molecular Mechanisms of Disease, Cambridge Institute for Medical Research, Wellcome/MRC Building, Addenbrooke's Hospital, Hills Road, Cambridge CB2 2XY, UK.

    Objectives: Recent data suggest that wild type huntingtin can protect against apoptosis in the testis of mice expressing full length huntingtin transgenes with expanded CAG repeats. It is not clear if this protective effect was confined to particular cell types, or if wild type huntingtin exerted its protective effect in this model by simply reducing the formation of toxic proteolytic fragments from mutant huntingtin.

    Methods: We cotransfected neuronal (SK-N-SH, human neuroblastoma) and non-neuronal (COS-7, monkey kidney) cell lines with HD exon 1 (containing either 21 or 72 CAG repeats) construct DNA and either full length wild type huntingtin or pFLAG (control vector).

    Results: Full length wild type huntingtin significantly reduced cell death resulting from the mutant HD exon 1 fragments containing 72 CAG repeats in both cell lines. Wild type huntingtin did not significantly modulate cell death caused by transfection of HD exon 1 fragments containing 21 CAG repeats in either cell line.

    Conclusions: Our results suggest that wild type huntingtin can significantly reduce the cellular toxicity of mutant HD exon 1 fragments in both neuronal and non-neuronal cell lines. This suggests that wild type huntingtin can be protective in different cell types and that it can act against the toxicity caused by a mutant huntingtin fragment as well as against a full length transgene.

    Journal of medical genetics 2001;38;7;450-2

  • Tissue transglutaminase selectively modifies proteins associated with truncated mutant huntingtin in intact cells.

    Chun W, Lesort M, Tucholski J, Faber PW, MacDonald ME, Ross CA and Johnson GV

    Department of Psychiatry and Behavioral Neurobiology, University of Alabama at Birmingham, Birmingham, Alabama 35294-0017, USA.

    The cause of Huntington's disease (HD) is a pathological expansion of the polyglutamine domain within the N-terminal region of huntingtin. Neuronal intranuclear inclusions and cytoplasmic aggregates composed of the mutant huntingtin within certain neuronal populations are a characteristic hallmark of HD. However, how the expanded polyglutamine repeats of mutant huntingtin cause HD is not known. Because in vitro expanded polyglutamine repeats are excellent glutaminyl-donor substrates of tissue transglutaminase (tTG), it has been hypothesized that tTG may contribute to the formation of these aggregates in HD. However, an association between huntingtin and tTG or modification of huntingtin by tTG has not been demonstrated in cells. To examine the interactions between tTG and huntingtin human neuroblastoma SH-SY5Y cells were stably transfected with full-length huntingtin containing 23 (FL-Q23) (wild type) or 82 (FL-Q82) (mutant) glutamine repeats or a truncated N-terminal huntingtin construct containing 23 (Q23) (wild type) or 62 (Q62) (mutant) glutamine repeats. Aggregates were rarely observed in the cells expressing full-length mutant huntingtin, and no specific colocalization of full-length huntingtin and tTG was observed. In contrast, in cells expressing truncated mutant huntingtin (Q62) there were numerous complexes of truncated mutant huntingtin and many of these complexes co-localized with tTG. However, the complexes were not insoluble structures. Further, truncated huntingtin coimmunoprecipitated with tTG, and this association increased when tTG was activated. Activation of tTG did not result in the modification of either truncated or full-length huntingtin, however proteins that were associated with truncated mutant huntingtin were selectively modified by tTG. This study is the first to demonstrate that tTG specifically interacts with a truncated form of huntingtin, and that activated tTG selectively modifies mutant huntingtin-associated proteins. These data suggest that proteolysis of full-length mutant huntingtin likely precedes its interaction with tTG and this process may facilitate the modification of huntingtin-associated proteins and thus contribute to the etiology of HD.

    Funded by: NIA NIH HHS: AG12396

    Neurobiology of disease 2001;8;3;391-404

  • Interference by huntingtin and atrophin-1 with cbp-mediated transcription leading to cellular toxicity.

    Nucifora FC, Sasaki M, Peters MF, Huang H, Cooper JK, Yamada M, Takahashi H, Tsuji S, Troncoso J, Dawson VL, Dawson TM and Ross CA

    Division of Neurobiology, Department of Psychiatry, The Johns Hopkins University School of Medicine, Baltimore, MD 21205-2196, USA.

    Expanded polyglutamine repeats have been proposed to cause neuronal degeneration in Huntington's disease (HD) and related disorders, through abnormal interactions with other proteins containing short polyglutamine tracts such as the transcriptional coactivator CREB binding protein, CBP. We found that CBP was depleted from its normal nuclear location and was present in polyglutamine aggregates in HD cell culture models, HD transgenic mice, and human HD postmortem brain. Expanded polyglutamine repeats specifically interfere with CBP-activated gene transcription, and overexpression of CBP rescued polyglutamine-induced neuronal toxicity. Thus, polyglutamine-mediated interference with CBP-regulated gene transcription may constitute a genetic gain of function, underlying the pathogenesis of polyglutamine disorders.

    Funded by: NINDS NIH HHS: NS16375, NS34172, NS37090, NS38144

    Science (New York, N.Y.) 2001;291;5512;2423-8

  • Trinucleotide repeat analysis of Huntington's disease gene in Singapore.

    Law HY, Ng IS, Yoon CS, Zhao Y and Wong MC

    DNA Diagnostic and Research Laboratory, Genetics Service, Department of Paediatric Medicine, KK Women's and Children's Hospital, 100 Bukit Timah Road, Singapore 229899. hylaw@kkh.com.sg

    Introduction: Huntington's disease (HD) is an inherited neurodegenerative disorder characterised by chorea and progressive dementia. The mutation causing the disease has been identified as an unstable expansion of a trinucleotide (CAG)n. We have assessed the (CAG)n repeats in the patients and controls in our population.

    Polymerase chain reactions (PCRs) for the repeat region were carried out for 116 individuals: 10 were asymptomatic at-risk members from 5 families; 53 symptomatic patients from various hospitals; and 53 normal unrelated Singaporeans. Estimation of the number of repeats was based on Metaphor gel electrophoresis, sizing using the GeneScan on ABI 310 Genetic Analyzer, and sequencing using the same equipment.

    Results: Metaphor gel sizing generally gives an over-estimation, and GeneScan gives an under-estimation of repeat numbers compared with sequencing which is the gold standard. Of the 63 patients and family members tested, 25 had one expanded allele of 40 to 54 CAG repeats and the other allele in the normal range of 15 to 30 repeats. One patient had an allele in the intermediate range (38).

    Conclusion: The range of CAG repeats in the normal and HD alleles in our population is similar to those reported elsewhere. An accurate sizing can only be obtained with sequencing. For allele sizes in the intermediate range (37-40), sequencing should be carried out to confirm the carrier status of a patient.

    Annals of the Academy of Medicine, Singapore 2001;30;2;122-7

  • The Gln-Ala repeat transcriptional activator CA150 interacts with huntingtin: neuropathologic and genetic evidence for a role in Huntington's disease pathogenesis.

    Holbert S, Denghien I, Kiechle T, Rosenblatt A, Wellington C, Hayden MR, Margolis RL, Ross CA, Dausset J, Ferrante RJ and Néri C

    Laboratory of Genomic Biology, Fondation Jean Dausset, Centre d'Etude du Polymorphisme Humain, 75010 Paris, France.

    Huntington's disease (HD) is a neurodegenerative disease caused by polyglutamine expansion in the protein huntingtin (htt). Pathogenesis in HD appears to involve the formation of ubiquitinated neuronal intranuclear inclusions containing N-terminal mutated htt, abnormal protein interactions, and the aggregate sequestration of a variety of proteins (noticeably, transcription factors). To identify novel htt-interacting proteins in a simple model system, we used a yeast two-hybrid screen with a Caenorhabditis elegans activation domain library. We found a predicted WW domain protein (ZK1127.9) that interacts with N-terminal fragments of htt in two-hybrid tests. A human homologue of ZK1127.9 is CA150, a transcriptional coactivator with a N-terminal insertion that contains an imperfect (Gln-Ala)(38) tract encoded by a polymorphic repeat DNA. CA150 interacted in vitro with full-length htt from lymphoblastoid cells. The expression of CA150, measured immunohistochemically, was markedly increased in human HD brain tissue compared with normal age-matched human brain tissue, and CA150 showed aggregate formation with partial colocalization to ubiquitin-positive aggregates. In 432 HD patients, the CA150 repeat length explains a small, but statistically significant, amount of the variability in the onset age. Our data suggest that abnormal expression of CA150, mediated by interaction with polyglutamine-expanded htt, may alter transcription and have a role in HD pathogenesis.

    Funded by: NIA NIH HHS: AG13846, P01 AG012992, P30 AG013846; NINDS NIH HHS: NS35255, NS37102

    Proceedings of the National Academy of Sciences of the United States of America 2001;98;4;1811-6

  • Isolation of a 40-kDa Huntingtin-associated protein.

    Peters MF and Ross CA

    Division of Neurobiology, Department of Psychiatry, Department of Neuroscience, and The Program in Cellular and Molecular Medicine, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205-2196, USA. mfpeters@jhmi.edu

    Huntington's disease is caused by an expanded CAG trinucleotide repeat coding for a polyglutamine stretch within the huntingtin protein. Currently, the function of normal huntingtin and the mechanism by which expanded huntingtin causes selective neurotoxicity remain unknown. Clues may come from the identification of huntingtin-associated proteins (HAPs). Here, we show that huntingtin copurifies with a single novel 40-kDa protein termed HAP40. HAP40 is encoded by the open reading frame factor VIII-associated gene A (F8A) located within intron 22 of the factor VIII gene. In transfected cell extracts, HAP40 coimmunoprecipitates with full-length huntingtin but not with an N-terminal huntingtin fragment. Recombinant HAP40 is cytoplasmic in the presence of huntingtin but is actively targeted to the nucleus in the absence of huntingtin. These data indicate that HAP40 is likely to contribute to the function of normal huntingtin and is a candidate for involvement in the aberrant nuclear localization of mutant huntingtin found in degenerating neurons in Huntington's disease.

    Funded by: NINDS NIH HHS: NS16375, NS34172, NS38144

    The Journal of biological chemistry 2001;276;5;3188-94

  • Modeling Huntington's disease in cells, flies, and mice.

    Sipione S and Cattaneo E

    Department of Pharmacological Sciences, University of Milano, Center of Excellence on Neurodegenerative Diseases, Italy.

    A milestone in Huntington's disease (HD) research is represented by the identification of the causative gene. With the genetics at hand, a series of transgenic cellular and animal models has been developed, which has greatly contributed to understanding of HD. All these models are described in this review, and are compared to each other, along with the information they have generated. Although the mechanism by which progressive loss of striatal neurons occurs in HD remains uncertain, hypotheses on mutant huntingtin toxicity involve impaired vescicular trafficking, transcriptional dysregulation, and/or activation of apoptotic pathways. The development of inducible HD mice has shown that neurodegeneration in HD may be at least partially blocked. Although traditionally considered a "gain-of-function" disease, the recent finding that normal huntingtin has an important role in neuronal survival suggests that loss of function of the normal protein might contribute to HD as well, also discloseing new perspectives on the therapeutical approach to the pathology.

    Molecular neurobiology 2001;23;1;21-51

  • Presymptomatic tests in Huntington's disease and dominant ataxias.

    Cannella M, Simonelli M, D'Alessio C, Pierelli F, Ruggieri S and Squitieri F

    Department of Molecular Pathology, IRCCS INM Neuromed, Pozzilli, Italy.

    Huntington's disease (HD) and dominant ataxias (SCA) represent neurodegenerative hereditary diseases dominantly transmitted for which a direct and accurate genetic test is now available for molecular confirmation and presymptomatic test. Predictive testing programs, according to published international guidelines, are available worldwide. A large number of subjects (n=165) required a predictive HD diagnosis, although only 36% completed the program flow-chart and received the final genetic result (26 had a positive, 34 negative result for mutation). In 4 cases, an allele of intermediate range (33-34 CAGs) was found. Two of these shared the intermediate allele with an expanded repeat. In this case, we estimated the patient's risk to have affected children over the usually reported 50%. In 4 cases, the presymptomatic diagnosis was requested by persons at-risk for SCA1 and SCA3/Machado-Joseph disease. There were no adverse events to results of both HD and SCA presymptomatic diagnoses.

    Neurological sciences : official journal of the Italian Neurological Society and of the Italian Society of Clinical Neurophysiology 2001;22;1;55-6

  • FIP-2, a coiled-coil protein, links Huntingtin to Rab8 and modulates cellular morphogenesis.

    Hattula K and Peränen J

    Institute of Biotechnology, Program in Cellular Biotechnology, P.O. Box 56 (Viikinkaari 9), FIN-00014, University of Helsinki, Finland.

    Huntington's disease is characterised by the death of cortical and striatal neurons, and is the result of an expanded polyglutamine tract in the Huntingtin protein [1]. Huntingtin is present on both endocytic and secretory membrane organelles but its function is unclear [2,3]. Rab GTPases regulate both of these transport pathways [4]. We have previously shown that Rab8 controls polarised membrane transport by modulating cell morphogenesis [5]. To understand Rab8-mediated processes, we searched for Rab8-interacting proteins by the yeast two-hybrid system. Here, we report that Huntingtin is linked to the Rab8 protein through FIP-2, a tumour necrosis factor-alpha (TNF-alpha)-inducible coiled-coil protein related to the NEMO protein [6,7]. The activated form of Rab8 interacted with the amino-terminal region of FIP-2, whereas dominant-negative Rab8 did not. Huntingtin bound to the carboxy-terminal region of FIP-2. Coexpressed FIP-2 and Huntingtin enhanced the recruitment of Huntingtin to Rab8-positive vesicular structures, and FIP-2 promoted cell polarisation in a similar way to Rab8. We propose a model in which Huntingtin, together with FIP-2 and Rab8, are part of a protein network that regulates membrane trafficking and cellular morphogenesis.

    Current biology : CB 2000;10;24;1603-6

  • Comparison of the number of triplets in SCA1, MJD/SCA3, HD, SBMA, DRPLA, MD, FRAXA and FRDA genes in schizophrenic patients and a healthy population.

    Culjković B, Stojković O, Savić D, Zamurović N, Nesić M, Major T, Keckarevi D, Romac S, Zamurovi B and Vukosavić S

    American journal of medical genetics 2000;96;6;884-7

  • Huntington disease: DNA analysis in Brazilian population.

    Raskin S, Allan N, Teive HA, Cardoso F, Haddad MS, Levi G, Boy R, Lerena Junior J, Sotomaior VS, Janzen-Dück M, Jardim LB, Fellander FR and Andrade LA

    Centro deAconselhamento e Laboratório Genetika-Curitiba, PR, Brazil.

    Huntington disease (HD) is associated with expansions of a CAG trinucleotide repeat in the HD gene. Accurate measurement of a specific CAG repeat sequence in the HD gene in 92 Brazilian controls without HD, 44 Brazilian subjects with clinical findings suggestive of HD and 40 individuals from 6 putative HD families, showed a range from 7 to 33 repeats in normal subjects and 39 to 88 repeats in affected subjects. A trend between early age at onset of first symptoms and increasing number of repeats was seen. Major increase of repeat size through paternal inheritance than through maternal inheritance was observed. Data generated from this study may have significant implications for the etiology, knowledge of the incidence, diagnosis, prognosis, genetic counseling and treatment of HD Brazilian patients.

    Arquivos de neuro-psiquiatria 2000;58;4;977-85

  • Polymorphisms in the CAG repeat--a source of error in Huntington disease DNA testing.

    Yu S, Fimmel A, Fung D and Trent RJ

    Department of Molecular and Clinical Genetics, Royal Prince Alfred Hospital, Camperdown NSW, Australia.

    Five of 400 patients (1.3%), referred for Huntington disease DNA testing, demonstrated a single allele on CAG alone, but two alleles when the CAG + CCG repeats were measured. The PCR assay failed to detect one allele in the CAG alone assay because of single-base silent polymorphisms in the penultimate or the last CAG repeat. The region around and within the CAG repeat sequence in the Huntington disease gene is a hot-spot for DNA polymorphisms, which can occur in up to 1% of subjects tested for Huntington disease. These polymorphisms may interfere with amplification by PCR, and so have the potential to produce a diagnostic error.

    Clinical genetics 2000;58;6;469-72

  • Huntingtin: an iron-regulated protein essential for normal nuclear and perinuclear organelles.

    Hilditch-Maguire P, Trettel F, Passani LA, Auerbach A, Persichetti F and MacDonald ME

    Molecular Neurogenetics Unit, Massachusetts General Hospital, Building 149, 13th Street, Charlestown, MA 02129, USA.

    Huntington's disease (HD), with its selective neuronal cell loss, is caused by an elongated glutamine tract in the huntingtin protein. To discover the pathways that are candidates for the protein's normal and/or abnormal function, we surveyed 19 classes of organelle in Hdh(ex4/5)/Hdh(ex4/5) knock-out compared with wild-type embryonic stem cells to identify any that might be affected by huntingtin deficiency. Although the majority did not differ, dramatic changes in six classes revealed that huntingtin's function is essential for the normal nuclear (nucleoli, transcription factor-speckles) and perinuclear membrane (mitochondria, endoplasmic reticulum, Golgi and recycling endosomes) organelles and for proper regulation of the iron pathway. Moreover, upmodulation by deferoxamine mesylate implicates huntingtin as an iron-response protein. However, excess huntingtin produced abnormal organelles that resemble the deficiency phenotype, suggesting the importance of huntingtin level to the protein's normal pathway. Thus, organelles that require huntingtin to function suggest roles for the protein in RNA biogenesis, trafficking and iron homeostasis to be explored in HD pathogenesis.

    Funded by: NINDS NIH HHS: NS16367, NS32765; Telethon: E.0087

    Human molecular genetics 2000;9;19;2789-97

  • HIP12 is a non-proapoptotic member of a gene family including HIP1, an interacting protein with huntingtin.

    Chopra VS, Metzler M, Rasper DM, Engqvist-Goldstein AE, Singaraja R, Gan L, Fichter KM, McCutcheon K, Drubin D, Nicholson DW and Hayden MR

    Department of Medical Genetics, and Centre for Molecular Medicine and Therapeutics, University of British Columbia, Vancouver, Canada.

    Huntingtin-interacting protein I (HIP1) is a membrane-associated protein that interacts with huntingtin, the protein altered in Huntington disease. HIP1 shows homology to Sla2p, a protein essential for the assembly and function of the cytoskeleton and endocytosis in Saccharomyces cerevisiae. We have determined that the HIP1 gene comprises 32 exons spanning approximately 215 kb of genomic DNA and gives rise to two alternate splice forms termed HIP1-1 and HIP1-2. Additionally, we have identified a novel protein termed HIP12 with significant sequence and biochemical similarities to HIP1 and high sequence similarity to Sla2p. HIP12 differs from HIP1 in its pattern of expression both at the mRNA and protein level. However, HIP1 and HIP12 are both found within the brain and show a similar subcellular distribution pattern. In contrast to HIP1, which is toxic in cell culture, HIP12 does not confer toxicity in the same assay systems. Interestingly, HIP12 does not interact with huntingtin but can interact with HIP1. suggesting a potential interaction in vivo that may influence the function of each respective protein.

    Mammalian genome : official journal of the International Mammalian Genome Society 2000;11;11;1006-15

  • Identification and characterization of the miniature pig Huntington's disease gene homolog: evidence for conservation and polymorphism in the CAG triplet repeat.

    Matsuyama N, Hadano S, Onoe K, Osuga H, Showguchi-Miyata J, Gondo Y and Ikeda JE

    Department of Neurobiology, SLA Research, Inc., Bohseidai, Isehara, Kanagawa, Japan.

    Huntington's disease (HD) is associated with a significant expansion of a CAG trinucleotide repeat, which results in a lengthened polyglutamine tract in the single gene product, huntingtin, on human 4p16.3. We isolated cDNA clones that encompassed the entire coding sequence of the miniature pig HD gene (Sus HD) from two porcine testis cDNA libraries. The cDNA contig revealed a 12,749-nucleotide transcript coding for a 345-kDa protein (3139 amino acid residues), which exhibited 96% peptide sequence homology to human huntingtin. Northern blot analysis revealed that the Sus HD gene was ubiquitously expressed as two large transcripts of approximately 11 and 13 kb in size in all the tested tissues, much like the human HD gene. The CAG trinucleotide repeat was found to be interrupted by CAA triplets and to encode 17 or 18 consecutive glutamine residues. In our laboratory stock of miniature pig, three allotypes in the triplet repeat sequence were found. Thus, the Sus HD gene closely resembles its human counterpart in terms of sequence and expression pattern. In particular, human-miniature pig similarities in the normal length of the CAG triplet repeat as well as its repeat-number polymorphism may indicate that miniature pig would provide a good animal model for Huntington's disease.

    Genomics 2000;69;1;72-85

  • Huntingtin's WW domain partners in Huntington's disease post-mortem brain fulfill genetic criteria for direct involvement in Huntington's disease pathogenesis.

    Passani LA, Bedford MT, Faber PW, McGinnis KM, Sharp AH, Gusella JF, Vonsattel JP and MacDonald ME

    Molecular Neurogenetics Unit, Massachusetts General Hospital, Building 149, 13th Street, Charlestown, MA 02129, USA.

    An elongated glutamine tract in mutant huntingtin initiates Huntington's disease (HD) pathogenesis via a novel structural property that displays neuronal selectivity, glutamine progressivity and dominance over the normal protein based on genetic criteria. As this mechanism is likely to involve a deleterious protein interaction, we have assessed the major class of huntingtin interactors comprising three WW domain proteins. These are revealed to be related spliceosome proteins (HYPA/FBP-11 and HYPC) and a transcription factor (HYPB) that implicate huntingtin in mRNA biogenesis. In HD post-mortem brain, specific antibody reagents detect each partner in HD target neurons, in association with disease-related N-terminal morphologic deposits but not with filter trapped insoluble-aggregate. Glutathione S:-transferase partner 'pull-down' assays reveal soluble, aberrantly migrating, forms of full-length mutant huntingtin specific to HD target tissue. Importantly, these novel mutant species exhibit exaggerated WW domain binding that abrogates partner association with other huntingtin isoforms. Thus, each WW domain partner's association with huntingtin fulfills HD genetic criteria, supporting a direct role in pathogenesis. Our findings indicate that modification of mutant huntingtin in target neurons may promote an abnormal interaction with one, or all, of huntingtin's WW domain partners, perhaps altering ribonucleoprotein function with toxic consequences.

    Funded by: NIMH NIH HHS: MH/NS 31862; NINDS NIH HHS: NS16367, NS32765

    Human molecular genetics 2000;9;14;2175-82

  • Human huntingtin-associated protein (HAP-1) gene: genomic organisation and an intragenic polymorphism.

    Nasir J, Lafuente MJ, Duan K, Colomer V, Engelender S, Ingersoll R, Margolis RL, Ross CA and Hayden MR

    Human Genetics Unit, Molecular Medicine Centre, Western General Hospital, EH4 2XU, Edinburgh, UK. j.nasir@ed.ac.uk

    The huntingtin-associated protein (HAP-1) interacts with the Huntington disease gene product, huntingtin. It is predominantly expressed in the brain and shows an increased affinity for mutant huntingtin. We have sequenced an 18,656bp genomic region encompassing the entire human HAP-1 gene and determined its genomic organisation, with 11 exons spanning 12.1kb. We have also found an intragenic polymorphism within intron 6 of HAP-1. We have recently shown that HAP-1 maps to a region of the genome which has been implicated in a variety of neurological conditions, including progressive supranuclear palsy (PSP), a late-onset atypical parkinsonian disorder. The detailed characterisation of the genomic organisation of HAP-1 and the presence of an intragenic polymorphism will be helpful in evaluating its role in different disorders, using candidate gene approaches.

    Funded by: NINDS NIH HHS: NS16375

    Gene 2000;254;1-2;181-7

  • Prevalence of Huntington disease in New South Wales in 1996.

    McCusker EA, Casse RF, Graham SJ, Williams DB and Lazarus R

    Neurology Department, Westmead Hospital, Sydney, NSW. elizabeth_mccusker@mail.wmi.usyd.edu.au

    Objective: To estimate the prevalence of Huntington disease (HD) in New South Wales on Australian Census Day (6 August) 1996.

    Design: Survey of records of the Huntington Disease Service and major hospitals, and of neurologists, psychiatrists, clinical geneticists and genetic counsellors.

    All patients in NSW who, on Census Day 1996, either had a definite diagnosis of HD (motor signs of chorea or ataxia and family history of HD or positive DNA test result) or would have had signs and later received a definite diagnosis (assessed 1 April 1997 to 1 July 1999).

    Prevalence (HD patients per 100,000 population); patient characteristics; year and basis of diagnosis.

    Results: 380 patients with definite HD were identified, giving a prevalence of HD in NSW in 1996 of 6.29 per 100,000 population (95% CI, 5.68-6.96). A third of HD patients were aged 60 years or older. Diagnosis was confirmed by DNA testing for 171 patients (45%), including 30 (8%) with no recorded family history. Average numbers of new diagnoses per year were 11.8 (1984-1988), 21.8 (1989-1993) and 28.6 (1994-1998). Estimated number of people with a 50% risk of inheriting the HD mutation was 25.2 per 100,000 population. Estimated incidence of HD in 1996 was 0.65 per 100,000 population.

    Conclusions: Prevalence of HD in NSW is similar to estimated prevalence in other Australian and Western populations. Increasing numbers of cases are being diagnosed, and the 18 chronic care beds currently designated for HD patients in NSW are unlikely to be sufficient.

    The Medical journal of Australia 2000;173;4;187-90

  • Inhibiting caspase cleavage of huntingtin reduces toxicity and aggregate formation in neuronal and nonneuronal cells.

    Wellington CL, Singaraja R, Ellerby L, Savill J, Roy S, Leavitt B, Cattaneo E, Hackam A, Sharp A, Thornberry N, Nicholson DW, Bredesen DE and Hayden MR

    Centre for Molecular Medicine and Therapeutics, University of British Columbia, Vancouver, British Columbia V5Z 4H4, Canada.

    Huntington's disease is a neurodegenerative disorder caused by CAG expansion that results in expansion of a polyglutamine tract at the extreme N terminus of huntingtin (htt). htt with polyglutamine expansion is proapoptotic in different cell types. Here, we show that caspase inhibitors diminish the toxicity of htt. Additionally, we define htt itself as an important caspase substrate by generating a site-directed htt mutant that is resistant to caspase-3 cleavage at positions 513 and 530 and to caspase-6 cleavage at position 586. In contrast to cleavable htt, caspase-resistant htt with an expanded polyglutamine tract has reduced toxicity in apoptotically stressed neuronal and nonneuronal cells and forms aggregates at a much reduced frequency. These results suggest that inhibiting caspase cleavage of htt may therefore be of potential therapeutic benefit in Huntington's disease.

    Funded by: NCI NIH HHS: CA69381; NIA NIH HHS: AG12282; NINDS NIH HHS: NS40251A

    The Journal of biological chemistry 2000;275;26;19831-8

  • Activation of MLK2-mediated signaling cascades by polyglutamine-expanded huntingtin.

    Liu YF, Dorow D and Marshall J

    Department of Pharmaceutical Sciences, Northeastern University, Boston, Massachusetts 02115, USA. yafliu@lynx.neu.edu

    We previously reported that expression of polyglutamine-expanded huntingtin induces apoptosis via c-Jun amino-terminal kinase (JNK) activation in HN33 cells (Liu, Y. F. (1998) J. Biol. Chem. 273, 28873-28822). Extending this study, we now demonstrate a role of mixed-lineage kinase 2 (MLK2), a JNK activator, in polyglutamine-expanded huntingtin-mediated neuronal toxicity. We find that normal huntingtin interacts with MLK2, whereas the polyglutamine expansion interferes with this interaction. Similar to the expression of polyglutamine-expanded huntingtin, expression of MLK2 also induces JNK activation and apoptosis in HN33 cells. Co-expression of dominant negative MLK2 significantly attenuates neuronal apoptosis induced by the mutated huntingtin. Furthermore, over-expression of the N terminus of normal huntingtin partially rescues the neuronal toxicity induced by MLK2. Our results suggest that activation of MLK2-mediated signaling cascades may be partially involved in neuronal death induced by polyglutamine-expanded huntingtin.

    The Journal of biological chemistry 2000;275;25;19035-40

  • The Huntington's disease protein interacts with p53 and CREB-binding protein and represses transcription.

    Steffan JS, Kazantsev A, Spasic-Boskovic O, Greenwald M, Zhu YZ, Gohler H, Wanker EE, Bates GP, Housman DE and Thompson LM

    Department of Biological Chemistry, D240 Medical Sciences I, University of California, Irvine, CA 92697-1700, USA.

    Huntington's Disease (HD) is caused by an expansion of a polyglutamine tract within the huntingtin (htt) protein. Pathogenesis in HD appears to include the cytoplasmic cleavage of htt and release of an amino-terminal fragment capable of nuclear localization. We have investigated potential consequences to nuclear function of a pathogenic amino-terminal region of htt (httex1p) including aggregation, protein-protein interactions, and transcription. httex1p was found to coaggregate with p53 in inclusions generated in cell culture and to interact with p53 in vitro and in cell culture. Expanded httex1p represses transcription of the p53-regulated promoters, p21(WAF1/CIP1) and MDR-1. httex1p was also found to interact in vitro with CREB-binding protein (CBP) and mSin3a, and CBP to localize to neuronal intranuclear inclusions in a transgenic mouse model of HD. These results raise the possibility that expanded repeat htt causes aberrant transcriptional regulation through its interaction with cellular transcription factors which may result in neuronal dysfunction and cell death in HD.

    Funded by: NCI NIH HHS: P01 CA042063, P01-CA42063

    Proceedings of the National Academy of Sciences of the United States of America 2000;97;12;6763-8

  • Aberrant interactions of transcriptional repressor proteins with the Huntington's disease gene product, huntingtin.

    Boutell JM, Thomas P, Neal JW, Weston VJ, Duce J, Harper PS and Jones AL

    Institute of Medical Genetics, University of Wales College of Medicine, Cardiff, UK.

    We detected an interaction of the N-terminus of huntingtin (htt171) with the C-terminal region of the nuclear receptor co-repressor (N-CoR) using the yeast two-hybrid system. This interaction was repeat length dependent and specific to htt171; the co-repressor did not interact with the repeat carrying a section of atrophin 1 nor with the androgen receptor or polyglutamine alone. The interaction was confirmed using His-tagged Escherichia coli -expressed C-terminal human and rat co-repressor protein which pulled full-length huntingtin out of homogenized rat brain and in pull-down assays. The N-CoR represses transcription from sequence-specific ligand-activated receptors such as the retinoid X-thyroid hormone receptor dimers and other nuclear receptors including Mad-Max receptor dimers. The mechanism of this repression appears to be through the formation of a complex of repressor proteins including the N-CoR, mSin3 and histone deacetylases. We have used N-CoR and mSin3A antibodies in immunohistochemical studies and find that in Huntington's disease (HD) cortex and caudate, the cellular localization of these proteins is exclusively cytoplasmic whilst in control brain they are localized in the nucleus as well as the cytoplasm; mSin3A immunoreactivity also occurred in a subset of huntingtin positive intranuclear inclusions. The relocalization of repressor proteins in HD brain may alter transcription and be involved in the pathology of the disease.

    Funded by: Medical Research Council: G9810900

    Human molecular genetics 1999;8;9;1647-55

  • The localization and interactions of huntingtin.

    Jones AL

    Institute of Medical Genetics, University of Wales College of Medicine, Cardiff, UK. jonesl1@cf.ac.uk

    Huntingtin was localized by using a series of antibodies that detected different areas of the protein from the immediate N-terminus to the C-terminal region of the protein. The more C-terminal antibodies gave a cytoplasmic localization in neurons of the brain in controls and cases of Huntington's disease (HD). The N-terminal antibody, however, gave a distinctive pattern of immunoreactivity in the HD brain, with marked staining of axon tracts and white matter and the detection of densely staining intranuclear inclusions. This implies some processing differences between mutated and normal huntingtin. We have also localized two interacting proteins, cystathionine beta-synthase and the nuclear receptor co-repressor (N-CoR), in brain. Cystathionine beta-synthase was not relocalized in HD brain, but the N-CoR was excluded from neuronal nuclei in HD brain, and a further protein that exists in the same repression complex, mSin3, was similarly excluded. We conclude that the co-repressor might have a part in HD pathology.

    Philosophical transactions of the Royal Society of London. Series B, Biological sciences 1999;354;1386;1021-7

  • PQBP-1, a novel polyglutamine tract-binding protein, inhibits transcription activation by Brn-2 and affects cell survival.

    Waragai M, Lammers CH, Takeuchi S, Imafuku I, Udagawa Y, Kanazawa I, Kawabata M, Mouradian MM and Okazawa H

    Group of Molecular Neurobiology, Department of Neurology, Graduate School of Medicine, University of Tokyo, Japan.

    A novel gene, designated PQBP-1, which encodes a 265 residue protein that binds to the polyglutamine tract of the brain-specific transcription factor Brn-2, was identified. PQBP-1, which also interacts with the polyglutamine tract of triplet repeat disease gene products, binds with a higher affinity to an expanded polyglutamine tract. PQBP-1 has several functional domains, including hepta- and di-amino acid repeat sequences rich in polar residues essential for its interaction with the polyglutamine tract, a WWP/WW domain which binds to proline-rich motifs in other proteins, a putative nuclear localization signal sequence and a C2domain implicated in Ca2+-dependent phospholipid signaling. PQBP-1 is located in the nucleus and inhibits transcriptional activation by Brn-2. Overexpression of PQBP-1 in P19 embryonic carcinoma cells suppresses their growth rate and enhances their susceptibility to various stresses including serum deprivation, retinoic acid treatment and UV irradiation. Northern blot and in situ hybridization analyses revealed that PQBP-1 is a ubiquitous protein and is expressed primarily in neurons throughout the brain, with abundant levels in hippocampus, cerebellar cortex and olfactory bulb. These results suggest that PQBP-1 mediates important cellular functions under physiological and pathological conditions via its interaction with polyglutamine tracts.

    Human molecular genetics 1999;8;6;977-87

  • Inhibition of caspase-1 slows disease progression in a mouse model of Huntington's disease.

    Ona VO, Li M, Vonsattel JP, Andrews LJ, Khan SQ, Chung WM, Frey AS, Menon AS, Li XJ, Stieg PE, Yuan J, Penney JB, Young AB, Cha JH and Friedlander RM

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

    Huntington's disease is an autosomal-dominant progressive neurodegenerative disorder resulting in specific neuronal loss and dysfunction in the striatum and cortex. The disease is universally fatal, with a mean survival following onset of 15-20 years and, at present, there is no effective treatment. The mutation in patients with Huntington's disease is an expanded CAG/polyglutamine repeat in huntingtin, a protein of unknown function with a relative molecular mass of 350,000 (M(r) 350K). The length of the CAG/polyglutamine repeat is inversely correlated with the age of disease onset. The molecular pathways mediating the neuropathology of Huntington's disease are poorly understood. Transgenic mice expressing exon 1 of the human huntingtin gene with an expanded CAG/polyglutamine repeat develop a progressive syndrome with many of the characteristics of human Huntington's disease. Here we demonstrate evidence of caspase-1 activation in the brains of mice and humans with the disease. In this transgenic mouse model of Huntington's disease, expression of a dominant-negative caspase-1 mutant extends survival and delays the appearance of neuronal inclusions, neurotransmitter receptor alterations and onset of symptoms, indicating that caspase-1 is important in the pathogenesis of the disease. In addition, we demonstrate that intracerebroventricular administration of a caspase inhibitor delays disease progression and mortality in the mouse model of Huntington's disease.

    Nature 1999;399;6733;263-7

  • Huntingtin acts in the nucleus to induce apoptosis but death does not correlate with the formation of intranuclear inclusions.

    Saudou F, Finkbeiner S, Devys D and Greenberg ME

    Department of Neurology, Children's Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA.

    The mechanisms by which mutant huntingtin induces neurodegeneration were investigated using a cellular model that recapitulates features of neurodegeneration seen in Huntington's disease. When transfected into cultured striatal neurons, mutant huntingtin induces neurodegeneration by an apoptotic mechanism. Antiapoptotic compounds or neurotrophic factors protected neurons against mutant huntingtin. Blocking nuclear localization of mutant huntingtin suppressed its ability to form intranuclear inclusions and to induce neurodegeneration. However, the presence of inclusions did not correlate with huntingtin-induced death. The exposure of mutant huntingtin-transfected striatal neurons to conditions that suppress the formation of inclusions resulted in an increase in mutant huntingtin-induced death. These findings suggest that mutant huntingtin acts within the nucleus to induce neurodegeneration. However, intranuclear inclusions may reflect a cellular mechanism to protect against huntingtin-induced cell death.

    Funded by: NICHD NIH HHS: P30-HD 18655; NINDS NIH HHS: NS 28829

    Cell 1998;95;1;55-66

  • SH3GL3 associates with the Huntingtin exon 1 protein and promotes the formation of polygln-containing protein aggregates.

    Sittler A, Wälter S, Wedemeyer N, Hasenbank R, Scherzinger E, Eickhoff H, Bates GP, Lehrach H and Wanker EE

    Max-Planck-Institut für Molekulare Genetik, Berlin, Germany.

    The mechanism by which aggregated polygins cause the selective neurodegeneration in Huntington's disease (HD) is unknown. Here, we show that the SH3GL3 protein, which is preferentially expressed in brain and testis, selectively interacts with the HD exon 1 protein (HDex1p) containing a glutamine repeat in the pathological range and promotes the formation of insoluble polyglutamine-containing aggregates in vivo. The C-terminal SH3 domain in SH3GL3 and the proline-rich region in HDex1p are essential for the interaction. Coimmunoprecipitations and immunofluorescence studies revealed that SH3GL3 and HDex1p colocalize in transfected COS cells. Additionally, an anti-SH3GL3 antibody was also able to coimmunoprecipitate the full-length huntingtin from an HD human brain extract. The characteristics of the interaction between SH3GL3 and huntingtin and the colocalization of the two proteins suggest that SH3GL3 could be involved in the selective neuronal cell death in HD.

    Molecular cell 1998;2;4;427-36

  • Huntingtin interacts with a family of WW domain proteins.

    Faber PW, Barnes GT, Srinidhi J, Chen J, Gusella JF and MacDonald ME

    Molecular Neurogenetics Unit, Massachusetts General Hospital East, Building 149, 13th Street, Charlestown, MA 02129, USA.

    The hallmark neuropathology of Huntington's disease (HD) is due to elongation of a polyglutamine segment in huntingtin, a novel approximately 350 kDa protein of unknown function. We used a yeast two-hybrid interactor screen to identify proteins whose association with huntingtin might be altered in the pathogenic process. Surprisingly, no interactors were found with internal and C-terminal segments of huntingtin. In contrast, huntingtin's N-terminus detected 13 distinct proteins, seven novel and six reported previously. Among these, we identified a major interactor class, comprising three distinct WW domain proteins, HYPA, HYPB and HYPC, that bind normal and mutant huntingtin in extracts of HD lymphoblastoid cells. This interaction is mediated by huntingtin's proline-rich region and is enhanced by lengthening the adjacent glutamine tract. Although HYPB and HYPC are novel, HYPA is human FBP-11, a protein implicated in spliceosome function. The emergence of this class of proteins as huntingtin partners argues that a WW domain-mediated process, such as non-receptor signaling, protein degradation or pre-mRNA splicing, may participate in HD pathogenesis.

    Funded by: NINDS NIH HHS: NS16367, NS32765

    Human molecular genetics 1998;7;9;1463-74

  • A human HAP1 homologue. Cloning, expression, and interaction with huntingtin.

    Li SH, Hosseini SH, Gutekunst CA, Hersch SM, Ferrante RJ and Li XJ

    Department of Genetics, Emory University School of Medicine, Atlanta, Georgia 30322, USA.

    Huntington's disease (HD) is caused by the expansion of a glutamine repeat in the protein huntingtin. The expanded glutamine repeat is thought to mediate a gain of function by causing huntingtin to abnormally interact with other proteins. We previously identified a rat huntingtin-associated protein (HAP1) that binds to huntingtin; HAP1 binds more tightly to huntingtin with an expanded glutamine repeat than to wild type huntingtin. Identification of the human homologue of HAP1 is necessary for investigation of the potential role of HAP1 in HD pathology. Here, we report the cloning of a human HAP1 homologue (hHAP) that shares 62% identity with rat HAP1 over its entire sequence and 82% amino acid identity in the putative huntingtin-binding region. The hHAP gene encodes a 4.1-kilobase transcript and a 75-kDa protein which are specifically expressed in human brain tissues. Its expression in Huntington's disease brains is reduced in parallel with a decreased expression of huntingtin. While two isoforms of rat HAP1 are expressed at similar levels in rat brain, only a single major form of hHAP is found in primate brains. In vitro binding, immunoprecipitation, and coexpression studies confirm the interaction of hHAP with huntingtin. The in vitro binding of hHAP to huntingtin is enhanced by lengthening the glutamine repeat. Despite similar binding properties of rat HAP1 and hHAP, differences in the sequences and expression of hHAP may contribute to a specific role for its interaction with huntingtin in humans.

    Funded by: NINDS NIH HHS: NS 16375, NS36232

    The Journal of biological chemistry 1998;273;30;19220-7

  • Amyloid formation by mutant huntingtin: threshold, progressivity and recruitment of normal polyglutamine proteins.

    Huang CC, Faber PW, Persichetti F, Mittal V, Vonsattel JP, MacDonald ME and Gusella JF

    Molecular Neurogenetics Unit, Massachusetts General Hospital, Charlestown 02129, USA.

    Huntington's disease (HD) is caused by an expanded CAG trinucleotide repeat encoding a tract of consecutive glutamines near the amino terminus of huntingtin, a large protein of unknown function. It has been proposed that the expanded polyglutamine stretch confers a new property on huntingtin and thereby causes cell and region-specific neurodegeneration. Genotype-phenotype correlations predict that this novel property appears above a threshold length (approximately 38 glutamines), becomes progressively more evident with increasing polyglutamine length, is completely dominant over normal huntingtin and is not appreciably worsened by a double genetic dose in HD homozygotes. Recently, an amino terminal fragment of mutant huntingtin has been found to form self-initiated fibrillar aggregates in vitro. We have tested the capacity for aggregation to assess whether this property matches the criteria expected for a fundamental role in HD pathogenesis. We find that that in vitro aggregation displays a threshold and progressivity for polyglutamine length remarkably similar to the HD disease process. Moreover, the mutant huntingtin amino terminus is capable of recruiting into aggregates normal glutamine tract proteins, such as the amino terminal segments of both normal huntingtin and of TATA-binding protein (TBP). Our examination of in vivo aggregates from HD post-mortem brains indicates that they contain an amino terminal segment of huntingtin of between 179 and 595 residues. They also contain non-huntingtin protein, as evidenced by immunostaining for TBP. Interestingly, like the in vitro aggregates, aggregates from HD brain display Congo red staining with green birefringence characteristic of amyloid. Our data support the view that the expanded polyglutamine segment confers on huntingtin a new property that plays a determining role in HD pathogenesis and could be a target for treatment. Moreover, the new property might have its toxic consequences by interaction with one or more normal polyglutamine-containing proteins essential for the survival of target neurons.

    Funded by: NIMH NIH HHS: MH/NS31862; NINDS NIH HHS: NS16367, NS32765

    Somatic cell and molecular genetics 1998;24;4;217-33

  • HAP1-huntingtin interactions do not contribute to the molecular pathology in Huntington's disease transgenic mice.

    Bertaux F, Sharp AH, Ross CA, Lehrach H, Bates GP and Wanker E

    Division of Medical and Molecular Genetics, UMDS, Guy's Hospital, London, UK.

    HAP1 (huntingtin associated protein) has previously been found to interact with huntingtin (htt) in a glutamine length dependent manner and has been proposed to play a role in the cell specific neurodegeneration observed in Huntington's disease (HD). We have isolated mouse HAP1 (hap1) and have shown that expression is not enriched in areas specifically affected in HD. We have used the yeast two hybrid system to demonstrate that htt amino acids 171-230 are necessary for the hap1-htt binding and that hapl does not interact with the transgene exon 1 protein in a transgenic model of HD.

    Funded by: NINDS NIH HHS: NS16375; Wellcome Trust

    FEBS letters 1998;426;2;229-32

  • Caspase cleavage of gene products associated with triplet expansion disorders generates truncated fragments containing the polyglutamine tract.

    Wellington CL, Ellerby LM, Hackam AS, Margolis RL, Trifiro MA, Singaraja R, McCutcheon K, Salvesen GS, Propp SS, Bromm M, Rowland KJ, Zhang T, Rasper D, Roy S, Thornberry N, Pinsky L, Kakizuka A, Ross CA, Nicholson DW, Bredesen DE and Hayden MR

    Centre for Molecular Medicine and Therapeutics and Department of Medical Genetics, University of British Columbia, Vancouver, British Columbia V6T 1Z4, Canada.

    The neurodegenerative diseases Huntington disease, dentatorubropallidoluysian atrophy, spinocerebellar atrophy type 3, and spinal bulbar muscular atrophy are caused by expansion of a polyglutamine tract within their respective gene products. There is increasing evidence that generation of truncated proteins containing an expanded polyglutamine tract may be a key step in the pathogenesis of these disorders. We now report that, similar to huntingtin, atrophin-1, ataxin-3, and the androgen receptor are cleaved in apoptotic extracts. Furthermore, each of these proteins is cleaved by one or more purified caspases, cysteine proteases involved in apoptotic death. The CAG length does not modulate susceptibility to cleavage of any of the full-length proteins. Our results suggest that by generation of truncated polyglutamine-containing proteins, caspase cleavage may represent a common step in the pathogenesis of each of these neurodegenerative diseases.

    Funded by: NIA NIH HHS: AG12282; NIMH NIH HHS: MH01275-01A1; NINDS NIH HHS: NS34172; ...

    The Journal of biological chemistry 1998;273;15;9158-67

  • Huntingtin interacts with cystathionine beta-synthase.

    Boutell JM, Wood JD, Harper PS and Jones AL

    Institute of Medical Genetics, University of Wales College of Medicine, Cardiff CF4 4XN, UK.

    We have screened a rat brain library to identify proteins which interact with the 5'-end of huntingtin (amino acids 1-171), including the polyglutamine tract, in the yeast two-hybrid system. We detected an interaction with cystathionine beta-synthase (CBS) [L-serine hydrolyase (adding homocysteine), EC], which was confirmed in vitro using His-tagged CBS expressed in Escherichia coli , which was able to specifically bind both rat and human full-length huntingtin. Neither normal nor expanded polyglutamine repeat alone interacted with CBS in the yeast two-hybrid system and nor did constructs containing SBMA or DRPLA with normal or expanded polyglutamine tracts. CBS therefore appears to bind specifically to huntingtin. CBS deficiency is associated with homocystinuria, which is known to affect various physiological systems, including the central nervous system. Homocysteine, one of the substrates of CBS, is known to accumulate in homocystinuria and is metabolized to homocysteate and homocysteine sulphinate, both known to be powerful excitotoxic amino acids. It has been suggested that Huntington's disease involves the action of excitotoxic amino acids and this interaction with CBS may suggest a mechanism for such excitotoxic damage.

    Human molecular genetics 1998;7;3;371-8

  • Interaction of huntingtin-associated protein with dynactin P150Glued.

    Li SH, Gutekunst CA, Hersch SM and Li XJ

    Genetics, Emory University School of Medicine, Atlanta, Georgia 30322, USA.

    Huntingtin is the protein product of the gene for Huntington's disease (HD) and carries a polyglutamine repeat that is expanded in HD (>36 units). Huntingtin-associated protein (HAP1) is a neuronal protein and binds to huntingtin in association with the polyglutamine repeat. Like huntingtin, HAP1 has been found to be a cytoplasmic protein associated with membranous organelles, suggesting the existence of a protein complex including HAP1, huntingtin, and other proteins. Using the yeast two-hybrid system, we found that HAP1 also binds to dynactin P150(Glued) (P150), an accessory protein for cytoplasmic dynein that participates in microtubule-dependent retrograde transport of membranous organelles. An in vitro binding assay showed that both huntingtin and P150 selectively bound to a glutathione transferase (GST)-HAP1 fusion protein. An immunoprecipitation assay demonstrated that P150 and huntingtin coprecipitated with HAP1 from rat brain cytosol. Western blot analysis revealed that HAP1 was enriched in rat brain microtubules and comigrated with P150 and huntingtin in sucrose gradients. Immunofluorescence showed that transfected HAP1 colocalized with P150 and huntingtin in human embryonic kidney (HEK) 293 cells. We propose that HAP1, P150, and huntingtin are present in a protein complex that may participate in dynein-dynactin-associated intracellular transport.

    Funded by: NINDS NIH HHS: NS01624

    The Journal of neuroscience : the official journal of the Society for Neuroscience 1998;18;4;1261-9

  • SH3 domain-dependent association of huntingtin with epidermal growth factor receptor signaling complexes.

    Liu YF, Deth RC and Devys D

    Center for Neurological Disease, Brigham and Women's Hospital and Department of Neurology, Harvard Medical School, Boston, Massachusetts 02114, USA.

    Based on the presence of multiple proline-rich motifs in the huntingtin sequence, we tested its possible association with epidermal growth factor (EGF) receptor signaling complexes through SH3 domain-containing modules. We found that huntingtin is associated with Grb2, RasGAP, and tyrosine-phosphorylated EGF receptor. These associations are regulated by activation of the EGF receptor, suggesting that they may be part of EGF receptor-mediated cellular signaling cascade. In vitro binding studies indicate that SH3 domains of Grb2 or RasGAP are required for their binding to huntingtin. Our results suggest that huntingtin may be a unique adapter protein for EGF receptor-mediated signaling and may be involved in the regulation of Ras-dependent signaling pathways.

    The Journal of biological chemistry 1997;272;13;8121-4

  • HIP-I: a huntingtin interacting protein isolated by the yeast two-hybrid system.

    Wanker EE, Rovira C, Scherzinger E, Hasenbank R, Wälter S, Tait D, Colicelli J and Lehrach H

    Max Planck Institut für Molekulare Genetik, Berlin (Dahlem), Germany.

    We report the discovery of the huntingtin interacting protein I (HIP-I) which binds specifically to the N-terminus of human huntingtin, both in the two-hybrid screen and in in vitro binding experiments. For the interaction in vivo, a protein region downstream of the polyglutamine stretch in huntingtin is essential. The HIP1 cDNA isolated by the two-hybrid screen encodes a 55 kDa fragment of a novel protein. Using an affinity-purified polyclonal antibody raised against recombinant HIP-I, a protein of 116 kDa was detected in brain extracts by Western blot analysis. The predicted amino acid sequence of the HIP-I fragment exhibits significant similarity to cytoskeleton proteins, suggesting that HIP-I and huntingtin play a functional role in the cell filament networks. The HIP1 gene is ubiquitously expressed in different brain regions at low level. HIP-I is enriched in human brain but can also be detected in other human tissues as well as in mouse brain. HIP-I and huntingtin behave almost identically during subcellular fractionation and both proteins are enriched in the membrane containing fractions.

    Human molecular genetics 1997;6;3;487-95

  • Huntingtin is ubiquitinated and interacts with a specific ubiquitin-conjugating enzyme.

    Kalchman MA, Graham RK, Xia G, Koide HB, Hodgson JG, Graham KC, Goldberg YP, Gietz RD, Pickart CM and Hayden MR

    Department of Medical Genetics, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z4.

    Using the yeast two-hybrid system, we have identified a human ubiquitin-conjugating enzyme (hE2-25K) as a protein that interacts with the gene product for Huntington disease (HD) (Huntingtin). This protein has complete amino acid identity with the bovine E2-25K protein and has striking similarity to the UBC-1, -4 and -5 enzymes of Saccharomyces cerevisiae. This protein is highly expressed in brain and a slightly larger protein recognized by an anti-E2-25K polyclonal antibody is selectively expressed in brain regions affected in HD. The huntingtin-E2-25K interaction is not obviously modulated by CAG length. We also demonstrate that huntingtin is ubiquitinated. These findings have implications for the regulated catabolism of the gene product for HD.

    The Journal of biological chemistry 1996;271;32;19385-94

  • Cleavage of huntingtin by apopain, a proapoptotic cysteine protease, is modulated by the polyglutamine tract.

    Goldberg YP, Nicholson DW, Rasper DM, Kalchman MA, Koide HB, Graham RK, Bromm M, Kazemi-Esfarjani P, Thornberry NA, Vaillancourt JP and Hayden MR

    Department of Medical Genetics, University of British Columbia, Vancouver, British Columbia, Canada.

    Apoptosis has recently been recognized as a mode of cell death in Huntington disease (HD). Apopain, a human counterpart of the nematode cysteine protease death-gene product, CED-3, has a key role in proteolytic events leading to apoptosis. Here we show that apoptotic extracts and apopain itself specifically cleave the HD gene product, huntingtin. The rate of cleavage increases with the length of the huntingtin polyglutamine tract, providing an explanation for the gain-of-function associated with CAG expansion. Our results show that huntingtin is cleaved by cysteine proteases and suggest that HD might be a disorder of inappropriate apoptosis.

    Nature genetics 1996;13;4;442-9

  • Huntingtin and DRPLA proteins selectively interact with the enzyme GAPDH.

    Burke JR, Enghild JJ, Martin ME, Jou YS, Myers RM, Roses AD, Vance JM and Strittmatter WJ

    Department of Medicine (Neurology), Duke University Medical Center, Durham, North Carolina 27710, USA.

    At least five adult-onset neurodegenerative diseases, including Huntingtin disease (HD), and dentatorubral-pallidoluysian atrophy (DRPLA) are produced by genes containing a variably increased CAG repeat within the coding region. The size range of the repeats is similar in all diseases; unaffected individuals have fewer than 30 CAG repeats, whereas affected patients usually have more than 40 repeats. The size of the inherited CAG repeat correlates with the severity and age of disease onset. The CAG triplet repeat produces a polyglutamine domain in the expressed proteins. All of these diseases are inherited in a dominant fashion, and a pathologic gain of function in gene carriers has been proposed. We sought to identify proteins in the brain that selectively interact with polyglutamine-domain proteins, hypothesizing that the polyglutamine domain may determine protein-protein interactions.

    Funded by: NINDS NIH HHS: NS 01533, NS 26630

    Nature medicine 1996;2;3;347-50

  • A huntingtin-associated protein enriched in brain with implications for pathology.

    Li XJ, Li SH, Sharp AH, Nucifora FC, Schilling G, Lanahan A, Worley P, Snyder SH and Ross CA

    Department of Psychiatry, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.

    Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder caused by an expanding polyglutamine repeat in the IT15 or huntingtin gene. Although this gene is widely expressed and is required for normal development, the pathology of HD is restricted to the brain, for reasons that remain poorly understood. The huntingtin gene product is expressed at similar levels in patients and controls, and the genetics of the disorder suggest that the expansion of the polyglutamine repeat induces a toxic gain of function, perhaps through interactions with other cellular proteins. Here we report the identification of a protein (huntingtin-associated protein (HAP)-1) that binds to huntingtin. This binding is enhanced by an expanded polyglutamine repeat, the length of which is also known to correlate with the age of disease onset. The HAP-1 protein is enriched in the brain, suggesting a possible basis for the selective brain pathology of HD.

    Nature 1995;378;6555;398-402

  • Identification and localization of huntingtin in brain and human lymphoblastoid cell lines with anti-fusion protein antibodies.

    Gutekunst CA, Levey AI, Heilman CJ, Whaley WL, Yi H, Nash NR, Rees HD, Madden JJ and Hersch SM

    Department of Neurology, Emory University School of Medicine, Atlanta, GA 30322, USA.

    The Huntington disease (HD) phenotype is associated with expansion of a trinucleotide repeat in the IT15 gene, which is predicted to encode a 348-kDa protein named huntington. We used polyclonal and monoclonal anti-fusion protein antibodies to identify native huntingtin in rat, monkey, and human. Western blots revealed a protein with the expected molecular weight which is present in the soluble fraction of rat and monkey brain tissues and lymphoblastoid cells from control cases. In lymphoblastoid cell lines from juvenile-onset heterozygote HD cases, both normal and mutant huntingtin are expressed, and increasing repeat expansion leads to lower levels of the mutant protein. Immunocytochemistry indicates that huntingtin is located in neurons throughout the brain, with the highest levels evident in larger neurons. In the human striatum, huntingtin is enriched in a patch-like distribution, potentially corresponding to the first areas affected in HD. Subcellular localization of huntingtin is consistent with a cytosolic protein primarily found in somatodendritic regions. Huntingtin appears to particularly associate with microtubules, although some is also associated with synaptic vesicles. On the basis of the localization of huntingtin in association with microtubules, we speculate that the mutation impairs the cytoskeletal anchoring or transport of mitochondria, vesicles, or other organelles or molecules.

    Funded by: NCRR NIH HHS: M01-RR00039; NIDA NIH HHS: DA05002; NINDS NIH HHS: NS01624

    Proceedings of the National Academy of Sciences of the United States of America 1995;92;19;8710-4

  • Targeted disruption of the Huntington's disease gene results in embryonic lethality and behavioral and morphological changes in heterozygotes.

    Nasir J, Floresco SB, O'Kusky JR, Diewert VM, Richman JM, Zeisler J, Borowski A, Marth JD, Phillips AG and Hayden MR

    Department of Medical Genetics, University of British Columbia, Vancouver, Canada.

    Huntington's disease (HD) is an incurable neuropsychiatric disease associated with CAG repeat expansion within a widely expressed gene that causes selective neuronal death. To understand its normal function, we have created a targeted disruption in exon 5 of Hdh (Hdhex5), the murine homolog of the HD gene. Homozygotes die before embryonic day 8.5, initiate gastrulation, but do not proceed to the formation of somites or to organogenesis. Mice heterozygous for the Hdhex5 mutation display increased motor activity and cognitive deficits. Neuropathological assessment of two heterozygous mice shows significant neuronal loss in the subthalamic nucleus. These studies show that the HD gene is essential for postimplantation development and that it may play an important role in normal functioning of the basal ganglia.

    Cell 1995;81;5;811-23

  • Cellular localization of the Huntington's disease protein and discrimination of the normal and mutated form.

    Trottier Y, Devys D, Imbert G, Saudou F, An I, Lutz Y, Weber C, Agid Y, Hirsch EC and Mandel JL

    Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), CNRS, INSERM, ULP, Illkirch, France.

    Huntington's disease (HD) results from the expansion of a polyglutamine encoding CAG repeat in a gene of unknown function. The wide expression of this transcript does not correlate with the pattern of neuropathology in HD. To study the HD gene product (huntingtin), we have developed monoclonal antibodies raised against four different regions of the protein. On western blots, these monoclonals detect the approximately 350 kD huntingtin protein in various human cell lines and in neural and non-neural rodent tissues. In cell lines from HD patients, a doublet protein is detected corresponding to the mutated and normal huntingtin. Immunohistochemical studies in the human brain using two of these antibodies detects the huntingtin in perikarya of some neurons, neuropiles, varicosities and as punctate staining likely to be nerve endings.

    Nature genetics 1995;10;1;104-10

  • Structural analysis of the 5' region of mouse and human Huntington disease genes reveals conservation of putative promoter region and di- and trinucleotide polymorphisms.

    Lin B, Nasir J, Kalchman MA, McDonald H, Zeisler J, Goldberg YP and Hayden MR

    Department of Medical Genetics, University of British Columbia, Vancouver, Canada.

    We have previously cloned and characterized the murine homologue of the Huntington disease (HD) gene and shown that it maps to mouse chromosome 5 within a region of conserved synteny with human chromosome 4p16.3. Here we present a detailed comparison of the sequence of the putative promoter and the organization of the 5' genomic region of the murine (Hdh) and human HD genes encompassing the first five exons. We show that in this region these two genes share identical exon boundaries, but have different-size introns. Two dinucleotide (CT) and one trinucleotide intronic polymorphism in Hdh and an intronic CA polymorphism in the HD gene were identified. Comparison of 940-bp sequence 5' to the putative translation start site reveals a highly conserved region (78.8% nucleotide identity) between Hdh and the HD gene from nucleotide -56 to -206 (of Hdh). Neither Hdh nor the HD gene have typical TATA or CCAAT elements, but both show one putative AP2 binding site and numerous potential Sp1 binding sites. The high sequence identity between Hdh and the HD gene for approximately 200 bp 5' to the putative translation start site indicates that these sequences may play a role in regulating expression of the Huntington disease gene.

    Genomics 1995;25;3;707-15

  • Mutational bias provides a model for the evolution of Huntington's disease and predicts a general increase in disease prevalence.

    Rubinsztein DC, Amos W, Leggo J, Goodburn S, Ramesar RS, Old J, Bontrop R, McMahon R, Barton DE and Ferguson-Smith MA

    East Anglian Regional Genetics Service Molecular Genetics Laboratory, Addenbrooke's NHS Trust, Cambridge, UK.

    Huntington's disease (HD) correlates with abnormal expansion in a block of CAG repeats in the Huntington's disease gene. We have investigated HD evolution by typing CAG alleles in several human populations and in a variety of primates. We find that human alleles have expanded from a shorter ancestral state and exhibit unusual asymmetric length distributions. Computer simulations are used to show that the human state can be derived readily from a primate ancestor, without the need to invoke natural selection. The key element is a simple length-dependent mutational bias towards longer alleles. Our model can explain a number of empirical observations, and predicts an ever-increasing incidence of HD.

    Nature genetics 1994;7;4;525-30

  • Structure and expression of the Huntington's disease gene: evidence against simple inactivation due to an expanded CAG repeat.

    Ambrose CM, Duyao MP, Barnes G, Bates GP, Lin CS, Srinidhi J, Baxendale S, Hummerich H, Lehrach H, Altherr M et al.

    Molecular Neurogenetics Unit, Massachusetts General Hospital, Boston 02114.

    Huntington's disease, a neurodegenerative disorder characterized by loss of striatal neurons, is caused by an expanded, unstable trinucleotide repeat in a novel 4p16.3 gene. To lay the foundation for exploring the pathogenic mechanism in HD, we have determined the structure of the disease gene and examined its expression. The HD locus spans 180 kb and consists of 67 exons ranging in size from 48 bp to 341 bp with an average of 138 bp. Scanning of the HD transcript failed to reveal any additional sequence alterations characteristic of HD chromosomes. A codon loss polymorphism in linkage disequilibrium with the disorder revealed that both normal and HD alleles are represented in the mRNA population in HD heterozygotes, indicating that the defect does not eliminate transcription. The gene is ubiquitously expressed as two alternatively polyadenylated forms displaying different relative abundance in various fetal and adult tissues, suggesting the operation of interacting factors in determining specificity of cell loss. The HD gene was disrupted in a female carrying a balanced translocation with a breakpoint between exons 40 and 41. The absence of any abnormal phenotype in this individual argues against simple inactivation of the gene as the mechanism by which the expanded trinucleotide repeat causes HD. Taken together, these observations suggest that the dominant HD mutation either confers a new property on the mRNA or, more likely, alters an interaction at the protein level.

    Funded by: NINDS NIH HHS: NS16367; Wellcome Trust

    Somatic cell and molecular genetics 1994;20;1;27-38

  • Differential 3' polyadenylation of the Huntington disease gene results in two mRNA species with variable tissue expression.

    Lin B, Rommens JM, Graham RK, Kalchman M, MacDonald H, Nasir J, Delaney A, Goldberg YP and Hayden MR

    Department of Medical Genetics, University of British Columbia, Vancouver, Canada.

    Recently a novel gene containing a CAG trinucleotide repeat that is expanded on HD chromosomes has been identified(1). This gene was shown to detect a single transcript of 10-11 kb by RNA hybridization. We have however, previously identified three cDNAs which are part of the same gene that have been shown to detect two distinct transcripts of 10 kb and one that is significantly larger(2,3). These different mRNA species could be due to use of alternate transcription start sites, alternate splicing or selection of different polyadenylation sites. We have identified cDNA clones spanning the HD gene including two (HD12 and HD14) that share identical protein coding sequences but differ in size and sequence of their 3' untranslated region. HD14 has 3,360 base pairs of additional sequence distal to the previously published 3' end (1). RNA hybridization has revealed that the larger 13.7 kb fragment is the predominant transcript in human brain. cDNA fragments unique to HD14 detected only the larger transcript. Sequence analysis identified two different putative polyadenylation sequences at position 10,326 and 13,645 of the HD14 cDNA. These findings indicate that the two observed mRNA species originate from a single gene and that differential polyadenylation leads to transcripts of different size. The relative increased abundance of the larger transcript in human brain may provide some insights into the mechanism by which a widely expressed gene may exert tissue specific effects.

    Human molecular genetics 1993;2;10;1541-5

  • A novel gene containing a trinucleotide repeat that is expanded and unstable on Huntington's disease chromosomes. The Huntington's Disease Collaborative Research Group.

    No authors listed

    The Huntington's disease (HD) gene has been mapped in 4p16.3 but has eluded identification. We have used haplotype analysis of linkage disequilibrium to spotlight a small segment of 4p16.3 as the likely location of the defect. A new gene, IT15, isolated using cloned trapped exons from the target area contains a polymorphic trinucleotide repeat that is expanded and unstable on HD chromosomes. A (CAG)n repeat longer than the normal range was observed on HD chromosomes from all 75 disease families examined, comprising a variety of ethnic backgrounds and 4p16.3 haplotypes. The (CAG)n repeat appears to be located within the coding sequence of a predicted approximately 348 kd protein that is widely expressed but unrelated to any known gene. Thus, the HD mutation involves an unstable DNA segment, similar to those described in fragile X syndrome, spino-bulbar muscular atrophy, and myotonic dystrophy, acting in the context of a novel 4p16.3 gene to produce a dominant phenotype.

    Funded by: NINDS NIH HHS: NS16367, NS22031, NS25631; Wellcome Trust

    Cell 1993;72;6;971-83

  • The Huntington's disease candidate region exhibits many different haplotypes.

    MacDonald ME, Novelletto A, Lin C, Tagle D, Barnes G, Bates G, Taylor S, Allitto B, Altherr M, Myers R et al.

    Neurogenetics Laboratory, Massachusetts General Hospital, Boston.

    Analysis of 78 Huntington's disease (HD) chromosomes with multi-allele markers revealed 26 different haplotypes, suggesting a variety of independent HD mutations. The most frequent haplotype, accounting for about one third of disease chromosomes, suggests that the disease gene is between D4S182 and D4S180. However, the paucity of an expected class of chromosomes that can be related to this major haplotype by assuming single crossovers may reflect the operation of other mechanisms in creating haplotype diversity. Some of these mechanisms sustain alternative scenarios that do not require a multiple mutational origin for HD and/or its positioning between D4S182 and D4S180.

    Nature genetics 1992;1;2;99-103

OMIM - other

Gene lists (1)

Gene List Source Species Name Description Gene count
L00000069 G2C Homo sapiens BAYES-COLLINS-HUMAN-PSD-FULL Human cortex biopsy PSD full list 1461
© G2C 2014. The Genes to Cognition Programme received funding from The Wellcome Trust and the EU FP7 Framework Programmes:
EUROSPIN (FP7-HEALTH-241498), SynSys (FP7-HEALTH-242167) and GENCODYS (FP7-HEALTH-241995).

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