G2Cdb::Gene report

Gene id
G00000859
Gene symbol
App (MGI)
Species
Mus musculus
Description
amyloid beta (A4) precursor protein
Orthologue
G00002108 (Homo sapiens)

Databases (9)

Gene
ENSMUSG00000022892 (Ensembl mouse gene)
11820 (Entrez Gene)
195 (G2Cdb plasticity & disease)
Gene Expression
NM_007471 (Allen Brain Atlas)
EMAGE:1132 (EMAGE)
11820 (Genepaint)
Literature
104760 (OMIM)
Marker Symbol
MGI:88059 (MGI)
Protein Sequence
P12023 (UniProt)

Synonyms (6)

  • Abeta
  • Adap
  • Cvap
  • appican
  • betaAPP
  • protease nexin II

Literature (588)

Pubmed - other

  • The amyloid precursor protein intracellular domain alters gene expression and induces neuron-specific apoptosis.

    Ohkawara T, Nagase H, Koh CS and Nakayama K

    Department of Anatomy, Shinshu University, School of Medicine, Matsumoto, Nagano, Japan.

    Although amyloid precursor protein (APP) plays a central role in Alzheimer's disease, the physiological functions of this protein have yet to be fully elucidated. As previously reported, we established an embryonic carcinoma P19 cell line expressing the intracellular domain of APP (AICD). While neurons were differentiated from these cell lines with retinoic acid treatment, expression of AICD induced neuron-specific apoptosis. As the first step to identify the genes involved in this process, we evaluated AICD-induced changes in gene expression through cell death. The levels of expression of 41,256 transcripts were monitored by DNA microarray analysis. The expression of 277 genes showed up-regulation by more than 10-fold in the presence of AICD. Conversely, the expression of 341 genes showed down-regulation to less than one-tenth of the original level. Reverse transcription-polymerase chain reaction of 17 selected genes showed excellent agreement with the microarray results. These results suggest that AICD induces dynamic changes in gene expression, which may be closely correlated with AICD-induced neuron-specific apoptosis.

    Gene 2011;475;1;1-9

  • Comparative transcriptome profiling of amyloid precursor protein family members in the adult cortex.

    Aydin D, Filippov MA, Tschäpe JA, Gretz N, Prinz M, Eils R, Brors B and Müller UC

    Department of Bioinformatics and Functional Genomics, Institute of Pharmacy and Molecular Biotechnology, Heidelberg University, Im Neuenheimer Feld 364, D-69120 Heidelberg, Germany.

    Background: The β-amyloid precursor protein (APP) and the related β-amyloid precursor-like proteins (APLPs) undergo complex proteolytic processing giving rise to several fragments. Whereas it is well established that Aβ accumulation is a central trigger for Alzheimer's disease, the physiological role of APP family members and their diverse proteolytic products is still largely unknown. The secreted APPsα ectodomain has been shown to be involved in neuroprotection and synaptic plasticity. The γ-secretase-generated APP intracellular domain (AICD) functions as a transcriptional regulator in heterologous reporter assays although its role for endogenous gene regulation has remained controversial.

    Results: To gain further insight into the molecular changes associated with knockout phenotypes and to elucidate the physiological functions of APP family members including their proposed role as transcriptional regulators, we performed DNA microarray transcriptome profiling of prefrontal cortex of adult wild-type (WT), APP knockout (APP-/-), APLP2 knockout (APLP2-/-) and APPsα knockin mice (APPα/α) expressing solely the secreted APPsα ectodomain. Biological pathways affected by the lack of APP family members included neurogenesis, transcription, and kinase activity. Comparative analysis of transcriptome changes between mutant and wild-type mice, followed by qPCR validation, identified co-regulated gene sets. Interestingly, these included heat shock proteins and plasticity-related genes that were both down-regulated in knockout cortices. In contrast, we failed to detect significant differences in expression of previously proposed AICD target genes including Bace1, Kai1, Gsk3b, p53, Tip60, and Vglut2. Only Egfr was slightly up-regulated in APLP2-/- mice. Comparison of APP-/- and APPα/α with wild-type mice revealed a high proportion of co-regulated genes indicating an important role of the C-terminus for cellular signaling. Finally, comparison of APLP2-/- on different genetic backgrounds revealed that background-related transcriptome changes may dominate over changes due to the knockout of a single gene.

    Conclusion: Shared transcriptome profiles corroborated closely related physiological functions of APP family members in the adult central nervous system. As expression of proposed AICD target genes was not altered in adult cortex, this may indicate that these genes are not affected by lack of APP under resting conditions or only in a small subset of cells.

    BMC genomics 2011;12;160

  • A single tyrosine residue in the amyloid precursor protein intracellular domain is essential for developmental function.

    Barbagallo AP, Wang Z, Zheng H and D'Adamio L

    Department of Microbiology and Immunology, Einstein College of Medicine, Bronx, New York 10461, USA.

    The Aβ-precursor protein (APP) intracellular domain is highly conserved and contains many potentially important residues, in particular the (682)YENPTY(687) motif. To dissect the functions of this sequence in vivo, we created an APP knock-in allele mutating Tyr(682) to Gly (Y682G). Crossing this allele to APP-like protein 2 (APLP2) knock-out background showed that mutation of Tyr(682) results in postnatal lethality and neuromuscular synapse defects similar to doubly deficient APP/APLP2 mice. Our results demonstrate that a single residue in the APP intracellular region, Tyr(682), is indispensable for the essential function of APP in developmental regulation.

    Funded by: NIA NIH HHS: AG020670, AG032051, AG033007, R01 AG020670, R01 AG032051, R01 AG033007, R01 AG033007-01A1, R01 AG033007-02, R01 AG033007-03, R01 AG033007-03S1, R01 AG033007-04

    The Journal of biological chemistry 2011;286;11;8717-21

  • Impaired attention in the 3xTgAD mouse model of Alzheimer's disease: rescue by donepezil (Aricept).

    Romberg C, Mattson MP, Mughal MR, Bussey TJ and Saksida LM

    Department of Experimental Psychology, University of Cambridge, Cambridge CB2 3EB, United Kingdom. carola.romberg@gmail.com

    Several mouse models of Alzheimer's disease (AD) with abundant β-amyloid and/or aberrantly phosphorylated tau develop memory impairments. However, multiple non-mnemonic cognitive domains such as attention and executive control are also compromised early in AD individuals. Currently, it is unclear whether mutations in the β-amyloid precursor protein (APP) and tau are sufficient to cause similar, AD-like attention deficits in mouse models of the disease. To address this question, we tested 3xTgAD mice (which express APPswe, PS1M146V, and tauP301L mutations) and wild-type control mice on a newly developed touchscreen-based 5-choice serial reaction time test of attention and response control. The 3xTgAD mice attended less accurately to short, spatially unpredictable stimuli when the attentional demand of the task was high, and also showed a general tendency to make more perseverative responses than wild-type mice. The attentional impairment of 3xTgAD mice was comparable to that of AD patients in two aspects: first, although 3xTgAD mice initially responded as accurately as wild-type mice, they subsequently failed to sustain their attention over the duration of the task; second, the ability to sustain attention was enhanced by the cholinesterase inhibitor donepezil (Aricept). These findings demonstrate that familial AD mutations not only affect memory, but also cause significant impairments in attention, a cognitive domain supported by the prefrontal cortex and its afferents. Because attention deficits are likely to affect memory encoding and other cognitive abilities, our findings have important consequences for the assessment of disease mechanisms and therapeutics in animal models of AD.

    Funded by: Intramural NIH HHS: Z01 AG000312-08, Z01 AG000313-08, Z01 AG000314-08, Z01 AG000317-08; Medical Research Council: G0001354; Wellcome Trust

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2011;31;9;3500-7

  • Lipoprotein lipase is a novel amyloid beta (Abeta)-binding protein that promotes glycosaminoglycan-dependent cellular uptake of Abeta in astrocytes.

    Nishitsuji K, Hosono T, Uchimura K and Michikawa M

    Section of Pathophysiology and Neurobiology, National Center for Geriatrics and Gerontology, Obu, Aichi 474-8511, Japan.

    Lipoprotein lipase (LPL) is a member of a lipase family known to hydrolyze triglyceride molecules in plasma lipoprotein particles. LPL also plays a role in the binding of lipoprotein particles to cell-surface molecules, including sulfated glycosaminoglycans (GAGs). LPL is predominantly expressed in adipose and muscle but is also highly expressed in the brain where its specific roles are unknown. It has been shown that LPL is colocalized with senile plaques in Alzheimer disease (AD) brains, and its mutations are associated with the severity of AD pathophysiological features. In this study, we identified a novel function of LPL; that is, LPL binds to amyloid β protein (Aβ) and promotes cell-surface association and uptake of Aβ in mouse primary astrocytes. The internalized Aβ was degraded within 12 h, mainly in a lysosomal pathway. We also found that sulfated GAGs were involved in the LPL-mediated cellular uptake of Aβ. Apolipoprotein E was dispensable in the LPL-mediated uptake of Aβ. Our findings indicate that LPL is a novel Aβ-binding protein promoting cellular uptake and subsequent degradation of Aβ.

    The Journal of biological chemistry 2011;286;8;6393-401

  • Overexpression of glutaminyl cyclase, the enzyme responsible for pyroglutamate A{beta} formation, induces behavioral deficits, and glutaminyl cyclase knock-out rescues the behavioral phenotype in 5XFAD mice.

    Jawhar S, Wirths O, Schilling S, Graubner S, Demuth HU and Bayer TA

    Department of Molecular Psychiatry. Graduate School, University Medicine Goettingen, 37075 Goettingen, Germany.

    Pyroglutamate-modified Aβ (AβpE3-42) peptides are gaining considerable attention as potential key players in the pathology of Alzheimer disease (AD) due to their abundance in AD brain, high aggregation propensity, stability, and cellular toxicity. Overexpressing AβpE3-42 induced a severe neuron loss and neurological phenotype in TBA2 mice. In vitro and in vivo experiments have recently proven that the enzyme glutaminyl cyclase (QC) catalyzes the formation of AβpE3-42. The aim of the present work was to analyze the role of QC in an AD mouse model with abundant AβpE3-42 formation. 5XFAD mice were crossed with transgenic mice expressing human QC (hQC) under the control of the Thy1 promoter. 5XFAD/hQC bigenic mice showed significant elevation in TBS, SDS, and formic acid-soluble AβpE3-42 peptides and aggregation in plaques. In 6-month-old 5XFAD/hQC mice, a significant motor and working memory impairment developed compared with 5XFAD. The contribution of endogenous QC was studied by generating 5XFAD/QC-KO mice (mouse QC knock-out). 5XFAD/QC-KO mice showed a significant rescue of the wild-type mice behavioral phenotype, demonstrating the important contribution of endogenous mouse QC and transgenic overexpressed QC. These data clearly demonstrate that QC is crucial for modulating AβpE3-42 levels in vivo and prove on a genetic base the concept that reduction of QC activity is a promising new therapeutic approach for AD.

    The Journal of biological chemistry 2011;286;6;4454-60

  • p75NTR regulates Abeta deposition by increasing Abeta production but inhibiting Abeta aggregation with its extracellular domain.

    Wang YJ, Wang X, Lu JJ, Li QX, Gao CY, Liu XH, Sun Y, Yang M, Lim Y, Evin G, Zhong JH, Masters C and Zhou XF

    Department of Human Physiology and Centre for Neuroscience, Flinders University, Adelaide 5001, South Australia, Australia. yanjiang_wang@tmmu.edu.cn

    Accumulation of toxic amyloid-β (Aβ) in the cerebral cortex and hippocampus is a major pathological feature of Alzheimer's disease (AD). The neurotrophin receptor p75NTR has been proposed to mediate Aβ-induced neurotoxicity; however, its role in the development of AD remains to be clarified. The p75NTR/ExonIII-/- mice and APPSwe/PS1dE9 mice were crossed to generate transgenic AD mice with deletion of p75NTR gene. In APPSwe/PS1dE9 transgenic mice, p75NTR expression was localized in the basal forebrain neurons and degenerative neurites in neocortex, increased with aging, and further activated by Aβ accumulation. Deletion of the p75NTR gene in APPSwe/PS1dE9 mice reduced soluble Aβ levels in the brain and serum, but increased the accumulation of insoluble Aβ and Aβ plaque formation. There was no change in the levels of amyloid precursor protein (APP) and its proteolytic derivatives, or α-, β-, and γ-secretase activities, or in levels of BACE1, neprilysin (NEP), and insulin-degrading enzyme (IDE) proteins. Aβ production by cortical neurons of APPSwe/PS1dE9 mice was reduced by deletion of p75NTR gene in vitro. Recombinant extracellular domain of p75NTR attenuated the oligomerization and fibrillation of synthetic Aβ(42) peptide in vitro, and reduced local Aβ plaques after hippocampus injection in vivo. In addition, deletion of p75NTR attenuated microgliosis but increased the microhemorrhage profiles in the brain. The deletion of p75NTR did not significantly change the cognitive function of the mice up to the age of 9 months. Our data suggest that p75NTR plays a critical role in regulating Aβ levels by both increasing Aβ production and attenuating its aggregation, and they caution that a therapeutic intervention simply reducing p75NTR may exacerbate AD pathology.

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2011;31;6;2292-304

  • Sphingolipid storage affects autophagic metabolism of the amyloid precursor protein and promotes Abeta generation.

    Tamboli IY, Hampel H, Tien NT, Tolksdorf K, Breiden B, Mathews PM, Saftig P, Sandhoff K and Walter J

    Department of Neurology, University of Bonn, 53127 Bonn, Germany.

    Deposition of amyloid β peptides (Aβs) in extracellular amyloid plaques within the human brain is a hallmark of Alzheimer's disease (AD). Aβ derives from proteolytic processing of the amyloid precursor protein (APP) by β- and γ-secretases. The initial cleavage by β-secretase results in shedding of the APP ectodomain and generation of APP C-terminal fragments (APP-CTFs), which can then be further processed within the transmembrane domain by γ-secretase, resulting in release of Aβ. Here, we demonstrate that accumulation of sphingolipids (SLs), as occurs in lysosomal lipid storage disorders (LSDs), decreases the lysosome-dependent degradation of APP-CTFs and stimulates γ-secretase activity. Together, this results in increased generation of both intracellular and secreted Aβ. Notably, primary fibroblasts from patients with different SL storage diseases show strong accumulation of potentially amyloidogenic APP-CTFs. By using biochemical, cell biological, and genetic approaches, we demonstrate that SL accumulation affects autophagic flux and impairs the clearance of APP-CTFs. Thus, accumulation of SLs might not only underlie the pathogenesis of LSDs, but also trigger increased generation of Aβ and contribute to neurodegeneration in sporadic AD.

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2011;31;5;1837-49

  • Depletion of CXCR2 inhibits γ-secretase activity and amyloid-β production in a murine model of Alzheimer's disease.

    Bakshi P, Margenthaler E, Reed J, Crawford F and Mullan M

    Laboratories of Chemical Biology and Drug Discovery in Neurology, Roskamp Institute, 2040 Whitfield Avenue, Sarasota, FL 34243, United States. Pbakshi@rfdn.org

    Alzheimer's disease (AD) is a neurodegenerative disorder that leads to progressive cognitive decline. Recent studies from our group and others have suggested that certain G-protein coupled receptors (GPCRs) can influence the processing of the amyloid precursor protein (APP). Earlier, we demonstrated that stimulation of a chemokine receptor, CXCR2, results in enhanced γ-secretase activity and in increased amyloid-beta (Aβ) production. Taken together, results obtained from in vitro studies indicate that therapeutic targeting of CXCR2 might aid in lowering Aβ levels in the AD brain. To better understand the precise function and to predict the consequences of CXCR2 depletion in the AD brain, we have crossed CXCR2 knockout mice with mice expressing presenilin (PS1 M146L) and APPsw mutations (PSAPP). Our present study confirms that CXCR2 depletion results in reduction of Aβ with concurrent increases of γ-secretase substrates. At the mechanistic level, the effect of CXCR2 on γ-secretase was not found to occur via their direct interaction. Furthermore, we provide evidence that Aβ promotes endocytosis of CXCR2 via increasing levels of CXCR2 ligands. In conclusion, our current study confirms the regulatory role of CXCR2 in APP processing, and poses it as a potential target for developing novel therapeutics for intervention in AD.

    Cytokine 2011;53;2;163-9

  • The purinergic receptor P2X7 triggers alpha-secretase-dependent processing of the amyloid precursor protein.

    Delarasse C, Auger R, Gonnord P, Fontaine B and Kanellopoulos JM

    CRICM, INSERM UMR_S975, 75013 Paris, France.

    The amyloid precursor protein (APP) is cleaved by β- and γ-secretases to generate the β-amyloid (Aβ) peptides, which are present in large amounts in the amyloid plaques of Alzheimer disease (AD) patient brains. Non-amyloidogenic processing of APP by α-secretases leads to proteolytic cleavage within the Aβ peptide sequence and shedding of the soluble APP ectodomain (sAPPα), which has been reported to be endowed with neuroprotective properties. In this work, we have shown that activation of the purinergic receptor P2X7 (P2X7R) stimulates sAPPα release from mouse neuroblastoma cells expressing human APP, from human neuroblastoma cells and from mouse primary astrocytes or neural progenitor cells. sAPPα shedding is inhibited by P2X7R antagonists or knockdown of P2X7R with specific small interfering RNA (siRNA) and is not observed in neural cells from P2X7R-deficient mice. P2X7R-dependent APP-cleavage is independent of extracellular calcium and strongly inhibited by hydroxamate-based metalloprotease inhibitors, TAPI-2 and GM6001. However, knockdown of a disintegrin and metalloproteinase-9 (ADAM9), ADAM10 and ADAM17 by specific siRNA, known to have α-secretase activity, does not block the P2X7R-dependent non-amyloidogenic pathway. Using several specific pharmacological inhibitors, we demonstrate that the mitogen-activated protein kinase modules Erk1/2 and JNK are involved in P2X7R-dependent α-secretase activity. Our study suggests that P2X7R, which is expressed in hippocampal neurons and glial cells, is a potential therapeutic target in AD.

    The Journal of biological chemistry 2011;286;4;2596-606

  • Noninvasive magnetic resonance imaging detection of cerebral amyloid angiopathy-related microvascular alterations using superparamagnetic iron oxide particles in APP transgenic mouse models of Alzheimer's disease: application to passive Abeta immunotherapy.

    Beckmann N, Gérard C, Abramowski D, Cannet C and Staufenbiel M

    Global Imaging Group, Novartis Institutes for BioMedical Research, CH-4056 Basel, Switzerland. nicolau.beckmann@novartis.com

    Cerebral amyloid angiopathy (CAA) is a common feature of Alzheimer's disease (AD). More advanced stages are accompanied by microhemorrhages and vasculitis. Peripheral blood-borne macrophages are intimately linked to cerebrovascular pathology coincident with AD. Magnetic resonance imaging (MRI) was used to noninvasively study microvascular lesions in amyloid precursor protein transgenic mouse AD models. Foci of signal attenuation were detected in cortical and thalamic brain regions of aged APP23 mice. Their strength and number was considerably enhanced by intravenous administration of iron oxide nanoparticles, which are taken up by macrophages through absorptive endocytosis, 24 h before image acquisition. The number of cortical sites displaying signal attenuation increased with age. Histology at these sites demonstrated the presence of iron-containing macrophages in the vicinity of CAA-affected blood vessels. A fraction of the sites additionally showed thickened vessel walls and vasculitis. Consistent with the visualization of CAA-associated lesions, MRI detected a much smaller number of attenuated signal sites in APP23xPS45 mice, for which a strong presenilin mutation caused a shift toward amyloid β(42), thus reducing vascular amyloid. Similar results were obtained with APP24 and APP51 mice, which develop significantly less CAA and microvascular pathology than APP23. In a longitudinal study, we noninvasively demonstrated the reinforced formation of microvascular pathology during passive amyloid β immunotherapy of APP23 mice. Histology confirmed that foci of signal attenuation reflected an increase in CAA-related lesions. Our data demonstrate that MRI has the sensitivity to noninvasively monitor the development of vascular pathology and its possible enhancement by amyloid β immunotherapy in transgenic mice modeling AD.

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2011;31;3;1023-31

  • A high-resolution anatomical atlas of the transcriptome in the mouse embryo.

    Diez-Roux G, Banfi S, Sultan M, Geffers L, Anand S, Rozado D, Magen A, Canidio E, Pagani M, Peluso I, Lin-Marq N, Koch M, Bilio M, Cantiello I, Verde R, De Masi C, Bianchi SA, Cicchini J, Perroud E, Mehmeti S, Dagand E, Schrinner S, Nürnberger A, Schmidt K, Metz K, Zwingmann C, Brieske N, Springer C, Hernandez AM, Herzog S, Grabbe F, Sieverding C, Fischer B, Schrader K, Brockmeyer M, Dettmer S, Helbig C, Alunni V, Battaini MA, Mura C, Henrichsen CN, Garcia-Lopez R, Echevarria D, Puelles E, Garcia-Calero E, Kruse S, Uhr M, Kauck C, Feng G, Milyaev N, Ong CK, Kumar L, Lam M, Semple CA, Gyenesei A, Mundlos S, Radelof U, Lehrach H, Sarmientos P, Reymond A, Davidson DR, Dollé P, Antonarakis SE, Yaspo ML, Martinez S, Baldock RA, Eichele G and Ballabio A

    Telethon Institute of Genetics and Medicine, Naples, Italy.

    Ascertaining when and where genes are expressed is of crucial importance to understanding or predicting the physiological role of genes and proteins and how they interact to form the complex networks that underlie organ development and function. It is, therefore, crucial to determine on a genome-wide level, the spatio-temporal gene expression profiles at cellular resolution. This information is provided by colorimetric RNA in situ hybridization that can elucidate expression of genes in their native context and does so at cellular resolution. We generated what is to our knowledge the first genome-wide transcriptome atlas by RNA in situ hybridization of an entire mammalian organism, the developing mouse at embryonic day 14.5. This digital transcriptome atlas, the Eurexpress atlas (http://www.eurexpress.org), consists of a searchable database of annotated images that can be interactively viewed. We generated anatomy-based expression profiles for over 18,000 coding genes and over 400 microRNAs. We identified 1,002 tissue-specific genes that are a source of novel tissue-specific markers for 37 different anatomical structures. The quality and the resolution of the data revealed novel molecular domains for several developing structures, such as the telencephalon, a novel organization for the hypothalamus, and insight on the Wnt network involved in renal epithelial differentiation during kidney development. The digital transcriptome atlas is a powerful resource to determine co-expression of genes, to identify cell populations and lineages, and to identify functional associations between genes relevant to development and disease.

    Funded by: Medical Research Council: MC_U127527203; Telethon: TGM11S03

    PLoS biology 2011;9;1;e1000582

  • A role for FE65 in controlling GnRH-1 neurogenesis.

    Forni PE, Fornaro M, Guénette S and Wray S

    Cellular and Developmental Neurobiology Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892, USA.

    Gonadotropin-releasing hormone-1 (GnRH-1) neurons migrate from the nasal placode to the forebrain where they control gonadal function via the hypothalamic-pituitary-gonadal axis. The birth of GnRH-1-expressing neurons is one of the first neurogenic events in the developing nasal placode. By gene expression screening on single GnRH-1 neurons, amyloid precursor binding protein-1 (FE65) was identified in migratory GnRH-1 neurons. FE65 has been shown to modulate β1-integrin dynamics, actin cytoskeleton, cell motility, and FE65/amyloid precursor protein signaling has been described in neuro/glial cell fate determination as well as in modulating neurogenesis. Analysis of two mouse lines, one deficient for the 97 kDa FE65 isoform and a second deficient for the 97 and 60 kDa forms of FE65, showed overlapping phenotypes. In both lines, no migratory defects of the GnRH-1 neurons were observed, but a 25% increase in GnRH-1 neuronal number during embryonic development was found. Bromodeoxyuridine birth tracing and spatiotemporal tracking of GnRH-1 cell precursors demonstrated that the lack of the N-terminal portion of FE65, which includes part of the functional nuclear translocation/gene transcription domain of FE65 (WW domain), extends the timing of GnRH-1 neurogenesis in the developing nasal placode without affecting proliferation of GnRH-1 neuronal progenitors or cell death. The observed changes in the dynamics of GnRH-1 neurogenesis highlight a unique role for the 97 kDa isoform of FE65 and suggest that GnRH-1 cells, which have a short neurogenic window, originate from multipotent progenitors able to generate distinct cell types as GnRH-1 neurogenesis declines in response to environmental changes.

    Funded by: Intramural NIH HHS: ZIA NS002824-20

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2011;31;2;480-91

  • Effect of trehalose on the interaction of Alzheimer's Aβ-peptide and anionic lipid monolayers.

    Izmitli A, Schebor C, McGovern MP, Reddy AS, Abbott NL and de Pablo JJ

    Chemical and Biological Engineering, University of Wisconsin-Madison, 1415 Engineering Drive, Madison, WI 53706, USA.

    The interaction of amyloid β-peptide (Aβ) with cell membranes is believed to play a central role in the pathogenesis of Alzheimer's disease. In particular, recent experimental evidence indicates that bilayer and monolayer membranes accelerate the aggregation and amyloid fibril formation rate of Aβ. Understanding that interaction could help develop therapeutic strategies for treatment of the disease. Trehalose, a disaccharide of glucose, has been shown to be effective in preventing the aggregation of numerous proteins. It has also been shown to delay the onset of certain amyloid-related diseases in a mouse model. Using Langmuir monolayers and molecular simulations of the corresponding system, we study several thermodynamic and kinetic aspects of the insertion of Aβ peptide into DPPG monolayers in water and trehalose subphases. In the water subphase, the insertion of the Aβ peptide into the monolayer exhibits a lag time which decreases with increasing temperature of the subphase. In the presence of trehalose, the lag time is completely eliminated and peptide insertion is completed within a shorter time period compared to that observed in pure water. Molecular simulations show that more peptide is inserted into the monolayer in the water subphase, and that such insertion is deeper. The peptide at the monolayer interface orients itself parallel to the monolayer, while it inserts with an angle of 50° in the trehalose subphase. Simulations also show that trehalose reduces the conformational change that the peptide undergoes when it inserts into the monolayer. This observation helps explain the experimentally observed elimination of the lag time by trehalose and the temperature dependence of the lag time in the water subphase.

    Biochimica et biophysica acta 2011;1808;1;26-33

  • In vivo regulation of amyloid precursor protein neuronal splicing by microRNAs.

    Smith P, Al Hashimi A, Girard J, Delay C and Hébert SS

    Centre de recherche du CHUQ (CHUL), Axe Neurosciences, Québec, Canada.

    The β-amyloid peptide that accumulate in Alzheimer's disease (AD) brain derive from proteolytic processing of the amyloid precursor protein (APP). Recent evidence suggest that microRNAs (miRNAs) participate in the post-transcriptional regulation of APP expression. Because gene dosage effects of the APP gene can cause genetic AD, dysregulation of the miRNA network could contribute significantly to disease. Here, we present evidence that, besides APP expression regulation, miRNAs are equally involved in the regulation of neuronal APP mRNA alternative splicing. Lack of miRNAs in post-mitotic neurons in vivo is associated with APP exons 7 and 8 inclusion, while ectopic expression of miR-124, an abundant neuronal-specific miRNA, reversed these effects in cultured neurons. Similar results were obtained by depletion of endogenous polypyrimidine tract binding protein 1 (PTBP1) in cells, a recognized miR-124 target gene. Furthermore, PTBP1 levels correlate with the presence of APP exons 7 and 8, while PTBP2 levels correlate with the skipping of these exons during neuronal differentiation. Finally, we show that miR-124 is down-regulated in AD brain. In sum, our results suggest that specific miRNAs are involved in the fine-tuning of APP alternative splicing in neurons. Since abnormal neuronal splicing of APP affects β-amyloid peptide production, these results could contribute to the understanding of the implication of miRNAs in brain health and disease.

    Journal of neurochemistry 2011;116;2;240-7

  • Mitochondrial γ-secretase participates in the metabolism of mitochondria-associated amyloid precursor protein.

    Pavlov PF, Wiehager B, Sakai J, Frykman S, Behbahani H, Winblad B and Ankarcrona M

    Karolinska Institutet and Dainippon Sumitomo Pharma Alzheimer Center, Department of Neurobiology, Care Sciences and Society, Karolinska Institutet, Huddinge, Sweden.

    Intracellular amyloid-β peptide (Aβ) has been implicated in the pathogenesis of Alzheimer's disease (AD). Mitochondria were found to be the target both for amyloid precursor protein (APP) that accumulates in the mitochondrial import channels and for Aβ that interacts with several proteins inside mitochondria and leads to mitochondrial dysfunction. Here, we have studied the role of mitochondrial γ-secretase in processing different substrates. We found that a significant proportion of APP is associated with mitochondria in cultured cells and that γ-secretase cleaves the shedded C-terminal part of APP identified as C83 associated with the outer membrane of mitochondria (OMM). Moreover, we have established the topology of the C83 in the OMM and found the APP intracellular domain (AICD) to be located inside mitochondria. Our data show for the first time that APP is a substrate for the mitochondrial γ-secretase and that AICD is produced inside mitochondria. Thus, we provide a mechanistic view of the mitochondria-associated APP metabolism where AICD, P3 peptide and potentially Aβ are produced locally and may contribute to mitochondrial dysfunction in AD.

    FASEB journal : official publication of the Federation of American Societies for Experimental Biology 2011;25;1;78-88

  • Neprilysin-2 is an important β-amyloid degrading enzyme.

    Hafez D, Huang JY, Huynh AM, Valtierra S, Rockenstein E, Bruno AM, Lu B, DesGroseillers L, Masliah E and Marr RA

    Department of Neuroscience, Rosalind Franklin University of Medicine and Science, North Chicago, Illinois 60064, USA.

    Proteases that degrade the amyloid-β peptide (Aβ) are important in protecting against Alzheimer's disease (AD), and understanding these proteases is critical to understanding AD pathology. Endopeptidases sensitive to inhibition by thiorphan and phosphoramidon are especially important, because these inhibitors induce dramatic Aβ accumulation (∼30- to 50-fold) and pathological deposition in rodents. The Aβ-degrading enzyme neprilysin (NEP) is the best known target of these inhibitors. However, genetic ablation of NEP results in only modest increases (∼1.5- to 2-fold) in Aβ, indicating that other thiorphan/phosphoramidon-sensitive endopeptidases are at work. Of particular interest is the NEP homolog neprilysin 2 (NEP2), which is thiorphan/phosphoramidon-sensitive and degrades Aβ. We investigated the role of NEP2 in Aβ degradation in vivo through the use of gene knockout and transgenic mice. Mice deficient for the NEP2 gene showed significant elevations in total Aβ species in the hippocampus and brainstem/diencephalon (∼1.5-fold). Increases in Aβ accumulation were more dramatic in NEP2 knockout mice crossbred with APP transgenic mice. In NEP/NEP2 double-knockout mice, Aβ levels were marginally increased (∼1.5- to 2-fold), compared with NEP(-/-)/NEP2(+/+) controls. Treatment of these double-knockout mice with phosphoramidon resulted in elevations of Aβ, suggesting that yet other NEP-like Aβ-degrading endopeptidases are contributing to Aβ catabolism.

    The American journal of pathology 2011;178;1;306-12

  • The transcriptionally active amyloid precursor protein (APP) intracellular domain is preferentially produced from the 695 isoform of APP in a {beta}-secretase-dependent pathway.

    Belyaev ND, Kellett KA, Beckett C, Makova NZ, Revett TJ, Nalivaeva NN, Hooper NM and Turner AJ

    Proteolysis Research Group, Faculty of Biological Sciences, Institute of Molecular and Cellular Biology, University of Leeds, Leeds LS2 9JT, United Kingdom.

    Amyloidogenic processing of the amyloid precursor protein (APP) by β- and γ-secretases generates several biologically active products, including amyloid-β (Aβ) and the APP intracellular domain (AICD). AICD regulates transcription of several neuronal genes, especially the Aβ-degrading enzyme, neprilysin (NEP). APP exists in several alternatively spliced isoforms, APP(695), APP(751), and APP(770). We have examined whether each isoform can contribute to AICD generation and hence up-regulation of NEP expression. Using SH-SY5Y neuronal cells stably expressing each of the APP isoforms, we observed that only APP(695) up-regulated nuclear AICD levels (9-fold) and NEP expression (6-fold). Increased NEP expression was abolished by a β- or γ-secretase inhibitor but not an α-secretase inhibitor. This correlated with a marked increase in both Aβ(1-40) and Aβ(1-42) in APP(695) cells as compared with APP(751) or APP(770) cells. Similar phenomena were observed in Neuro2a but not HEK293 cells. SH-SY5Y cells expressing the Swedish mutant of APP(695) also showed an increase in Aβ levels and NEP expression as compared with wild-type APP(695) cells. Chromatin immunoprecipitation revealed that AICD was associated with the NEP promoter in APP(695), Neuro2a, and APP(Swe) cells but not APP(751) nor APP(770) cells where AICD was replaced by histone deacetylase 1 (HDAC1). AICD occupancy of the NEP promoter was replaced by HDAC1 after treatment of the APP(695) cells with a β- but not an α-secretase inhibitor. The increased AICD and NEP levels were significantly reduced in cholesterol-depleted APP(695) cells. In conclusion, Aβ and functional AICD appear to be preferentially synthesized through β-secretase action on APP(695).

    Funded by: Medical Research Council: G0501565, G0802189, G9824728

    The Journal of biological chemistry 2010;285;53;41443-54

  • GRK5 deficiency accelerates {beta}-amyloid accumulation in Tg2576 mice via impaired cholinergic activity.

    Cheng S, Li L, He S, Liu J, Sun Y, He M, Grasing K, Premont RT and Suo WZ

    Laboratory for Alzheimer's Disease and Aging Research, Kansas City Veterans Affairs Medical Center, Kansas City, Missouri 64128, USA.

    Membrane G protein-coupled receptor kinase 5 (GRK5) deficiency is linked to Alzheimer disease, yet its precise roles in the disease pathogenesis remain to be delineated. We have previously demonstrated that GRK5 deficiency selectively impairs desensitization of presynaptic M2 autoreceptors, which causes presynaptic M2 hyperactivity and inhibits acetylcholine release. Here we report that inactivation of one copy of Grk5 gene in transgenic mice overexpressing β-amyloid precursor protein (APP) carrying Swedish mutations (Tg2576 or APPsw) resulted in significantly increased β-amyloid (Aβ) accumulation, including increased Aβ(+) plaque burdens and soluble Aβ in brain lysates and interstitial fluid (ISF). In addition, secreted β-APP fragment (sAPPβ) also increased, whereas full-length APP level did not change, suggesting an alteration in favor of β-amyloidogenic APP processing in these animals. Reversely, perfusion of methoctramine, a selective M2 antagonist, fully corrected the difference between the control and GRK5-deficient APPsw mice for ISF Aβ. In contrast, a cholinesterase inhibitor, eserine, although significantly decreasing the ISF Aβ in both control and GRK5-deficient APPsw mice, failed to correct the difference between them. However, combining eserine with methoctramine additively reduced the ISF Aβ further in both animals. Altogether, these findings indicate that GRK5 deficiency accelerates β-amyloidogenic APP processing and Aβ accumulation in APPsw mice via impaired cholinergic activity and that presynaptic M2 hyperactivity is the specific target for eliminating the pathologic impact of GRK5 deficiency. Moreover, a combination of an M2 antagonist and a cholinesterase inhibitor may reach the maximal disease-modifying effect for both amyloid pathology and cholinergic dysfunction.

    The Journal of biological chemistry 2010;285;53;41541-8

  • Identification of low molecular weight pyroglutamate A{beta} oligomers in Alzheimer disease: a novel tool for therapy and diagnosis.

    Wirths O, Erck C, Martens H, Harmeier A, Geumann C, Jawhar S, Kumar S, Multhaup G, Walter J, Ingelsson M, Degerman-Gunnarsson M, Kalimo H, Huitinga I, Lannfelt L and Bayer TA

    Department of Molecular Psychiatry and Alzheimer, Graduate School, University Medicine Goettingen, 37075 Goettingen, Germany.

    N-terminally truncated Aβ peptides starting with pyroglutamate (AβpE3) represent a major fraction of all Aβ peptides in the brain of Alzheimer disease (AD) patients. AβpE3 has a higher aggregation propensity and stability and shows increased toxicity compared with full-length Aβ. In the present work, we generated a novel monoclonal antibody (9D5) that selectively recognizes oligomeric assemblies of AβpE3 and studied the potential involvement of oligomeric AβpE3 in vivo using transgenic mouse models as well as human brains from sporadic and familial AD cases. 9D5 showed an unusual staining pattern with almost nondetectable plaques in sporadic AD patients and non-demented controls. Interestingly, in sporadic and familial AD cases prominent intraneuronal and blood vessel staining was observed. Using a novel sandwich ELISA significantly decreased levels of oligomers in plasma samples from patients with AD compared with healthy controls were identified. Moreover, passive immunization of 5XFAD mice with 9D5 significantly reduced overall Aβ plaque load and AβpE3 levels, and normalized behavioral deficits. These data indicate that 9D5 is a therapeutically and diagnostically effective monoclonal antibody targeting low molecular weight AβpE3 oligomers.

    The Journal of biological chemistry 2010;285;53;41517-24

  • Alzheimer's disease peptide beta-amyloid interacts with fibrinogen and induces its oligomerization.

    Ahn HJ, Zamolodchikov D, Cortes-Canteli M, Norris EH, Glickman JF and Strickland S

    Laboratory of Neurobiology and Genetics, The Rockefeller University, New York, NY 10065, USA.

    Increasing evidence supports a vascular contribution to Alzheimer's disease (AD), but a direct connection between AD and the circulatory system has not been established. Previous work has shown that blood clots formed in the presence of the β-amyloid peptide (Aβ), which has been implicated in AD, have an abnormal structure and are resistant to degradation in vitro and in vivo. In the present study, we show that Aβ specifically interacts with fibrinogen with a K(d) of 26.3 ± 6.7 nM, that the binding site is located near the C terminus of the fibrinogen β-chain, and that the binding causes fibrinogen to oligomerize. These results suggest that the interaction between Aβ and fibrinogen modifies fibrinogen's structure, which may then lead to abnormal fibrin clot formation. Overall, our study indicates that the interaction between Aβ and fibrinogen may be an important contributor to the vascular abnormalities found in AD.

    Funded by: NINDS NIH HHS: NS050537, R01 NS050537

    Proceedings of the National Academy of Sciences of the United States of America 2010;107;50;21812-7

  • Human intravenous immunoglobulin provides protection against Aβ toxicity by multiple mechanisms in a mouse model of Alzheimer's disease.

    Magga J, Puli L, Pihlaja R, Kanninen K, Neulamaa S, Malm T, Härtig W, Grosche J, Goldsteins G, Tanila H, Koistinaho J and Koistinaho M

    Department of Neurobiology, A, I, Virtanen Institute for Molecular Sciences, University of Eastern Finland, Kuopio, Finland. Johanna.Magga@uef.fi

    Background: Purified intravenous immunoglobulin (IVIG) obtained from the plasma of healthy humans is indicated for the treatment of primary immunodeficiency disorders associated with defects in humoral immunity. IVIG contains naturally occurring auto-antibodies, including antibodies (Abs) against β-amyloid (Aβ) peptides accumulating in the brains of Alzheimer's disease (AD) patients. IVIG has been shown to alleviate AD pathology when studied with mildly affected AD patients. Although its mechanisms-of-action have been broadly studied, it remains unresolved how IVIG affects the removal of natively formed brain Aβ deposits by primary astrocytes and microglia, two major cell types involved in the neuroinflammatory responses.

    Methods: We first determined the effect of IVIG on Aβ toxicity in primary neuronal cell culture. The mechanisms-of-action of IVIG in reduction of Aβ burden was analyzed with ex vivo assay. We studied whether IVIG solubilizes natively formed Aβ deposits from brain sections of APP/PS1 mice or promotes Aβ removal by primary glial cells. We determined the role of lysosomal degradation pathway and Aβ Abs in the IVIG-promoted reduction of Aβ. Finally, we studied the penetration of IVIG into the brain parenchyma and interaction with brain deposits of human Aβ in a mouse model of AD in vivo.

    Results: IVIG was protective against Aβ toxicity in a primary mouse hippocampal neuron culture. IVIG modestly inhibited the fibrillization of synthetic Aβ1-42 but did not solubilize natively formed brain Aβ deposits ex vivo. IVIG enhanced microglia-mediated Aβ clearance ex vivo, with a mechanism linked to Aβ Abs and lysosomal degradation. The IVIG-enhanced Aβ clearance appears specific for microglia since IVIG did not affect Aβ clearance by astrocytes. The cellular mechanisms of Aβ clearance we observed have potential relevance in vivo since after peripheral administration IVIG penetrated to mouse brain tissue reaching highest concentrations in the hippocampus and bound selectively to Aβ deposits in co-localization with microglia.

    Conclusions: Our results demonstrate that IVIG promotes recognition and removal of natively formed brain Aβ deposits by primary microglia involving natural Aβ Abs in IVIG. These findings may have therapeutic relevance in vivo as IVIG penetrates through the blood-brain barrier and specifically binds to Aβ deposits in brain parenchyma.

    Journal of neuroinflammation 2010;7;90

  • Mutation analysis of the presenilin 1 N-terminal domain reveals a broad spectrum of gamma-secretase activity toward amyloid precursor protein and other substrates.

    Gong P, Vetrivel KS, Nguyen PD, Meckler X, Cheng H, Kounnas MZ, Wagner SL, Parent AT and Thinakaran G

    Department of Neurobiology, University of Chicago, Chicago, Illinois 60637, USA.

    The γ-secretase protein complex executes the intramembrane proteolysis of amyloid precursor protein (APP), which releases Alzheimer disease β-amyloid peptide. In addition to APP, γ-secretase also cleaves several other type I membrane protein substrates including Notch1 and N-cadherin. γ-Secretase is made of four integral transmembrane protein subunits: presenilin (PS), nicastrin, APH1, and PEN2. Multiple lines of evidence indicate that a heteromer of PS-derived N- and C-terminal fragments functions as the catalytic subunit of γ-secretase. Only limited information is available on the domains within each subunit involved in the recognition and recruitment of diverse substrates and the transfer of substrates to the catalytic site. Here, we performed mutagenesis of two domains of PS1, namely the first luminal loop domain (LL1) and the second transmembrane domain (TM2), and analyzed PS1 endoproteolysis as well as the catalytic activities of PS1 toward APP, Notch, and N-cadherin. Our results show that distinct residues within LL1 and TM2 domains as well as the length of the LL1 domain are critical for PS1 endoproteolysis, but not for PS1 complex formation with nicastrin, APH1, and PEN2. Furthermore, our experimental PS1 mutants formed γ-secretase complexes with distinct catalytic properties toward the three substrates examined in this study; however, the mutations did not affect PS1 interaction with the substrates. We conclude that the N-terminal LL1 and TM2 domains are critical for PS1 endoproteolysis and the coordination between the putative substrate-docking site and the catalytic core of the γ-secretase.

    Funded by: NIA NIH HHS: AG019070, AG021495, R01 AG019070, R01 AG021495; NINDS NIH HHS: NS055223, R01 NS055223

    The Journal of biological chemistry 2010;285;49;38042-52

  • Amyloid β accelerates phosphorylation of tau and neurofibrillary tangle formation in an amyloid precursor protein and tau double-transgenic mouse model.

    Seino Y, Kawarabayashi T, Wakasaya Y, Watanabe M, Takamura A, Yamamoto-Watanabe Y, Kurata T, Abe K, Ikeda M, Westaway D, Murakami T, Hyslop PS, Matsubara E and Shoji M

    Department of General Medicine, Mutsu General Hospital, Mutsu, Japan.

    In Alzheimer's disease, Aβ deposits are considered the initial cardinal events that induce tauopathy secondarily. However, the relationship between Aβ amyloidosis and tauopathy has not been determined in detail. We produced double transgenic mice, 2×TgTau(+/-) APP(+/-) , by mating Tg2576 mice that exhibit Aβ amyloidosis and TgTauP301L mice that show tauopathy, and statistically analyzed the effect of Aβ accumulation on tauopathy. There was no significant difference in theprogression of Aβ accumulation among 2×TgTau(+/-) APP(+/-) and 1×TgTau(-/-) APP(+/-) , and tau accumulation among 2×TgTau(+/-) APP(+/-) and 1×Tg Tau(+/-) APP(-/-) . The appearance rates of phosphorylated tau developing in neurons and processes were significantly accelerated in 2×TgTau(+/-) APP(+/-) mice compared with those in 1×TgTau(+/-) APP(-/-) mice at 23 months of age. Accumulation of phosphorylated and confomationally altered tau and GSK3β in neuronal processes was accelerated in the white matter in 2×TgTau(+/-) APP(+/-) . The level of phosphorylated tau in the sarkosyl-insoluble fraction was increased in 2×TgTau(+/-) APP(+/-) brains compared with that in 1×TgTau(+/-) APP(-/-) brains. Thus, Aβ amyloid partially enhances tauopathy through accumulation of insoluble, phosphorylated, and conformationally changed tau in neuronal cytoplasm and processes in the late stage.

    Funded by: Wellcome Trust: 081864

    Journal of neuroscience research 2010;88;16;3547-54

  • Transgenic mice overexpressing APP and transforming growth factor-beta1 feature cognitive and vascular hallmarks of Alzheimer's disease.

    Ongali B, Nicolakakis N, Lecrux C, Aboulkassim T, Rosa-Neto P, Papadopoulos P, Tong XK and Hamel E

    Laboratory of Cerebrovascular Research, Montreal Neurological Institute, McGill University, Montréal, QC, Canada H3A 2B4.

    High brain levels of amyloid-β (Aβ) and transforming growth factor-β1 (TGF-β1) have been implicated in the cognitive and cerebrovascular alterations of Alzheimer's disease (AD). We sought to investigate the impact of combined increases in Aβ and TGF-β1 on cerebrovascular, neuronal, and mnemonic function using transgenic mice overproducing these peptides (A/T mice). In particular, we measured cerebrovascular reactivity, evoked cerebral blood flow and glucose uptake during brain activation, cholinergic status, and spatial memory, along with cerebrovascular fibrosis, amyloidosis, and astrogliosis, and their evolution with age. An assessment of perfusion and metabolic responses was considered timely, given ongoing efforts for their validation as AD biomarkers. Relative to wild-type littermates, A/T mice displayed an early progressive decline in cerebrovascular dilatory ability, preserved contractility, and reduction in constitutive nitric oxide synthesis that establishes resting vessel tone. Altered levels of vasodilator-synthesizing enzymes and fibrotic proteins, resistance to antioxidant treatment, and unchanged levels of the antioxidant enzyme, superoxide dismutase-2, accompanied these impairments. A/T mice featured deficient neurovascular and neurometabolic coupling to whisker stimulation, cholinergic denervation, cerebral and cerebrovascular Aβ deposition, astrocyte activation, and impaired Morris water maze performance, which gained severity with age. The combined Aβ- and TGF-β1-driven pathology recapitulates salient cerebrovascular, neuronal, and cognitive AD landmarks and yields a versatile model toward highly anticipated diagnostic and therapeutic tools for patients featuring Aβ and TGF-β1 increments.

    Funded by: Canadian Institutes of Health Research: MOP-84275

    The American journal of pathology 2010;177;6;3071-80

  • Macroautophagy is not directly involved in the metabolism of amyloid precursor protein.

    Boland B, Smith DA, Mooney D, Jung SS, Walsh DM and Platt FM

    Laboratory for Neurodegenerative Research, School of Biomolecular and Biomedical Science, Conway Institute, University College Dublin, Dublin 4, Ireland. barry.boland@ucd.ie

    Alterations in the metabolism of amyloid precursor protein (APP) are believed to play a central role in Alzheimer disease pathogenesis. Burgeoning data indicate that APP is proteolytically processed in endosomal-autophagic-lysosomal compartments. In this study, we used both in vivo and in vitro paradigms to determine whether alterations in macroautophagy affect APP metabolism. Three mouse models of glycosphingolipid storage diseases, namely Niemann-Pick type C1, GM1 gangliosidosis, and Sandhoff disease, had mTOR-independent increases in the autophagic vacuole (AV)-associated protein, LC3-II, indicative of impaired lysosomal flux. APP C-terminal fragments (APP-CTFs) were also increased in brains of the three mouse models; however, discrepancies between LC3-II and APP-CTFs were seen between primary (GM1 gangliosidosis and Sandhoff disease) and secondary (Niemann-Pick type C1) lysosomal storage models. APP-CTFs were proportionately higher than LC3-II in cerebellar regions of GM1 gangliosidosis and Sandhoff disease, although LC3-II increased before APP-CTFs in brains of NPC1 mice. Endogenous murine Aβ40 from RIPA-soluble extracts was increased in brains of all three mice. The in vivo relationship between AV and APP-CTF accumulation was also seen in cultured neurons treated with agents that impair primary (chloroquine and leupeptin + pepstatin) and secondary (U18666A and vinblastine) lysosomal flux. However, Aβ secretion was unaffected by agents that induced autophagy (rapamycin) or impaired AV clearance, and LC3-II-positive AVs predominantly co-localized with degradative LAMP-1-positive lysosomes. These data suggest that neuronal macroautophagy does not directly regulate APP metabolism but highlights the important anti-amyloidogenic role of lysosomal proteolysis in post-secretase APP-CTF catabolism.

    Funded by: Wellcome Trust: 069883/B/02/Z

    The Journal of biological chemistry 2010;285;48;37415-26

  • Statins promote the degradation of extracellular amyloid {beta}-peptide by microglia via stimulation of exosome-associated insulin-degrading enzyme (IDE) secretion.

    Tamboli IY, Barth E, Christian L, Siepmann M, Kumar S, Singh S, Tolksdorf K, Heneka MT, Lütjohann D, Wunderlich P and Walter J

    Departments of Neurology, University of Bonn, Sigmund-Freud-Strasse 25, 53127 Bonn, Germany.

    Epidemiological studies indicate that intake of statins decrease the risk of developing Alzheimer disease. Cellular and in vivo studies suggested that statins might decrease the generation of the amyloid β-peptide (Aβ) from the β-amyloid precursor protein. Here, we show that statins potently stimulate the degradation of extracellular Aβ by microglia. The statin-dependent clearance of extracellular Aβ is mainly exerted by insulin-degrading enzyme (IDE) that is secreted in a nonconventional pathway in association with exosomes. Stimulated IDE secretion and Aβ degradation were also observed in blood of mice upon peripheral treatment with lovastatin. Importantly, increased IDE secretion upon lovastatin treatment was dependent on protein isoprenylation and up-regulation of exosome secretion by fusion of multivesicular bodies with the plasma membrane. These data demonstrate a novel pathway for the nonconventional secretion of IDE via exosomes. The modulation of this pathway could provide a new strategy to enhance the extracellular clearance of Aβ.

    The Journal of biological chemistry 2010;285;48;37405-14

  • Activation of extrasynaptic, but not synaptic, NMDA receptors modifies amyloid precursor protein expression pattern and increases amyloid-ß production.

    Bordji K, Becerril-Ortega J, Nicole O and Buisson A

    Unité Mixte de Recherche, Centre National de la Recherche Scientifique, Caen, France. bordji@cyceron.fr

    Calcium is a key mediator controlling essential neuronal functions depending on electrical activity. Altered neuronal calcium homeostasis affects metabolism of amyloid precursor protein (APP), leading to increased production of β-amyloid (Aβ), and contributing to the initiation of Alzheimer's disease (AD). A linkage between excessive glutamate receptor activation and neuronal Aβ release was established, and recent reports suggest that synaptic and extrasynaptic NMDA receptor (NMDAR) activation may have distinct consequences in plasticity, gene regulation, and neuronal death. Here, we report for the first time that prolonged activation of extrasynaptic NMDAR, but not synaptic NMDAR, dramatically increased the neuronal production of Aβ. This effect was preceded by a shift from APP695 to Kunitz protease inhibitory domain (KPI) containing APPs (KPI-APPs), isoforms exhibiting an important amyloidogenic potential. Conversely, after synaptic NMDAR activation, we failed to detect any KPI-APP expression and neuronal Aβ production was not modified. Calcium imaging data showed that intracellular calcium concentration after extrasynaptic NMDAR stimulation was lower than after synaptic activation. This suggests distinct signaling pathways for each pool of receptors. We found that modification of neuronal APP expression pattern triggered by extrasynaptic NMDAR activation was regulated at an alternative splicing level involving calcium-/calmodulin-dependent protein kinase IV, but overall APP expression remained identical. Finally, memantine dose-dependently inhibited extrasynaptic NMDAR-induced KPI-APPs expression as well as neuronal Aβ release. Altogether, these data suggest that a chronic activation of extrasynaptic NMDAR promotes amyloidogenic KPI-APP expression leading to neuronal Aβ release, representing a causal risk factor for developing AD.

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2010;30;47;15927-42

  • Multiple events lead to dendritic spine loss in triple transgenic Alzheimer's disease mice.

    Bittner T, Fuhrmann M, Burgold S, Ochs SM, Hoffmann N, Mitteregger G, Kretzschmar H, LaFerla FM and Herms J

    Center of Neuropathology and Prion Research, Ludwig Maximilians University, Munich, Germany.

    The pathology of Alzheimer's disease (AD) is characterized by the accumulation of amyloid-β (Aβ) peptide, hyperphosphorylated tau protein, neuronal death, and synaptic loss. By means of long-term two-photon in vivo imaging and confocal imaging, we characterized the spatio-temporal pattern of dendritic spine loss for the first time in 3xTg-AD mice. These mice exhibit an early loss of layer III neurons at 4 months of age, at a time when only soluble Aβ is abundant. Later on, dendritic spines are lost around amyloid plaques once they appear at 13 months of age. At the same age, we observed spine loss also in areas apart from amyloid plaques. This plaque independent spine loss manifests exclusively at dystrophic dendrites that accumulate both soluble Aβ and hyperphosphorylated tau intracellularly. Collectively, our data shows that three spatio-temporally independent events contribute to a net loss of dendritic spines. These events coincided either with the occurrence of intracellular soluble or extracellular fibrillar Aβ alone, or the combination of intracellular soluble Aβ and hyperphosphorylated tau.

    PloS one 2010;5;11;e15477

  • Tyr(682) in the intracellular domain of APP regulates amyloidogenic APP processing in vivo.

    Barbagallo AP, Weldon R, Tamayev R, Zhou D, Giliberto L, Foreman O and D'Adamio L

    Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York, United States of America.

    Background: The pathogenesis of Alzheimer's disease is attributed to misfolding of Amyloid-β (Aβ) peptides. Aβ is generated during amyloidogenic processing of Aβ-precursor protein (APP). Another characteristic of the AD brain is increased phosphorylation of APP amino acid Tyr(682). Tyr(682) is part of the Y(682)ENPTY(687) motif, a docking site for interaction with cytosolic proteins that regulate APP metabolism and signaling. For example, normal Aβ generation and secretion are dependent upon Tyr(682) in vitro. However, physiological functions of Tyr(682) are unknown.

    To this end, we have generated an APP Y682G knock-in (KI) mouse to help dissect the role of APP Tyr(682) in vivo. We have analyzed proteolytic products from both the amyloidogenic and non-amyloidogenic processing of APP and measure a profound shift towards non-amyloidogenic processing in APP KI mice. In addition, we demonstrate the essential nature of amino acid Tyr(682) for the APP/Fe65 interaction in vivo.

    Together, these observations point to an essential role of APP intracellular domain for normal APP processing and function in vivo, and provide rationale for further studies into physiological functions associated with this important phosphorylation site.

    Funded by: NIA NIH HHS: R01 AG033007, R01 AG033007-01A1, R01 AG033007-02, R01 AG033007-03, R01 AG033007-03S1, R01 AG033007-04, R01AG033007; NIGMS NIH HHS: T32 GM007288, T32 GM007491

    PloS one 2010;5;11;e15503

  • Altered synaptic plasticity in the mossy fibre pathway of transgenic mice expressing mutant amyloid precursor protein.

    Witton J, Brown JT, Jones MW and Randall AD

    School of Physiology and Pharmacology, University of Bristol, University Walk, Bristol, BS8 1TD, UK.

    Aβ peptides derived from the cleavage of amyloid precursor protein are widely believed to play an important role in the pathophysiology of Alzheimer's disease. A common way to study the impact of these molecules on CNS function is to compare the physiology of transgenic mice that overproduce Aβ with non-transgenic animals. In the hippocampus, this approach has been frequently applied to the investigation of synaptic transmission and plasticity in the perforant and Schaffer collateral commissural pathways, the first and third components of the classical hippocampal trisynaptic circuit, respectively. Similar studies however have not been carried out on the remaining component of the trisynaptic circuit, the mossy fibre pathway. Using transverse hippocampal slices prepared from ~2 year old animals we have compared mossy fibre synaptic function in wild-type mice and their Tg2576 littermates which age-dependently overproduce Aβ. Input-output curves were not altered in slices from Tg2576 mice, but these animals exhibited a significant loss of the prominent frequency-facilitation expressed by the mossy fibre pathway. In addition to this change in short term synaptic plasticity, high frequency stimulation-induced, NMDA-receptor-independent LTP was absent in slices from the transgenic mice. These data represent the first description of functional deficits in the mossy fibre pathway of Aβ-overproducing transgenic mice.

    Funded by: Medical Research Council: G0501146

    Molecular brain 2010;3;32

  • Amyloid precursor protein mediates monocyte adhesion in AD tissue and apoE(-)/(-) mice.

    Austin SA and Combs CK

    Department of Pharmacology, Physiology & Therapeutics, University of North Dakota, School of Medicine and Health Sciences, 504 Hamline St., Room 116, Grand Forks, ND 58203, United States.

    Amyloid precursor protein (APP) is a type 1 integral membrane protein, which is highly conserved and ubiquitously expressed. Numerous data suggest it functions in cellular adhesion. For example, APP binds components of the extracellular matrix to propagate intracellular signaling responses. In order to investigate adhesion-related changes in inflamed vasculature, brains from apolipoprotein E(-/-) (apoE(-/-)) mice were examined for changes related to APP then compared to human Alzheimer's disease (AD) brains. Cerebrovasculature from mouse apoE(-)/(-) and human AD brains revealed strong immunoreactivity for APP, APP phosphorylated at tyrosine residue 682 (pAPP) and Aβ. Further, Western blot analyses from mouse apoE(-/-) and AD brains showed statistically higher protein levels of APP, pAPP and increased APP association with the tyrosine kinase, Src. Lastly, utilizing a modified Stamper-Woodruff adhesion assay, we demonstrated that adhesion of monocytic cells to apoE(-/-) and AD brain endothelium is partially APP dependent. These data suggest that endothelial APP function coupled with increased Aβ production are involved in the vascular dysfunction associated with atherosclerosis and AD.

    Funded by: NCRR NIH HHS: 1 P20RR17699-01, 2P20RR017600-06, C06 RR017600, P20 RR017699; NIA NIH HHS: 1R01AG026330-01A2, R01 AG026330

    Neurobiology of aging 2010;31;11;1854-66

  • Effect of VEGF and its receptor antagonist SU-5416, an inhibitor of angiogenesis, on processing of the β-amyloid precursor protein in primary neuronal cells derived from brain tissue of Tg2576 mice.

    Bürger S, Yafai Y, Bigl M, Wiedemann P and Schliebs R

    Paul Flechsig Institute for Brain Research, Medical Faculty, University of Leipzig, Germany.

    A large number of Alzheimer patients demonstrate cerebrovascular pathology, which has been assumed to be related to β-amyloid (Aβ) deposition. Aβ peptides have been described to inhibit angiogenesis both in vitro and in vivo, and deregulation of angiogenic factors may contribute to various neurological disorders including neurodegeneration. One of the key angiogenic factor is the vascular endothelial growth factor (VEGF). Increased levels of VEGF have been observed in brains of Alzheimer patients, while the functional significance of VEGF up-regulation in the pathogenesis and progression of AD is still a matter of debate. To test whether VEGF may affect neuronal APP processing, primary neuronal cells derived from brain tissue of E16 embryos of Tg2576 mice were exposed with 1 ng/ml VEGF for 6, 12, and 24h, followed by monitoring formation and secretion of soluble Aβ peptides, release of the human APP cleavage products, sAPPβswe and sAPPα, into the culture medium as well as the activities of α- and β-secretases in neuronal cell extracts. Exposure of primary neuronal cells by VEGF for 24h led to slightly reduced sAPPβ release, accompanied by decreased β-secretase activity 12h after VEGF exposure. Incubation of neurons by the VEGF receptor antagonist and angiogenesis inhibitor SU-5416 for 24h resulted in increased release of sAPPβswe, and strikingly enhanced secretion of Aβ peptides into the culture medium, which was accompanied by a significant increase in β-secretase activity, as compared to control incubations. The SU-5416-induced effects on APP processing could not be suppressed by the additional presence of VEGF, suggesting that SU-5416 affects pathways that are apparently independent of VEGF receptor signaling. The data obtained indicate that VEGF-driven mechanisms may affect APP processing, suggesting a link of angiogenesis and pathogenesis of Alzheimer's disease.

    International journal of developmental neuroscience : the official journal of the International Society for Developmental Neuroscience 2010;28;7;597-604

  • In vivo effects of APP are not exacerbated by BACE2 co-overexpression: behavioural characterization of a double transgenic mouse model.

    Azkona G, Levannon D, Groner Y and Dierssen M

    Neurobehavioural Phenotyping of Mouse Models of Disease, Genes and Disease Program, Center for Genomic Regulation (CRG), Barcelona Biomedical Research Park (PRBB), Dr. Aiguader, 88, 08003, Barcelona, Catalonia, Spain.

    Down syndrome, the most common genetic disorder leading to mental retardation, is caused by the presence of all or part of an extra copy of chromosome 21. At relatively early ages, Down syndrome patients develop progressive formation and extracellular aggregation of amyloid-β peptide, considered as one of the causal factors for the pathogenesis of Alzheimer's disease. This neuropathological hallmark has been attributed to the overexpression of APP but could also be contributed by other HSA21 genes. BACE2 maps to HSA21 and is homologous to BACE1, a β-secretase involved in the amyloidogenic pathway of APP proteolysis, and thus it has been hypothesized that the co-overexpression of both genes could contribute to Alzheimer's like neuropathology present in Down syndrome. The aim of the present study has been to analyse the impact of the co-overexpression of BACE2 and APP, using a double transgenic mouse model. Double transgenic mice did not present any neurological or sensorimotor alterations, nor genotype-dependent anxiety-like behaviour or age-associated cognitive dysfunction. Interestingly, TgBACE2-APP mice showed deregulation of BACE2 expression levels that were significantly increased with respect to single TgBACE2 mice. Co-overexpression of BACE2 and APP did not increase amyloid-β peptide concentration in brain. Our results suggest that the in vivo effects of APP are not exacerbated by BACE2 co-overexpression but may have some protective effects in specific behavioural and cognitive domains in transgenic mice.

    Amino acids 2010;39;5;1571-80

  • Megalin interacts with APP and the intracellular adapter protein FE65 in neurons.

    Alvira-Botero X, Pérez-Gonzalez R, Spuch C, Vargas T, Antequera D, Garzón M, Bermejo-Pareja F and Carro E

    Laboratory of Neuroscience, Research Center, Hospital 12 de Octubre, 28041 Madrid, Spain.

    Increasing evidence has implicated megalin, a low-density lipoprotein receptor-related protein, in the pathogenesis of Alzheimer's disease (AD). In the brain, megalin is expressed in brain capillaries, ependymal cells and choroid plexus, where it participates in the clearance of brain amyloid β-peptide (Aβ) complex. Recently, megalin has also been detected in oligodendrocytes and astrocytes. In this study we demonstrate that megalin is widely distributed in neurons throughout the brain. Additionally, given that FE65 mediates the interaction between the low density lipoprotein receptor-related protein-1 and the amyloid precursor protein (APP) to modulate the rate of APP internalization from the cell surface, we hypothesize that megalin could also interact with APP in neurons. Our results confirm that megalin interacts with APP and FE65, suggesting that these three proteins form a tripartite complex. Moreover, our findings imply that megalin may participate in neurite branching. Taken together, these results indicate that megalin has an important role in Aβ-mediated neurotoxicity, and therefore may be involved in the neurodegenerative processes that occur in AD.

    Molecular and cellular neurosciences 2010;45;3;306-15

  • ATP-binding cassette transporter A1 mediates the beneficial effects of the liver X receptor agonist GW3965 on object recognition memory and amyloid burden in amyloid precursor protein/presenilin 1 mice.

    Donkin JJ, Stukas S, Hirsch-Reinshagen V, Namjoshi D, Wilkinson A, May S, Chan J, Fan J, Collins J and Wellington CL

    Department of Pathology and Laboratory Medicine, Child and Family Research Institute, University of British Columbia, Vancouver, British Columbia V5Z 4H4, Canada.

    The cholesterol transporter ATP-binding cassette transporter A1 (ABCA1) moves lipids onto apolipoproteins including apolipoprotein E (apoE), which is the major cholesterol carrier in the brain and an established genetic risk factor for late-onset Alzheimer disease (AD). In amyloid mouse models of AD, ABCA1 deficiency exacerbates amyloidogenesis, whereas ABCA1 overexpression ameliorates amyloid load, suggesting a role for ABCA1 in Aβ metabolism. Agonists of liver X receptors (LXR), including GW3965, induce transcription of several genes including ABCA1 and apoE, and reduce Aβ levels and improve cognition in AD mice. However, the specific role of ABCA1 in mediating beneficial responses to LXR agonists in AD mice is unknown. We evaluated behavioral and neuropathogical outcomes in GW3965-treated female APP/PS1 mice with and without ABCA1. Treatment of APP/PS1 mice with GW3965 increased ABCA1 and apoE protein levels. ABCA1 was required to observe significantly elevated apoE levels in brain tissue and cerebrospinal fluid upon therapeutic (33 mg/kg/day) GW3965 treatment. At 33 mg/kg/day, GW3965 was also associated with a trend toward redistribution of Aβ to the carbonate-soluble pool independent of ABCA1. APP/PS1 mice treated with either 2.5 or 33 mg/kg/day of GW3965 showed a clear trend toward reduced amyloid burden in hippocampus and whole brain, whereas APP/PS1-treated mice lacking ABCA1 failed to display reduced amyloid load in the whole brain and showed trends toward increased hippocampal amyloid. Treatment of APP/PS1 mice with either dose of GW3965 completely restored novel object recognition memory to wild-type levels, which required ABCA1. These results suggest that ABCA1 contributes to several beneficial effects of the LXR agonist GW3965 in APP/PS1 mice.

    Funded by: Canadian Institutes of Health Research

    The Journal of biological chemistry 2010;285;44;34144-54

  • Gene expression profiling in the stress control brain region hypothalamic paraventricular nucleus reveals a novel gene network including amyloid beta precursor protein.

    Tsolakidou A, Czibere L, Pütz B, Trümbach D, Panhuysen M, Deussing JM, Wurst W, Sillaber I, Landgraf R, Holsboer F and Rein T

    Max-Planck Institute of Psychiatry, Munich, Germany.

    Background: The pivotal role of stress in the precipitation of psychiatric diseases such as depression is generally accepted. This study aims at the identification of genes that are directly or indirectly responding to stress. Inbred mouse strains that had been evidenced to differ in their stress response as well as in their response to antidepressant treatment were chosen for RNA profiling after stress exposure. Gene expression and regulation was determined by microarray analyses and further evaluated by bioinformatics tools including pathway and cluster analyses.

    Results: Forced swimming as acute stressor was applied to C57BL/6J and DBA/2J mice and resulted in sets of regulated genes in the paraventricular nucleus of the hypothalamus (PVN), 4 h or 8 h after stress. Although the expression changes between the mouse strains were quite different, they unfolded in phases over time in both strains. Our search for connections between the regulated genes resulted in potential novel signalling pathways in stress. In particular, Guanine nucleotide binding protein, alpha inhibiting 2 (GNAi2) and amyloid β (A4) precursor protein (APP) were detected as stress-regulated genes, and together with other genes, seem to be integrated into stress-responsive pathways and gene networks in the PVN.

    Conclusions: This search for stress-regulated genes in the PVN revealed its impact on interesting genes (GNAi2 and APP) and a novel gene network. In particular the expression of APP in the PVN that is governing stress hormone balance, is of great interest. The reported neuroprotective role of this molecule in the CNS supports the idea that a short acute stress can elicit positive adaptational effects in the brain.

    BMC genomics 2010;11;546

  • Tau reduction prevents Abeta-induced defects in axonal transport.

    Vossel KA, Zhang K, Brodbeck J, Daub AC, Sharma P, Finkbeiner S, Cui B and Mucke L

    Gladstone Institute of Neurological Disease, San Francisco, CA 94158, USA. kvossel@gladstone.ucsf.edu

    Amyloid-β (Aβ) peptides, derived from the amyloid precursor protein, and the microtubule-associated protein tau are key pathogenic factors in Alzheimer's disease (AD). How exactly they impair cognitive functions is unknown. We assessed the effects of Aβ and tau on axonal transport of mitochondria and the neurotrophin receptor TrkA, cargoes that are critical for neuronal function and survival and whose distributions are altered in AD. Aβ oligomers rapidly inhibited axonal transport of these cargoes in wild-type neurons. Lowering tau levels prevented these defects without affecting baseline axonal transport. Thus, Aβ requires tau to impair axonal transport, and tau reduction protects against Aβ-induced axonal transport defects.

    Funded by: NCRR NIH HHS: C06 RR018928; NIA NIH HHS: AG011385, R01 AG011385, R01 AG011385-07, R37 AG011385; NINDS NIH HHS: K99 NS057906, NS041787, NS057906, R00 NS057906, R00 NS057906-05, R01 NS041787, R01 NS041787-09

    Science (New York, N.Y.) 2010;330;6001;198

  • Soluble amyloid precursor protein (APP) regulates transthyretin and Klotho gene expression without rescuing the essential function of APP.

    Li H, Wang B, Wang Z, Guo Q, Tabuchi K, Hammer RE, Südhof TC and Zheng H

    Huffington Center on Aging, Baylor College of Medicine, Houston, TX 77030, USA.

    Amyloidogenic processing of the amyloid precursor protein (APP) generates a large secreted ectodomain fragment (APPsβ), β-amyloid (Aβ) peptides, and an APP intracellular domain (AICD). Whereas Aβ is viewed as critical for Alzheimer's disease pathogenesis, the role of other APP processing products remains enigmatic. Of interest, the AICD has been implicated in transcriptional regulation, and N-terminal cleavage of APPsβ has been suggested to produce an active fragment that may mediate axonal pruning and neuronal cell death. We previously reported that mice deficient in APP and APP-like protein 2 (APLP2) exhibit early postnatal lethality and neuromuscular synapse defects, whereas mice with neuronal conditional deletion of APP and APLP2 are viable. Using transcriptional profiling, we now identify transthyretin (TTR) and Klotho as APP/APLP2-dependent genes whose expression is decreased in loss-of-function states but increased in gain-of-function states. Significantly, by creating an APP knockin allele that expresses only APPsβ protein, we demonstrate that APPsβ is not normally cleaved in vivo and is fully capable of mediating the APP-dependent regulation of TTR and Klotho gene expression. Despite being an active regulator of gene expression, APPsβ did not rescue the lethality and neuromuscular synapse defects of APP and APLP2 double-KO animals. Our studies identify TTR and Klotho as physiological targets of APP that are regulated by soluble APPsβ independent of developmental APP functions. This unexpected APP-mediated signaling pathway may play an important role in maintaining TTR and Klotho levels and their respective functions in Aβ sequestration and aging.

    Funded by: Howard Hughes Medical Institute; NIA NIH HHS: AG032051, AG033467, R01 AG032051, R01 AG033467; NICHD NIH HHS: P30 HD024064, P30HD024064; NIMH NIH HHS: MH52804, R37 MH052804

    Proceedings of the National Academy of Sciences of the United States of America 2010;107;40;17362-7

  • Genetic dissection of the amyloid precursor protein in developmental function and amyloid pathogenesis.

    Li H, Wang Z, Wang B, Guo Q, Dolios G, Tabuchi K, Hammer RE, Südhof TC, Wang R and Zheng H

    Huffington Center on Aging, Baylor College of Medicine, Houston, Texas 77030, USA.

    Proteolytic processing of the amyloid precursor protein (APP) generates large soluble APP derivatives, β-amyloid (Aβ) peptides, and APP intracellular domain. Expression of the extracellular sequences of APP or its Caenorhabditis elegans counterpart has been shown to be sufficient in partially rescuing the CNS phenotypes of the APP-deficient mice and the lethality of the apl-1 null C. elegans, respectively, leaving open the question as what is the role of the highly conserved APP intracellular domain? To address this question, we created an APP knock-in allele in which the mouse Aβ sequence was replaced by the human Aβ. A frameshift mutation was introduced that replaced the last 39 residues of the APP sequence. We demonstrate that the C-terminal mutation does not overtly affect APP processing and amyloid pathology. In contrast, crossing the mutant allele with APP-like protein 2 (APLP2)-null mice results in similar neuromuscular synapse defects and early postnatal lethality as compared with mice doubly deficient in APP and APLP2, demonstrating an indispensable role of the APP C-terminal domain in these development activities. Our results establish an essential function of the conserved APP intracellular domain in developmental regulation, and this activity can be genetically uncoupled from APP processing and Aβ pathogenesis.

    Funded by: Howard Hughes Medical Institute; NIA NIH HHS: AG020670, AG032051, R01 AG020670, R01 AG032051; NICHD NIH HHS: HD024064, P30 HD024064; NIMH NIH HHS: MH52804, R37 MH052804; NINDS NIH HHS: NS061777, P30 NS061777

    The Journal of biological chemistry 2010;285;40;30598-605

  • Circulating neprilysin clears brain amyloid.

    Liu Y, Studzinski C, Beckett T, Murphy MP, Klein RL and Hersh LB

    Department of Molecular and Cellular Biochemistry, University of Kentucky, College of Medicine, Lexington, KY 40536-0509, USA.

    The use of the peptidase neprilysin (NEP) as a therapeutic for lowering brain amyloid burden is receiving increasing attention. We have previously demonstrated that peripheral expression of NEP on the surface of hindlimb muscle lowers brain amyloid burden in a transgenic mouse model of Alzheimer's disease. In this study we now show that using adeno-associated virus expressing a soluble secreted form of NEP (secNEP-AAV8), NEP secreted into plasma is effective in clearing brain Abeta. Soluble NEP expression in plasma was sustained over the 3-month time period it was measured. Secreted NEP decreased plasma Abeta by 30%, soluble brain Abeta by approximately 28%, insoluble brain Abeta by approximately 55%, and Abeta oligomersby 12%. This secNEP did not change plasma levels of substance P or bradykinin, nor did it alter blood pressure. No NEP was detected in CSF, nor did the AAV virus produce brain expression of NEP. Thus the lowering of brain Abeta was due to plasma NEP which altered blood-brain Abeta transport dynamics. Expressing NEP in plasma provides a convenient way to monitor enzyme activity during the course of its therapeutic testing.

    Funded by: NCRR NIH HHS: P20 RR020171, P20RR02017; NIA NIH HHS: R21 AG024899; NIDA NIH HHS: DA 02243, R01 DA002243

    Molecular and cellular neurosciences 2010;45;2;101-7

  • Iron-export ferroxidase activity of β-amyloid precursor protein is inhibited by zinc in Alzheimer's disease.

    Duce JA, Tsatsanis A, Cater MA, James SA, Robb E, Wikhe K, Leong SL, Perez K, Johanssen T, Greenough MA, Cho HH, Galatis D, Moir RD, Masters CL, McLean C, Tanzi RE, Cappai R, Barnham KJ, Ciccotosto GD, Rogers JT and Bush AI

    Mental Health Research Institute, The University of Melbourne, Parkville, Victoria 3052, Australia.

    Alzheimer's Disease (AD) is complicated by pro-oxidant intraneuronal Fe(2+) elevation as well as extracellular Zn(2+) accumulation within amyloid plaque. We found that the AD β-amyloid protein precursor (APP) possesses ferroxidase activity mediated by a conserved H-ferritin-like active site, which is inhibited specifically by Zn(2+). Like ceruloplasmin, APP catalytically oxidizes Fe(2+), loads Fe(3+) into transferrin, and has a major interaction with ferroportin in HEK293T cells (that lack ceruloplasmin) and in human cortical tissue. Ablation of APP in HEK293T cells and primary neurons induces marked iron retention, whereas increasing APP695 promotes iron export. Unlike normal mice, APP(-/-) mice are vulnerable to dietary iron exposure, which causes Fe(2+) accumulation and oxidative stress in cortical neurons. Paralleling iron accumulation, APP ferroxidase activity in AD postmortem neocortex is inhibited by endogenous Zn(2+), which we demonstrate can originate from Zn(2+)-laden amyloid aggregates and correlates with Aβ burden. Abnormal exchange of cortical zinc may link amyloid pathology with neuronal iron accumulation in AD.

    Funded by: NIA NIH HHS: 1R01AG12686, R01 AG012686, R01 AG012686-10

    Cell 2010;142;6;857-67

  • The functional neurophysiology of the amyloid precursor protein (APP) processing pathway.

    Randall AD, Witton J, Booth C, Hynes-Allen A and Brown JT

    MRC Centre for Synaptic Plasticity, Department of Anatomy, University of Bristol School of Medical Sciences, Bristol, UK. a.d.randall@bristol.ac.uk

    Amyloid beta (Abeta) peptides derived from proteolytic cleavage of amyloid precursor protein (APP) are thought to be a pivotal toxic species in the pathogenesis of Alzheimer's disease (AD). Furthermore, evidence has been accumulating that components of APP processing pathway are involved in non-pathological normal function of the CNS. In this review we aim to cover the extensive body of research aimed at understanding how components of this pathway contribute to neurophysiological function of the CNS in health and disease. We briefly outline changes to clinical neurophysiology seen in AD patients before discussing functional changes in mouse models of AD which range from changes to basal synaptic transmission and synaptic plasticity through to abnormal synchronous network activity. We then describe the various neurophysiological actions that are produced by application of exogenous Abeta in various forms, and finally discuss a number or other neurophysiological aspects of the APP pathway, including functional activities of components of secretase complexes other than Abeta production.

    Neuropharmacology 2010;59;4-5;243-67

  • ADDLs and the signaling web that leads to Alzheimer's disease.

    Krafft GA and Klein WL

    Acumen Pharmaceuticals, Glenview, IL 60025, USA. gkrafft@acumenpharm.com

    Today, it is widely accepted that ADDLs, soluble oligomeric assemblies of the amyloid beta peptide, play a prominent role in triggering the cognitive deficits and neurodegeneration that constitute Alzheimer's disease (AD). Within the past decade, the longstanding emphasis on fibrillar deposits and neuronal death has given way to a new paradigm involving ADDL-triggered aberrant synaptic signaling and consequent memory malfunction and neurodegeneration. As with any paradigm shift in biology, not all molecular details have been elucidated, and not all AD scientists are fully subscribed. Nevertheless, the ADDL paradigm affords a promising framework for ongoing AD research and for development of the first therapeutics endowed with the dual capabilities of immediate symptom reversal and long-term disease modification. In this review we provide a brief account of the discovery of ADDLs, followed by a summary of key results that address questions concerning ADDL structure and assembly, biological activity and therapeutic possibilities.

    Neuropharmacology 2010;59;4-5;230-42

  • DHEA prevents Aβ25-35-impaired survival of newborn neurons in the dentate gyrus through a modulation of PI3K-Akt-mTOR signaling.

    Li L, Xu B, Zhu Y, Chen L, Sokabe M and Chen L

    Department of Physiology, Nanjing Medical University, Jiangsu, China.

    Infusion (i.c.v.) of beta-amyloid 25-35 (Abeta(25-35)) stimulates proliferation of progenitor cells in the hippocampal dentate gyrus (DG) of adult male mice, but a large population of the newborn cells will die in the 2nd week after birth, a critical period for neurite growth. Neurosteroid dehydroepiandrosterone (DHEA) has been demonstrated to promote neurite growth. Herein, we report that the DHEA-treatment on 6-12 days after BrdU-injection (BrdU-D(6-12)) dose-dependently attenuates the loss of newborn neurons induced by Abeta(25-35)-infusion. The DHEA-neuroprotection was blocked by the sigma(1) receptor antagonist NE100 and mimicked by the sigma(1) receptor agonist PRE084 when administered on BrdU-D(6-12). The DHEA-action was sensitive to the PI3K inhibitor LY294002 and the mammalian target of rapamycin (mTOR) inhibitor rapamycin. The Abeta(25-35)-infusion decreased the levels of Akt, mTOR and p70S6k phosphorylation, which could be rescued by DHEA-treatment in a sigma(1) receptor-dependent manner. Furthermore, the Abeta(25-35)-infusion led to a decrease in the dendritic density and length of doublecortin positive cells in the DG, which also was improved by the DHEA-treatment on BrdU-D(6-12). These findings suggest that DHEA prevents the Abeta(25-35)-impaired survival and dendritic growth of newborn neurons through a sigma(1) receptor-mediated modulation of PI3K-Akt-mTOR-p70S6k signaling.

    Neuropharmacology 2010;59;4-5;323-33

  • The prion protein as a receptor for amyloid-beta.

    Kessels HW, Nguyen LN, Nabavi S and Malinow R

    Center for Neural Circuits and Behavior, 9500 Gilman Drive 0634, University of California at San Diego, La Jolla, California 92093, USA.

    Increased levels of brain amyloid-beta, a secreted peptide cleavage product of amyloid precursor protein (APP), is believed to be critical in the aetiology of Alzheimer's disease. Increased amyloid-beta can cause synaptic depression, reduce the number of spine protrusions (that is, sites of synaptic contacts) and block long-term synaptic potentiation (LTP), a form of synaptic plasticity; however, the receptor through which amyloid-beta produces these synaptic perturbations has remained elusive. Laurén et al. suggested that binding between oligomeric amyloid-beta (a form of amyloid-beta thought to be most active) and the cellular prion protein (PrP(C)) is necessary for synaptic perturbations. Here we show that PrP(C) is not required for amyloid-beta-induced synaptic depression, reduction in spine density, or blockade of LTP; our results indicate that amyloid-beta-mediated synaptic defects do not require PrP(c).

    Funded by: NIA NIH HHS: R01 AG032132, R01 AG032132-14, R01 AG032132-15, R01 AG032132-17, R01 AG032132-18; NIGMS NIH HHS: T32 GM008444; NIMH NIH HHS: R01 MH049159, R01 MH049159-09, R01 MH049159-21, R01 MH049159-22

    Nature 2010;466;7308;E3-4; discussion E4-5

  • Beta amyloid-independent role of amyloid precursor protein in generation and maintenance of dendritic spines.

    Lee KJ, Moussa CE, Lee Y, Sung Y, Howell BW, Turner RS, Pak DT and Hoe HS

    Department of Pharmacology, Georgetown University Medical Center, 3900 Reservoir Road NW, Washington, DC 20057-1464, USA.

    Synapse loss induced by amyloid beta (Abeta) is thought to be a primary contributor to cognitive decline in Alzheimer's disease. Abeta is generated by proteolysis of amyloid precursor protein (APP), a synaptic receptor whose physiological function remains unclear. In the present study, we investigated the role of APP in dendritic spine formation, which is known to be important for learning and memory. We found that overexpression of APP increased spine number, whereas knockdown of APP reduced spine density in cultured hippocampal neurons. This spine-promoting effect of APP required both the extracellular and intracellular domains of APP, and was accompanied by specific upregulation of the GluR2, but not the GluR1, subunit of AMPA receptors. In an in vivo experiment, we found that cortical layers II/III and hippocampal CA1 pyramidal neurons in 1 year-old APP-deficient mice had fewer and shorter dendritic spines than wild-type littermates. In contrast, transgenic mice overexpressing mutant APP exhibited increased spine density compared to control animals, though only at a young age prior to overaccumulation of soluble amyloid. Additionally, increased glutamate synthesis was observed in young APP transgenic brains, whereas glutamate levels were decreased and GABA levels were increased in APP-deficient mice. These results demonstrate that APP is important for promoting spine formation and is required for proper spine development.

    Funded by: NIA NIH HHS: AG026478, AG032330, AG032330-02S1, AG30378, K01 AG030378, R01 AG026478, R03 AG032330, R03 AG034253; NINDS NIH HHS: NS5048085, R01 NS048085, R01 NS048085-05

    Neuroscience 2010;169;1;344-56

  • APP intracellular domain impairs adult neurogenesis in transgenic mice by inducing neuroinflammation.

    Ghosal K, Stathopoulos A and Pimplikar SW

    Department of Neurosciences, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio, United States of America.

    Background: A devastating aspect of Alzheimer's disease (AD) is the progressive deterioration of memory due to neuronal loss. Amyloid precursor protein (APP) occupies a central position in AD and APP-derived amyloid-beta (Abeta) peptides are thought to play a pivotal role in disease pathogenesis. Nonetheless, it is becoming clear that AD etiology is highly complex and that factors other than Abeta also contribute to AD pathogenesis. APP intracellular domain (AICD) is generated together with Abeta and we recently showed that AICD transgenic mice recapitulate pathological features of AD such as tau hyperphosphorylation, memory deficits and neurodegeneration without increasing the Abeta levels. Since impaired adult neurogenesis is shown to augment memory deficits in AD mouse models, here we examined the status of adult neurogenesis in AICD transgenic mice.

    We previously generated transgenic mice co-expressing 59-residue long AICD fragment and its binding partner Fe65. Hippocampal progenitor cell proliferation was determined by BrdU incorporation at 1.5, 3 and 12 months of age. Only male transgenic and their respective wilt type littermate control mice were used. We find age-dependent decrease in BrdU incorporation and doublecortin-positive cells in the dentate gyrus of AICD transgenic mice suggesting impaired adult neurogenesis. This deficit resulted from decreased proliferation and survival, whereas neuronal differentiation remained unaffected. Importantly, this impairment was independent of Abeta since APP-KO mice expressing AICD also exhibit reduced neurogenesis. The defects in adult neurogenesis are prevented by long-term treatment with the non-steroidal anti-inflammatory agents ibuprofen or naproxen suggesting that neuroinflammation is critically involved in impaired adult neurogenesis in AICD transgenic mice.

    Since adult neurogenesis is crucial for spatial memory, which is particularly vulnerable in AD, these findings suggest that AICD can exacerbate memory defects in AD by impairing adult neurogenesis. Our findings further establish that AICD, in addition to Abeta, contributes to AD pathology and that neuroinflammation plays a much broader role in AD pathogenesis than previously thought.

    Funded by: NIA NIH HHS: R01 AG026146, R01-AG026146

    PloS one 2010;5;7;e11866

  • Low-density lipoprotein receptor-related protein 1 (LRP1) mediates neuronal Abeta42 uptake and lysosomal trafficking.

    Fuentealba RA, Liu Q, Zhang J, Kanekiyo T, Hu X, Lee JM, LaDu MJ and Bu G

    Department of Pediatrics, Washington University School of Medicine, St. Louis, Missouri, United States of America.

    Background: Alzheimer's disease (AD) is characterized by the presence of early intraneuronal deposits of amyloid-beta 42 (Abeta42) that precede extracellular amyloid deposition in vulnerable brain regions. It has been hypothesized that endosomal/lysosomal dysfunction might be associated with the pathological accumulation of intracellular Abeta42 in the brain. Our previous findings suggest that the LDL receptor-related protein 1 (LRP1), a major receptor for apolipoprotein E, facilitates intraneuronal Abeta42 accumulation in mouse brain. However, direct evidence of neuronal endocytosis of Abeta42 through LRP1 is lacking.

    Here we show that LRP1 endocytic function is required for neuronal Abeta42 uptake. Overexpression of a functional LRP1 minireceptor, mLRP4, increases Abeta42 uptake and accumulation in neuronal lysosomes. Conversely, knockdown of LRP1 expression significantly decreases neuronal Abeta42 uptake. Disruptions of LRP1 endocytic function by either clathrin knockdown or by removal of its cytoplasmic tail decreased both uptake and accumulation of Abeta42 in neurons. Finally, we show that LRP1-mediated neuronal accumulation of Abeta42 is associated with increased cellular toxicity.

    These results demonstrate that LRP1 endocytic function plays an important role in the uptake and accumulation of Abeta42 in neuronal lysosomes. These findings emphasize the central function of LRP1 in neuronal Abeta metabolism.

    Funded by: NIA NIH HHS: P01 AG021184, P01 AG030128, P01 AG030128-02, P01AG021184, R01 AG019121, R01 AG027924, R01 AG035355, R01AG027924, R01AG19121; NINDS NIH HHS: P30 NS057105, P30NS057105

    PloS one 2010;5;7;e11884

  • SIRT1 suppresses beta-amyloid production by activating the alpha-secretase gene ADAM10.

    Donmez G, Wang D, Cohen DE and Guarente L

    Paul F. Glenn Laboratory and Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.

    A hallmark of Alzheimer's disease (AD) is the accumulation of plaques of Abeta 1-40 and 1-42 peptides, which result from the sequential cleavage of APP by the beta and gamma-secretases. The production of Abeta peptides is avoided by alternate cleavage of APP by the alpha and gamma-secretases. Here we show that production of beta-amyloid and plaques in a mouse model of AD are reduced by overexpressing the NAD-dependent deacetylase SIRT1 in brain, and are increased by knocking out SIRT1 in brain. SIRT1 directly activates the transcription of the gene encoding the alpha-secretase, ADAM10. SIRT1 deacetylates and coactivates the retinoic acid receptor beta, a known regulator of ADAM10 transcription. ADAM10 activation by SIRT1 also induces the Notch pathway, which is known to repair neuronal damage in the brain. Our findings indicate SIRT1 activation is a viable strategy to combat AD and perhaps other neurodegenerative diseases.

    Funded by: NIA NIH HHS: R01 AG015339, R01 AG015339-08

    Cell 2010;142;2;320-32

  • Oligemic hypoperfusion differentially affects tau and amyloid-{beta}.

    Koike MA, Green KN, Blurton-Jones M and Laferla FM

    Department of Neurobiology and Behavior, Institute for Memory Impairments and Neurological Disorders, University of California, Irvine, 3212 Biological Sciences III, Irvine, CA 92697-4545, USA.

    Decreased blood flow to the brain in humans is associated with altered Alzheimer's disease (AD)-related pathology, although the underlying mechanisms by which hypoperfusion influences AD neuropathology remains unknown. To try to address this question, we developed an oligemic model of cerebral hypoperfusion in the 3xTg-AD mouse model of AD. We bilaterally and transiently occluded the common carotid artery and then examined the molecular and cellular pathways by which hypoperfusion influenced tau and amyloid-beta proteins. We report the novel finding that a single, mild, transient hypoperfusion insult acutely increases Abeta levels by enhancing beta-secretase protein expression. In contrast, transient hypoperfusion markedly decreases total tau levels, coincident with activation of macroautophagy and ubiquitin-proteosome pathways. Furthermore, we find that oligemia results in a significant increase specifically in tau phosphorylated at serine(212) and threonine(214), a tau epitope associated with paired helical filaments in AD patients. Despite the mild and transient nature of this hypoperfusion injury, the pattern of decreased total tau, altered phosphorylated tau, and increased amyloid-beta persisted for several weeks postoligemia. Our study indicates that a single, mild, cerebral hypoperfusion event produces profound and long lasting effects on both tau and amyloid-beta. This finding may have implications for the pathogenesis of AD, as it indicates for the first time that total tau and amyloid-beta are differentially impacted by mild hypoperfusion.

    Funded by: NIA NIH HHS: AG-021982, AG-029378, K01 AG029378, K01 AG029378-03, R01 AG021982; NINDS NIH HHS: 1F31NS063650-01A1, F31 NS063650

    The American journal of pathology 2010;177;1;300-10

  • Regulation of cholesterol efflux by amyloid beta secretion.

    Umeda T, Mori H, Zheng H and Tomiyama T

    Department of Neuroscience, Osaka City University Graduate School of Medicine, Osaka, Japan.

    Amyloid beta (Abeta) is a key molecule in the pathogenesis of Alzheimer's disease, but its physiological function remains unclear. Abeta is produced from amyloid precursor protein (APP) by beta- and gamma-secretases, which is enhanced by high levels of cellular cholesterol, so cholesterol is a risk factor for Alzheimer's disease. This linkage led us to hypothesize that Abeta is produced to regulate cellular cholesterol levels in response to high-cholesterol stimulation. Here we show that Abeta production caused a reduction of cellular cholesterol levels in transfected HEK293 cells and neuronal IMR-32 and Neuro2a cells, which was accompanied by an increase in efflux of cholesterol from cells. Fractionation of the culture media by ultracentrifugation and subsequent immunoelectron microscopic observation revealed that Abeta assembled high-density lipoprotein-like particles with cellular cholesterol during its secretion. This assembly was mediated by the ATP-binding cassette transporter A1. APP transgenic and knockout mice exhibited lower and higher levels of cellular cholesterol in their brains, suggesting that Abeta-mediated regulation of cellular cholesterol is physiological. Furthermore, we found that, when injected into mouse cerebral ventricle, reconstituted lipoproteins with Abeta were excreted into the peripheral tissues more efficiently than those without Abeta. This result suggests that Abeta mediates cholesterol transport from the brain to the circulation. We propose, based on these findings, a novel, apolipoprotein-like function for Abeta that is involved in maintenance of cellular and cerebral cholesterol homeostasis.

    Journal of neuroscience research 2010;88;9;1985-94

  • LGI3 interacts with flotillin-1 to mediate APP trafficking and exosome formation.

    Okabayashi S and Kimura N

    Laboratory of Disease Control, Tsukuba Primate Research Center, National Institute of Biomedical Innovation, Tsukuba-shi, Ibaraki 305-0843, Japan. kimura@nibio.go.jp

    We recently showed that leucine-rich glioma inactivated 3 (LGI3) mediates the internalization of beta-amyloid protein and transferrin, a well-known marker for clathrin-dependent endocytosis, in neural cells. These findings strongly suggest that LGI3 is involved in the endocytosis system in the brain; however, the precise function of LGI3 remains unclear. Here, we show that LGI3 interacts with flotillin-1 (Flo1), and RNA interference analysis shows that LGI3 stabilized Flo1, and Flo1 also stabilized LGI3 vice versa. Moreover, the downregulation of the LGI3/Flo1 complex altered beta-amyloid precursor protein trafficking directly to late endosomes and disrupted exosome formation, suggesting that LGI3 is involved not only in endocytosis but also in another intracellular transport system through binding with its co-factor such as Flo1.

    Neuroreport 2010;21;9;606-10

  • Lowering beta-amyloid levels rescues learning and memory in a Down syndrome mouse model.

    Netzer WJ, Powell C, Nong Y, Blundell J, Wong L, Duff K, Flajolet M and Greengard P

    Laboratory of Molecular and Cellular Neuroscience, The Rockefeller University, New York, New York, USA. netzerw@rockefeller.edu

    beta-amyloid levels are elevated in Down syndrome (DS) patients throughout life and are believed to cause Alzheimer's disease (AD) in adult members of this population. However, it is not known if beta-amyloid contributes to intellectual disability in younger individuals. We used a gamma-secretase inhibitor to lower beta-amyloid levels in young mice that model DS. This treatment corrected learning deficits characteristic of these mice, suggesting that beta-amyloid-lowering therapies might improve cognitive function in young DS patients.

    Funded by: NIA NIH HHS: AG09464, P01 AG009464; NICHD NIH HHS: R21 HD065290, R21HD065290; NIMH NIH HHS: K08 MH065975, R01 MH081164, R01MH081164

    PloS one 2010;5;6;e10943

  • Increased tau phosphorylation and beta amyloid in the hipocampus of mouse pups by early life lead exposure.

    Li N, Yu ZL, Wang L, Zheng YT, Jia JX, Wang Q, Zhu MJ, Liu XL, Xia X and Li WJ

    Zhengzhou University College of Public Health Zhengzhou 450001 China.

    The aim of this study was to investigate the effects of maternal lead exposure on the learning and memory ability and expression of tau protein phosphorylation (P-tau) and beta amyloid protein (Abeta) in hippocampus of mice offspring. Pb exposure initiated from beginning of gestation to weaning. Pb acetate administered in drinking solutions was dissolved in distilled deionized water at the concentrations of 0.1%, 0.5% and 1% groups. On the 21 th of postnatal day, the learning and memory ability of the mouse pups was tested by Water Maze test and the Pb levels in blood and hippocampus of the offspring were also determined. The expression of P-tau and Abeta in hippocampus was measured by immunohistochemistry and Western blotting. The Pb levels in blood and hippocampus of all exposure groups were significantly higher than that of the control group ( P < 0.05). In Water Maze test, the performances of 0.5% and 1% groups were worse than that of the control group ( P < 0.05). The expression of P-tau and Abeta was increased in Pb exposed groups than that of the control group ( P < 0.05). Tau hyper-phosphorylation and Abeta increase in the hippocampus of pups may contribute to the impairment of learning and memory associated with maternal Pb exposure.

    Acta biologica Hungarica 2010;61;2;123-34

  • Interaction of a novel mitochondrial protein, 4-nitrophenylphosphatase domain and non-neuronal SNAP25-like protein homolog 1 (NIPSNAP1), with the amyloid precursor protein family.

    Tummala H, Li X and Homayouni R

    College of Pharmacy, South Dakota State University, IM 108, Brookings, SD 57006, USA. hemachand.tummala@sdstate.edu

    Amyloid precursor protein (APP) and its paralogs, amyloid precursor-like protein-1 and amyloid precursor-like protein-2, appear to have redundant but essential role(s) during development. To gain insights into the physiological and possibly pathophysiological functions of APP, we used a functional proteomic approach to identify proteins that interact with the highly conserved C-terminal region of APP family proteins. Previously, we characterized an interaction between APP and ubiquitous mitochondrial creatine kinase. Here, we describe an interaction between APP and a novel protein, 4-nitrophenylphosphatase domain and non-neuronal SNAP25-like protein homolog 1 (NIPSNAP1). The interaction between APP and NIPSNAP1 was confirmed both in transiently transfected COS7 cells and in the mouse brain, where NIPSNAP1 is expressed at a high level. We demonstrate that NIPSNAP1 is targeted to the mitochondria via its N-terminal targeting sequence, and interacts with mitochondrial chaperone translocase of the outer membrane 22. Mitochondrial localization of NIPSNAP1 appears to be critical for its interaction with APP, and overexpression of APP appeared to disrupt NIPSNAP1 mitochondrial localization. Moreover, APP overexpression resulted in downregulation of NIPSNAP1 levels in cultured cells. Our data suggest that APP may affect mitochondrial function through a direct interaction with NIPSNAP1 as well as with other mitochondrial proteins.

    Funded by: NLM NIH HHS: LM07292

    The European journal of neuroscience 2010;31;11;1926-34

  • Abnormal post-translational and extracellular processing of brevican in plaque-bearing mice over-expressing APPsw.

    Ajmo JM, Bailey LA, Howell MD, Cortez LK, Pennypacker KR, Mehta HN, Morgan D, Gordon MN and Gottschall PE

    Department of Molecular Pharmacology and Physiology, University of South Florida College of Medicine, Tampa, Florida, USA.

    Aggregation of amyloid-beta (Abeta) in the forebrain of Alzheimer's disease (AD) subjects may disturb the molecular organization of the extracellular microenvironment that modulates neural and synaptic plasticity. Proteoglycans are major components of this extracellular environment. To test the hypothesis that Abeta, or another amyloid precursor protein (APP) dependent mechanism modifies the accumulation and/or turnover of extracellular proteoglycans, we examined whether the expression and processing of brevican, an abundant extracellular, chondroitin sulfate (CS)-bearing proteoglycan, were altered in brains of Abeta-depositing transgenic mice (APPsw - APP gene bearing the Swedish mutation) as a model of AD. The molecular size of CS chains attached to brevican was smaller in hippocampal tissue from APPsw mice bearing Abeta deposits compared to non-transgenic mice, likely because of changes in the CS chains. Also, the abundance of the major proteolytic fragment of brevican was markedly diminished in extracts from several telencephalic regions of APPsw mice compared to non-transgenic mice, yet these immunoreactive fragments appeared to accumulate adjacent to the plaque edge. These results suggest that Abeta or APP exert inhibitory effects on proteolytic cleavage mechanisms responsible for synthesis and turnover of proteoglycans. As proteoglycans stabilize synaptic structure and inhibit molecular plasticity, defective brevican processing observed in Abeta-bearing mice and potentially end-stage human AD, may contribute to deficient neural plasticity.

    Funded by: NIA NIH HHS: AG015490, AG018478, AG022101, R01 AG015490, R01 AG018478, R01 AG022101, R01 AG022101-04

    Journal of neurochemistry 2010;113;3;784-95

  • Profile for amyloid-beta and tau expression in primary cortical cultures from 3xTg-AD mice.

    Vale C, Alonso E, Rubiolo JA, Vieytes MR, LaFerla FM, Giménez-Llort L and Botana LM

    Departamento de Farmacología, Facultad de Veterinaria, Universidad de Santiago de Compostela, 27003, Lugo, Spain.

    Advances in transgenic technology as well as in the genetics of Alzheimer disease (AD) have allowed the establishment of animal models that reproduce amyloid-beta plaques and neurofibrillary tangles, the main pathological hallmarks of AD. Among these models, 3xTg-AD mice harboring PS1 (M146V), APP (Swe) and tau (P301L) human transgenes provided the model that most closely mimics human AD features. Although cortical cultures from 3xTg-AD mice have been shown to present disturbances in intracellular [Ca(2+)] homeostasis, the development of AD pathology in vitro has not been previously evaluated. In the current work, we determined the temporal profile for amyloid precursor protein, amyloid-beta and tau expression in primary cortical cultures from 3xTg-AD mice. Immunocytochemistry and Western blot analysis showed an increased expression of these proteins as well as several phosphorylated tau isoforms with time in culture. Alterations in calcium homeostasis and cholinergic and glutamatergic responses were also observed early in vitro. Thus, 3x-TgAD cortical neurons in vitro provide an exceptional tool to investigate pharmacological approaches as well as the cellular basis for AD and related diseases.

    Cellular and molecular neurobiology 2010;30;4;577-90

  • Molecular interplay between mammalian target of rapamycin (mTOR), amyloid-beta, and Tau: effects on cognitive impairments.

    Caccamo A, Majumder S, Richardson A, Strong R and Oddo S

    Department of Physiology, University of Texas Health Science Center, San Antonio, Texas 78245, USA.

    Accumulation of amyloid-beta (Abeta) and Tau is an invariant feature of Alzheimer disease (AD). The upstream role of Abeta accumulation in the disease pathogenesis is widely accepted, and there is strong evidence showing that Abeta accumulation causes cognitive impairments. However, the molecular mechanisms linking Abeta to cognitive decline remain to be elucidated. Here we show that the buildup of Abeta increases the mammalian target of rapamycin (mTOR) signaling, whereas decreasing mTOR signaling reduces Abeta levels, thereby highlighting an interrelation between mTOR signaling and Abeta. The mTOR pathway plays a central role in controlling protein homeostasis and hence, neuronal functions; indeed mTOR signaling regulates different forms of learning and memory. Using an animal model of AD, we show that pharmacologically restoring mTOR signaling with rapamycin rescues cognitive deficits and ameliorates Abeta and Tau pathology by increasing autophagy. Indeed, we further show that autophagy induction is necessary for the rapamycin-mediated reduction in Abeta levels. The results presented here provide a molecular basis for the Abeta-induced cognitive deficits and, moreover, show that rapamycin, an FDA approved drug, improves learning and memory and reduces Abeta and Tau pathology.

    Funded by: NIA NIH HHS: 1P30-AG-13319, AG29729-4

    The Journal of biological chemistry 2010;285;17;13107-20

  • Diabetes-accelerated memory dysfunction via cerebrovascular inflammation and Abeta deposition in an Alzheimer mouse model with diabetes.

    Takeda S, Sato N, Uchio-Yamada K, Sawada K, Kunieda T, Takeuchi D, Kurinami H, Shinohara M, Rakugi H and Morishita R

    Department of Clinical Gene Therapy, Graduate School of Medicine, Osaka University, Osaka 565-0871, Japan.

    Recent epidemiological studies suggest that diabetes mellitus is a strong risk factor for Alzheimer disease. However, the underlying mechanisms remain largely unknown. In this study, to investigate the pathophysiological interaction between these diseases, we generated animal models that reflect the pathologic conditions of both diseases. We crossed Alzheimer transgenic mice (APP23) with two types of diabetic mice (ob/ob and NSY mice), and analyzed their metabolic and brain pathology. The onset of diabetes exacerbated Alzheimer-like cognitive dysfunction without an increase in brain amyloid-beta burden in double-mutant (APP(+)-ob/ob) mice. Notably, APP(+)-ob/ob mice showed cerebrovascular inflammation and severe amyloid angiopathy. Conversely, the cross-bred mice showed an accelerated diabetic phenotype compared with ob/ob mice, suggesting that Alzheimer amyloid pathology could aggravate diabetes. Similarly, APP(+)-NSY fusion mice showed more severe glucose intolerance compared with diabetic NSY mice. Furthermore, high-fat diet feeding induced severe memory deficits in APP(+)-NSY mice without an increase in brain amyloid-beta load. Here, we created Alzheimer mouse models with early onset of cognitive dysfunction. Cerebrovascular changes and alteration in brain insulin signaling might play a pivotal role in this relationship. These findings could provide insights into this intensely debated association.

    Proceedings of the National Academy of Sciences of the United States of America 2010;107;15;7036-41

  • Aph-1 associates directly with full-length and C-terminal fragments of gamma-secretase substrates.

    Chen AC, Guo LY, Ostaszewski BL, Selkoe DJ and LaVoie MJ

    Center for Neurologic Diseases, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115, USA.

    Gamma-secretase is a ubiquitous, multiprotein enzyme composed of presenilin, nicastrin, Aph-1, and Pen-2. It mediates the intramembrane proteolysis of many type 1 proteins, plays an essential role in numerous signaling pathways, and helps drive the pathogenesis of Alzheimer disease by excising the hydrophobic, aggregation-prone amyloid beta-peptide from the beta-amyloid precursor protein. A central unresolved question is how its many substrates bind and enter the gamma-secretase complex. Here, we provide evidence that both the beta-amyloid precursor protein holoprotein and its C-terminal fragments, the immediate substrates of gamma-secretase, can associate with Aph-1 at overexpressed as well as endogenous protein levels. This association was observed using bi-directional co-immunoprecipitation in multiple systems and detergent conditions, and an beta-amyloid precursor protein-Aph-1 complex was specifically isolated following in situ cross-linking in living cells. In addition, another endogenous canonical gamma-substrate, Jagged, showed association of both its full-length and C-terminal fragment forms with Aph-1. We were also able to demonstrate that this interaction with substrates was conserved across the multiple isoforms of Aph-1 (beta, alphaS, and alphaL), as they were all able to bind beta-amyloid precursor protein with similar affinity. Finally, two highly conserved intramembrane histidines (His-171 and His-197) within Aph-1, which were recently shown to be important for gamma-secretase activity, are required for efficient binding of substrates. Taken together, our data suggest a dominant role for Aph-1 in interacting with gamma-secretase substrates prior to their processing by the proteolytic complex.

    Funded by: NIA NIH HHS: AG015379, AG023094, K01 AG023094, P01 AG015379, R37 AG006173

    The Journal of biological chemistry 2010;285;15;11378-91

  • Age-related loss of phospholipid asymmetry in APP(NLh)/APP(NLh) x PS-1(P264L)/PS-1(P264L) human double mutant knock-in mice: relevance to Alzheimer disease.

    Bader Lange ML, St Clair D, Markesbery WR, Studzinski CM, Murphy MP and Butterfield DA

    Department of Chemistry, University of Kentucky, Lexington, KY 40506-0055, USA.

    Using APP(NLh)/APP(NLh) x PS-1(P246L)/PS-1(P246L) human double knock-in (APP/PS-1) mice, we examined whether phosphatidylserine (PtdSer) asymmetry is significantly altered in brain of this familial Alzheimer disease mouse model in an age-dependent manner as a result of oxidative stress, toxic Abeta(1-42) oligomer production, and/or apoptosis. Annexin V (AV) and NBD-PS fluorescence in synaptosomes of wild-type (WT) and APP/PS-1 mice were used to determine PtdSer exposure with age, while Mg(2+) ATPase activity was determined to correlate PtdSer asymmetry changes with PtdSer translocase, flippase, activity. AV and NBD-PS results demonstrated significant PtdSer exposure beginning at 9 months compared to 1-month-old WT controls for both assays, a trend that was exacerbated in synaptosomes of APP/PS-1 mice. Decreasing Mg(2+) ATPase activity confirms that the age-related loss of PtdSer asymmetry is likely due to loss of flippase activity, more prominent in APP/PS-1 brain. Two-site sandwich ELISA on SDS- and FA-soluble APP/PS-1 brain fractions were conducted to correlate Abeta(1-40) and Abeta(1-42) levels with age-related trends determined from the AV, NBD-PS, and Mg(2+) ATPase assays. ELISA revealed a significant increase in both SDS- and FA-soluble Abeta(1-40) and Abeta(1-42) with age, consistent with PtdSer and flippase assay trends. Lastly, because PtdSer exposure is affected by pro-apoptotic caspase-3, levels of both latent and active forms were measured. Western blotting results demonstrated an increase in both active fragments of caspase-3 with age, while levels of pro-caspase-3 decrease. These results are discussed with relevance to loss of lipid asymmetry and consequent neurotoxicity in brain of subjects with Alzheimer disease.

    Funded by: NIA NIH HHS: AG-005119, AG-029839, AG-05119, AG-10836, P01 AG005119, P01 AG005119-110001, P01 AG005119-120001, P01 AG005119-12S20001, P01 AG005119-130001, P01 AG005119-13S10001, P01 AG005119-140001, P01 AG005119-150006, P01 AG005119-20A10010, P01 AG005119-210010, P01 AG010836, P01 AG010836-110011, P01 AG010836-120011, P01 AG010836-130011, P01 AG010836-140011, P01 AG010836-150011, R01 AG029839; NINDS NIH HHS: NS-058382, R01 NS058382, R01 NS058382-03

    Neurobiology of disease 2010;38;1;104-15

  • Cell-type dependent modulation of Notch signaling by the amyloid precursor protein.

    Oh SY, Chen CD and Abraham CR

    Department of Biochemistry, Boston University School of Medicine, Boston, Massachusetts, USA.

    The amyloid precursor protein is a ubiquitously expressed transmembrane protein that has been long implicated in the pathogenesis of Alzheimer's disease but its normal biological function has remained elusive despite extensive effort. We have previously reported the identification of Notch2 as an amyloid precursor protein interacting protein in E18 rat neurons. Here, we sought to reveal the physiologic consequences of this interaction. We report a functional relationship between amyloid precursor protein and Notch1, which does not affect Delta ligand binding. First, we observed interactions between the amyloid precursor protein and Notch in mouse embryonic stem cells lacking both presenilin 1 and presenilin 2, the active proteolytic components of the gamma-secretase complex, suggesting that these two transmembrane proteins can interact in the absence of presenilin. Next, we demonstrated that the amyloid precursor protein affects Notch signaling by using Notch-dependent luciferase assays in two cell lines, the human embryonic kidney 293 and the monkey kidney, COS7. We found that the amyloid precursor protein exerts opposing effects on Notch signaling in human embryonic kidney 293 vs. COS7 cells. Finally, we show that more Notch Intracellular Domain is found in the nucleus in the presence of exogenous amyloid precursor protein or its intracellular domain, suggesting the mechanism by which the amyloid precursor protein affects Notch signaling in certain cells. Our results provide evidence of potentially important communications between the amyloid precursor protein and Notch.

    Journal of neurochemistry 2010;113;1;262-74

  • Increased food intake leads to obesity and insulin resistance in the tg2576 Alzheimer's disease mouse model.

    Kohjima M, Sun Y and Chan L

    Department of Medicine, Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77030, USA.

    Recent studies suggest that hyperinsulinemia and insulin resistance are linked to Alzheimer's disease (AD). In this study, we used Tg2576 transgenic (Tg) mice, a widely used transgenic mouse model for AD, to explore the relationship between increased amyloid beta-peptide (Abeta) and insulin resistance. When fed a high-fat diet (HFD), Tg mice developed obesity and insulin resistance at 16 wk of age. Furthermore, HFD-fed Tg mice displayed abnormal feeding behavior and increased caloric intake with time. Although caloric intake of HFD-fed Tg mice was similar to that of normal diet-fed Tg or wild-type mice during 4 to 8 wk of age, it increased sharply at 12 wk, and went up further at 16 wk, which paralleled changes in the level of Abeta40 and Abeta42 in the brain of these mice. Limiting food intake in HFD-fed Tg mice by pair-feeding a caloric intake identical with that of normal diet-fed mice completely prevented the obesity and insulin intolerance of HFD-fed Tg mice. The hypothalamus of HFD-fed Tg mice had a significant decrease in the expression of the anorexigenic neuropeptide, brain-derived neurotrophic factor, at both the mRNA and protein levels. These findings suggest that the increased Abeta in the brain of HFD-fed Tg2576 mice is associated with reduced brain-derived neurotrophic factor expression, which led to abnormal feeding behavior and increased food intake, resulting in obesity and insulin resistance in these animals.

    Funded by: NHLBI NIH HHS: HL-51586, R01 HL051586; NIDDK NIH HHS: P30 DK079638, P30DK079638

    Endocrinology 2010;151;4;1532-40

  • Neurons generated from APP/APLP1/APLP2 triple knockout embryonic stem cells behave normally in vitro and in vivo: lack of evidence for a cell autonomous role of the amyloid precursor protein in neuronal differentiation.

    Bergmans BA, Shariati SA, Habets RL, Verstreken P, Schoonjans L, Müller U, Dotti CG and De Strooper B

    Laboratory of Neuronal Cell Biology and Gene Transfer, Leuven, Belgium.

    Alzheimer's disease amyloid precursor protein (APP) has been implicated in many neurobiologic processes, but supporting evidence remains indirect. Studies are confounded by the existence of two partially redundant APP homologues, APLP1 and APLP2. APP/APLP1/APLP2 triple knockout (APP tKO) mice display cobblestone lissencephaly and are perinatally lethal. To circumvent this problem, we generated APP triple knockout embryonic stem (ES) cells and differentiated these to APP triple knockout neurons in vitro and in vivo. In comparison with wild-type (WT) ES cell-derived neurons, APP tKO neurons formed equally pure neuronal cultures, had unaltered in vitro migratory capacities, had a similar acquisition of polarity, and were capable of extending long neurites and forming active excitatory synapses. These data were confirmed in vivo in chimeric mice with APP tKO neurons expressing the enhanced green fluorescent protein (eGFP) present in a WT background brain. The results suggest that the loss of the APP family of proteins has no major effect on these critical neuronal processes and that the apparent multitude of functions in which APP has been implicated might be characterized by molecular redundancy. Our stem cell culture provides an excellent tool to circumvent the problem of lack of viability of APP/APLP triple knockout mice and will help to explore the function of this intriguing protein further in vitro and in vivo.

    Stem cells (Dayton, Ohio) 2010;28;3;399-406

  • TAG-1 is an inhibitor of TGFbeta2-induced neuronal death via amyloid beta precursor protein.

    Tachi N, Hashimoto Y, Nawa M and Matsuoka M

    Department of Pharmacology, Tokyo Medical University, 6-1-1 Shinjuku, Tokyo 160-8402, Japan.

    Our earlier studies indicated that TGFbeta2-induced neuronal cell death by binding to the extracellular domain of amyloid beta precursor protein (APP) on the cell surface and by triggering an intracellular death signal pathway, mediated by a heterotrimeric G protein Go, Rac1/cdc42, ASK1, JNK, NADPH oxidase, and caspases in this order. Recently, transient axonal glycoprotein-1 (TAG-1), a glycophosphatidylinositol-linked protein, was identified as another natural ligand of APP. TAG-1 increases APP intracellular domain release and triggers FE65-dependent transcriptional activity in a gamma-secretase-dependent manner by binding to APP. In this study, we show that TAG-1 inhibits TGFbeta2-mediated neuronal cell death via APP by attenuating the binding of TGFbeta2 to APP in a gamma-secretase-independent manner. TAG-1 is expressed in murine hippocampal neurons at 8 weeks of age, but its expression is reduced at 8 and 20 months. These findings suggest that an age-related reduction of TAG-1 expression may predispose neurons to cell death, induced by the binding of TGFbeta2 to APP. This mechanism may contribute to the onset and the progression of Alzheimer's disease-relevant neuronal cell death.

    Biochemical and biophysical research communications 2010;394;1;119-25

  • Synaptic plasticity in the hippocampus of a APP/PS1 mouse model of Alzheimer's disease is impaired in old but not young mice.

    Gengler S, Hamilton A and Hölscher C

    School of Biomed Sciences, Ulster University, Coleraine, United Kingdom.

    Background: Alzheimer disease (AD) is a neurodegenerative disorder for which there is no cure. We have investigated synaptic plasticity in area CA1 in a novel AD mouse model (APPPS1-21) which expresses the Swedish mutation of APP and the L166P mutation of human PS-1. This model shows initial plaque formation at 2 months in the neocortex and 4 months in the hippocampus and displays beta-amyloid-associated pathologies and learning impairments.

    We tested long-term potentiation (LTP) and short term potentiation (paired-pulse facilitation, PPF) of synaptic transmission in vivo in area CA1 of the hippocampus. There was no difference in LTP or PPF at 4-5 months of age in APPPS1-21 mice compared to littermate controls. At 6 months of age there was also no difference in LTP but APPPS1-21 mice showed slightly increased PPF (p<0.03). In 8 months old mice, LTP was greatly impaired in APPPS-21 animals (p<0.0001) while PPF was not changed. At 15 months of age, APPPS1-21 mice showed again impaired LTP compared to littermate controls (p<0.005), and PPF was also significantly reduced at 80 ms (p<0.005) and 160 ms (p<0.01) interstimulus interval. Immunohistological analysis showed only modest amyloid deposition in the hippocampus at 4 and 6 months with a robust increase up to 15 months of age.

    Conclusions: Our results suggest that increased formation and aggregation of beta amyloid with aging is responsible for the impaired LTP with aging in this mouse model, while the transient increase of PPF at 6 months of age is caused by some other mechanism.

    PloS one 2010;5;3;e9764

  • Loss of function of ATXN1 increases amyloid beta-protein levels by potentiating beta-secretase processing of beta-amyloid precursor protein.

    Zhang C, Browne A, Child D, Divito JR, Stevenson JA and Tanzi RE

    Department of Neurology, Genetics and Aging Research Unit, MassGeneral Institute for Neurodegenerative Diseases, Massachusetts General Hospital, Harvard Medical School, Charlestown, Massachusetts 02129-2060, USA.

    Alzheimer disease (AD) is a devastating neurodegenerative disease with complex and strong genetic inheritance. Four genes have been established to either cause familial early onset AD (APP, PSEN1, and PSEN2) or to increase susceptibility for late onset AD (APOE). To date approximately 80% of the late onset AD genetic variance remains elusive. Recently our genome-wide association screen identified four novel late onset AD candidate genes. Ataxin 1 (ATXN1) is one of these four AD candidate genes and has been indicated to be the disease gene for spinocerebellar ataxia type 1, which is also a neurodegenerative disease. Mounting evidence suggests that the excessive accumulation of Abeta, the proteolytic product of beta-amyloid precursor protein (APP), is the primary AD pathological event. In this study, we ask whether ATXN1 may lead to AD pathogenesis by affecting Abeta and APP processing utilizing RNA interference in a human neuronal cell model and mouse primary cortical neurons. We show that knock-down of ATXN1 significantly increases the levels of both Abeta40 and Abeta42. This effect could be rescued with concurrent overexpression of ATXN1. Moreover, overexpression of ATXN1 decreased Abeta levels. Regarding the underlying molecular mechanism, we show that the effect of ATXN1 expression on Abeta levels is modulated via beta-secretase cleavage of APP. Taken together, ATXN1 functions as a genetic risk modifier that contributes to AD pathogenesis through a loss-of-function mechanism by regulating beta-secretase cleavage of APP and Abeta levels.

    Funded by: NCI NIH HHS: P01 CA095616; NIMH NIH HHS: R37 MH060009, R37 MH060009-10; NINDS NIH HHS: U24 NS050606, U24NS050606

    The Journal of biological chemistry 2010;285;12;8515-26

  • Blocking beta2-adrenergic receptor attenuates acute stress-induced amyloid beta peptides production.

    Yu NN, Wang XX, Yu JT, Wang ND, Lu RC, Miao D, Tian Y and Tan L

    Department of Neurology, Qingdao Municipal Hospital, School of Medicine, Qingdao University, No.5 Donghai Middle Road, Qingdao, Shandong Province 266071, PR China.

    Environmental factors play an important role in the Alzheimer's disease (AD) development and stress may accelerate the progression of AD. Beta-adrenergic receptors are activated by stress and may influence different aspects of cognitive function. So, it was hypothesized that stress may accelerate the pathological progression of AD by the activation of beta(2)-adrenergic receptor (beta(2)-AR). We have investigated the role of acute stress and activation of beta(2)-AR in amyloid beta (Abeta) peptides production in a mouse model of acute restraint stress. Injections of the beta(2)-AR-selective agonist clenbuterol hydrochloride enhanced the production of acute stress-induced Abeta peptides production; the beta(2)-AR-selective antagonist ICI 118,551 reduced Abeta peptides production. It is suggested that acute stress induces abnormal activation of beta(2)-AR which subsequently enhances Abeta peptides (the main neuropathological hallmarks of AD) production possibly resulting in the onset of AD. The findings indicate that new therapeutic strategies designed to blocking beta(2)-AR might be valuable for the prevention and treatment of AD.

    Brain research 2010;1317;305-10

  • Generation of conditional null alleles for APP and APLP2.

    Mallm JP, Tschäpe JA, Hick M, Filippov MA and Müller UC

    Department of Bioinformatics and Functional Genomics, Institute of Pharmacy and Molecular Biotechnology, University of Heidelberg, Heidelberg, Germany.

    Proteolytical cleavage of the beta-amyloid precursor protein (APP) generates beta-amyloid, which is deposited in the brains of patients suffering from Alzheimer's disease (AD). Despite the well-established key role of APP for AD pathogenesis, the physiological function of APP and its close homologues APLP1 and APLP2 remains poorly understood. Previously, we generated APP(-/-) mice that proved viable, whereas APP(-/-)APLP2(-/-) mice and triple knockouts died shortly after birth, likely due to deficits of neuromuscular synaptic transmission. Here, we generated conditional knockout alleles for both APP and APLP2 in which the promoter and exon1 were flanked by loxP sites. No differences in expression were detectable between wt and floxed alleles, whereas null alleles were obtained upon crossing with Cre-transgenic deleter mice. These mice will now allow for tissue and time-point controlled knockout of both genes.

    Genesis (New York, N.Y. : 2000) 2010;48;3;200-6

  • Evidence for dimeric BACE-mediated APP processing.

    Jin S, Agerman K, Kolmodin K, Gustafsson E, Dahlqvist C, Jureus A, Liu G, Fälting J, Berg S, Lundkvist J and Lendahl U

    Department of Cell and Molecular Biology, Karolinska Institute, SE-171 77 Stockholm, Sweden.

    beta-Secretase (BACE) is an aspartyl protease, which proteolytically processes amyloid precursor protein, making BACE an interesting pharmacological target in Alzheimer's disease. To study the enzymatic function of BACE, we mutated either of the two aspartic acid residues in the active site of BACE. This rendered BACE functionally inactive without affecting the degree of glycosylation or endosomal localization. In contrast, substituting both active site aspartic acid residues produced a functionally inactive, endoplasmic reticulum-retained and partially glycosylated BACE. Interestingly, co-expression of the two single active site mutants partially restored beta-site cleavage of amyloid precursor protein, and the restored activity was inhibited with similar dose-dependency and potency as wildtype BACE by a small molecule inhibitor raised against BACE. In sum, our data suggest that two different active site mutants can complement each other in a partially functional BACE dimer and mediate APP processing.

    Biochemical and biophysical research communications 2010;393;1;21-7

  • Beta-amyloid protein (25-35) disrupts hippocampal network activity: role of Fyn-kinase.

    Peña F, Ordaz B, Balleza-Tapia H, Bernal-Pedraza R, Márquez-Ramos A, Carmona-Aparicio L and Giordano M

    Departamento de Farmacobiología, Centro de Investigación y de Estudios Avanzados Sede Sur, México, D.F., México. jfpena@cinvestav.mx

    Early cognitive deficit characteristic of early Alzheimer's disease seems to be produced by the soluble forms of beta-amyloid protein. Such cognitive deficit correlates with neuronal network dysfunction that is reflected as alterations in the electroencephalogram of both Alzheimer patients and transgenic murine models of such disease. Correspondingly, recent studies have demonstrated that chronic exposure to betaAP affects hippocampal oscillatory properties. However, it is still unclear if such neuronal network dysfunction results from a direct action of betaAP on the hippocampal circuit or it is secondary to the chronic presence of the protein in the brain. Therefore, we aimed to explore the effect of acute exposure to betaAP(25-35) on hippocampal network activity both in vitro and in vivo, as well as on intrinsic and synaptic properties of hippocampal neurons. We found that betaAP(25-35), reversibly, affects spontaneous hippocampal population activity in vitro. Such effect is not produced by the inverse sequence betaAP(35-25) and is reproduced by the full-length peptide betaAP(1-42). Correspondingly betaAP(25-35), but not the inverse sequence betaAP(35-25), reduces theta-like activity recorded from the hippocampus in vivo. The betaAP(25-35)-induced disruption in hippocampal network activity correlates with a reduction in spontaneous neuronal activity and synaptic transmission, as well as with an inhibition in the subthreshold oscillations produced by pyramidal neurons in vitro. Finally, we studied the involvement of Fyn-kinase on the betaAP(25-35)-induced disruption in hippocampal network activity in vitro. Interestingly, we found that such phenomenon is not observed in slices obtained from Fyn-knockout mice. In conclusion, our data suggest that betaAP acutely affects proper hippocampal function through a Fyn-dependent mechanism. We propose that such alteration might be related to the cognitive impairment observed, at least, during the early phases of Alzheimer's disease.

    Hippocampus 2010;20;1;78-96

  • Co-localization of the amyloid precursor protein and Notch intracellular domains in nuclear transcription factories.

    Konietzko U, Goodger ZV, Meyer M, Kohli BM, Bosset J, Lahiri DK and Nitsch RM

    Division of Psychiatry Research, University of Zürich, August Forel Street 1, 8008 Zürich, Switzerland. uwekon@bli.uzh.ch

    The beta-amyloid precursor protein (APP) plays a major role in Alzheimer's disease. The APP intracellular domain (AICD), together with Fe65 and Tip60, localizes to spherical nuclear AFT complexes, which may represent sites of transcription. Despite a lack of co-localization with several described nuclear compartments, we have identified a close apposition between AFT complexes and splicing speckles, Cajal bodies and PML bodies. Live imaging revealed that AFT complexes were highly mobile within nuclei and following pharmacological inhibition of transcription fused into larger assemblies. We have previously shown that AICD regulates the expression of its own precursor APP. In support of our earlier findings, transfection of APP promoter plasmids as substrates resulted in cytosolic AFT complex formation at labeled APP promoter plasmids. In addition, identification of chromosomal APP or KAI1 gene loci by fluorescence in situ hybridization showed their close association with nuclear AFT complexes. The transcriptional activator Notch intracellular domain (NICD) localized to the same nuclear spots as occupied by AFT complexes suggesting that these nuclear compartments correspond to transcription factories. Fe65 and Tip60 also co-localized with APP in the neurites of primary neurons. Pre-assembled AFT complexes may serve to assist fast nuclear signaling upon endoproteolytic APP cleavage.

    Funded by: NIA NIH HHS: R01 AG018379-09, R01 AG018884-07

    Neurobiology of aging 2010;31;1;58-73

  • Genetic reductions of beta-site amyloid precursor protein-cleaving enzyme 1 and amyloid-beta ameliorate impairment of conditioned taste aversion memory in 5XFAD Alzheimer's disease model mice.

    Devi L and Ohno M

    Center for Dementia Research, Nathan Kline Institute, New York University School of Medicine, 140 Old Orangeburg Road, Orangeburg, NY 10962, USA.

    Although transgenic mouse models of Alzheimer's disease (AD) recapitulate amyloid-beta (Abeta)-related pathologies and cognitive impairments, previous studies have mainly evaluated their hippocampus-dependent memory dysfunctions using behavioral tasks such as the water maze and fear conditioning. However, multiple memory systems become impaired in AD as the disease progresses and it is important to test whether other forms of memory are affected in AD models. This study was designed to use conditioned taste aversion (CTA) and contextual fear conditioning paradigms to compare the phenotypes of hippocampus-independent and -dependent memory functions, respectively, in 5XFAD amyloid precursor protein/presenilin-1 transgenic mice that harbor five familial AD mutations. Although both types of memory were significantly impaired in 5XFAD mice, the onset of CTA memory deficits ( approximately 9 months of age) was delayed compared with that of contextual memory deficits ( approximately 6 months of age). Furthermore, 5XFAD mice that were genetically engineered to have reduced levels of beta-site amyloid precursor protein-cleaving enzyme 1 (BACE1) (BACE1(+/-).5XFAD) exhibited improved CTA memory, which was equivalent to the performance of wild-type controls. Importantly, elevated levels of cerebral beta-secretase-cleaved C-terminal fragment (C99) and Abeta peptides in 5XFAD mice were significantly reduced in BACE1(+/-).5XFAD mice. Furthermore, Abeta deposition in the insular cortex and basolateral amygdala, two brain regions that are critically involved in CTA performance, was also reduced in BACE1(+/-).5XFAD compared with 5XFAD mice. Our findings indicate that the CTA paradigm is useful for evaluating a hippocampus-independent form of memory defect in AD model mice, which is sensitive to rescue by partial reductions of the beta-secretase BACE1 and consequently of cerebral Abeta.

    Funded by: NIMH NIH HHS: R01 MH067251, R01 MH067251-05

    The European journal of neuroscience 2010;31;1;110-8

  • Genetic regulatory network analysis for app based on genetical genomics approach.

    Wang X, Chen Y, Wang X and Lu L

    Institute of Bioinformatics, Zhejiang University, Hangzhou, P.R. China.

    A number of studies have shown that amyloid precursor protein (App) plays a critical role in Alzheimer's disease (AD); however, little is known about the genetic regulatory network. In this study, the authors combined array analysis and quantitative trait loci (QTL) mapping to characterize the genetic variation and genetic regulatory network for App using hippocampus of BXD recombinant inbred (RI) mice. The variation in expression level of App is conspicuous across the 78 BXD RI strains. Moreover, the expression level of App is significantly higher in DBA/2J than the level in C57BL/6J (p < .001). Quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) analysis has further confirmed the significant difference between the two parental strains C57BL/6J and DBA/2J. The authors performed an interval mapping for App gene expression and found that it is cis regulated with highly significant likelihood-ratio statistic (LRS) score (LRS = 19; p < .05). Four SNPs and two InDels (insertions or deletions) were identified in the promoter, and one of the SNPs is located in the pax2 motif. Genetic regulatory network analysis showed that App coregulated with many AD-related genes, including Gsk3b, Falz, Mef2a, Tlk2, Rtn, and Prkca. The genetical genomics approach demonstrates the importance and the potential power of the expression quantitative trait loci (eQTL) studies in identifying regulatory network that contribute to complex traits, such as AD.

    Funded by: NIAAA NIH HHS: U01-AA014425

    Experimental aging research 2010;36;1;79-93

  • Protective effect of N-glycan bisecting GlcNAc residues on beta-amyloid production in Alzheimer's disease.

    Akasaka-Manya K, Manya H, Sakurai Y, Wojczyk BS, Kozutsumi Y, Saito Y, Taniguchi N, Murayama S, Spitalnik SL and Endo T

    Department of Glycobiology, Tokyo Metropolitan Institute of Gerontology, Foundation for Research on Aging and Promotion of Human Welfare, Itabashi-ku, Tokyo 173-0015, Japan.

    Alteration of glycoprotein glycans often changes various properties of the target glycoprotein and contributes to a wide variety of diseases. Here, we focused on the N-glycans of amyloid precursor protein whose cleaved fragment, beta-amyloid, is thought to cause much of the pathology of Alzheimer's disease (AD). We previously determined the N-glycan structures of normal and mutant amyloid precursor proteins (the Swedish type and the London type). In comparison with normal amyloid precursor protein, mutant amyloid precursor proteins had higher contents of bisecting GlcNAc residues. Because N-acetylglucosaminyltransferase III (GnT-III) is the glycosyltransferase responsible for synthesizing a bisecting GlcNAc residue, the current report measured GnT-III mRNA expression levels in the brains of AD patients. Interestingly, GnT-III mRNA expression was increased in AD brains. Furthermore, beta-amyloid treatment increased GnT-III mRNA expression in Neuro2a mouse neuroblastoma cells. We then examined the influence of bisecting GlcNAc on the production of beta-amyloid. Both beta-amyloid 40 and beta-amyloid 42 were significantly decreased in GnT-III-transfected cells. When secretase activities were analyzed in GnT-III transfectant cells, alpha-secretase activity was increased. Taken together, these results suggest that upregulation of GnT-III in AD brains may represent an adaptive response to protect them from additional beta-amyloid production.

    Glycobiology 2010;20;1;99-106

  • Amyloid-beta decreases cell-surface AMPA receptors by increasing intracellular calcium and phosphorylation of GluR2.

    Liu SJ, Gasperini R, Foa L and Small DH

    Menzies Research Institute, University of Tasmania, Hobart, Tasmania, Australia.

    alpha-Amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptors (AMPARs) are key regulators of synaptic function and cognition. In Alzheimer's disease (AD), cell-surface AMPARs are downregulated, however the reason for this downregulation is not clear. In the present study, we found that Abeta significantly decreased levels of the cell-surface AMPA-type glutamate receptor subunit 2 (GluR2), and increased the concentration of free cytosolic calcium ion ([Ca2+]i) in hippocampal neurons. Ion channel blockers (nifedipine, tetrodotoxin, SKF96365) decreased [Ca2+ and increased the level of cell-surface GluR2, whereas Bay K 8644, an activator of L-type voltage-gated calcium channels increased [Ca2+]i and decreased cell-surface GluR2. Abeta and Bay K 8644 increased phosphorylation of serine-880 (S880) on GluR2, whereas the nifedipine. tetrodotoxin and SKF96365 decreased S880 phosphorylation. Finally, we found that bisindolylmeimide I (GF 109203X, GFX), an inhibitor of protein kinase C (PKC) blocked both the decrease in cell-surface GluR2 and the increase in phospho-S880 induced by Abeta and Bay K 8644. Taken together, these results demonstrate that Abeta decreases cell-surface GluR2 by increasing PKC-mediated phosphorylation of S880. Our study supports the view that a rise in cytosolic [Ca2+]i induced by Abeta could impair synaptic function by decreasing the availability of AMPARs at the synapse. This decrease in AMPARs may contribute to the decline in cognitive function seen in AD.

    Journal of Alzheimer's disease : JAD 2010;21;2;655-66

  • Differential accumulation of secreted AbetaPP metabolites in ocular fluids.

    Prakasam A, Muthuswamy A, Ablonczy Z, Greig NH, Fauq A, Rao KJ, Pappolla MA and Sambamurti K

    Department of Neurosciences, Medical University of South Carolina, Charleston, SC, USA.

    Amyloid-beta (Abeta) accumulates in several types of retinal degeneration and in Alzheimer's disease (AD), but its source has been unclear. We detected the neuronal 695 amino acid form of amyloid-beta protein precursor (AbetaPP) in the normal retina and AbetaPP751 in the retinal pigment epithelium (RPE) and anterior eye tissues. Similar to the brain, alpha- and beta-secretases cleaved AbetaPP to soluble derivatives (sAbetaPP) alpha or beta and membrane-bound C-terminal fragments alpha or beta in the retina and RPE. Levels of sAbetaPP were particularly high in the vitreous and low in aqueous humor revealing a molecular barrier for AbetaPP. In contrast, Abeta40 and Abeta42 levels were only 50% lower in the aqueous than the vitreous humor, indicating relatively barrier-free movement of Abeta. These studies demonstrated a relatively high yield of AbetaPP and Abeta in the ocular fluids, which may serve as a trackable marker for AD. In addition, failure of free clearance from the eye may trigger retina degeneration in a manner similar to Abeta-related neurodegeneration in AD.

    Funded by: Intramural NIH HHS: Z01 AG000311-08, Z01 AG000315-08; NEI NIH HHS: R01 EY019065; NIA NIH HHS: AG023055, AG028544, R01 AG023055, R03 AG028544

    Journal of Alzheimer's disease : JAD 2010;20;4;1243-53

  • Plaque deposition dependent decrease in 5-HT2A serotonin receptor in AbetaPPswe/PS1dE9 amyloid overexpressing mice.

    Holm P, Ettrup A, Klein AB, Santini MA, El-Sayed M, Elvang AB, Stensbøl TB, Mikkelsen JD, Knudsen GM and Aznar S

    Neurobiology Research Unit and Center for Integrated Molecular Brain Imaging, Copenhagen University Hospital Rigshospitalet, Copenhagen, Denmark.

    Intrahippocampal injections of aggregated amyloid-beta (Abeta)1-42 in rats result in memory impairment and in reduction of hippocampal 5-HT2A receptor levels. In order to investigate how changes in 5-HT2A levels and functionality relate to the progressive accumulation of Abeta protein, we studied 5-HT2A receptor regulation in double transgenic AbetaPPswe/PS1dE9 mice which display excess production of Abeta and age-dependent increase in amyloid plaques. Three different age-groups, 4-month-old, 8- month-old, and 11-month-old were included in the study. [3H]-MDL100907, [3H]-escitalopram, and [11C]-PIB autoradiography was performed for measuring 5-HT2A receptor, serotonin transporter (SERT), and Abeta plaque levels in medial prefrontal cortex (mPFC), prefrontal cortex (PFC), frontoparietal cortex (FPC), dorsal and ventral hippocampus, and somatosensory cortex. To investigate 5-HT2A receptor functionality, animals were treated with the 5-HT2A receptor agonist DOI and head-twitch response (HTR) subsequently recorded. Expression level of the immediate early gene c-fos was measured by in situ hybridization. We found that the age-related increase in Abeta plaque burden was accompanied by a significant decrease in 5-HT2A receptor binding in mPFC in the 11-month-old group. The changes in 5-HT2A receptor binding correlated negatively with [11C]-PIB binding and were not accompanied by decreases in SERT binding. Correspondingly, 11-month-old transgenic mice showed diminished DOI-induced HTR and reduced increase in expression of c-fos mRNA in mPFC and FPC. These observations point towards a direct association between Abeta accumulation and changes in 5-HT2A receptor expression that is independent of upstream changes in the serotonergic system.

    Journal of Alzheimer's disease : JAD 2010;20;4;1201-13

  • Sex- and brain region-specific acceleration of β-amyloidogenesis following behavioral stress in a mouse model of Alzheimer's disease.

    Devi L, Alldred MJ, Ginsberg SD and Ohno M

    Center for Dementia Research, Nathan Kline Institute, Orangeburg, New York 10962, USA.

    Background: It is hypothesized that complex interactions between multiple environmental factors and genetic factors are implicated in sporadic Alzheimer's disease (AD); however, the underlying mechanisms are poorly understood. Importantly, recent evidence reveals that expression and activity levels of the β-site APP cleaving enzyme 1 (BACE1), which initiates amyloid-β (Aβ) production, are elevated in AD brains. In this study, we investigated a molecular mechanism by which sex and stress interactions may accelerate β-amyloidogenesis and contribute to sporadic AD.

    Results: We applied 5-day restraint stress (6 h/day) to the male and female 5XFAD transgenic mouse model of AD at the pre-pathological stage of disease, which showed little amyloid deposition under non-stressed control conditions. Exposure to the relatively brief behavioral stress increased levels of neurotoxic Aβ42 peptides, the β-secretase-cleaved C-terminal fragment (C99) and plaque burden in the hippocampus of female 5XFAD mice but not in that of male 5XFAD mice. In contrast, significant changes in the parameters of β-amyloidosis were not observed in the cerebral cortex of stressed male or female 5XFAD mice. We found that this sex- and brain region-specific acceleration of β-amyloidosis was accounted for by elevations in BACE1 and APP levels in response to adverse stress. Furthermore, not only BACE1 mRNA but also phosphorylation of the translation initiation factor eIF2α (a proposed mediator of the post-transcriptional upregulation of BACE1) was elevated in the hippocampus of stressed female 5XFAD mice.

    Conclusions: Our results suggest that the higher prevalence of sporadic AD in women may be attributable to the vulnerability of female brains (especially, the hippocampus) to stressful events, which alter APP processing to favor the β-amyloidogenesis through the transcriptional and translational upregulation of BACE1 combined with elevations in its substrate APP.

    Funded by: NIMH NIH HHS: R01 MH067251

    Molecular brain 2010;3;34

  • Amyloid precursor protein regulates Cav1.2 L-type calcium channel levels and function to influence GABAergic short-term plasticity.

    Yang L, Wang Z, Wang B, Justice NJ and Zheng H

    Huffington Center on Aging, Baylor College of Medicine, Houston, Texas 77030, USA. liy@bcm.edu

    Amyloid precursor protein (APP) has been strongly implicated in the pathogenesis of Alzheimer's disease (AD). Although impaired synaptic function is believed to be an early and causative event in AD, how APP physiologically regulates synaptic properties remains poorly understood. Here, we report a critical role for APP in the regulation of L-type calcium channels (LTCC) in GABAergic inhibitory neurons in striatum and hippocampus. APP deletion in mice leads to an increase in the levels of Ca(v)1.2, the pore-forming subunit of LTCCs, and subsequent increases in GABAergic calcium currents (I(Ca(2+))) that can be reversed by reintroduction of APP. Upregulated levels of Ca(v)1.2 result in reduced GABAergic paired-pulse inhibition and increased GABAergic post-tetanic potentiation in both striatal and hippocampal neurons, indicating that APP modulates synaptic properties of GABAergic neurons by regulating Ca(v)1.2. Furthermore, APP physically interacts with Ca(v)1.2, suggesting a mechanism in which loss of APP leads to an inappropriate accumulation and aberrant activity of Ca(v)1.2. These results provide a direct link between APP and calcium signaling and might help explain how altered APP regulation leads to changes in synaptic function that occur with AD.

    Funded by: NIA NIH HHS: AG032051, AG033467, R01 AG032051, R01 AG032051-01, R01 AG032051-02, R01 AG033467, R01 AG033467-01, R01 AG033467-02

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2009;29;50;15660-8

  • Amyloid-beta expression in retrosplenial cortex of triple transgenic mice: relationship to cholinergic axonal afferents from medial septum.

    Robertson RT, Baratta J, Yu J and LaFerla FM

    Department of Anatomy and Neurobiology, University of California, Irvine, CA 92697, USA. rtrobert@uci.edu

    Triple transgenic (3xTg-AD) mice harboring the presenilin 1, amyloid precursor protein, and tau transgenes (Oddo et al., 2003b) display prominent levels of amyloid-beta (Abeta) immunoreactivity in forebrain regions. The Abeta immunoreactivity is first seen intracellularly in neurons and later as extracellular plaque deposits. The present study examined Abeta immunoreactivity that occurs in layer III of the granular division of retrosplenial cortex (RSg). This pattern of Abeta immunoreactivity in layer III of RSg develops relatively late, and is seen in animals older than 14 months. The appearance of the Abeta immunoreactivity is similar to an axonal terminal field and thus may offer a unique opportunity to study the relationship between afferent projections and the formation of Abeta deposits. Axonal tract tracing techniques demonstrated that the pattern of axon terminal labeling in layer III of RSg, following placement of DiI in medial septum, is remarkably similar to the pattern of cholinergic axons in RSg, as detected by acetylcholinesterase histochemical staining, choline acetyltransferase immunoreactivity, or p75 receptor immunoreactivity; this pattern also is strikingly similar to the band of Abeta immunoreactivity. In animals sustaining early damage to the medial septal nucleus (prior to the advent of Abeta immunoreactivity), the band of Abeta in layer III of RSg does not develop; the corresponding band of cholinergic markers also is eliminated. In older animals (after the appearance of the Abeta immunoreactivity) damage to cholinergic afferents by electrolytic lesions, immunotoxin lesions, or cutting the cingulate bundle, result in a rapid loss of the cholinergic markers and a slower reduction of Abeta immunoreactivity. These results suggest that the septal cholinergic axonal projections transport Abeta or amyloid precursor protein (APP) to layer III of RSg.

    Funded by: NIA NIH HHS: AG-02754; NINDS NIH HHS: NS 30109, P01 NS030109-05S10001

    Neuroscience 2009;164;3;1334-46

  • Brain-derived neurotrophic factor reduces amyloidogenic processing through control of SORLA gene expression.

    Rohe M, Synowitz M, Glass R, Paul SM, Nykjaer A and Willnow TE

    Max Delbrueck Center for Molecular Medicine, Charité-Universitätsmedizin Berlin, D-13125 Berlin, Germany.

    Sorting protein-related receptor with A-type repeats (SORLA) is a major risk factor in cellular processes leading to Alzheimer's disease (AD). It acts as sorting receptor for the amyloid precursor protein (APP) that regulates intracellular trafficking and processing into amyloidogenic-beta peptides (A beta). Overexpression of SORLA in neurons reduces while inactivation of gene expression (as in knock-out mouse models) accelerates amyloidogenic processing and senile plaque formation. The current study aimed at identifying molecular pathways that control SORLA gene transcription in vivo and that may contribute to low levels of receptor expression in the brain of patients with AD. Using screening approaches in primary neurons, we identified brain-derived neurotrophic factor (BDNF) as a major inducer of Sorla that activates receptor gene transcription through the ERK (extracellular regulated kinase) pathway. In line with a physiological role as regulator of Sorla, expression of the receptor is significantly impaired in mouse models with genetic (Bdnf(-/-)) or disease-related loss of BDNF activity in the brain (Huntington's disease). Intriguingly, exogenous application of BDNF reduced A beta production in primary neurons and in the brain of wild-type mice in vivo, but not in animals genetically deficient for Sorla. These findings demonstrate that the beneficial effects ascribed to BDNF in APP metabolism act through induction of Sorla that encodes a negative regulator of neuronal APP processing.

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2009;29;49;15472-8

  • Fibril fragmentation enhances amyloid cytotoxicity.

    Xue WF, Hellewell AL, Gosal WS, Homans SW, Hewitt EW and Radford SE

    Astbury Centre for Structural Molecular Biology, Institute of Molecular and Cellular Biology, University of Leeds, Leeds LS2 9JT, United Kingdom.

    Fibrils associated with amyloid disease are molecular assemblies of key biological importance, yet how cells respond to the presence of amyloid remains unclear. Cellular responses may not only depend on the chemical composition or molecular properties of the amyloid fibrils, but their physical attributes such as length, width, or surface area may also play important roles. Here, we report a systematic investigation of the effect of fragmentation on the structural and biological properties of amyloid fibrils. In addition to the expected relationship between fragmentation and the ability to seed, we show a striking finding that fibril length correlates with the ability to disrupt membranes and to reduce cell viability. Thus, despite otherwise unchanged molecular architecture, shorter fibrillar samples show enhanced cytotoxic potential than their longer counterparts. The results highlight the importance of fibril length in amyloid disease, with fragmentation not only providing a mechanism by which fibril load can be rapidly increased but also creating fibrillar species of different dimensions that can endow new or enhanced biological properties such as amyloid cytotoxicity.

    Funded by: Biotechnology and Biological Sciences Research Council: BB/526502/1; Wellcome Trust: 075675

    The Journal of biological chemistry 2009;284;49;34272-82

  • Residues at P2-P1 positions of epsilon- and zeta-cleavage sites are important in formation of beta-amyloid peptide.

    Tan J, Mao G, Cui MZ, Lamb B, Sy MS and Xu X

    Department of Pathobiology, College of Veterinary Medicine, The University of Tennessee, 2407 River Drive, Knoxville, TN 37996, USA.

    Most of the Alzheimer's disease (AD)-linked mutations in amyloid precursor protein (APP), which cause abnormal production of beta-amyloid (Abeta), are localized at the major beta-secretase-and gamma-secretase cleavage sites. In this study, using an APP-knockout mouse neuronal cell line, our data demonstrated that at the P2-P1 positions of the epsilon-cleavage site at Abeta49 and the zeta-cleavage site at Abeta46, aromatic amino acids caused a strong reduction in total Abeta. On the other hand, residues with a long side chain caused a decrease in Abeta(40) and a concomitant increase in Abeta(42) and Abeta(38). These findings indicate that the structures of the substituting residues at these key positions strongly determine the efficiency and preference of gamma-secretase-mediated APP processing, which determines the ratio of different secreted Abeta species, a crucial factor in the disease development. Our findings provide new insight into the mechanisms of gamma-secretase-mediated APP processing and, specifically, into why most AD-linked APP mutations are localized at major gamma-secretase cleavage sites. This information may contribute to the development of methods of prevention and treatment of Alzheimer's disease aimed at modulating gamma-secretase activity.

    Funded by: NIA NIH HHS: R01 AG026640, R01AG26640, R21 AG039596; NINDS NIH HHS: R01 NS042314

    Neurobiology of disease 2009;36;3;453-60

  • Depletion of vitamin E increases amyloid beta accumulation by decreasing its clearances from brain and blood in a mouse model of Alzheimer disease.

    Nishida Y, Ito S, Ohtsuki S, Yamamoto N, Takahashi T, Iwata N, Jishage K, Yamada H, Sasaguri H, Yokota S, Piao W, Tomimitsu H, Saido TC, Yanagisawa K, Terasaki T, Mizusawa H and Yokota T

    Department of Neurology and Neurological Science, Graduate School, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519. Japan.

    Increased oxidative damage is a prominent and early feature in Alzheimer disease. We previously crossed Alzheimer disease transgenic (APPsw) model mice with alpha-tocopherol transfer protein knock-out (Ttpa(-/-)) mice in which lipid peroxidation in the brain was significantly increased. The resulting double-mutant (Ttpa(-/-)APPsw) mice showed increased amyloid beta (Abeta) deposits in the brain, which was ameliorated with alpha-tocopherol supplementation. To investigate the mechanism of the increased Abeta accumulation, we here studied generation, degradation, aggregation, and efflux of Abeta in the mice. The clearance of intracerebral-microinjected (125)I-Abeta(1-40) from brain was decreased in Ttpa(-/-) mice to be compared with wild-type mice, whereas the generation of Abeta was not increased in Ttpa(-/-)APPsw mice. The activity of an Abeta-degrading enzyme, neprilysin, did not decrease, but the expression level of insulin-degrading enzyme was markedly decreased in Ttpa(-/-) mouse brain. In contrast, Abeta aggregation was accelerated in Ttpa(-/-) mouse brains compared with wild-type brains, and well known molecules involved in Abeta transport from brain to blood, low density lipoprotein receptor-related protein-1 (LRP-1) and p-glycoprotein, were up-regulated in the small vascular fraction of Ttpa(-/-) mouse brains. Moreover, the disappearance of intravenously administered (125)I-Abeta(1-40) was decreased in Ttpa(-/-) mice with reduced translocation of LRP-1 in the hepatocytes. These results suggest that lipid peroxidation due to depletion of alpha-tocopherol impairs Abeta clearances from the brain and from the blood, possibly causing increased Abeta accumulation in Ttpa(-/-)APPsw mouse brain and plasma.

    The Journal of biological chemistry 2009;284;48;33400-8

  • Receptor-associated protein interacts with amyloid-beta peptide and promotes its cellular uptake.

    Kanekiyo T and Bu G

    Department of Pediatrics, Washington University School of Medicine, St. Louis, Missouri 63110, USA.

    Brain amyloid-beta (Abeta) peptide accumulation and aggregation are critical events in the pathogenesis of Alzheimer disease. Increasing evidence has demonstrated that LRP1 is involved in Alzheimer disease pathogenesis. The physiological ligands of LRP1, including apoE, play significant roles in the cellular clearance of Abeta. The receptor-associated protein (RAP) is a specialized chaperone for members of the low density lipoprotein receptor family. RAP shares structural and receptor-binding properties with apoE. Here, we show that RAP binds to both Abeta40 and Abeta42 in a concentration-dependent manner and forms complexes with them. Fluorescence-activated cell sorter analysis showed that RAP significantly enhances the cellular internalization of Abeta in different cell types, including brain vascular smooth muscle, neuroblastoma, glioblastoma, and Chinese hamster ovary cells. This effect of RAP was confirmed by fluorescence microscopy and enzyme-linked immunosorbent assay. RAP binds to both LRP1 and heparin; however, the ability of RAP to enhance Abeta cellular uptake was blocked by heparin and heparinase treatment but not by LRP1 deficiency. Furthermore, the effects of RAP were significantly decreased in heparan sulfate proteoglycan-deficient Chinese hamster ovary cells. Our findings reveal that RAP is a novel Abeta-binding protein that promotes cellular internalization of Abeta.

    Funded by: NIA NIH HHS: AG027924, AG031784, P01 AG030128, R01 AG027924, R01 AG031784

    The Journal of biological chemistry 2009;284;48;33352-9

  • Amyloid-beta and tau synergistically impair the oxidative phosphorylation system in triple transgenic Alzheimer's disease mice.

    Rhein V, Song X, Wiesner A, Ittner LM, Baysang G, Meier F, Ozmen L, Bluethmann H, Dröse S, Brandt U, Savaskan E, Czech C, Götz J and Eckert A

    Neurobiology Laboratory for Brain Aging and Mental Health, Psychiatric University Clinics, University of Basel, 4025 Basel, Switzerland.

    Alzheimer's disease (AD) is characterized by amyloid-beta (Abeta)-containing plaques, neurofibrillary tangles, and neuron and synapse loss. Tangle formation has been reproduced in P301L tau transgenic pR5 mice, whereas APP(sw)PS2(N141I) double-transgenic APP152 mice develop Abeta plaques. Cross-breeding generates triple transgenic ((triple)AD) mice that combine both pathologies in one model. To determine functional consequences of the combined Abeta and tau pathologies, we performed a proteomic analysis followed by functional validation. Specifically, we obtained vesicular preparations from (triple)AD mice, the parental strains, and nontransgenic mice, followed by the quantitative mass-tag labeling proteomic technique iTRAQ and mass spectrometry. Within 1,275 quantified proteins, we found a massive deregulation of 24 proteins, of which one-third were mitochondrial proteins mainly related to complexes I and IV of the oxidative phosphorylation system (OXPHOS). Notably, deregulation of complex I was tau dependent, whereas deregulation of complex IV was Abeta dependent, both at the protein and activity levels. Synergistic effects of Abeta and tau were evident in 8-month-old (triple)AD mice as only they showed a reduction of the mitochondrial membrane potential at this early age. At the age of 12 months, the strongest defects on OXPHOS, synthesis of ATP, and reactive oxygen species were exhibited in the (triple)AD mice, again emphasizing synergistic, age-associated effects of Abeta and tau in perishing mitochondria. Our study establishes a molecular link between Abeta and tau protein in AD pathology in vivo, illustrating the potential of quantitative proteomics.

    Proceedings of the National Academy of Sciences of the United States of America 2009;106;47;20057-62

  • Early-onset and robust amyloid pathology in a new homozygous mouse model of Alzheimer's disease.

    Willuweit A, Velden J, Godemann R, Manook A, Jetzek F, Tintrup H, Kauselmann G, Zevnik B, Henriksen G, Drzezga A, Pohlner J, Schoor M, Kemp JA and von der Kammer H

    Evotec Neurosciences GmbH, Hamburg, Germany. antje.willuweit@evotec.com

    Background: Transgenic mice expressing mutated amyloid precursor protein (APP) and presenilin (PS)-1 or -2 have been successfully used to model cerebral beta-amyloidosis, one of the characteristic hallmarks of Alzheimer's disease (AD) pathology. However, the use of many transgenic lines is limited by premature death, low breeding efficiencies and late onset and high inter-animal variability of the pathology, creating a need for improved animal models. Here we describe the detailed characterization of a new homozygous double-transgenic mouse line that addresses most of these issues.

    The transgenic mouse line (ARTE10) was generated by co-integration of two transgenes carrying the K670N/M671L mutated amyloid precursor protein (APP(swe)) and the M146V mutated presenilin 1 (PS1) both under control of a neuron-specific promoter. Mice, hemi- as well as homozygous for both transgenes, are viable and fertile with good breeding capabilities and a low rate of premature death. They develop robust AD-like cerebral beta-amyloid plaque pathology with glial inflammation, signs of neuritic dystrophy and cerebral amyloid angiopathy. Using our novel image analysis algorithm for semi-automatic quantification of plaque burden, we demonstrate an early onset and progressive plaque deposition starting at 3 months of age in homozygous mice with low inter-animal variability and 100%-penetrance of the phenotype. The plaques are readily detected in vivo by PiB, the standard human PET tracer for AD. In addition, ARTE10 mice display early loss of synaptic markers and age-related cognitive deficits. By applying a gamma-secretase inhibitor we show a dose dependent reduction of soluble amyloid beta levels in the brain.

    Conclusions: ARTE10 mice develop a cerebral beta-amyloidosis closely resembling the beta-amyloid-related aspects of human AD neuropathology. Unifying several advantages of previous transgenic models, this line particularly qualifies for the use in target validation and for evaluating potential diagnostic or therapeutic agents targeting the amyloid pathology of AD.

    PloS one 2009;4;11;e7931

  • Allosteric modulation of PS1/gamma-secretase conformation correlates with amyloid beta(42/40) ratio.

    Uemura K, Lill CM, Li X, Peters JA, Ivanov A, Fan Z, DeStrooper B, Bacskai BJ, Hyman BT and Berezovska O

    Alzheimer Research Unit, MassGeneral Institute for Neurodegenerative Diseases, Massachusetts General Hospital, Charlestown, Massachusetts, USA.

    Background: Presenilin 1(PS1) is the catalytic subunit of gamma-secretase, the enzyme responsible for the Abeta C-terminal cleavage site, which results in the production of Abeta peptides of various lengths. Production of longer forms of the Abeta peptide occur in patients with autosomal dominant Alzheimer disease (AD) due to mutations in presenilin. Many modulators of gamma-secretase function have been described. We hypothesize that these modulators act by a common mechanism by allosterically modifying the structure of presenilin.

    To test this hypothesis we generated a genetically encoded GFP-PS1-RFP (G-PS1-R) FRET probe that allows monitoring of the conformation of the PS1 molecule in its native environment in live cells. We show that G-PS1-R can be incorporated into the gamma-secretase complex, reconstituting its activity in PS1/2 deficient cells. Using Förster resonance energy transfer (FRET)-based approaches we show that various pharmacological and genetic manipulations that target either gamma-secretase components (PS1, Pen2, Aph1) or gamma-secretase substrate (amyloid precursor protein, APP) and are known to change Abeta(42) production are associated with a consistent conformational change in PS1.

    These results strongly support the hypothesis that allosteric changes in PS1 conformation underlie changes in the Abeta(42/40) ratio. Direct measurement of physiological and pathological changes in the conformation of PS1/gamma-secretase may provide insight into molecular mechanism of Abeta(42) generation, which could be exploited therapeutically.

    Funded by: NIA NIH HHS: AG 15379, AG026593, P01 AG015379, R01 AG026593; NIBIB NIH HHS: EB000768, R01 EB000768

    PloS one 2009;4;11;e7893

  • Expression of complement system components during aging and amyloid deposition in APP transgenic mice.

    Reichwald J, Danner S, Wiederhold KH and Staufenbiel M

    Novartis Institutes for BioMedical Research, Forum1, Novartis Campus, CH-4056 Basel, Switzerland. julia.reichwald@novartis.com

    Background: A causal role of the complement system in Alzheimer's disease pathogenesis has been postulated based on the identification of different activated components up to the membrane attack complex at amyloid plaques in brain. However, histological studies of amyloid plaque bearing APP transgenic mice provided only evidence for an activation of the early parts of the complement cascade. To better understand the contribution of normal aging and amyloid deposition to the increase in complement activation we performed a detailed characterization of the expression of the major mouse complement components.

    Methods: APP23 mice expressing human APP751 with the Swedish double mutation as well as C57BL/6 mice were used at different ages. mRNA was quantified by Realtime PCR and the age- as well as amyloid induced changes determined. The protein levels of complement C1q and C3 were analysed by Western blotting. Histology was done to test for amyloid plaque association and activation of the complement cascade.

    Results: High mRNA levels were detected for C1q and some inhibitory complement components. The expression of most activating components starting at C3 was low. Expression of C1q, C3, C4, C5 and factor B mRNA increased with age in control C57BL/6 mice. C1q and C3 mRNA showed a substantial additional elevation during amyloid formation in APP23 mice. This increase was confirmed on the protein level using Western blotting, whereas immunohistology indicated a recruitment of complement to amyloid plaques up to the C3 convertase.

    Conclusion: Early but not late components of the mouse complement system show an age-dependent increase in expression. The response to amyloid deposition is comparatively smaller. The low expression of C3 and C5 and failure to upregulate C5 and downstream components differs from human AD brain and likely contributes to the lack of full complement activation in APP transgenic mice.

    Journal of neuroinflammation 2009;6;35

  • Amyloid-beta dynamics are regulated by orexin and the sleep-wake cycle.

    Kang JE, Lim MM, Bateman RJ, Lee JJ, Smyth LP, Cirrito JR, Fujiki N, Nishino S and Holtzman DM

    Department of Neurology, Washington University, St. Louis, MO 63110, USA.

    Amyloid-beta (Abeta) accumulation in the brain extracellular space is a hallmark of Alzheimer's disease. The factors regulating this process are only partly understood. Abeta aggregation is a concentration-dependent process that is likely responsive to changes in brain interstitial fluid (ISF) levels of Abeta. Using in vivo microdialysis in mice, we found that the amount of ISF Abeta correlated with wakefulness. The amount of ISF Abeta also significantly increased during acute sleep deprivation and during orexin infusion, but decreased with infusion of a dual orexin receptor antagonist. Chronic sleep restriction significantly increased, and a dual orexin receptor antagonist decreased, Abeta plaque formation in amyloid precursor protein transgenic mice. Thus, the sleep-wake cycle and orexin may play a role in the pathogenesis of Alzheimer's disease.

    Funded by: NIA NIH HHS: AG025824, AG029524, AG030946, K01 AG029524, K01 AG029524-03, K23 AG030946, K23 AG030946-03, P50 AG005681, R01 AG025824, R01 AG025824-03; NIDDK NIH HHS: P30 DK056341, P30 DK056341-09; NIMH NIH HHS: MH072525, R01 MH072525, R01 MH072525-04; NINDS NIH HHS: NS065667, P30 NS057105, P30 NS057105-04, R01 NS065667, R01 NS065667-02

    Science (New York, N.Y.) 2009;326;5955;1005-7

  • Amyloid-peptide vaccinations reduce {beta}-amyloid plaques but exacerbate vascular deposition and inflammation in the retina of Alzheimer's transgenic mice.

    Liu B, Rasool S, Yang Z, Glabe CG, Schreiber SS, Ge J and Tan Z

    Department of Neurology, University of California Irvine School of Medicine, USA.

    Alzheimer's disease (AD) is pathologically characterized by accumulation of beta-amyloid (Abeta) protein deposits and/or neurofibrillary tangles in association with progressive cognitive deficits. Although numerous studies have demonstrated a relationship between brain pathology and AD progression, the Alzheimer's pathological hallmarks have not been found in the AD retina. A recent report showed Abeta plaques in the retinas of APPswe/PS1DeltaE9 transgenic mice. We now report the detection of Abeta plaques with increased retinal microvascular deposition of Abeta and neuroinflammation in Tg2576 mouse retinas. The majority of Abeta-immunoreactive plaques were detected from the ganglion cell layer to the inner plexiform layer, and some plaques were observed in the outer nuclear layer, photoreceptor outer segment, and optic nerve. Hyperphosphorylated tau was labeled in the corresponding areas of the Abeta plaques in adjacent sections. Although Abeta vaccinations reduced retinal Abeta deposits, there was a marked increase in retinal microvascular Abeta deposition as well as local neuroinflammation manifested by microglial infiltration and astrogliosis linked with disruption of the retinal organization. These results provide evidence to support further investigation of the use of retinal imaging to diagnose AD and to monitor disease activity.

    The American journal of pathology 2009;175;5;2099-110

  • Microglia activated with the toll-like receptor 9 ligand CpG attenuate oligomeric amyloid {beta} neurotoxicity in in vitro and in vivo models of Alzheimer's disease.

    Doi Y, Mizuno T, Maki Y, Jin S, Mizoguchi H, Ikeyama M, Doi M, Michikawa M, Takeuchi H and Suzumura A

    Department of Neuroimmunology, Research Institute of Environmental Medicine, Nagoya University, Japan.

    Soluble oligomeric amyloid beta (oAbeta) 1-42 causes synaptic dysfunction and neuronal injury in Alzheimer's disease (AD). Although accumulation of microglia around senile plaques is a hallmark of AD pathology, the role of microglia in oAbeta1-42 neurotoxicity is not fully understood. Here, we showed that oAbeta but not fibrillar Abeta was neurotoxic, and microglia activated with unmethylated DNA CpG motif (CpG), a ligand for Toll-like receptor 9, attenuated oAbeta1-42 neurotoxicity in primary neuron-microglia co-cultures. CpG enhanced microglial clearance of oAbeta1-42 and induced higher levels of the antioxidant enzyme heme oxygenase-1 in microglia without producing neurotoxic molecules such as nitric oxide and glutamate. Among subclasses of CpGs, class B and class C activated microglia to promote neuroprotection. Moreover, intracerebroventricular administration of CpG ameliorated both the cognitive impairments induced by oAbeta1-42 and the impairment of associative learning in Tg2576 mouse model of AD. We propose that CpG may be an effective therapeutic strategy for limiting oAbeta1-42 neurotoxicity in AD.

    The American journal of pathology 2009;175;5;2121-32

  • Alzheimer's disease-like pathological features in transgenic mice expressing the APP intracellular domain.

    Ghosal K, Vogt DL, Liang M, Shen Y, Lamb BT and Pimplikar SW

    Department of Neurosciences, Lerner Research Institute, Cleveland Clinic, Cleveland, OH 44195, USA.

    The hypothesis that amyloid-beta (Abeta) peptides are the primary cause of Alzheimer's disease (AD) remains the best supported theory of AD pathogenesis. Yet, many observations are inconsistent with the hypothesis. Abeta peptides are generated when amyloid precursor protein (APP) is cleaved by presenilins, a process that also produces APP intracellular domain (AICD). We previously generated AICD-overexpressing transgenic mice that showed abnormal activation of GSK-3beta, a pathological feature of AD. We now report that these mice exhibit additional AD-like characteristics, including hyperphosphorylation and aggregation of tau, neurodegeneration and working memory deficits that are prevented by treatment with lithium, a GSK-3beta inhibitor. Consistent with its potential role in AD pathogenesis, we find AICD levels to be elevated in brains from AD patients. The in vivo findings that AICD can contribute to AD pathology independently of Abeta have important therapeutic implications and may explain some observations that are discordant with the amyloid hypothesis.

    Funded by: NIA NIH HHS: R01 AG026146, R01AG026146; NICHD NIH HHS: T32 HD007104

    Proceedings of the National Academy of Sciences of the United States of America 2009;106;43;18367-72

  • Degradation of amyloid beta protein by purified myelin basic protein.

    Liao MC, Ahmed M, Smith SO and Van Nostrand WE

    Department of Neurosurgery, Stony Brook University, Stony Brook, New York 11794-8122, USA.

    The progressive accumulation of beta-amyloid (Abeta) in senile plaques and in the cerebral vasculature is the hallmark of Alzheimer disease and related disorders. Impaired clearance of Abeta from the brain likely contributes to the prevalent sporadic form of Alzheimer disease. Several major pathways for Abeta clearance include receptor-mediated cellular uptake, blood-brain barrier transport, and direct proteolytic degradation. Myelin basic protein (MBP) is the major structural protein component of myelin and plays a functional role in the formation and maintenance of the myelin sheath. MBP possesses endogenous serine proteinase activity and can undergo autocatalytic cleavage liberating distinct fragments. Recently, we showed that MBP binds Abeta and inhibits Abeta fibril formation (Hoos, M. D., Ahmed, M., Smith, S. O., and Van Nostrand, W. E. (2007) J. Biol. Chem. 282, 9952-9961; Hoos, M. D., Ahmed, M., Smith, S. O., and Van Nostrand, W. E. (2009) Biochemistry 48, 4720-4727). Here we show that Abeta40 and Abeta42 peptides are degraded by purified human brain MBP and recombinant human MBP, but not an MBP fragment that lacks autolytic activity. MBP-mediated Abeta degradation is inhibited by serine proteinase inhibitors. Similarly, Cos-1 cells expressing MBP degrade exogenous Abeta40 and Abeta42. In addition, we demonstrate that purified MBP also degrades assembled fibrillar Abeta in vitro. Mass spectrometry analysis identified distinct degradation products generated from Abeta digestion by MBP. Lastly, we demonstrate in situ that purified MBP can degrade parenchymal amyloid plaques as well as cerebral vascular amyloid that form in brain tissue of Abeta precursor protein transgenic mice. Together, these findings indicate that purified MBP possesses Abeta degrading activity in vitro.

    Funded by: NIA NIH HHS: R01 AG027317, R01-AG027317; NIGMS NIH HHS: T32 GM008444; NINDS NIH HHS: R01 NS035781, R01-NS035781

    The Journal of biological chemistry 2009;284;42;28917-25

  • Induced dimerization of the amyloid precursor protein leads to decreased amyloid-beta protein production.

    Eggert S, Midthune B, Cottrell B and Koo EH

    Department of Neurosciences, University of California, San Diego, La Jolla, California 92093, USA.

    The amyloid precursor protein (APP) plays a central role in Alzheimer disease (AD) pathogenesis because sequential cleavages by beta- and gamma-secretase lead to the generation of the amyloid-beta (Abeta) peptide, a key constituent in the amyloid plaques present in brains of AD individuals. In several studies APP has recently been shown to form homodimers, and this event appears to influence Abeta generation. However, these studies have relied on APP mutations within the Abeta sequence itself that may affect APP processing by interfering with secretase cleavages independent of dimerization. Therefore, the impact of APP dimerization on Abeta production remains unclear. To address this question, we compared the approach of constitutive cysteine-induced APP dimerization with a regulatable dimerization system that does not require the introduction of mutations within the Abeta sequence. To this end we generated an APP chimeric molecule by fusing a domain of the FK506-binding protein (FKBP) to the C terminus of APP. The addition of the synthetic membrane-permeant drug AP20187 induces rapid dimerization of the APP-FKBP chimera. Using this system we were able to induce up to 70% APP dimers. Our results showed that controlled homodimerization of APP-FKBP leads to a 50% reduction in total Abeta levels in transfected N2a cells. Similar results were obtained with the direct precursor of beta-secretase cleavage, C99/SPA4CT-FKBP. Furthermore, there was no modulation of different Abeta peptide species after APP dimerization in this system. Taken together, our results suggest that APP dimerization can directly affect gamma-secretase processing and that dimerization is not required for Abeta production.

    Funded by: NIA NIH HHS: AG 12376, R01 AG012376

    The Journal of biological chemistry 2009;284;42;28943-52

  • AAV-tau mediates pyramidal neurodegeneration by cell-cycle re-entry without neurofibrillary tangle formation in wild-type mice.

    Jaworski T, Dewachter I, Lechat B, Croes S, Termont A, Demedts D, Borghgraef P, Devijver H, Filipkowski RK, Kaczmarek L, Kügler S and Van Leuven F

    Experimental Genetics Group, Department of Human Genetics, KULeuven-Campus, Leuven, Belgium.

    In Alzheimer's disease tauopathy is considered secondary to amyloid, and the duality obscures their relation and the definition of their respective contributions.Transgenic mouse models do not resolve this problem conclusively, i.e. the relative hierarchy of amyloid and tau pathology depends on the actual model and the genes expressed or inactivated. Here, we approached the problem in non-transgenic models by intracerebral injection of adeno-associated viral vectors to express protein tau or amyloid precursor protein in the hippocampus in vivo. AAV-APP mutant caused neuronal accumulation of amyloid peptides, and eventually amyloid plaques at 6 months post-injection, but with only marginal hippocampal cell-death. In contrast, AAV-Tau, either wild-type or mutant P301L, provoked dramatic degeneration of pyramidal neurons in CA1/2 and cortex within weeks. Tau-mediated neurodegeneration proceeded without formation of large fibrillar tau-aggregates or tangles, but with increased expression of cell-cycle markers.We present novel AAV-based models, which demonstrate that protein tau mediates pyramidal neurodegeneration in vivo. The data firmly support the unifying hypothesis that post-mitotic neurons are forced to re-enter the cell-cycle in primary and secondary tauopathies, including Alzheimer's disease.

    PloS one 2009;4;10;e7280

  • Amyloid beta and impairment in multiple memory systems in older transgenic APP TgCRND8 mice.

    Hanna A, Horne P, Yager D, Eckman C, Eckman E and Janus C

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

    The relationship between amyloid beta and cognitive dysfunction in mouse models of Alzheimer's disease has been evaluated predominantly with the spatial reference memory version of the water maze task. However, as Alzheimer's disease encompasses decline in multiple memory systems, it is important to also utilize non-spatial tasks to fully characterize the role of amyloid on behaviour in animal models. We used the TgCRND8 mouse model of Alzheimer's disease to evaluate the effect of amyloid on spatial reference memory, as well as on the non-spatial task of acquisition of conditioned taste aversion, and on the procedural task of swimming to a visible platform. We demonstrate that 8- to 12-month-old TgCRND8 mice are significantly impaired in all three tasks, and that the levels of soluble amyloid beta are significantly correlated with impairment in spatial reference memory, but not with impairment in conditioned taste aversion or swimming to a visible platform. Insoluble fractions of amyloid, which correspond closely to amyloid plaque burden in the brain, are not associated with any behavioural measure. Our study extends the characterization of the model to stages of advanced amyloid pathology and demonstrates that older TgCRND8 mice are impaired in multiple memory systems, including procedural tasks, which are spared at younger ages. The lack of association between amyloid plaques and memory decline supports clinical findings in Alzheimer's patients.

    Funded by: NINDS NIH HHS: NS048554

    Genes, brain, and behavior 2009;8;7;676-84

  • Amyloid beta-derived neuroplasticity in bone marrow-derived mesenchymal stem cells is mediated by NPY and 5-HT2B receptors via ERK1/2 signalling pathways.

    Jin HK, Bae JS, Furuya S and Carter JE

    Department of Laboratory Animal Medicine, College of Veterinary Medicine, Kyungpook National University, Jung-Gu, Daegu, South Korea.

    Objective: In Alzheimer's disease, toxic soluble and insoluble forms of amyloid beta (Abeta) cause synaptic dysfunction and neuronal loss. Given its potential role in producing a toxic host microenvironment for transplanted donor stem cells, we investigated the interaction between Abeta and proliferation, survival, and differentiation of bone marrow-derived mesenchymal stem cells (BM-MSC) in culture.

    We used BM-MSC that had been isolated from mouse bone marrow and cultured, and we also assessed relevant reaction mechanisms using gene microarray, immunocytochemistry, and inhibitors of potential signalling molecules, such as mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK)1/2 and tyrosine protein kinase.

    Interestingly, we found that treatment with aggregated (1-40 or 1-42) and oligomeric (1-42) Abeta promoted neuronal-like differentiation of BM-MSC without toxic effects. This was not dependent on soluble factors released from BM-MSC progeny nor solely on formation of Abeta fibrils. The effect of Abeta is mediated by G-protein coupled receptors, neuropeptide Y1 (NPY1R) and serotonin (5-hydroxytryptamine) receptor 2B, via phosphatidylinositol-3-OH kinase-dependent activation of the MAPK/ERK1/2. Our results lend support to the idea that reciprocal donor stem cell-host interactions may promote a regenerative response that can be exploited by epigenetic modulation of NPY/serotonergic gene expression, for stem cell therapy, in Alzheimer's disease.

    Cell proliferation 2009;42;5;571-86

  • Amyloid precursor family proteins are expressed by thymic and lymph node stromal cells but are not required for lymphocyte development.

    Laky K, Annaert W and Fowlkes BJ

    Laboratory of Cellular and Molecular Immunology, National Institutes of Allergy and Infectious Disease, National Institutes of Health, Bethesda, MD 20892-0420, USA.

    Pharmacological inhibitors that block amyloid precursor protein (APP) cleavage and the formation of senile plaques are under development for the treatment of familial Alzheimer's disease. Unfortunately, many inhibitors that block gamma-secretase-mediated cleavage of APP also have immunosuppressive side effects. In addition to APP, numerous other proteins undergo gamma-secretase-mediated cleavage. In order to develop safer inhibitors, it is necessary to determine which of the gamma-secretase substrates contribute to the immunosuppressive effects. Because APP family members are widely expressed and are reported to influence calcium flux, transcription and apoptosis, they could be important for normal lymphocyte maturation. We find that APP and amyloid precursor-like protein 2 are expressed by stromal cells of thymus and lymph nodes, but not by lymphocytes. Although signals provided by thymic stromal cells are critical for normal T cell differentiation, lymphocyte development proceeds unperturbed in mice deficient for these APP family members.

    Funded by: Intramural NIH HHS

    International immunology 2009;21;10;1163-74

  • Genetic and pharmacological evidence of intraneuronal Abeta accumulation in APP transgenic mice.

    Philipson O, Lannfelt L and Nilsson LN

    Department of Public Health and Caring Sciences, Uppsala University, SE-751 85 Uppsala, Sweden.

    Intraneuronal punctate immunostaining in Alzheimer's disease brain and amyloid-beta precursor protein (APP) transgenic mice has been suggested to represent Abeta, but this is somewhat controversial. Here we show that both biochemical Abeta levels and intraneuronal immunostaining are reduced in APP transgenic mice when gamma-secretase is inhibited. Moreover, BACE-1 deficient APP transgenic mice show neither Abeta production nor intraneuronal immunostaining. Our findings suggest that the punctate immunostaining with APP antibodies is due to Abeta that has accumulated inside neurons. Similar type of intraneuronal Abeta accumulation, which precedes senile plaque formation, may link Abeta to tauopathy and neurodegeneration in Alzheimer's disease pathogenesis.

    FEBS letters 2009;583;18;3021-6

  • Reelin signaling antagonizes beta-amyloid at the synapse.

    Durakoglugil MS, Chen Y, White CL, Kavalali ET and Herz J

    Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.

    Abnormal processing of the amyloid precursor protein (APP) and beta-amyloid (Abeta) plaque accumulation are defining features of Alzheimer disease (AD), a genetically complex neurodegenerative disease that is characterized by progressive synapse loss and neuronal cell death. Abeta induces synaptic dysfunction in part by altering the endocytosis and trafficking of AMPA and NMDA receptors. Reelin is a neuromodulator that increases glutamatergic neurotransmission by signaling through the postsynaptic ApoE receptors Apoer2 and Vldlr and thereby potently enhances synaptic plasticity. Here we show that Reelin can prevent the suppression of long-term potentiation and NMDA receptors, which is induced by levels of Abeta comparable to those present in an AD-afflicted brain. This reversal is dependent upon the activation of Src family tyrosine kinases. At high concentrations of Abeta peptides, Reelin can no longer overcome the Abeta induced functional suppression and this coincides with a complete blockade of the Reelin-dependent phosphorylation of NR2 subunits. We propose a model in which Abeta, Reelin, and ApoE receptors modulate neurotransmission and thus synaptic stability as opposing regulators of synaptic gain control.

    Funded by: NHLBI NIH HHS: R37 HL063762

    Proceedings of the National Academy of Sciences of the United States of America 2009;106;37;15938-43

  • Non-fibrillar amyloid-beta peptide reduces NMDA-induced neurotoxicity, but not AMPA-induced neurotoxicity.

    Niidome T, Goto Y, Kato M, Wang PL, Goh S, Tanaka N, Akaike A, Kihara T and Sugimoto H

    Department of Neuroscience for Drug Discovery, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto 606-8501, Japan. tniidome@pharm.kyoto-u.ac.jp

    Amyloid-beta peptide (Abeta) is thought to be linked to the pathogenesis of Alzheimer's disease. Recent studies suggest that Abeta has important physiological roles in addition to its pathological roles. We recently demonstrated that Abeta42 protects hippocampal neurons from glutamate-induced neurotoxicity, but the relationship between Abeta42 assemblies and their neuroprotective effects remains largely unknown. In this study, we prepared non-fibrillar and fibrillar Abeta42 based on the results of the thioflavin T assay, Western blot analysis, and atomic force microscopy, and examined the effects of non-fibrillar and fibrillar Abeta42 on glutamate-induced neurotoxicity. Non-fibrillar Abeta42, but not fibrillar Abeta42, protected hippocampal neurons from glutamate-induced neurotoxicity. Furthermore, non-fibrillar Abeta42 decreased both neurotoxicity and increases in the intracellular Ca(2+) concentration induced by N-methyl-d-aspartate (NMDA), but not by alpha-amino-3-hydrozy-5-methyl-4-isoxazole propionic acid (AMPA). Our results suggest that non-fibrillar Abeta42 protects hippocampal neurons from glutamate-induced neurotoxicity through regulation of the NMDA receptor.

    Biochemical and biophysical research communications 2009;386;4;734-8

  • Presynaptic and postsynaptic interaction of the amyloid precursor protein promotes peripheral and central synaptogenesis.

    Wang Z, Wang B, Yang L, Guo Q, Aithmitti N, Songyang Z and Zheng H

    Huffington Center on Aging, Baylor College of Medicine, Houston, Texas 77030, USA.

    A critical role of the amyloid precursor protein (APP) in Alzheimer's disease (AD) pathogenesis has been well established. However, the physiological function of APP remains elusive and much debated. We reported previously that the APP family of proteins is essential in mediating the developing neuromuscular synapse. In the current study, we created a conditional allele of APP and deleted APP in presynaptic motor neuron or postsynaptic muscle. Crossing these alleles onto the APP-like protein 2-null background reveals that, unexpectedly, inactivating APP in either compartment results in neuromuscular synapse defects similar to the germline deletion and that postsynaptic APP is obligatory for presynaptic targeting of the high-affinity choline transporter and synaptic transmission. Using a HEK293 and primary hippocampus mixed-culture assay, we report that expression of APP in HEK293 cells potently promotes synaptogenesis in contacting axons. This activity is dependent on neuronal APP and requires both the extracellular and intracellular domains; the latter forms a complex with Mint1 and Cask and is replaceable by the corresponding SynCAM (synaptic cell adhesion molecule) sequences. These in vitro and in vivo studies identify APP as a novel synaptic adhesion molecule. We postulate that transsynaptic APP interaction modulates its synaptic function and that perturbed APP synaptic adhesion activity may contribute to synaptic dysfunction and AD pathogenesis.

    Funded by: NIA NIH HHS: AG032051, AG033467, R01 AG032051, R01 AG032051-01, R01 AG032051-02, R01 AG033467, R01 AG033467-01; NICHD NIH HHS: HD024064, P30 HD024064

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2009;29;35;10788-801

  • Age-dependent dysregulation of brain amyloid precursor protein in the Ts65Dn Down syndrome mouse model.

    Choi JH, Berger JD, Mazzella MJ, Morales-Corraliza J, Cataldo AM, Nixon RA, Ginsberg SD, Levy E and Mathews PM

    Center for Dementia Research, Nathan Kline Institute, Orangeburg, New York 10962, USA. jchoi@nki.rfmh.org

    Individuals with Down syndrome develop beta-amyloid deposition characteristic of early-onset Alzheimer's disease (AD) in mid-life, presumably because of an extra copy of the chromosome 21-located amyloid precursor protein (App) gene. App mRNA and APP metabolite levels were assessed in the brains of Ts65Dn mice, a mouse model of Down syndrome, using quantitative PCR, western blot analysis, immunoprecipitation, and ELISAs. In spite of the additional App gene copy, App mRNA, APP holoprotein, and all APP metabolite levels in the brains of 4-month-old trisomic mice were not increased compared with the levels seen in diploid littermate controls. However starting at 10 months of age, brain APP levels were increased proportional to the App gene dosage imbalance reflecting increased App message levels in Ts65Dn mice. Similar to APP levels, soluble amino-terminal fragments of APP (sAPPalpha and sAPPbeta) were increased in Ts65Dn mice compared with diploid mice at 12 months but not at 4 months of age. Brain levels of both Abeta40 and Abeta42 were not increased in Ts65Dn mice compared with diploid mice at all ages examined. Therefore, multiple mechanisms contribute to the regulation towards diploid levels of APP metabolites in the Ts65Dn mouse brain.

    Funded by: NIA NIH HHS: AG017617, AG029787, F31 AG029787, F31 AG029787-03, P01 AG017617, P01 AG017617-10, R01 AG043375; NINDS NIH HHS: NS045205, R01 NS045205

    Journal of neurochemistry 2009;110;6;1818-27

  • Amyloid precursor protein, heat-shock proteins, and Bcl-2 form a complex in mitochondria and modulate mitochondria function and apoptosis in N2a cells.

    Yang TT, Hsu CT and Kuo YM

    China Medical University, Taichung, Taiwan. tingting@mail.cmu.edu.tw

    Neurons that degenerate in the brains of persons with Alzheimer's disease accumulate mitochondrial amyloid precursor protein (APP), which is thought to negatively affect mitochondrial function and cellular homeostasis. Because proteins that enter mitochondria require assistance from chaperone proteins, we hypothesized that heat-shock proteins (HSPs) help accumulate APP in mitochondria. We found that APP overexpression in N2a cells (APP cells) did not elicit mitochondrial dysfunction. Because cerebral hypoperfusion-associated energy deficiency is an important etiology for Alzheimer's disease, we also challenged the cells with serum starvation. APP/HSP/Bcl-2 complexes formed within the mitochondria of serum-starved APP cells, but not control cells. Mitochondria containing APP/HSP/Bcl-2 complexes induced apoptosis. We hypothesize that APP/HSP/Bcl-2 complexes diminish the functional capacities of HSPs and Bcl-2, which leads to mitochondrial injury and apoptosis.

    Mechanisms of ageing and development 2009;130;9;592-601

  • Gamma-secretase inhibition reduces spine density in vivo via an amyloid precursor protein-dependent pathway.

    Bittner T, Fuhrmann M, Burgold S, Jung CK, Volbracht C, Steiner H, Mitteregger G, Kretzschmar HA, Haass C and Herms J

    Center of Neuropathology and Prion Research, Ludwig-Maximilians-Universität, Munich, Germany.

    Alzheimer's disease (AD) represents the most common age-related neurodegenerative disorder. It is characterized by the invariant accumulation of the beta-amyloid peptide (Abeta), which mediates synapse loss and cognitive impairment in AD. Current therapeutic approaches concentrate on reducing Abeta levels and amyloid plaque load via modifying or inhibiting the generation of Abeta. Based on in vivo two-photon imaging, we present evidence that side effects on the level of dendritic spines may counteract the beneficial potential of these approaches. Two potent gamma-secretase inhibitors (GSIs), DAPT (N-[N-(3,5-difluorophenacetyl-L-alanyl)]-S-phenylglycine t-butyl ester) and LY450139 (hydroxylvaleryl monobenzocaprolactam), were found to reduce the density of dendritic spines in wild-type mice. In mice deficient for the amyloid precursor protein (APP), both GSIs had no effect on dendritic spine density, demonstrating that gamma-secretase inhibition decreases dendritic spine density via APP. Independent of the effects of gamma-secretase inhibition, we observed a twofold higher density of dendritic spines in the cerebral cortex of adult APP-deficient mice. This observation further supports the notion that APP is involved in the modulation of dendritic spine density--shown here for the first time in vivo.

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2009;29;33;10405-9

  • Amyloid beta-protein stimulates trafficking of cholesterol and caveolin-1 from the plasma membrane to the Golgi complex in mouse primary astrocytes.

    Igbavboa U, Sun GY, Weisman GA, He Y and Wood WG

    Department of Pharmacology, University of Minnesota School of Medicine and Geriatric Research, Education and Clinical Center, VA Medical Center, 6-120 Jackson Hall, 321 Church Street Southeast, Minneapolis, MN 55455, USA. igbav001@umn.edu

    The Golgi complex plays a key role in cholesterol trafficking in cells. Our earlier study demonstrated amyloid beta-protein (Abeta) alters cholesterol distribution and abundance in the Golgi complex of astrocytes. We now test the hypothesis that the Abeta-induced increase in Golgi complex cholesterol is due to retrograde movement of the cholesterol carrier protein caveolin-1 from the cell plasma membrane to the Golgi complex in astrocytes. Results with mouse primary astrocytes indicated that Abeta(1-42)-induced increase in cholesterol and caveolin abundance in the Golgi complex was accompanied by a reduction in cholesterol and caveolin levels in the plasma membrane. Transfected rat astrocytes (DITNC1) with siRNA directed at caveolin-1 mRNA inhibited the Abeta(1-42)-induced redistribution of both cholesterol and caveolin from the plasma membrane to the Golgi complex. In astrocytes not treated with Abeta(1-42), suppression of caveolin-1 expression also significantly reduced cholesterol abundance in the Golgi complex, further demonstrating the role for caveolin in retrograde transport of cholesterol from the plasma membrane to the Golgi complex. Perturbation of this process by Abeta(1-42) could have consequences on membrane structure and cellular functions requiring optimal levels of cholesterol.

    Funded by: NIA NIH HHS: AG-18357, AG-23524, P01 AG018357, P01 AG018357-08, R01 AG023524

    Neuroscience 2009;162;2;328-38

  • Paradoxical condensation of copper with elevated beta-amyloid in lipid rafts under cellular copper deficiency conditions: implications for Alzheimer disease.

    Hung YH, Robb EL, Volitakis I, Ho M, Evin G, Li QX, Culvenor JG, Masters CL, Cherny RA and Bush AI

    Oxidation Biology Laboratory, The Mental Health Research Institute of Victoria, Parkville, Victoria, Australia.

    Redox-active copper is implicated in the pathogenesis of Alzheimer disease (AD), beta-amyloid peptide (Abeta) aggregation, and amyloid formation. Abeta.copper complexes have been identified in AD and catalytically oxidize cholesterol and lipid to generate H2O2 and lipid peroxides. The site and mechanism of this abnormality is not known. Growing evidence suggests that amyloidogenic processing of the beta-amyloid precursor protein (APP) occurs in lipid rafts, membrane microdomains enriched in cholesterol. beta- and gamma-secretases, and Abeta have been identified in lipid rafts in cultured cells, human and rodent brains, but the role of copper in lipid raft amyloidogenic processing is presently unknown. In this study, we found that copper modulates flotillin-2 association with cholesterol-rich lipid raft domains, and consequently Abeta synthesis is attenuated via copper-mediated inhibition of APP endocytosis. We also found that total cellular copper is associated inversely with lipid raft copper levels, so that under intracellular copper deficiency conditions, Abeta.copper complexes are more likely to form. This explains the paradoxical hypermetallation of Abeta with copper under tissue copper deficiency conditions in AD.

    The Journal of biological chemistry 2009;284;33;21899-907

  • RAGE regulates BACE1 and Abeta generation via NFAT1 activation in Alzheimer's disease animal model.

    Cho HJ, Son SM, Jin SM, Hong HS, Shin DH, Kim SJ, Huh K and Mook-Jung I

    Department of Biochemistry and Biomedical Sciences, Seoul National University College of Medicine, 28 Yungun-dong, Jongro-gu, Seoul, 110-799, Korea.

    The receptor for advanced glycation end products (RAGE) is a multiligand cell surface receptor, and amyloid beta peptide (Abeta) is one of the ligands for RAGE. Because RAGE is a transporter of Abeta from the blood to the brain, RAGE is believed to play an important role in Alzheimer's disease (AD) pathogenesis. In the present study, the role of RAGE in Abeta production was examined in the brain tissue of an AD animal model, Tg2576 mice, as well as cultured cells. Because beta-site APP-cleaving enzyme 1 (BACE1), an essential protease for Abeta production, is up-regulated in cells overexpressing RAGE and in RAGE-injected brains of Tg2576 mice, the molecular mechanisms underlying RAGE, BACE1 expression, and Abeta production were examined. Because RAGE stimulates intracellular calcium, nuclear factor of activated T-cells 1 (NFAT1) was examined. NFAT1 was activated following RAGE-induced BACE1 expression followed by Abeta generation. Injection of soluble RAGE (sRAGE), which acts as a competitor with full-length RAGE (fRAGE), into aged Tg2576 mouse brains reduced the levels of plaques, Abeta, BACE1, and the active form of NFAT1 compared with fRAGE-injected Tg2576 mice. Taken together, RAGE stimulates functional BACE1 expression through NFAT1 activation, resulting in more Abeta production and deposition in the brain.

    FASEB journal : official publication of the Federation of American Societies for Experimental Biology 2009;23;8;2639-49

  • SLP-76-ADAP adaptor module regulates LFA-1 mediated costimulation and T cell motility.

    Wang H, Wei B, Bismuth G and Rudd CE

    Cell Signaling Section, Department of Pathology, Tennis Court Road, University of Cambridge, Cambridge CB2 1QP, United Kingdom.

    Although adaptor ADAP (FYB) and its binding to SLP-76 has been implicated in TcR-induced "inside-out" signaling for LFA-1 activation in T cells, little is known regarding its role in LFA-1-mediated "outside-in" signaling. In this study, we demonstrate that ADAP and SLP-76-ADAP binding are coupled to LFA-1 costimulation of IL-2 production, F-actin clustering, cell polarization, and T cell motility. LFA-1 enhancement of anti-CD3-induced IL-2 production was completely dependent on SLP-76-ADAP binding. Further, anti-CD3 was found to require CD11a ligation by antibody or ICAM1 to cause T cell polarization. ADAP augmented this polarization induced by anti-CD3/CD11a, but not by anti-CD3 alone. ADAP expression with LFA-1 ligation alone was sufficient to polarize T cells directly and to increase T cell motility whereas the loss of ADAP in ADAP-/- primary T cells reduced motility. A mutant lacking SLP-76-binding sites (M12) blocked LFA-1 costimulation of IL-2 production, polarization, and motility. LFA-1-ADAP polarization was also dependent on src kinases, Rho GTPases, phospholipase C, and phosphoinositol 3-kinase. Our findings provide evidence of an obligatory role for the SLP-76-ADAP module in LFA-1-mediated costimulation in T cells.

    Funded by: Wellcome Trust

    Proceedings of the National Academy of Sciences of the United States of America 2009;106;30;12436-41

  • Decreased brain-derived neurotrophic factor depends on amyloid aggregation state in transgenic mouse models of Alzheimer's disease.

    Peng S, Garzon DJ, Marchese M, Klein W, Ginsberg SD, Francis BM, Mount HT, Mufson EJ, Salehi A and Fahnestock M

    Department of Psychiatry and Behavioural Neurosciences, McMaster University, Hamilton, Ontario L8N 3Z5, Canada.

    Downregulation of brain-derived neurotrophic factor (BDNF) in the cortex occurs early in the progression of Alzheimer's disease (AD). Since BDNF plays a critical role in neuronal survival, synaptic plasticity, and memory, BDNF reduction may contribute to synaptic and cellular loss and memory deficits characteristic of AD. In vitro evidence suggests that amyloid-beta (A beta) contributes to BDNF downregulation in AD, but the specific A beta aggregation state responsible for this downregulation in vivo is unknown. In the present study, we examined cortical levels of BDNF mRNA in three different transgenic AD mouse models harboring mutations in APP resulting in A beta overproduction, and in a genetic mouse model of Down syndrome. Two of the three A beta transgenic strains (APP(NLh) and TgCRND8) exhibited significantly decreased cortical BDNF mRNA levels compared with wild-type mice, whereas neither the other strain (APP(swe)/PS-1) nor the Down syndrome mouse model (Ts65Dn) was affected. Only APP(NLh) and TgCRND8 mice expressed high A beta(42)/A beta(40) ratios and larger SDS-stable A beta oligomers (approximately 115 kDa). TgCRND8 mice exhibited downregulation of BDNF transcripts III and IV; transcript IV is also downregulated in AD. Furthermore, in all transgenic mouse strains, there was a correlation between levels of large oligomers, A beta(42)/A beta(40), and severity of BDNF decrease. These data show that the amount and species of A beta vary among transgenic mouse models of AD and are negatively correlated with BDNF levels. These findings also suggest that the effect of A beta on decreased BDNF expression is specific to the aggregation state of A beta and is dependent on large oligomers.

    Funded by: Canadian Institutes of Health Research: 85042; NIA NIH HHS: AG10688, P01 AG014449, P01 AG017617, R01 AG043375; NINDS NIH HHS: P01 NS048447, R01 NS043939

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2009;29;29;9321-9

  • Thyroid hormones reverse the UV-induced repression of APP in neuroblastoma cells.

    Cuesta A, Zambrano A, López E and Pascual A

    Instituto de Investigaciones Biomédicas (C.S.I.C.), Consejo Superior de Investigaciones Científicas, Arturo Duperier, 4, 28029 Madrid, Spain.

    As a precursor of the neurotoxic amyloid-beta peptide, APP plays a central role in Alzheimer's disease. We have recently reported that the tumor suppressor p53 inhibits APP gene transcription through the same DNA sequences that mediate an inhibitory effect of thyroid hormones. Now, we have analyzed whether the thyroid hormone T3 can modulate the effects of p53 on APP expression. Exposition to UVC radiation leads to a marked decrease of intracellular APP levels that is paradoxically reversed by T3. Repression by UVC and reversion by the hormone are not observed in cells depleted of p53, demonstrating a p53-dependent mechanism. These results suggest the existence of a cross-talk between p53 and T3 that could play an important role in Alzheimer s disease.

    FEBS letters 2009;583;14;2401-6

  • Insulin receptor dysfunction impairs cellular clearance of neurotoxic oligomeric a{beta}.

    Zhao WQ, Lacor PN, Chen H, Lambert MP, Quon MJ, Krafft GA and Klein WL

    Department of Neurobiology and Physiology, Northwestern University, Evanston, Illinois 60208, USA. wei-qin_zhao@merck.com

    Accumulation of amyloid beta (Abeta) oligomers in the brain is toxic to synapses and may play an important role in memory loss in Alzheimer disease. However, how these toxins are built up in the brain is not understood. In this study we investigate whether impairments of insulin and insulin-like growth factor-1 (IGF-1) receptors play a role in aggregation of Abeta. Using primary neuronal culture and immortal cell line models, we show that expression of normal insulin or IGF-1 receptors confers cells with abilities to reduce exogenously applied Abeta oligomers (also known as ADDLs) to monomers. In contrast, transfection of malfunctioning human insulin receptor mutants, identified originally from patient with insulin resistance syndrome, or inhibition of insulin and IGF-1 receptors via pharmacological reagents increases ADDL levels by exacerbating their aggregation. In healthy cells, activation of insulin and IGF-1 receptor reduces the extracellular ADDLs applied to cells via seemingly the insulin-degrading enzyme activity. Although insulin triggers ADDL internalization, IGF-1 appears to keep ADDLs on the cell surface. Nevertheless, both insulin and IGF-1 reduce ADDL binding, protect synapses from ADDL synaptotoxic effects, and prevent the ADDL-induced surface insulin receptor loss. Our results suggest that dysfunctions of brain insulin and IGF-1 receptors contribute to Abeta aggregation and subsequent synaptic loss.

    Funded by: Intramural NIH HHS; NIA NIH HHS: R01 AG022547, R01-AG022547

    The Journal of biological chemistry 2009;284;28;18742-53

  • Human and rodent amyloid-beta peptides differentially bind heme: relevance to the human susceptibility to Alzheimer's disease.

    Atamna H, Frey WH and Ko N

    Nutrition and Metabolism Center, Children's Hospital Oakland Research Institute, Oakland, CA 94609, USA. hatamna@tcmedc.org

    Amyloid-beta (Abeta) peptides are implicated in the neurodegeneration of Alzheimer's disease (AD). We previously investigated the mechanism of neurotoxicity of Abeta and found that human Abeta (huAbeta) binds and depletes heme, forming an Abeta-heme complex with peroxidase activity. Rodent Abeta (roAbeta) is identical to huAbeta, except for three amino acids within the proposed heme-binding motif (Site-H). We studied and compared heme-binding between roAbeta and huAbeta. Unlike roAbeta, huAbeta binds heme tightly (K(d)=140+/-60 nM) and forms a peroxidase. The plot of bound (huAbeta-heme) vs. unbound heme fits best to a two site binding hyperbola, suggesting huAbeta possesses two heme-binding sites. Consistently, a second high affinity heme-binding site was identified in the lipophilic region (site-L) of huAbeta (K(d)=210+/-80 nM). The plot of (roAbeta-heme) vs. unbound heme, on the other hand, was different as it fits best to a sigmoidal binding curve, indicating different binding and lower affinity of roAbeta for heme (K(d)=1 microM). The effect of heme-binding to site-H on heme-binding to site-L in roAbeta and huAbeta is discussed. While both roAbeta and huAbeta form aggregates equally, rodents lack AD-like neuropathology. High huAbeta/heme ratio increases the peroxidase activity. These findings suggest that depletion of regulatory heme and formation of Abeta-heme peroxidase contribute to huAbeta's neurotoxicity in the early stages of AD. Phylogenic variations in the amino acid sequence of Abeta explain tight heme-binding to huAbeta and likely contribute to the increased human susceptibility to AD.

    Archives of biochemistry and biophysics 2009;487;1;59-65

  • Gene expression profiles of APP and BACE1 in Tg SOD1G93A cortical cells.

    Spadoni O, Crestini A, Piscopo P, Malvezzi-Campeggi L, Carunchio I, Pieri M, Zona C and Confaloni A

    Department of Cellular Biology and Neuroscience, Istituto Superiore di Sanità, Viale Regina, Elena 299, Rome, Italy.

    Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease defined by motor neuron loss. Transgenic mouse model (Tg SOD1G93A) shows pathological features that closely mimic those seen in ALS patients. An hypothetic link between AD and ALS was suggested by finding an higher amount of amyloid precursor protein (APP) in the spinal cord anterior horn neurons, and of Abeta peptides in ALS patients skin. In this work, we have investigated the expression of some genes involved in Alzheimer's disease, as APP, beta- and gamma-secretase, in an animal model of ALS, to understand some possible common molecular mechanisms between these two pathologies. For gene expression analysis, we carried out a quantitative RT-PCR in ALS mice and in transgenic mice over-expressing human wild-type SOD1 (Tg hSOD1). We found that APP and BACE1 mRNA levels were increased 1.5-fold in cortical cells of Tg SOD1G93A mice respect to Tg hSOD1, whereas the expression of gamma-secretase genes, as PSEN1, PSEN2, Nicastrin, and APH1a, showed no statistical differences between wild-type and ALS mice. Biochemical analysis carried out by immunostaining and western blotting, did not show any significant modulation of the protein expression compared to the genes, suggesting the existence of post-translational mechanisms that modify protein levels.

    Cellular and molecular neurobiology 2009;29;5;635-41

  • Hypoxia increases Abeta generation by altering beta- and gamma-cleavage of APP.

    Li L, Zhang X, Yang D, Luo G, Chen S and Le W

    Institute of Health Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences & Shanghai Jiao Tong University School of Medicine, Shanghai, PR China.

    Environmental factors are significant contributors for the development of Alzheimer's disease (AD). The greatly increased incidence of AD following stroke and cerebral ischemia suggests that hypoxia is a risk factor which may accelerate AD pathogenesis by altering amyloid precursor protein (APP) processing. However, the molecular mechanisms underlying the hypoxia mediated AD pathogenesis have not been fully elucidated. In the present study we demonstrated that repeated hypoxia increased beta-amyloid (Abeta) generation and neuritic plaques formation by elevating beta-cleavage of APP in APP(swe)+PS1(A246E) transgenic mice. We also found that hypoxia enhanced the expression of APH-1a, a component of gamma-secretase complex, which in turn may lead to increase in gamma-cleavage activity. Furthermore, we demonstrated that repeated hypoxia treatment can activate macroautophagy, which may contribute to the increases in Abeta production since pretreatment with macroautophagy inhibitor 3-methyladenine significantly blocked chemical hypoxic condition-induced increase in Abeta production in SH-SY5Y cells. Taken together, our results suggest an important role of hypoxia in modulating the APP processing by facilitating both beta- and gamma-cleavage which may result in a significant increase of Abeta generation.

    Neurobiology of aging 2009;30;7;1091-8

  • Pharmacological evidences for DFK167-sensitive presenilin-independent gamma-secretase-like activity.

    Sevalle J, Ayral E, Hernandez JF, Martinez J and Checler F

    Institut de Pharmacologie Moléculaire et Cellulaire and Institut de NeuroMédecine Moléculaire, UMR6097 CNRS/UNSA, Equipe labellisée Fondation pour la Recherche Médicale, Sophia-Antipolis, Valbonne, France.

    Amyloid-beta (Abeta) peptides production is thought to be a key event in the neurodegenerative process ultimately leading to Alzheimer's disease (AD) pathology. A bulk of studies concur to propose that the C-terminal moiety of Abeta is released from its precursor beta-amyloid precursor protein by a high molecular weight enzymatic complex referred to as gamma-secretase, that is composed of at least, nicastrin (NCT), Aph-1, Pen-2, and presenilins (PS) 1 or 2. They are thought to harbor the gamma-secretase catalytic activity. However, several lines of evidence suggest that additional gamma-secretase-like activities could potentially contribute to Abeta production. By means of a quenched fluorimetric substrate (JMV2660) mimicking the beta-amyloid precursor protein sequence targeted by gamma-secretase, we first show that as expected, this probe allows monitoring of an activity detectable in several cell systems including the neuronal cell line telencephalon specific murine neurons (TSM1). This activity is reduced by DFK167, N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT), and LY68458, three inhibitors known to functionally interact with PS. Interestingly, JMV2660 but not the unrelated peptide JMV2692, inhibits Abeta production in an in vitrogamma-secretase assay as expected from a putative substrate competitor. This activity is enhanced by PS1 and PS2 mutations known to be responsible for familial forms of AD and reduced by aspartyl mutations inactivating PS or in cells devoid of PS or NCT. However, we clearly establish that residual JMV2660-hydrolysing activity could be recovered in PS- and NCT-deficient fibroblasts and that this activity remained inhibited by DFK167. Overall, our study describes the presence of a proteolytic activity displaying gamma-secretase-like properties but independent of PS and still blocked by DFK167, suggesting that the PS-dependent complex could not be the unique gamma-secretase activity responsible for Abeta production and delineates PS-independent gamma-secretase activity as a potential additional therapeutic target to fight AD pathology.

    Journal of neurochemistry 2009;110;1;275-83

  • Rac1 inhibition negatively regulates transcriptional activity of the amyloid precursor protein gene.

    Wang PL, Niidome T, Akaike A, Kihara T and Sugimoto H

    Department of Neuroscience for Drug Discovery, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto, Japan.

    Rac1, a member of the Rho family GTPases, participates in a variety of cellular functions including lamellipodia formation, actin cytoskeleton organization, cell growth, apoptosis, and neuronal development. Recent studies have implicated Rac1 in cytoskeletal abnormalities, production of reactive oxygen species, and generation of the amyloid beta-peptide (Abeta) observed in Alzheimer's disease. In this study, we examined the relationship between Rac1 and amyloid precursor protein (APP), because the abnormal proteolytic processing of APP is a pathologic feature of Alzheimer's disease. In primary hippocampal neurons, the Rac1-specific inhibitor NSC23766 decreased both Rac1 activity and APP protein levels in a concentration-dependent manner. To elucidate how NSC23766 decreases APP protein levels, we examined the effects of NSC23766 on APP processing, degradation, and biosynthesis. NSC23766 did not increase the levels of the proteolytic products of APP, sAPPalpha, Abeta40, and Abeta42. The proteasome inhibitor lactacystin did not reverse the NSC23766-induced decrease in APP protein levels. NSC23766 did, however, decrease the levels of both APP mRNA and APP protein. Decreased levels of APP mRNA and protein were also observed when HEK293 cells were transfected with an expression vector containing a dominant-negative Rac1 mutant or with siRNA targeting Rac1. By overexpressing progressively deleted fragments of the APP promoter in HEK293 cells, we identified a Rac1 response site at positions -233 to -41 bp in the APP promoter. Taken together, our results suggest that Rac1 regulates transcription of the APP gene in primary hippocampal neurons.

    Journal of neuroscience research 2009;87;9;2105-14

  • Repeated novel cage exposure-induced improvement of early Alzheimer's-like cognitive and amyloid changes in TASTPM mice is unrelated to changes in brain endocannabinoids levels.

    Pardon MC, Sarmad S, Rattray I, Bates TE, Scullion GA, Marsden CA, Barrett DA, Lowe J and Kendall DA

    University of Nottingham Medical School, Institute of Neuroscience, School of Biomedical Sciences, Queen's Medical Centre, Nottingham, United Kingdom. marie.pardon@nottingham.ac.uk

    Environmental factors (e.g. stress, exercise, enrichment) are thought to play a role in the development of Alzheimer's disease later in life. We investigated the influence of repeated novel cage exposure on the development of early Alzheimer's-like pathology in adult (4 months old) double transgenic mice over-expressing the amyloid precursor protein and presenilin-1 genes (TASTPM mouse line). The procedure involves the repeated placement of the animal into a novel clean cage, a manipulation which induces a stress response and exploratory activity and, as such, can also be seen as a mild form of enrichment. Before and after exposure to the novel cage procedure, separate groups of mice were evaluated for locomotor performance and short-term contextual memory in the fear-conditioning test. Repeated novel cage exposure prevented the onset of a short-term memory deficit that was apparent in 5.5- but not 4-month-old TASTPM mice, without reversing the deficit in extinction already evident at 4 months of age. Brain regional levels of soluble and insoluble amyloid and of endocannabinoids were quantified. Novel cage exposure attenuated soluble and insoluble amyloid accumulation in the hippocampus and frontal cortex, without affecting the age-related increases in regional brain endocannabinoids levels. These beneficial effects are likely to be the consequence of the increase in physical and exploratory activity induced by novel cage exposure and suggest that the impact of environmental factors on Alzheimer's-like changes may be dependent on the degree of activation of stress pathways.

    Neurobiology of aging 2009;30;7;1099-113

  • Interaction of reelin with amyloid precursor protein promotes neurite outgrowth.

    Hoe HS, Lee KJ, Carney RS, Lee J, Markova A, Lee JY, Howell BW, Hyman BT, Pak DT, Bu G and Rebeck GW

    Department of Neuroscience, Georgetown University Medical Center, Washington, DC 20057-1464, USA.

    The processing of amyloid precursor protein (APP) to Abeta is an important event in the pathogenesis of Alzheimer's disease, but the physiological function of APP is not well understood. Our previous work has shown that APP processing and Abeta production are regulated by the extracellular matrix protein Reelin. In the present study, we examined whether Reelin interacts with APP, and the functional consequences of that interaction in vitro. Using coimmunoprecipitation, we found that Reelin interacted with APP through the central domain of Reelin (repeats 3-6) and the E1 extracellular domain of APP. Reelin increased cell surface levels of APP and decreased endocytosis of APP in hippocampal neurons in vitro. In vivo, Reelin levels were increased in brains of APP knock-out mice and decreased in APP-overexpressing mice. RNA interference knockdown of APP decreased neurite outgrowth in vitro and prevented Reelin from increasing neurite outgrowth. Knock-out of APP or Reelin decreased dendritic arborization in cortical neurons in vivo, and APP overexpression increased dendritic arborization. APP and Reelin have previously been shown to promote neurite outgrowth through interactions with integrins. We confirmed that APP interacted with alpha3beta1 integrin, and alpha3beta1 integrin altered APP trafficking and processing. Addition of an alpha3beta1 integrin antibody prevented APP and Reelin-induced neurite outgrowth. These findings demonstrate that Reelin interacts with APP, potentially having important effects on neurite development.

    Funded by: NIA NIH HHS: AG014473, AG032330, P01 AG030128, R01 AG014473, R01 AG014473-13, R01 AG027924, R01 AG035355, R03 AG030060, R03 AG030060-02, R03 AG032330, R29 AG014473, R37 AG012406; NINDS NIH HHS: R01 NS048085, R01 NS048085-04

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2009;29;23;7459-73

  • Omi is a mammalian heat-shock protein that selectively binds and detoxifies oligomeric amyloid-beta.

    Liu ML, Liu MJ, Shen YF, Ryu H, Kim HJ, Klupsch K, Downward J and Hong ST

    Laboratory of Genetics, Department of Microbiology and Immunology and Institute for Medical Sciences, Chonbuk National University Medical School, Chonju 561-712, South Korea.

    The cellular generation of toxic metabolites and subsequent detoxification failure can cause the uncontrolled accumulation of these metabolites in cells, leading to cellular dysfunction. Amyloid-beta protein (Abeta), a normal metabolite of neurons, tends to form toxic oligomeric structures that cause neurodegeneration. It is unclear how healthy neurons control the levels of intracellular oligomeric Abeta in order to avoid neurodegeneration. Using immunochemical and biochemical studies, we show that the Abeta-binding serine protease Omi is a stress-relieving heat-shock protein that protects neurons against neurotoxic oligomeric Abeta. Through its PDZ domain, Omi binds preferentially to neurotoxic oligomeric forms of Abeta rather than non-toxic monomeric forms to detoxify oligomeric Abeta by disaggregation. This specific interaction leads not only to mutual detoxification of the pro-apoptotic activity of Omi and Abeta-induced neurotoxicity, but also to a reduction of neurotoxic-Abeta accumulation. The neuroprotective role of Omi is further supported by its upregulation during normal neurogenesis and neuronal maturation in mice, which could be in response to the increase in the generation of oligomeric Abeta during these processes. These findings provide novel and important insights into the detoxification pathway of intraneuronal oligomeric Abeta in mammals and the protective roles of Omi in neurodegeneration, suggesting a novel therapeutic target in neurodegenerative diseases.

    Journal of cell science 2009;122;Pt 11;1917-26

  • Organoselenium (Sel-Plex diet) decreases amyloid burden and RNA and DNA oxidative damage in APP/PS1 mice.

    Lovell MA, Xiong S, Lyubartseva G and Markesbery WR

    Department of Chemistry, University of Kentucky, Lexington, KY 40536, USA. malove2@email.uky.edu

    To evaluate potential antioxidant characteristics of organic selenium (Se), double knock-in transgenic mice expressing human mutations in the amyloid precursor protein (APP) and human presenilin-1 (PS1) were provided a Se-deficient diet, a Se-enriched diet (Sel-Plex), or a control diet from 4 to 9 months of age followed by a control diet until 12 months of age. Levels of DNA, RNA, and protein oxidation as well as lipid peroxidation markers were determined in all mice and amyloid beta-peptide (Abeta) plaques were quantified. APP/PS1 mice provided Sel-Plex showed significantly (P<0.05) lower levels of Abeta plaque deposition and significantly decreased levels of DNA and RNA oxidation. Sel-Plex-treated mice showed no significant differences in levels of lipid peroxidation or protein oxidation compared to APP/PS1 mice on a control diet. To determine if diminished oxidative damage was associated with increased antioxidant enzyme activities, brain glutathione peroxidase (GSH-Px), glutathione reductase, and glutathione transferase activities were measured. Sel-Plex-treated mice showed a modest but significant increase in GSH-Px activity compared to mice on a normal diet (P<0.5). Overall, these data suggest that organic Se can reduce Abeta burden and minimize DNA and RNA oxidation and support a role for it as a potential therapeutic agent in neurologic disorders with increased oxidative stress.

    Funded by: NIA NIH HHS: 5-P01-AG05119, 5-P30-AG028383, P01 AG005119, P01 AG005119-20A1, P30 AG028383, P30 AG028383-03

    Free radical biology & medicine 2009;46;11;1527-33

  • Intracellular sAPP retention in response to Abeta is mapped to cytoskeleton-associated structures.

    Henriques AG, Vieira SI, Crespo-López ME, Guiomar de Oliveira MA, da Cruz e Silva EF and da Cruz e Silva OA

    Laboratório de Neurociências, Centro de Biologia Celular, Universidade de Aveiro, Aveiro, Portugal.

    Amyloid beta (Abeta) contributes to neurodegeneration in Alzheimer's disease and provides a close association between molecular events and pathology, although the underlying molecular mechanisms are unclear. In the work described here, Abeta did not induce amyloid precursor protein (APP) expression, but APP processing/trafficking was markedly affected. In COS-7 cells, Abeta provokes retention of intracellular sAPPalpha (isAPPalpha). Intracellular holo-APP levels remain unchanged, and extracellular total sAPP increases, although extracellular sAPPalpha alone was not altered significantly. In primary neuronal cultures and PC12 cells, isAPP also increased, but this was mirrored by a decrease in extracellular total sAPP. The isAPP retention was particularly associated with the cytoskeletal fraction. The retention "per se" occurred in vesicular-like densities, negative for a C-terminal antibody and strongly positive for the 6E10 antibody, clearly showing abnormal intracellular accumulation of sAPPalpha in response to Abeta. Our data support a dynamic model for intracellular retention of sAPPalpha as an early response to Abeta exposure. Particularly noteworthy was the observation that removal of Abeta reversed the isAPP accumulation. Mechanistically, these findings disclose an attractive physiological response, revealing the capacity of cells to deal with adverse effects induced by Abeta.

    Journal of neuroscience research 2009;87;6;1449-61

  • Intracellular trafficking of presenilin 1 is regulated by beta-amyloid precursor protein and phospholipase D1.

    Liu Y, Zhang YW, Wang X, Zhang H, You X, Liao FF and Xu H

    Burnham Institute for Medical Research, La Jolla, California 92037, USA.

    Excessive accumulation of beta-amyloid peptides in the brain is a major cause for the pathogenesis of Alzheimer disease. beta-Amyloid is derived from beta-amyloid precursor protein (APP) through sequential cleavages by beta- and gamma-secretases, whose enzymatic activities are tightly controlled by subcellular localization. Delineation of how intracellular trafficking of these secretases and APP is regulated is important for understanding Alzheimer disease pathogenesis. Although APP trafficking is regulated by multiple factors including presenilin 1 (PS1), a major component of the gamma-secretase complex, and phospholipase D1 (PLD1), a phospholipid-modifying enzyme, regulation of intracellular trafficking of PS1/gamma-secretase and beta-secretase is less clear. Here we demonstrate that APP can reciprocally regulate PS1 trafficking; APP deficiency results in faster transport of PS1 from the trans-Golgi network to the cell surface and increased steady state levels of PS1 at the cell surface, which can be reversed by restoring APP levels. Restoration of APP in APP-deficient cells also reduces steady state levels of other gamma-secretase components (nicastrin, APH-1, and PEN-2) and the cleavage of Notch by PS1/gamma-secretase that is more highly correlated with cell surface levels of PS1 than with APP overexpression levels, supporting the notion that Notch is mainly cleaved at the cell surface. In contrast, intracellular trafficking of beta-secretase (BACE1) is not regulated by APP. Moreover, we find that PLD1 also regulates PS1 trafficking and that PLD1 overexpression promotes cell surface accumulation of PS1 in an APP-independent manner. Our results clearly elucidate a physiological function of APP in regulating protein trafficking and suggest that intracellular trafficking of PS1/gamma-secretase is regulated by multiple factors, including APP and PLD1.

    Funded by: NIA NIH HHS: R01 AG021173, R01 AG030197; NINDS NIH HHS: R01 NS046673, R01 NS054880

    The Journal of biological chemistry 2009;284;18;12145-52

  • Novel role of RanBP9 in BACE1 processing of amyloid precursor protein and amyloid beta peptide generation.

    Lakshmana MK, Yoon IS, Chen E, Bianchi E, Koo EH and Kang DE

    Department of Neurosciences, University of California, San Diego, La Jolla, California 92093, USA.

    Accumulation of the amyloid beta (Abeta) peptide derived from the proteolytic processing of amyloid precursor protein (APP) is the defining pathological hallmark of Alzheimer disease. We previously demonstrated that the C-terminal 37 amino acids of lipoprotein receptor-related protein (LRP) robustly promoted Abeta generation independent of FE65 and specifically interacted with Ran-binding protein 9 (RanBP9). In this study we found that RanBP9 strongly increased BACE1 cleavage of APP and Abeta generation. This pro-amyloidogenic activity of RanBP9 did not depend on the KPI domain or the Swedish APP mutation. In cells expressing wild type APP, RanBP9 reduced cell surface APP and accelerated APP internalization, consistent with enhanced beta-secretase processing in the endocytic pathway. The N-terminal half of RanBP9 containing SPRY-LisH domains not only interacted with LRP but also with APP and BACE1. Overexpression of RanBP9 resulted in the enhancement of APP interactions with LRP and BACE1 and increased lipid raft association of APP. Importantly, knockdown of endogenous RanBP9 significantly reduced Abeta generation in Chinese hamster ovary cells and in primary neurons, demonstrating its physiological role in BACE1 cleavage of APP. These findings not only implicate RanBP9 as a novel and potent regulator of APP processing but also as a potential therapeutic target for Alzheimer disease.

    Funded by: NIA NIH HHS: AG 005131-24S1

    The Journal of biological chemistry 2009;284;18;11863-72

  • AbetaPP/APLP2 family of Kunitz serine proteinase inhibitors regulate cerebral thrombosis.

    Xu F, Previti ML, Nieman MT, Davis J, Schmaier AH and Van Nostrand WE

    Department of Medicine, Stony Brook University, Stony Brook, New York 11794-8153, USA.

    The amyloid beta-protein precursor (AbetaPP) is best recognized as the precursor to the Abeta peptide that accumulates in the brains of patients with Alzheimer's disease, but less is known about its physiological functions. Isoforms of AbetaPP that contain a Kunitz-type serine proteinase inhibitor (KPI) domain are expressed in brain and, outside the CNS, in circulating blood platelets. Recently, we showed that KPI-containing forms of AbetaPP regulates cerebral thrombosis in vivo (Xu et al., 2005, 2007). Amyloid precursor like protein-2 (APLP2), a closely related homolog to AbetaPP, also possesses a highly conserved KPI domain. Virtually nothing is known of its function. Here, we show that APLP2 also regulates cerebral thrombosis risk. Recombinant purified KPI domains of AbetaPP and APLP2 both inhibit the plasma clotting in vitro. In a carotid artery thrombosis model, both AbetaPP(-/-) and APLP2(-/-) mice exhibit similar significantly shorter times to vessel occlusion compared with wild-type mice indicating a prothrombotic phenotype. Similarly, in an experimental model of intracerebral hemorrhage, both AbetaPP(-/-) and APLP2(-/-) mice produce significantly smaller hematomas with reduced brain hemoglobin content compared with wild-type mice. Together, these results indicate that AbetaPP and APLP2 share overlapping anticoagulant functions with regard to regulating thrombosis after cerebral vascular injury.

    Funded by: NHLBI NIH HHS: R01 HL052779, R01-HL052779; NINDS NIH HHS: R01 NS052533, R01 NS052533-03, R01-NS052533

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2009;29;17;5666-70

  • {beta}-Amyloid impairs AMPA receptor trafficking and function by reducing Ca2+/calmodulin-dependent protein kinase II synaptic distribution.

    Gu Z, Liu W and Yan Z

    Department of Physiology and Biophysics, State University of New York, School of Medicine and Biomedical Sciences, Buffalo, New York 14214, USA.

    A fundamental feature of Alzheimer disease (AD) is the accumulation of beta-amyloid (Abeta), a peptide generated from the amyloid precursor protein (APP). Emerging evidence suggests that soluble Abeta oligomers adversely affect synaptic function, which leads to cognitive failure associated with AD. The Abeta-induced synaptic dysfunction has been attributed to the synaptic removal of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors (AMPARs); however, it is unclear how Abeta induces the loss of AMPARs at the synapses. In this study we have examined the potential involvement of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII), a signaling molecule critical for AMPAR trafficking and function. We found that the synaptic pool of CaMKII was significantly decreased in cortical neurons from APP transgenic mice, and the density of CaMKII clusters at synapses was significantly reduced by Abeta oligomer treatment. In parallel, the surface expression of GluR1 subunit as well as AMPAR-mediated synaptic response and ionic current was selectively decreased in APP transgenic mice and Abeta-treated cultures. Moreover, the reducing effect of Abeta on AMPAR current density was mimicked and occluded by knockdown of CaMKII and blocked by overexpression of CaMKII. These results suggest that the Abeta-induced change in CaMKII subcellular distribution may underlie the removal of AMPARs from synaptic membrane by Abeta.

    Funded by: NIA NIH HHS: AG21923; NIMH NIH HHS: MH84233

    The Journal of biological chemistry 2009;284;16;10639-49

  • Synaptic NMDA receptor activation stimulates alpha-secretase amyloid precursor protein processing and inhibits amyloid-beta production.

    Hoey SE, Williams RJ and Perkinton MS

    King's College London, Wolfson Centre for Age-Related Diseases, London, United Kingdom.

    Altered amyloid precursor protein (APP) processing leading to increased production and oligomerization of Abeta may contribute to Alzheimer's disease (AD). Understanding how APP processing is regulated under physiological conditions may provide new insights into AD pathogenesis. Recent reports demonstrate that excitatory neural activity regulates APP metabolism and Abeta levels, although understanding of the molecular mechanisms involved is incomplete. We have investigated whether NMDA receptor activity regulates APP metabolism in primary cultured cortical neurons. We report that a pool of APP is localized to the postsynaptic compartment in cortical neurons and observed partial overlap of APP with both NR1 and PSD-95. NMDA receptor stimulation increased nonamyloidogenic alpha-secretase-mediated APP processing, as measured by a 2.5-fold increase in cellular alpha-C-terminal fragment (C83) levels after glutamate or NMDA treatment. This increase was blocked by the NMDA receptor antagonists d-AP5 and MK801 but not by the AMPA receptor antagonist CNQX or the L-type calcium channel blocker nifedipine, was prevented by chelation of extracellular calcium, and was blocked by the alpha-secretase inhibitor TAPI-1. Cotreatment of cortical neurons with bicuculline and 4-AP, which stimulates glutamate release and activates synaptic NMDA receptors, evoked an MK801-sensitive increase in C83 levels. Furthermore, NMDA receptor stimulation caused a twofold increase in the amount of soluble APP detected in the neuronal culture medium. Finally, NMDA receptor activity inhibited both Abeta1-40 release and Gal4-dependent luciferase activity induced by beta-gamma-secretase-mediated cleavage of an APP-Gal4 fusion protein. Altogether, these data suggest that calcium influx through synaptic NMDA receptors promotes nonamyloidogenic alpha-secretase-mediated APP processing.

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2009;29;14;4442-60

  • Disruption of fast axonal transport is a pathogenic mechanism for intraneuronal amyloid beta.

    Pigino G, Morfini G, Atagi Y, Deshpande A, Yu C, Jungbauer L, LaDu M, Busciglio J and Brady S

    Department of Anatomy and Cell Biology, University of Illinois, Chicago, IL 60612, USA.

    The pathological mechanism by which Abeta causes neuronal dysfunction and death remains largely unknown. Deficiencies in fast axonal transport (FAT) were suggested to play a crucial role in neuronal dysfunction and loss for a diverse set of dying back neuropathologies including Alzheimer's disease (AD), but the molecular basis for pathological changes in FAT were undetermined. Recent findings indicate that soluble intracellular oligomeric Abeta (oAbeta) species may play a critical role in AD pathology. Real-time analysis of vesicle mobility in isolated axoplasms perfused with oAbeta showed bidirectional axonal transport inhibition as a consequence of endogenous casein kinase 2 (CK2) activation. Conversely, neither unaggregated amyloid beta nor fibrillar amyloid beta affected FAT. Inhibition of FAT by oAbeta was prevented by two specific pharmacological inhibitors of CK2, as well as by competition with a CK2 substrate peptide. Furthermore, perfusion of axoplasms with active CK2 mimics the inhibitory effects of oAbeta on FAT. Both oAbeta and CK2 treatment of axoplasm led to increased phosphorylation of kinesin-1 light chains and subsequent release of kinesin from its cargoes. Therefore pharmacological modulation of CK2 activity may represent a promising target for therapeutic intervention in AD.

    Funded by: NIA NIH HHS: P01 AG030128, P01 AG030128-01A2; NINDS NIH HHS: NS23320, NS41170, NS43408, R01 NS023320, R01 NS023868, R01 NS023868-22, R01 NS023868-22S2, R01 NS041170, R01 NS043408

    Proceedings of the National Academy of Sciences of the United States of America 2009;106;14;5907-12

  • Protein kinase C-dependent alpha-secretory processing of the amyloid precursor protein is mediated by phosphorylation of myosin II-B.

    Argellati F, Domenicotti C, Passalacqua M, Janda E, Melloni E, Marinari UM, Pronzato MA and Ricciarelli R

    Department of Experimental Medicine, University of Genoa, Genoa, Italy.

    A substantial body of evidence indicates that protein kinase C (PKC) is involved in the alpha-secretory processing of the amyloid precursor protein (APP), an event that reduces the formation of the pathogenic amyloid-beta peptide. Recently, we have shown that trafficking and processing of APP are both impaired by knockdown of myosin II-B, one of the major neuronal motor proteins. Here, we provide evidence that the alpha-secretory processing of APP is mediated by PKC-dependent phosphorylation of myosin II-B. This signaling pathway provides an important link between APP and the neuronal cytoskeleton and might be crucial for the understanding of the biological and pathological roles of APP.

    FASEB journal : official publication of the Federation of American Societies for Experimental Biology 2009;23;4;1246-51

  • Fe65 is required for Tip60-directed histone H4 acetylation at DNA strand breaks.

    Stante M, Minopoli G, Passaro F, Raia M, Vecchio LD and Russo T

    CEINGE Centro d'Ingegneria Genetica Biotecnologie Avanzate, European School of Molecular Medicine, and Dipartimento di Biochimica e Biotecnologie Mediche, Università di Napoli Federico II, I-80145 Napoli, Italy.

    Fe65 is a binding partner of the Alzheimer's beta-amyloid precursor protein APP. The possible involvement of this protein in the cellular response to DNA damage was suggested by the observation that Fe65 null mice are more sensitive to genotoxic stress than WT counterpart. Fe65 associated with chromatin under basal conditions and its involvement in DNA damage repair requires this association. A known partner of Fe65 is the histone acetyltransferase Tip60. Considering the crucial role of Tip60 in DNA repair, we explored the hypothesis that the phenotype of Fe65 null cells depended on its interaction with Tip60. We demonstrated that Fe65 knockdown impaired recruitment of Tip60-TRRAP complex to DNA double strand breaks and decreased histone H4 acetylation. Accordingly, the efficiency of DNA repair was decreased upon Fe65 suppression. To explore whether APP has a role in this mechanism, we analyzed a Fe65 mutant unable to bind to APP. This mutant failed to rescue the phenotypes of Fe65 null cells; furthermore, APP/APLP2 suppression results in the impairment of recruitment of Tip60-TRRAP complex to DNA double strand breaks, decreased histone H4 acetylation and repair efficiency. On these bases, we propose that Fe65 and its interaction with APP play an important role in the response to DNA damage by assisting the recruitment of Tip60-TRRAP to DNA damage sites.

    Proceedings of the National Academy of Sciences of the United States of America 2009;106;13;5093-8

  • The effects of amyloid precursor protein on postsynaptic composition and activity.

    Hoe HS, Fu Z, Makarova A, Lee JY, Lu C, Feng L, Pajoohesh-Ganji A, Matsuoka Y, Hyman BT, Ehlers MD, Vicini S, Pak DT and Rebeck GW

    Departments of Neuroscience, Physiology and Biophysics, Pharmacology, and Neurology, Georgetown University Medical Center, Washington, D. C. 20057-1464, USA.

    The amyloid precursor protein (APP) is cleaved to produce the Alzheimer disease-associated peptide Abeta, but the normal functions of uncleaved APP in the brain are unknown. We found that APP was present in the postsynaptic density of central excitatory synapses and coimmunoprecipitated with N-methyl-d-aspartate receptors (NMDARs). The presence of APP in the postsynaptic density was supported by the observation that NMDARs regulated trafficking and processing of APP; overexpression of the NR1 subunit increased surface levels of APP, whereas activation of NMDARs decreased surface APP and promoted production of Abeta. We transfected APP or APP RNA interference into primary neurons and used electrophysiological techniques to explore the effects of APP on postsynaptic function. Reduction of APP decreased (and overexpression of APP increased) NMDAR whole cell current density and peak amplitude of spontaneous miniature excitatory postsynaptic currents. The increase in NMDAR current by APP was due to specific recruitment of additional NR2B-containing receptors. Consistent with these findings, immunohistochemical experiments demonstrated that APP increased the surface levels and decreased internalization of NR2B subunits. These results demonstrate a novel physiological role of postsynaptic APP in enhancing NMDAR function.

    Funded by: NIA NIH HHS: AG032330, AG12406, AG14473; NINDS NIH HHS: NS047700, NS48085, R01 NS048085, R01 NS048085-03

    The Journal of biological chemistry 2009;284;13;8495-506

  • Effect of amyloid beta on capacitive calcium entry in neural 2a cells.

    Niu Y, Su Z, Zhao C, Song B, Zhang X, Zhao N, Shen X and Gong Y

    State Key Laboratory of Biomembrane and Membrane Biotechnology, Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing 100084, China. niuy03@mails.tsinghua.edu.cn

    We studied the direct role of amyloid beta (Abeta) in regulating capacitive calcium entry (CCE), an important refilling mechanism for depleted intracellular calcium stores. For the first time, we found that Abeta can potentiate CCE. Neural 2a cells stably expressing Swedish mutant APP (APPswe), which can secrete large amounts of Abeta, have stronger CCE than its wild-type controls. Either reducing the Abeta in the medium by antibody binding or decreasing Abeta production by gamma secretase inhibitor treatment could significantly depress CCE in APPswe cells. The results demonstrated that the CCE potentiation in APPswe cells was caused by Abeta over-expression. Our research also revealed that the effect of Abeta on CCE potentiation could be decreased by Abeta channel blocker, which showed that the channels formed by Abeta are one of the ways through which Abeta causes CCE potentiation.

    Brain research bulletin 2009;78;4-5;152-7

  • Angiotensin II AT2 receptor oligomers mediate G-protein dysfunction in an animal model of Alzheimer disease.

    AbdAlla S, Lother H, el Missiry A, Langer A, Sergeev P, el Faramawy Y and Quitterer U

    Heinrich-Pette-Institute, Martinistrasse 52, D-20251 Hamburg, Germany.

    Progressive neurodegeneration and decline of cognitive functions are major hallmarks of Alzheimer disease (AD). Neurodegeneration in AD correlates with dysfunction of diverse signal transduction mechanisms, such as the G-protein-stimulated phosphoinositide hydrolysis mediated by Galphaq/11. We report here that impaired Galphaq/11-stimulated signaling in brains of AD patients and mice correlated with the appearance of cross-linked oligomeric angiotensin II AT2 receptors sequestering Galphaq/11. Amyloid beta (Abeta) was causal to AT2 oligomerization, because cerebral microinjection of Abeta triggered AT2 oligomerization in the hippocampus of mice in a dose-dependent manner. Abeta induced AT2 oligomerization by a two-step process of oxidative and transglutaminase-dependent cross-linking. The induction of AT2 oligomers in a transgenic mouse model with AD-like symptoms was associated with Galphaq/11 dysfunction and enhanced neurodegeneration. Vice versa, stereotactic inhibition of AT2 oligomers by RNA interference prevented the impairment of Galphaq/11 and delayed Tau phosphorylation. Thus, Abeta induces the formation of cross-linked AT2 oligomers that contribute to the dysfunction of Galphaq/11 in an animal model of Alzheimer disease.

    The Journal of biological chemistry 2009;284;10;6554-65

  • Cognitive and non-cognitive behaviors in an APPswe/PS1 bigenic model of Alzheimer's disease.

    Filali M, Lalonde R and Rivest S

    Laboratory of Molecular Endocrinology, CHUL Research Center and Department of Anatomy and Physiology, Laval University, Québec, Canada. mohammad.filali@crchul.ulaval.ca

    Neuropsychiatric signs are critical in primary caregiving of Alzheimer patients and yet have been relatively ignored in murine models. In the present study, APPswe/PS1 bigenic mice had higher levels of irritability than non-transgenic controls as measured in the touch escape test. Moreover, APPswe/PS1 mice showed poorer nest building than controls and a higher duration of immobility in the forced swimming assay. These results are concordant with the hypothesis of increased apathy and depression-like behavior in an Alzheimer's disease model. In addition, APPswe/PS1 bigenic mice were deficient in retention of passive avoidance learning and left-right discrimination learning, concordant with previous findings in other Alzheimer-like models.

    Genes, brain, and behavior 2009;8;2;143-8

  • Intestinal uptake of amyloid beta protein through columnar epithelial cells in suckling mice.

    Ano Y, Nakayama H, Sakudo A, Sawano Y, Tanokura M, Itohara S and Onodera T

    Department of Molecular Immunology, Graduate School of Agricultural and Life Sciences, the University of Tokyo, Yayoi, Bunkyo-ku, Tokyo, Japan.

    The mechanism of transmission of amyloid protein, especially the dynamics in the intestine, is still largely unknown. In the present study, a fusion protein (Abeta-EGFP) that combined enhanced green fluorescent protein with amyloid-beta protein (Abeta) was orally administered to mice before and after weaning, and the uptake and kinetics of amyloid protein within the intestine were elucidated through histopathology. Abeta-EGFP was incorporated into the cytoplasm of columnar epithelial cells, rather than M cells, at 3 h after administration and thereafter. Abeta-EGFP then accumulated in the crypt, Peyer's patch, and even the spleen. However, this uptake was not observed in weaned mice. These results suggest that a specific tolerant mechanism for incorporation of Abeta escaped from the digestion exists during suckling periods. This age-dependent uptake is important for estimating the risk of transmission.

    Histology and histopathology 2009;24;3;283-92

  • MicroRNA regulation of Alzheimer's Amyloid precursor protein expression.

    Hébert SS, Horré K, Nicolaï L, Bergmans B, Papadopoulou AS, Delacourte A and De Strooper B

    Center for Human Genetics, KULeuven, Belgium.

    Gene dosage effects of Amyloid precursor protein (APP) can cause familial AD. Recent evidence suggest that microRNA (miRNA) pathways, implicated in gene transcriptional control, could be involved in the development of sporadic Alzheimer's disease (AD). We therefore investigated whether miRNAs could participate in the regulation of APP gene expression. We show that miRNAs belonging to the miR-20a family (that is, miR-20a, miR-17-5p and miR-106b) could regulate APP expression in vitro and at the endogenous level in neuronal cell lines. A tight correlation between these miRNAs and APP was found during brain development and in differentiating neurons. We thus identify miRNAs as novel endogenous regulators of APP expression, suggesting that variations in miRNA expression could contribute to changes in APP expression in the brain during development and disease. This possibility is further corroborated by the observation that a statistically significant decrease in miR-106b expression was found in sporadic AD patients.

    Neurobiology of disease 2009;33;3;422-8

  • Effects of environmental enrichment and physical activity on neurogenesis in transgenic PS1/APP mice.

    Catlow BJ, Rowe AR, Clearwater CR, Mamcarz M, Arendash GW and Sanchez-Ramos J

    Department of Psychology, University of South Florida, Tampa, FL 33620, USA.

    Rodents exposed to environmental enrichment show many differences, including improved cognitive performance, when compared to those living in standard (impoverished) housing. The purpose of the present study was to determine if a selective increase in neurogenesis occurred in cognitively-protected Tg mice raised in an enriched environment compared to those reared in physical activity housing. At weaning, double Tg APP+PS1 mice were placed into one of three environments: complete environmental enrichment (CE), enhanced physical activity (PA), or individual, impoverished housing (IMP). At 9-10 months of age, Tg mice were injected with BrdU (100 mg/kg BID) followed by euthanasia either 24 h or 2 weeks after the last injection. Unbiased estimates of BrdU positive cells in the hippocampal subgranular zone revealed a significant increase in cellular proliferation in Tg mice raised in CE or PA compared to Tg mice reared in IMP housing. However, counts of BrdU birth-dated cells 2 weeks after labeling showed no difference among the three groups, indicating decreased survival of cells in those groups (CE and PA) with higher cellular proliferation rates in the neurogenic niche. Counts of calretinin-expressing cells, a marker of immature neurons, also indicated no difference among the three groups of mice. In view of our prior study showing that enhanced cognitive activity (but not enhanced physical activity) protects Tg mice against cognitive impairment, the present results indicate that increased generation and survival of new neurons in the hippocampal dentate gyrus is not involved with the cognitively-protective effects of complete CE in Alzheimer's transgenic mice.

    Funded by: NIA NIH HHS: P50 AG025711, P50 AG025711-03, P50AG025711

    Brain research 2009;1256;173-9

  • APP binds DR6 to trigger axon pruning and neuron death via distinct caspases.

    Nikolaev A, McLaughlin T, O'Leary DD and Tessier-Lavigne M

    Division of Research, Genentech, Inc., 1 DNA Way, South San Francisco, California 94080, USA.

    Naturally occurring axonal pruning and neuronal cell death help to sculpt neuronal connections during development, but their mechanistic basis remains poorly understood. Here we report that beta-amyloid precursor protein (APP) and death receptor 6 (DR6, also known as TNFRSF21) activate a widespread caspase-dependent self-destruction program. DR6 is broadly expressed by developing neurons, and is required for normal cell body death and axonal pruning both in vivo and after trophic-factor deprivation in vitro. Unlike neuronal cell body apoptosis, which requires caspase 3, we show that axonal degeneration requires caspase 6, which is activated in a punctate pattern that parallels the pattern of axonal fragmentation. DR6 is activated locally by an inactive surface ligand(s) that is released in an active form after trophic-factor deprivation, and we identify APP as a DR6 ligand. Trophic-factor deprivation triggers the shedding of surface APP in a beta-secretase (BACE)-dependent manner. Loss- and gain-of-function studies support a model in which a cleaved amino-terminal fragment of APP (N-APP) binds DR6 and triggers degeneration. Genetic support is provided by a common neuromuscular junction phenotype in mutant mice. Our results indicate that APP and DR6 are components of a neuronal self-destruction pathway, and suggest that an extracellular fragment of APP, acting via DR6 and caspase 6, contributes to Alzheimer's disease.

    Funded by: NEI NIH HHS: R01 EY007025, R01 EY007025-24, R01 EY07025; NIA NIH HHS: R01 AG025970

    Nature 2009;457;7232;981-9

  • Alzheimer disease Abeta production in the absence of S-palmitoylation-dependent targeting of BACE1 to lipid rafts.

    Vetrivel KS, Meckler X, Chen Y, Nguyen PD, Seidah NG, Vassar R, Wong PC, Fukata M, Kounnas MZ and Thinakaran G

    Department of Neurobiology, Neurology, and Pathology, The University of Chicago, Chicago, Illinois 60637, USA.

    Alzheimer disease beta-amyloid (Abeta) peptides are generated via sequential proteolysis of amyloid precursor protein (APP) by BACE1 and gamma-secretase. A subset of BACE1 localizes to cholesterol-rich membrane microdomains, termed lipid rafts. BACE1 processing in raft microdomains of cultured cells and neurons was characterized in previous studies by disrupting the integrity of lipid rafts by cholesterol depletion. These studies found either inhibition or elevation of Abeta production depending on the extent of cholesterol depletion, generating controversy. The intricate interplay between cholesterol levels, APP trafficking, and BACE1 processing is not clearly understood because cholesterol depletion has pleiotropic effects on Golgi morphology, vesicular trafficking, and membrane bulk fluidity. In this study, we used an alternate strategy to explore the function of BACE1 in membrane microdomains without altering the cellular cholesterol level. We demonstrate that BACE1 undergoes S-palmitoylation at four Cys residues at the junction of transmembrane and cytosolic domains, and Ala substitution at these four residues is sufficient to displace BACE1 from lipid rafts. Analysis of wild type and mutant BACE1 expressed in BACE1 null fibroblasts and neuroblastoma cells revealed that S-palmitoylation neither contributes to protein stability nor subcellular localization of BACE1. Surprisingly, non-raft localization of palmitoylation-deficient BACE1 did not have discernible influence on BACE1 processing of APP or secretion of Abeta. These results indicate that post-translational S-palmitoylation of BACE1 is not required for APP processing, and that BACE1 can efficiently cleave APP in both raft and non-raft microdomains.

    Funded by: NIA NIH HHS: AG019070, AG021495, R01 AG019070

    The Journal of biological chemistry 2009;284;6;3793-803

  • Collagen VI protects neurons against Abeta toxicity.

    Cheng JS, Dubal DB, Kim DH, Legleiter J, Cheng IH, Yu GQ, Tesseur I, Wyss-Coray T, Bonaldo P and Mucke L

    Gladstone Institute of Neurological Disease, 1650 Owens Street, San Francisco, California 94158, USA.

    Amyloid-beta (Abeta) peptides, widely presumed to cause Alzheimer's disease, increased mouse neuronal expression of collagen VI through a mechanism involving transforming growth factor signaling. Reduction of collagen VI augmented Abeta neurotoxicity, whereas treatment of neurons with soluble collagen VI blocked the association of Abeta oligomers with neurons, enhanced Abeta aggregation and prevented neurotoxicity. These results identify collagen VI as an important component of the neuronal injury response and demonstrate its neuroprotective potential.

    Funded by: Howard Hughes Medical Institute; NIA NIH HHS: P50 AG023501, P50 AG023501-066169

    Nature neuroscience 2009;12;2;119-21

  • Effects of short-term Western diet on cerebral oxidative stress and diabetes related factors in APP x PS1 knock-in mice.

    Studzinski CM, Li F, Bruce-Keller AJ, Fernandez-Kim SO, Zhang L, Weidner AM, Markesbery WR, Murphy MP and Keller JN

    Sanders-Brown Center on Aging, University of Kentucky, Lexington, USA.

    A chronic high fat Western diet (WD) promotes a variety of morbidity factors although experimental evidence for short-term WD mediating brain dysfunction remains to be elucidated. The amyloid precursor protein and presenilin-1 (APP x PS1) knock-in mouse model has been demonstrated to recapitulate some key features of Alzheimer's disease pathology, including amyloid-beta (Abeta) pathogenesis. In this study, we placed 1-month-old APP x PS1 mice and non-transgenic littermates on a WD for 4 weeks. The WD resulted in a significant elevation in protein oxidation and lipid peroxidation in the brain of APP x PS1 mice relative to non-transgenic littermates, which occurred in the absence of increased Abeta levels. Altered adipokine levels were also observed in APP x PS1 mice placed on a short-term WD, relative to non-transgenic littermates. Taken together, these data indicate that short-term WD is sufficient to selectively promote cerebral oxidative stress and metabolic disturbances in APP x PS1 knock-in mice, with increased oxidative stress preceding alterations in Abeta. These data have important implications for understanding how WD may potentially contribute to brain dysfunction and the development of neurodegenerative disorders such as Alzheimer's disease.

    Funded by: NCRR NIH HHS: P20 RR020171-057027, RR020171; NIDDK NIH HHS: P30 DK072476-04; NINDS NIH HHS: NS058382, R01 NS058382-02

    Journal of neurochemistry 2009;108;4;860-6

  • A new amyloid-like beta-aggregate with amyloid characteristics, except fibril morphology.

    Chang ES, Liao TY, Lim TS, Fann W and Chen RP

    Institute of Biological Chemistry, Academia Sinica, No. 128, Sec 2, Academia Road, Nankang, Taipei 115, Taiwan, ROC.

    Amyloid plaques, formed from amyloid beta (Abeta) peptides (mainly Abeta40 or Abeta42), are one of the most important pathological characteristics of Alzheimer's disease. Here, a single D-form proline substitution in the 40-amino-acid Abeta40 peptide can totally change the aggregation behavior of this peptide. The residue immediately preceding each glycine in Abeta40 (S8, V24, I32, and V36) was individually replaced by D-form proline ((D)Pro). The resulting (D)P-G sequence (the (D)Pro residue and the following Gly residue) was designed as a "structural clip" to force the formation of a bend in the peptide, as this sequence has been reported to be a strong promoter of beta-hairpin formation. The mutant peptide with Val24-to-(D)Pro substitution, named V24P, formed a new amyloid-like beta-aggregate at high peptide concentration. The aggregate has most of the characteristics of amyloid fibrils, except fibril morphology. Moreover, the mutant peptide V24P, when mixed with Abeta40, can attenuate the cytotoxicity of Abeta40.

    Journal of molecular biology 2009;385;4;1257-65

  • S-palmitoylation of gamma-secretase subunits nicastrin and APH-1.

    Cheng H, Vetrivel KS, Drisdel RC, Meckler X, Gong P, Leem JY, Li T, Carter M, Chen Y, Nguyen P, Iwatsubo T, Tomita T, Wong PC, Green WN, Kounnas MZ and Thinakaran G

    Department of Neurobiology, The University of Chicago, Chicago, Illinois 60637, USA.

    Proteolytic processing of amyloid precursor protein (APP) by beta- and gamma-secretases generates beta-amyloid (Abeta) peptides, which accumulate in the brains of individuals affected by Alzheimer disease. Detergent-resistant membrane microdomains (DRM) rich in cholesterol and sphingolipid, termed lipid rafts, have been implicated in Abeta production. Previously, we and others reported that the four integral subunits of the gamma-secretase associate with DRM. In this study we investigated the mechanisms underlying DRM association of gamma-secretase subunits. We report that in cultured cells and in brain the gamma-secretase subunits nicastrin and APH-1 undergo S-palmitoylation, the post-translational covalent attachment of the long chain fatty acid palmitate common in lipid raft-associated proteins. By mutagenesis we show that nicastrin is S-palmitoylated at Cys(689), and APH-1 is S-palmitoylated at Cys(182) and Cys(245). S-Palmitoylation-defective nicastrin and APH-1 form stable gamma-secretase complexes when expressed in knock-out fibroblasts lacking wild type subunits, suggesting that S-palmitoylation is not essential for gamma-secretase assembly. Nevertheless, fractionation studies show that S-palmitoylation contributes to DRM association of nicastrin and APH-1. Moreover, pulse-chase analyses reveal that S-palmitoylation is important for nascent polypeptide stability of both proteins. Co-expression of S-palmitoylation-deficient nicastrin and APH-1 in cultured cells neither affects Abeta40, Abeta42, and AICD production, nor intramembrane processing of Notch and N-cadherin. Our findings suggest that S-palmitoylation plays a role in stability and raft localization of nicastrin and APH-1, but does not directly modulate gamma-secretase processing of APP and other substrates.

    Funded by: NIA NIH HHS: AG019070, AG021495; NINDS NIH HHS: NS45150, P01 NS047308

    The Journal of biological chemistry 2009;284;3;1373-84

  • Increasing Cu bioavailability inhibits Abeta oligomers and tau phosphorylation.

    Crouch PJ, Hung LW, Adlard PA, Cortes M, Lal V, Filiz G, Perez KA, Nurjono M, Caragounis A, Du T, Laughton K, Volitakis I, Bush AI, Li QX, Masters CL, Cappai R, Cherny RA, Donnelly PS, White AR and Barnham KJ

    Department of Pathology, Centre for Neuroscience, School of Chemistry, and Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Melbourne, Victorial, 3010, Australia.

    Cognitive decline in Alzheimer's disease (AD) involves pathological accumulation of synaptotoxic amyloid-beta (Abeta) oligomers and hyperphosphorylated tau. Because recent evidence indicates that glycogen synthase kinase 3beta (GSK3beta) activity regulates these neurotoxic pathways, we developed an AD therapeutic strategy to target GSK3beta. The strategy involves the use of copper-bis(thiosemicarbazonoto) complexes to increase intracellular copper bioavailability and inhibit GSK3beta through activation of an Akt signaling pathway. Our lead compound Cu(II)(gtsm) significantly inhibited GSK3beta in the brains of APP/PS1 transgenic AD model mice. Cu(II)(gtsm) also decreased the abundance of Abeta trimers and phosphorylated tau, and restored performance of AD mice in the Y-maze test to levels expected for cognitively normal animals. Improvement in the Y-maze correlated directly with decreased Abeta trimer levels. This study demonstrates that increasing intracellular copper bioavailability can restore cognitive function by inhibiting the accumulation of neurotoxic Abeta trimers and phosphorylated tau.

    Proceedings of the National Academy of Sciences of the United States of America 2009;106;2;381-6

  • Activation of cell cycle proteins in transgenic mice in response to neuronal loss but not amyloid-beta and tau pathology.

    Lopes JP, Blurton-Jones M, Yamasaki TR, Agostinho P and LaFerla FM

    Center for Neuroscience and Cell Biology, Faculty of Medicine, Biochemistry Institute, University of Coimbra, Coimbra, Portugal. jpplopes@gmail.com

    Cell cycle proteins are elevated in the brain of patients and in transgenic models of Alzheimer's disease (AD), suggesting that aberrant cell cycle re-entry plays a key role in this disorder. However, the precise relationship between cell cycle reactivation and the hallmarks of AD, amyloid-beta (Abeta) plaques and tau-laden neurofibrillary tangles, remains unclear. We sought to determine whether cell cycle reactivation initiates in direct response to Abeta and tau accumulation or whether it occurs as a downstream consequence of neuronal death pathways. Therefore, we used a triple transgenic mouse model of AD (3xTg-AD) that develops plaques and tangles, but does not exhibit extensive neuronal loss, whereas to model hippocampal neuronal death a tetracycline-regulatable transgenic model of neuronal ablation (CaM/Tet-DT(A) mice) was used. Cell-cycle protein activation was determined in these two models of neurodegeneration, using biochemical and histological approaches. Our findings indicate that Cdk4, PCNA and phospho-Rb are significantly elevated in CaM/Tet-DT(A) mice following neuronal death. In contrast, no significant activation of cell-cycle proteins occurs in 3xTg-AD mice versus non-transgenic controls. Taken together, our data indicate that neuronal cell cycle reactivation is not a prominent feature induced by Abeta or tau pathology, but rather appears to be triggered by acute neuronal loss.

    Funded by: NIA NIH HHS: R01AG027544

    Journal of Alzheimer's disease : JAD 2009;16;3;541-9

  • Cardiomyocyte contractile dysfunction in the APPswe/PS1dE9 mouse model of Alzheimer's disease.

    Turdi S, Guo R, Huff AF, Wolf EM, Culver B and Ren J

    Division of Pharmaceutical Sciences & Center for Cardiovascular Research and Alternative Medicine, University of Wyoming College of Health Sciences, Laramie, WY, USA.

    Objectives: Ample clinical and experimental evidence indicated that patients with Alzheimer's disease display a high incidence of cardiovascular events. This study was designed to examine myocardial histology, cardiomyocyte shortening, intracellular Ca(2+) homeostasis and regulatory proteins, electrocardiogram, adrenergic response, endoplasmic reticulum (ER) stress and protein carbonyl formation in C57 wild-type (WT) mice and an APPswe/PS1dE9 transgenic (APP/PS1) model for Alzheimer's disease.

    Methods: Cardiomyocyte mechanical properties were evaluated including peak shortening (PS), time-to-PS (TPS), time-to-relengthening (TR), maximal velocity of shortening and relengthening (+/-dL/dt), intracellular Ca(2+) transient rise and decay.

    Results: Little histological changes were observed in APP/PS1 myocardium. Cardiomyocytes from APP/PS1 but not APP or PS1 single mutation mice exhibited depressed PS, reduced+/-dL/dt, normal TPS and TR compared with WT mice(.) Rise in intracellular Ca(2+) was lower accompanied by unchanged resting/peak intracellular Ca(2+) levels and intracellular Ca(2+) decay in APP/PS1 mice. Cardiomyocytes from APP/PS1 mice exhibited a steeper decline in PS at high frequencies. The responsiveness to adrenergic agonists was dampened although beta(1)-adrenergic receptor expression was unchanged in APP/PS1 hearts. Expression of the Ca(2+) regulatory protein phospholamban and protein carbonyl formation were downregulated and elevated, respectively, associated with unchanged SERCA2a, Na(+)-Ca(2+) exchanger and ER stress markers in APP/PS1 hearts. Our further study revealed that antioxidant N-acetylcysteine attenuated the contractile dysfunction in APP/PS1 mice.

    Conclusions: Our results depicted overt cardiomyocyte mechanical dysfunction in the APP/PS1 Alzheimer's disease model, possibly due to oxidative stress.

    Funded by: NCRR NIH HHS: 5 P20 RR016474

    PloS one 2009;4;6;e6033

  • In vivo turnover of tau and APP metabolites in the brains of wild-type and Tg2576 mice: greater stability of sAPP in the beta-amyloid depositing mice.

    Morales-Corraliza J, Mazzella MJ, Berger JD, Diaz NS, Choi JH, Levy E, Matsuoka Y, Planel E and Mathews PM

    Nathan Kline Institute for Psychiatric Research, Orangeburg, New York, USA. jmorales-corraliza@nki.rfmh.org

    The metabolism of the amyloid precursor protein (APP) and tau are central to the pathobiology of Alzheimer's disease (AD). We have examined the in vivo turnover of APP, secreted APP (sAPP), Abeta and tau in the wild-type and Tg2576 mouse brain using cycloheximide to block protein synthesis. In spite of overexpression of APP in the Tg2576 mouse, APP is rapidly degraded, similar to the rapid turnover of the endogenous protein in the wild-type mouse. sAPP is cleared from the brain more slowly, particularly in the Tg2576 model where the half-life of both the endogenous murine and transgene-derived human sAPP is nearly doubled compared to wild-type mice. The important Abeta degrading enzymes neprilysin and IDE were found to be highly stable in the brain, and soluble Abeta40 and Abeta42 levels in both wild-type and Tg2576 mice rapidly declined following the depletion of APP. The cytoskeletal-associated protein tau was found to be highly stable in both wild-type and Tg2576 mice. Our findings unexpectedly show that of these various AD-relevant protein metabolites, sAPP turnover in the brain is the most different when comparing a wild-type mouse and a beta-amyloid depositing, APP overexpressing transgenic model. Given the neurotrophic roles attributed to sAPP, the enhanced stability of sAPP in the beta-amyloid depositing Tg2576 mice may represent a neuroprotective response.

    Funded by: NIA NIH HHS: AG017617, AG022455; NINDS NIH HHS: NS045205

    PloS one 2009;4;9;e7134

  • Iron regulatory protein 2 is involved in brain copper homeostasis.

    Mueller C, Magaki S, Schrag M, Ghosh MC and Kirsch WM

    Neurosurgery Center for Research, Training and Education, Loma Linda University School of Medicine, Loma Linda, CA 92354, USA.

    Trace metal homeostasis is tightly controlled in the brain, as even a slight dysregulation may severely impact normal brain function. This is especially apparent in Alzheimer's disease, where brain homeostasis of trace metals such as copper and iron is dysregulated. As it is known that iron and copper metabolism are linked, we wanted to investigate if a common mechanism could explain the increase in iron and decrease in copper seen in Alzheimer's disease brain. Amyloid-beta protein precursor (AbetaPP) has been implicated in copper efflux from the brain. Furthermore, it was shown that iron regulatory proteins (IRP), which regulate iron homeostasis, can block AbetaPP mRNA translation. In a correlative study we have therefore compared brain regional copper levels and AbetaPP expression in mice with a targeted deletion of IRP2-/-. Compared with controls, six week old IRP2-/- mice had significantly less brain copper in the parietal cortex, hippocampus, ventral striatum, thalamus, hypothalamus, and whole brain, while AbetaPP was significantly upregulated in the hippocampus (p < 0.05) and showed a trend toward upregulation in the thalamus (p < 0.1). This is the first study to demonstrate that iron regulatory proteins affect brain copper levels, which has significant implications for neurodegenerative diseases.

    Funded by: NIA NIH HHS: AG20948, R01 AG020948

    Journal of Alzheimer's disease : JAD 2009;18;1;201-10

  • Molecular networks involved in mouse cerebral corticogenesis and spatio-temporal regulation of Sox4 and Sox11 novel antisense transcripts revealed by transcriptome profiling.

    Ling KH, Hewitt CA, Beissbarth T, Hyde L, Banerjee K, Cheah PS, Cannon PZ, Hahn CN, Thomas PQ, Smyth GK, Tan SS, Thomas T and Scott HS

    Molecular Medicine Division, The Walter and Eliza Hall Institute of Medical Research, Royal Parade, Parkville, Victoria 3052, Australia. michael.ling@imvs.sa.gov.au

    Background: Development of the cerebral cortex requires highly specific spatio-temporal regulation of gene expression. It is proposed that transcriptome profiling of the cerebral cortex at various developmental time points or regions will reveal candidate genes and associated molecular pathways involved in cerebral corticogenesis.

    Results: Serial analysis of gene expression (SAGE) libraries were constructed from C57BL/6 mouse cerebral cortices of age embryonic day (E) 15.5, E17.5, postnatal day (P) 1.5 and 4 to 6 months. Hierarchical clustering analysis of 561 differentially expressed transcripts showed regionalized, stage-specific and co-regulated expression profiles. SAGE expression profiles of 70 differentially expressed transcripts were validated using quantitative RT-PCR assays. Ingenuity pathway analyses of validated differentially expressed transcripts demonstrated that these transcripts possess distinctive functional properties related to various stages of cerebral corticogenesis and human neurological disorders. Genomic clustering analysis of the differentially expressed transcripts identified two highly transcribed genomic loci, Sox4 and Sox11, during embryonic cerebral corticogenesis. These loci feature unusual overlapping sense and antisense transcripts with alternative polyadenylation sites and differential expression. The Sox4 and Sox11 antisense transcripts were highly expressed in the brain compared to other mouse organs and are differentially expressed in both the proliferating and differentiating neural stem/progenitor cells and P19 (embryonal carcinoma) cells.

    Conclusions: We report validated gene expression profiles that have implications for understanding the associations between differentially expressed transcripts, novel targets and related disorders pertaining to cerebral corticogenesis. The study reports, for the first time, spatio-temporally regulated Sox4 and Sox11 antisense transcripts in the brain, neural stem/progenitor cells and P19 cells, suggesting they have an important role in cerebral corticogenesis and neuronal/glial cell differentiation.

    Genome biology 2009;10;10;R104

  • X11 proteins regulate the translocation of amyloid beta-protein precursor (APP) into detergent-resistant membrane and suppress the amyloidogenic cleavage of APP by beta-site-cleaving enzyme in brain.

    Saito Y, Sano Y, Vassar R, Gandy S, Nakaya T, Yamamoto T and Suzuki T

    Laboratory of Neuroscience, Graduate School of Pharmaceutical Sciences, Hokkaido University, Kita12-Nishi6, Sapporo 060-0812, Japan.

    X11 and X11-like proteins (X11L) are neuronal adaptor proteins whose association to the cytoplasmic domain of amyloid beta-protein precursor (APP) suppresses the generation of amyloid beta-protein (Abeta) implicated in Alzheimer disease pathogenesis. The amyloidogenic, but not amyloidolytic, metabolism of APP was selectively increased in the brain of mutant mice lacking X11L (Sano, Y., Syuzo-Takabatake, A., Nakaya, T., Saito, Y., Tomita, S., Itohara, S., and Suzuki, T. (2006) J. Biol. Chem. 281, 37853-37860). To reveal the actual role of X11 proteins (X11s) in suppressing amyloidogenic cleavage of APP in vivo, we generated X11 and X11L double knock-out mice and analyzed the metabolism of APP. The mutant mice showed enhanced beta-site cleavage of APP along with increased accumulation of Abeta in brain and increased colocalization of APP with beta-site APP-cleaving enzyme (BACE). In the brains of mice deficient in both X11 and X11L, the apparent relative subcellular distributions of both mature APP and its beta-C-terminal fragment were shifted toward the detergent-resistant membrane (DRM) fraction, an organelle in which BACE is active and both X11s are not nearly found. These results indicate that X11s associate primarily with APP molecules that are outside of DRM, that the dissociation of APP-X11/X11L complexes leads to entry of APP into DRM, and that cleavage of uncomplexed APP by BACE within DRM is enhanced by X11s deficiency. Present results lead to an idea that the dysfunction of X11L in the interaction with APP may recruit more APP into DRM and increase the generation of Abeta even if BACE activity did not increase in brain.

    Funded by: NIA NIH HHS: P01 AG010491, P01 AG010491-14, P01 AG10491

    The Journal of biological chemistry 2008;283;51;35763-71

  • The low density lipoprotein receptor-related protein 1 mediates uptake of amyloid beta peptides in an in vitro model of the blood-brain barrier cells.

    Yamada K, Hashimoto T, Yabuki C, Nagae Y, Tachikawa M, Strickland DK, Liu Q, Bu G, Basak JM, Holtzman DM, Ohtsuki S, Terasaki T and Iwatsubo T

    Department of Neuropathology and Neuroscience, Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo 113-0033, Japan.

    The metabolism of amyloid beta peptide (A beta) in the brain is crucial to the pathogenesis of Alzheimer disease. A body of evidence suggests that A beta is actively transported from brain parenchyma to blood across the blood-brain barrier (BBB), although the precise mechanism remains unclear. To unravel the cellular and molecular mechanism of A beta transport across the BBB, we established a new in vitro model of the initial internalization step of A beta transport using TR-BBB cells, a conditionally immortalized endothelial cell line from rat brain. We show that TR-BBB cells rapidly internalize A beta through a receptor-mediated mechanism. We also provide evidence that A beta internalization is mediated by LRP1 (low density lipoprotein receptor-related protein 1), since administration of LRP1 antagonist, receptor-associated protein, neutralizing antibody, or small interference RNAs all reduced A beta uptake. Despite the requirement of LRP1-dependent internalization, A beta does not directly bind to LRP1 in an in vitro binding assay. Unlike TR-BBB cells, mouse embryonic fibroblasts endogenously expressing functional LRP1 and exhibiting the authentic LRP1-mediated endocytosis (e.g. of tissue plasminogen activator) did not show rapid A beta uptake. Based on these data, we propose that the rapid LRP1-dependent internalization of A beta occurs under the BBB-specific cellular context and that TR-BBB is a useful tool for analyzing the molecular mechanism of the rapid transport of A beta across BBB.

    Funded by: NIA NIH HHS: AG13956, R01 AG027924

    The Journal of biological chemistry 2008;283;50;34554-62

  • Increased exploratory activity of APP23 mice in a novel environment is reversed by siRNA.

    Senechal Y, Prut L, Kelly PH, Staufenbiel M, Natt F, Hoyer D, Wiessner C and Dev KK

    Novartis Institutes for BioMedical Research, Basel, Switzerland.

    Genetic abnormalities in amyloid precursor protein (APP) are associated with Down's syndrome and familial Alzheimer's disease where hallmark plaques contain A beta peptides derived from APP. Both APP and its derivatives are implicated in neurodegenerative processes and may play important physiological and pathophysiological roles in synaptic function. Here, we show that young APP23 transgenic mice overexpressing human APP with the Swedish double mutation display altered novelty seeking behavior before the age of plaque onset. Using short interfering RNA (siRNA) targeted against APP, we investigate the direct contribution of APP and its derivatives to this behavioral deficit. After validating siRNAs targeting human APP in vitro, siRNAs were infused directly into the brain of APP23 mice for 2 weeks. Behavioral analysis shows that infusion of siRNA targeted against APP completely reverses increased exploratory activity in APP23 mice. Collectively, these data suggest that excessive APP and/or its derivatives, causes a hyperactive phenotype in APP23 mice when placed in a novel environment, which is fully reversible and not linked to plaque deposits.

    Brain research 2008;1243;124-33

  • apoE isoform-specific disruption of amyloid beta peptide clearance from mouse brain.

    Deane R, Sagare A, Hamm K, Parisi M, Lane S, Finn MB, Holtzman DM and Zlokovic BV

    Center for Neurodegenerative and Vascular Brain Disorders, Department of Neurosurgery, University of Rochester Medical School, Rochester, New York 14642, USA.

    Neurotoxic amyloid beta peptide (Abeta) accumulates in the brains of individuals with Alzheimer disease (AD). The APOE4 allele is a major risk factor for sporadic AD and has been associated with increased brain parenchymal and vascular amyloid burden. How apoE isoforms influence Abeta accumulation in the brain has, however, remained unclear. Here, we have shown that apoE disrupts Abeta clearance across the mouse blood-brain barrier (BBB) in an isoform-specific manner (specifically, apoE4 had a greater disruptive effect than either apoE3 or apoE2). Abeta binding to apoE4 redirected the rapid clearance of free Abeta40/42 from the LDL receptor-related protein 1 (LRP1) to the VLDL receptor (VLDLR), which internalized apoE4 and Abeta-apoE4 complexes at the BBB more slowly than LRP1. In contrast, apoE2 and apoE3 as well as Abeta-apoE2 and Abeta-apoE3 complexes were cleared at the BBB via both VLDLR and LRP1 at a substantially faster rate than Abeta-apoE4 complexes. Astrocyte-secreted lipo-apoE2, lipo-apoE3, and lipo-apoE4 as well as their complexes with Abeta were cleared at the BBB by mechanisms similar to those of their respective lipid-poor isoforms but at 2- to 3-fold slower rates. Thus, apoE isoforms differentially regulate Abeta clearance from the brain, and this might contribute to the effects of APOE genotype on the disease process in both individuals with AD and animal models of AD.

    Funded by: NIA NIH HHS: R37 AG013956, R37 AG023084, R37 AG13956; NINDS NIH HHS: R37 NS034467, R37 NS34467

    The Journal of clinical investigation 2008;118;12;4002-13

  • Valproic acid inhibits Abeta production, neuritic plaque formation, and behavioral deficits in Alzheimer's disease mouse models.

    Qing H, He G, Ly PT, Fox CJ, Staufenbiel M, Cai F, Zhang Z, Wei S, Sun X, Chen CH, Zhou W, Wang K and Song W

    Department of Psychiatry, Townsend Family Laboratories, Brain Research Center, University of British Columbia, Vancouver, BC, Canada.

    Neuritic plaques in the brains are one of the pathological hallmarks of Alzheimer's disease (AD). Amyloid beta-protein (Abeta), the central component of neuritic plaques, is derived from beta-amyloid precursor protein (APP) after beta- and gamma-secretase cleavage. The molecular mechanism underlying the pathogenesis of AD is not yet well defined, and there has been no effective treatment for AD. Valproic acid (VPA) is one of the most widely used anticonvulsant and mood-stabilizing agents for treating epilepsy and bipolar disorder. We found that VPA decreased Abeta production by inhibiting GSK-3beta-mediated gamma-secretase cleavage of APP both in vitro and in vivo. VPA treatment significantly reduced neuritic plaque formation and improved memory deficits in transgenic AD model mice. We also found that early application of VPA was important for alleviating memory deficits of AD model mice. Our study suggests that VPA may be beneficial in the prevention and treatment of AD.

    The Journal of experimental medicine 2008;205;12;2781-9

  • Oxidative damage in brain from human mutant APP/PS-1 double knock-in mice as a function of age.

    Abdul HM, Sultana R, St Clair DK, Markesbery WR and Butterfield DA

    Department of Chemistry, Center for Membrane Sciences, University of Kentucky, Lexington, KY 40506-0055, USA.

    Oxidative stress is strongly implicated in the progressive decline of cognition associated with aging and neurodegenerative disorders. In the brain, free radical-mediated oxidative stress plays a critical role in the age-related decline of cellular function as a result of the oxidation of proteins, lipids, and nucleic acids. A number of studies indicate that an increase in protein oxidation and lipid peroxidation is associated with age-related neurodegenerative diseases and cellular dysfunction observed in aging brains. Oxidative stress is one of the important factors contributing to Alzheimer's disease (AD), one of whose major hallmarks includes brain depositions of amyloid beta-peptide (Abeta) derived from amyloid precursor protein (APP). Mutation in APP and PS-1 genes, which increases production of the highly amyloidogenic amyloid beta-peptide (Abeta42), is the major cause of familial AD. In the present study, protein oxidation and lipid peroxidation in the brain from knock-in mice expressing human mutant APP and PS-1 were compared with brain from wild type, as a function of age. The results suggest that there is an increased oxidative stress in the brain of wild-type mice as a function of age. In APP/PS-1 mouse brain, there is a basal increase (at 1 month) in oxidative stress compared to the wild type (1 month), as measured by protein oxidation and lipid peroxidation. In addition, age-related elevation of oxidative damage was observed in APP/PS-1 mice brain compared to that of wild-type mice brain. These results are discussed with reference to the importance of Abeta42-associated oxidative stress in the pathogenesis of AD.

    Funded by: NIA NIH HHS: AG-029839, AG-05119, AG-10836, P01 AG005119, P01 AG005119-110001, P01 AG005119-120001, P01 AG005119-12S20001, P01 AG005119-130001, P01 AG005119-13S10001, P01 AG005119-140001, P01 AG005119-20A10010, P01 AG010836, P01 AG010836-110011, P01 AG010836-120011, P01 AG010836-130011, P01 AG010836-140011, P01 AG010836-150011, R01 AG029839

    Free radical biology & medicine 2008;45;10;1420-5

  • Loss of gamma-secretase function impairs endocytosis of lipoprotein particles and membrane cholesterol homeostasis.

    Tamboli IY, Prager K, Thal DR, Thelen KM, Dewachter I, Pietrzik CU, St George-Hyslop P, Sisodia SS, De Strooper B, Heneka MT, Filippov MA, Müller U, van Leuven F, Lütjohann D and Walter J

    Department of Neurology, University of Bonn, 53127 Bonn, Germany.

    Presenilins (PSs) are components of the gamma-secretase complex that mediates intramembranous cleavage of type I membrane proteins. We show that gamma-secretase is involved in the regulation of cellular lipoprotein uptake. Loss of gamma-secretase function decreased endocytosis of low-density lipoprotein (LDL) receptor. The decreased uptake of lipoproteins led to upregulation of cellular cholesterol biosynthesis by increased expression of CYP51 and enhanced metabolism of lanosterol. Genetic deletion of PS1 or transgenic expression of PS1 mutants that cause early-onset Alzheimer's disease led to accumulation of gamma-secretase substrates and mistargeting of adaptor proteins that regulate endocytosis of the LDL receptor. Consistent with decreased endocytosis of these receptors, PS1 mutant mice have elevated levels of apolipoprotein E in the brain. Thus, these data demonstrate a functional link between two major genetic factors that cause early-onset and late-onset Alzheimer's disease.

    Funded by: Wellcome Trust: 081864

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2008;28;46;12097-106

  • Amyloid precursor protein trafficking, processing, and function.

    Thinakaran G and Koo EH

    Department of Neurobiology, The University of Chicago, Chicago, Illinois 60637, USA. gopal@uchicago.edu

    Intracellular trafficking and proteolytic processing of amyloid precursor protein (APP) have been the focus of numerous investigations over the past two decades. APP is the precursor to the amyloid beta-protein (Abeta), the 38-43-amino acid residue peptide that is at the heart of the amyloid cascade hypothesis of Alzheimer disease (AD). Tremendous progress has been made since the initial identification of Abeta as the principal component of brain senile plaques of individuals with AD. Specifically, molecular characterization of the secretases involved in Abeta production has facilitated cell biological investigations on APP processing and advanced efforts to model AD pathogenesis in animal models. This minireview summarizes salient features of APP trafficking and amyloidogenic processing and discusses the putative biological functions of APP.

    Funded by: NIA NIH HHS: AG019070, AG021495, AG032179, AG12376, R01 AG019070, R01 AG021495

    The Journal of biological chemistry 2008;283;44;29615-9

  • Regulation of amyloid beta-protein precursor by phosphorylation and protein interactions.

    Suzuki T and Nakaya T

    Laboratory of Neuroscience, Graduate School of Pharmaceutical Sciences, Hokkaido University, Kita-ku, Sapporo 060-0812, Japan. tsuzuki@pharm.hokudai.ac.jp

    Amyloid beta-protein precursor (APP), a type I membrane protein, is cleaved by primary alpha-or beta-secretase and secondary gamma-secretase. Cleavage of APP by beta- and gamma-secretases generates amyloid beta-protein, the main constituent of the cerebrovascular amyloid that accompanies Alzheimer disease. The generation and aggregation of amyloid beta-protein in the brain are believed to be a primary cause of Alzheimer disease pathogenesis, and indeed, early onset Alzheimer disease is genetically linked to APP and also to presenilins 1 and 2, which are components of gamma-secretase. Proteolytic cleavage of APP has been investigated as a candidate target for Alzheimer disease therapy, but the mechanisms regulating APP metabolism are still unclear. APP is a type I membrane protein with a short cytoplasmic region consisting of 47 amino acids. Recent research has elucidated the significance of the cytoplasmic region in the metabolism, trafficking, and physiological function of APP. The structure and function of the APP cytoplasmic domain can be modified by phosphorylation and through interaction with cytoplasmic proteins. This minireview summarizes a large body of recent information on the regulation of APP by phosphorylation and protein interaction, along with some of the physiological functions of APP. Recent findings regarding the regulation of APP processing contribute to the development of novel drugs and/or therapies for Alzheimer disease.

    The Journal of biological chemistry 2008;283;44;29633-7

  • Microarray analysis of Foxa2 mutant mouse embryos reveals novel gene expression and inductive roles for the gastrula organizer and its derivatives.

    Tamplin OJ, Kinzel D, Cox BJ, Bell CE, Rossant J and Lickert H

    Program in Developmental and Stem Cell Biology, Research Institute, The Hospital for Sick Children, 555 University Avenue, Toronto, Ontario, M5G 1X8, Canada. owen.tamplin@utoronto.ca

    Background: The Spemann/Mangold organizer is a transient tissue critical for patterning the gastrula stage vertebrate embryo and formation of the three germ layers. Despite its important role during development, there are still relatively few genes with specific expression in the organizer and its derivatives. Foxa2 is a forkhead transcription factor that is absolutely required for formation of the mammalian equivalent of the organizer, the node, the axial mesoderm and the definitive endoderm (DE). However, the targets of Foxa2 during embryogenesis, and the molecular impact of organizer loss on the gastrula embryo, have not been well defined.

    Results: To identify genes specific to the Spemann/Mangold organizer, we performed a microarray-based screen that compared wild-type and Foxa2 mutant embryos at late gastrulation stage (E7.5). We could detect genes that were consistently down-regulated in replicate pools of mutant embryos versus wild-type, and these included a number of known node and DE markers. We selected 314 genes without previously published data at E7.5 and screened for expression by whole mount in situ hybridization. We identified 10 novel expression patterns in the node and 5 in the definitive endoderm. We also found significant reduction of markers expressed in secondary tissues that require interaction with the organizer and its derivatives, such as cardiac mesoderm, vasculature, primitive streak, and anterior neuroectoderm.

    Conclusion: The genes identified in this screen represent novel Spemann/Mangold organizer genes as well as potential Foxa2 targets. Further investigation will be needed to define these genes as novel developmental regulatory factors involved in organizer formation and function. We have placed these genes in a Foxa2-dependent genetic regulatory network and we hypothesize how Foxa2 may regulate a molecular program of Spemann/Mangold organizer development. We have also shown how early loss of the organizer and its inductive properties in an otherwise normal embryo, impacts on the molecular profile of surrounding tissues.

    BMC genomics 2008;9;511

  • Prevention of axonal injury using calpain inhibitor in chronic progressive experimental autoimmune encephalomyelitis.

    Hassen GW, Feliberti J, Kesner L, Stracher A and Mokhtarian F

    SUNY Downstate Medical Center, Brooklyn, New York, USA.

    Axonal injury is the major correlate of permanent disability in neurodegenerative diseases such as multiple sclerosis (MS), especially in secondary-progressive MS which follows relapsing-remitting disease course. Proteolytic enzyme, calpain, is a potential candidate for causing axonal injury. Most current treatment options only target the inflammatory component of MS. Previous work using calpain inhibitor CYLA in our laboratory showed significant reduction in clinical sign, demyelination and tissue calpain content in acute experimental autoimmune encephalomyelitis (EAE). Here we evaluated markers of axonal injury (amyloid precursor protein, Na(v)1.6 channels), neuronal calpain content and the effect of CYLA on axonal protection using histological methods in chronic EAE [myelin oligodendrocyte glycoprotein (MOG)-induced disease model of MS]. Intraperitoneal application of CYLA (2 mg/mouse/day) significantly reduced the clinical signs, tissue calpain content, demyelination and inflammatory infiltration of EAE. Similarly, markers for axonal injury were barely detectable in the treated mice. Thus, this novel drug, which markedly suppresses the disease course, axonal injury and its progression, is a candidate for the treatment of a neurodegenerative disease such as multiple sclerosis.

    Funded by: NINDS NIH HHS: R01 NS040157-01A2

    Brain research 2008;1236;206-15

  • Membrane microdomain switching: a regulatory mechanism of amyloid precursor protein processing.

    Sakurai T, Kaneko K, Okuno M, Wada K, Kashiyama T, Shimizu H, Akagi T, Hashikawa T and Nukina N

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

    Neuronal activity has an impact on beta cleavage of amyloid precursor protein (APP) by BACE1 to generate amyloid-beta peptide (Abeta). However, the molecular mechanisms underlying this effect remain to be elucidated. Cholesterol dependency of beta cleavage prompted us to analyze immunoisolated APP-containing detergent-resistant membranes from rodent brains. We found syntaxin 1 as a key molecule for activity-dependent regulation of APP processing in cholesterol-dependent microdomains. In living cells, APP associates with syntaxin 1-containing microdomains through X11-Munc18, which inhibits the APP-BACE1 interaction and beta cleavage via microdomain segregation. Phosphorylation of Munc18 by cdk5 causes a shift of APP to BACE1-containing microdomains. Neuronal hyperactivity, implicated in Abeta overproduction, promotes the switching of APP microdomain association as well as beta cleavage in a partially cdk5-dependent manner. We propose that microdomain switching is a mechanism of cholesterol- and activity-dependent regulation of APP processing in neurons.

    The Journal of cell biology 2008;183;2;339-52

  • Structure of the C-terminal phosphotyrosine interaction domain of Fe65L1 complexed with the cytoplasmic tail of amyloid precursor protein reveals a novel peptide binding mode.

    Li H, Koshiba S, Hayashi F, Tochio N, Tomizawa T, Kasai T, Yabuki T, Motoda Y, Harada T, Watanabe S, Inoue M, Hayashizaki Y, Tanaka A, Kigawa T and Yokoyama S

    Systems and Structural Biology Center, RIKEN Yokohama Institute, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan.

    Fe65L1, a member of the Fe65 family, is an adaptor protein that interacts with the cytoplasmic domain of Alzheimer amyloid precursor protein (APP) through its C-terminal phosphotyrosine interaction/phosphotyrosine binding (PID/PTB) domain. In the present study, the solution structures of the C-terminal PID domain of mouse Fe65L1, alone and in complex with a 32-mer peptide (DAAVTPEERHLSKMQQNGYENPTYKFFEQMQN) derived from the cytoplasmic domain of APP, were determined using NMR spectroscopy. The C-terminal PID domain of Fe65L1 alone exhibits a canonical PID/PTB fold, whereas the complex structure reveals a novel mode of peptide binding. In the complex structure, the NPTY motif forms a type-I beta-turn, and the residues immediately N-terminal to the NPTY motif form an antiparallel beta-sheet with the beta5 strand of the PID domain, the binding mode typically observed in the PID/PTB.peptide complex. On the other hand, the N-terminal region of the peptide forms a 2.5-turn alpha-helix and interacts extensively with the C-terminal alpha-helix and the peripheral regions of the PID domain, representing a novel mode of peptide binding that has not been reported previously for the PID/PTB.peptide complex. The indispensability of the N-terminal region of the peptide for the high affinity of the PID-peptide interaction is consistent with NMR titration and isothermal calorimetry data. The extensive binding features of the PID domain of Fe65L1 with the cytoplasmic domain of APP provide a framework for further understanding of the function, trafficking, and processing of APP modulated by adapter proteins.

    The Journal of biological chemistry 2008;283;40;27165-78

  • The effect of amyloidosis-beta and ageing on proliferation of neuronal progenitor cells in APP-transgenic mouse hippocampus and in culture.

    Kolecki R, Lafauci G, Rubenstein R, Mazur-Kolecka B, Kaczmarski W and Frackowiak J

    Lehigh University, Bethlehem, PA, USA.

    Stimulation of endogenous neurogenesis and transplantation of neuronal progenitors (NPs) are considered in therapy of neuronal loss associated with ageing and in neurodegenerative diseases with amyloidosis-beta, for example, Alzheimer's disease and Down syndrome. However, the influence of brain environment altered by ageing and deposits of amyloid-beta on proliferation of endogenous and transplanted NPs and their maturation into neurons is not understood. We studied the effect of ageing and development of amyloidosis-beta on proliferation of NPs (1) in the granular layer of dentate gyrus in the hippocampi of APP-transgenic mice (Tg9291) before and after development of amyloidosis-beta, that is, in mice aged 2-4 months and 9-12 months, respectively, and in age-matched controls; and (2) in culture of NPs isolated from brains of control and Tg9291 mice, aged 3 and 9 months. We found that the number of proliferating NPs was reduced in 9-12-months-old mice, in both control and Tg9291, as compared to 2-4-months-old mice. However, the 9-12-months-old Tg9291 mice with amyloid-beta deposits had significantly more proliferating NPs than the age-matched controls. NPs proliferation in culture did not depend on the age, presence of APP-transgene, and amyloidosis-beta in donors. The results indicate that the local brain environment influences proliferation of NPs, and development of amyloidosis-beta in the neurogenic regions attenuates the age-associated reduction of proliferation of NPs. Identification of the responsible mechanisms may be important for development of a successful therapy of neurodegeneration caused by amyloidosis-beta.

    Acta neuropathologica 2008;116;4;419-24

  • Reduced retinal function in amyloid precursor protein-over-expressing transgenic mice via attenuating glutamate-N-methyl-d-aspartate receptor signaling.

    Shimazawa M, Inokuchi Y, Okuno T, Nakajima Y, Sakaguchi G, Kato A, Oku H, Sugiyama T, Kudo T, Ikeda T, Takeda M and Hara H

    Department of Biofunctional Evaluation, Laboratory of Molecular Pharmacology, Gifu Pharmaceutical University, Gifu, Japan.

    Here, we examined whether amyloid-beta (Abeta) protein participates in cell death and retinal function using three types of transgenic (Tg) mice in vivo [human mutant amyloid precursor protein (APP) Tg (Tg 2576) mice, mutant presenilin-1 (PS-1) knock-in mice, and APP/PS-1 double Tg mice]. ELISA revealed that the insoluble form of Abeta(1-40) was markedly accumulated in the retinas of APP and APP/PS-1, but not PS-1 Tg, mice (vs. wild-type mice). In APP Tg and APP/PS-1 Tg mice, immunostaining revealed accumulations of intracellular Abeta(1-42) in retinal ganglion cells and in the inner and outer nuclear layers. APP Tg and APP/PS-1 Tg, but not PS-1 Tg, mice had less NMDA-induced retinal damage than wild-type mice, and the reduced damage in APP/PS-1 Tg mice was diminished by the pre-treatment of N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester, a gamma-secretase inhibitor. Furthermore, the number of TUNEL-positive cells was significantly less in ganglion cell layer of APP/PS-1 Tg mice than PS-1 Tg mice 24 h after NMDA injection. The phosphorylated form of calcium/calmodulin-dependent protein kinase IIalpha (CaMKIIalpha), but not total CaMKIIalpha or total NMDA receptor 1 (NR1) subunit, in total retinal extracts was decreased in non-treated retinas of APP/PS-1 Tg mice (vs. wild-type mice). CaMKIIalpha and NR2B proteins, but not NR1, in retinal membrane fraction were significantly decreased in APP/PS-1 Tg mice as compared with wild-type mice. The NMDA-induced increase in p-CaMKIIalpha in the retina was also lower in APP/PS-1 Tg mice than in wild-type mice. In electroretinogram and visual-evoked potential recordings, the implicit time to each peak from a light stimulus was prolonged in APP/PS-1 mice versus wild-type mice. Hence, Abeta may impair retinal function by reducing activation of NMDA-receptor signaling pathways.

    Journal of neurochemistry 2008;107;1;279-90

  • Differential regulation of neurogenesis in two neurogenic regions of APPswe/PS1dE9 transgenic mice.

    Niidome T, Taniuchi N, Akaike A, Kihara T and Sugimoto H

    Department of Neuroscience for Drug Discovery, Graduate School of Pharmaceutical Sciences, Kyoto University, Japan. tniidome@pharm.kyoto-u.ac.jp

    Neurogenesis occurs in two neurogenic regions of the adult mammalian brain: the subgranular zone and the subventricular zone. We have recently demonstrated that the number of bromodeoxyuridine-positive and doublecortin-positive cells is decreased in the subgranular zone of amyloid precursor protein with a Swedish mutation and presenilin-1 with a deletion of exon 9 transgenic mice, an animal model of Alzheimer's disease. In this study, we characterized neurogenesis in the subventricular zone of amyloid precursor protein with a Swedish mutation and presenilin-1 with a deletion of exon 9 transgenic mice at 9 months of age and compared it with neurogenesis in the subgranular zone. In the subventricular zone, the number of proliferating cell nuclear antigen-positive and bromodeoxyuridine-positive cells were normal. In the subgranular zone, the number of proliferating cell nuclear antigen-positive cells was normal; however, the number of bromodeoxyuridine-positive cells was significantly decreased. These results suggest that neurogenesis, probably reflecting the survival of neural progenitor cells, differs between the subgranular zone and the subventricular zone.

    Neuroreport 2008;19;14;1361-4

  • Receptor-associated protein (RAP) plays a central role in modulating Abeta deposition in APP/PS1 transgenic mice.

    Xu G, Karch C, Li N, Lin N, Fromholt D, Gonzales V and Borchelt DR

    Department of Neuroscience, McKnight Brain Institute, Santa Fe Health Alzheimer's Disease Research Center, University of Florida, Gainesville, Florida, United States of America.

    Background: Receptor associated protein (RAP) functions in the endoplasmic reticulum (ER) to assist in the maturation of several membrane receptor proteins, including low density lipoprotein receptor-related protein (LRP) and lipoprotein receptor 11 (SorLA/LR11). Previous studies in cell and mouse model systems have demonstrated that these proteins play roles in the metabolism of the amyloid precursor protein (APP), including processes involved in the generation, catabolism and deposition of beta-amyloid (Abeta) peptides.

    Mice transgenic for mutant APPswe and mutant presenilin 1 (PS1dE9) were mated to mice with homozygous deletion of RAP. Unexpectedly, mice that were homozygous null for RAP and transgenic for APPswe/PS1dE9 showed high post-natal mortality, necessitating a shift in focus to examine the levels of amyloid deposition in APPswe/PS1dE9 that were hemizygous null for RAP. Immunoblot analysis confirmed 50% reductions in the levels of RAP with modest reductions in the levels of proteins dependent upon RAP for maturation [LRP trend towards a 20% reduction ; SorLA/LR11 statistically significant 15% reduction (p<0.05)]. Changes in the levels of these proteins in the brains of [APPswe/PS1dE9](+/-)/RAP(+/-) mice correlated with 30-40% increases in amyloid deposition by 9 months of age.

    Partial reductions in the ER chaperone RAP enhance amyloid deposition in the APPswe/PS1dE9 model of Alzheimer amyloidosis. Partial reductions in RAP also affect the maturation of LRP and SorLA/LR11, which are each involved in several different aspects of APP processing and Abeta catabolism. Together, these findings suggest a central role for RAP in Alzheimer amyloidogenesis.

    PloS one 2008;3;9;e3159

  • Activity requires soluble amyloid precursor protein alpha to promote neurite outgrowth in neural stem cell-derived neurons via activation of the MAPK pathway.

    Gakhar-Koppole N, Hundeshagen P, Mandl C, Weyer SW, Allinquant B, Müller U and Ciccolini F

    Department of Neurobiology, Interdisciplinary Centre for Neurosciences, Heidelberg, Germany.

    It is known that activity modulates neuronal differentiation in the adult brain but the signalling mechanisms underlying this process remain to be identified. We show here that activity requires soluble amyloid precursor protein (sAPP) to enhance neurite outgrowth of young neurons differentiating from neural stem cells. Inhibition of sAPP secretion and anti-APP antibodies both abolished the effect of depolarization on neurite outgrowth, whereas exogenous sAPPalpha, similar to depolarization, induced neurite elongation. Depolarization and sAPPalpha both required active N-methyl-D-aspartic acid receptor (NMDAR) and mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) recruitment to induce neurite outgrowth. However, depolarization and sAPPalpha played different roles in modulating this signalling cascade. Depolarization induced ERK phosphorylation with fast kinetics via activation of NMDAR. By contrast, acute application of sAPPalpha did not lead to ERK activation. However, continuous generation of sAPPalpha was necessary for depolarization-induced ERK phosphorylation, indicating that sAPPalpha promotes MAPK/ERK recruitment by an indirect mechanism. In addition, we found that blockade of NMDAR down-regulated APP expression, whereas depolarization increased sAPPalpha, suggesting that activity may also act upstream of sAPP signalling by regulating the amount of cellular APP and extracellular sAPPalpha. Finally, we show that soluble amyloid precursor-like protein 2 (sAPLP2), but not sAPLP1, is functionally redundant to sAPP in promoting neurite outgrowth and that soluble members of the APP family require membrane-bound APP to enhance neurite outgrowth. In summary, these experiments indicate a novel role of APP family members in activity-dependent neuronal differentiation.

    The European journal of neuroscience 2008;28;5;871-82

  • Neuro-inflammation induced by lipopolysaccharide causes cognitive impairment through enhancement of beta-amyloid generation.

    Lee JW, Lee YK, Yuk DY, Choi DY, Ban SB, Oh KW and Hong JT

    College of Pharmacy and CBITRC, Chungbuk National University 12, Cheongju, Chungbuk, Korea. 2jaewoong@hanmail.net

    Background: Alzheimer's disease (AD) is characterized by extensive loss of neurons in the brain of AD patients. Intracellular accumulation of beta-amyloid peptide (Abeta) has also shown to occur in AD. Neuro-inflammation has been known to play a role in the pathogenesis of AD.

    Methods: In this study, we investigated neuro-inflammation and amyloidogenesis and memory impairment following the systemic inflammation generated by lipopolysaccharide (LPS) using immunohistochemistry, ELISA, behavioral tests and Western blotting.

    Results: Intraperitoneal injection of LPS, (250 microg/kg) induced memory impairment determined by passive avoidance and water maze tests in mice. Repeated injection of LPS (250 microg/kg, 3 or 7 times) resulted in an accumulation of Abeta1-42 in the hippocampus and cerebralcortex of mice brains through increased beta- and gamma-secretase activities accompanied with the increased expression of amyloid precursor protein (APP), 99-residue carboxy-terminal fragment of APP (C99) and generation of Abeta1-42 as well as activation of astrocytes in vivo. 3 weeks of pretreatment of sulindac sulfide (3.75 and 7.5 mg/kg, orally), an anti-inflammatory agent, suppressed the LPS-induced amyloidogenesis, memory dysfunction as well as neuronal cell death in vivo. Sulindac sulfide (12.5-50 microM) also suppressed LPS (1 microg/ml)-induced amyloidogenesis in cultured neurons and astrocytes in vitro.

    Conclusion: This study suggests that neuro-inflammatory reaction could contribute to AD pathology, and anti-inflammatory agent could be useful for the prevention of AD.

    Journal of neuroinflammation 2008;5;37

  • Non-cell-autonomous effects of presenilin 1 variants on enrichment-mediated hippocampal progenitor cell proliferation and differentiation.

    Choi SH, Veeraraghavalu K, Lazarov O, Marler S, Ransohoff RM, Ramirez JM and Sisodia SS

    Committee on Neurobiology, University of Chicago, Chicago, IL 60637, USA.

    Presenilin 1 (PS1) regulates environmental enrichment (EE)-mediated neural progenitor cell (NPC) proliferation and neurogenesis in the adult hippocampus. We now report that transgenic mice that ubiquitously express human PS1 variants linked to early-onset familial Alzheimer's disease (FAD) neither exhibit EE-induced proliferation, nor neuronal lineage commitment of NPCs. Remarkably, the proliferation and differentiation of cultured NPCs from standard-housed mice expressing wild-type PS1 or PS1 variants are indistinguishable. On the other hand, wild-type NPCs cocultured with primary microglia from mice expressing PS1 variants exhibit impaired proliferation and neuronal lineage commitment, phenotypes that are recapitulated with mutant microglia conditioned media in which we detect altered levels of selected soluble signaling factors. These findings lead us to conclude that factors secreted from microglia play a central role in modulating hippocampal neurogenesis, and argue for non-cell-autonomous mechanisms that govern FAD-linked PS1-mediated impairments in adult hippocampal neurogenesis.

    Funded by: NIA NIH HHS: AG021494, AG027854, R01 AG021494, R01 AG027854, R01 AG033570, R01 AG040185

    Neuron 2008;59;4;568-80

  • The amyloid-beta rise and gamma-secretase inhibitor potency depend on the level of substrate expression.

    Burton CR, Meredith JE, Barten DM, Goldstein ME, Krause CM, Kieras CJ, Sisk L, Iben LG, Polson C, Thompson MW, Lin XA, Corsa J, Fiedler T, Pierdomenico M, Cao Y, Roach AH, Cantone JL, Ford MJ, Drexler DM, Olson RE, Yang MG, Bergstrom CP, McElhone KE, Bronson JJ, Macor JE, Blat Y, Grafstrom RH, Stern AM, Seiffert DA, Zaczek R, Albright CF and Toyn JH

    Bristol-Myers Squibb Research and Development, Wallingford, Connecticut 06492, USA.

    The amyloid-beta (Abeta) peptide, which likely plays a key role in Alzheimer disease, is derived from the amyloid-beta precursor protein (APP) through consecutive proteolytic cleavages by beta-site APP-cleaving enzyme and gamma-secretase. Unexpectedly gamma-secretase inhibitors can increase the secretion of Abeta peptides under some circumstances. This "Abeta rise" phenomenon, the same inhibitor causing an increase in Abeta at low concentrations but inhibition at higher concentrations, has been widely observed. Here we show that the Abeta rise depends on the beta-secretase-derived C-terminal fragment of APP (betaCTF) or C99 levels with low levels causing rises. In contrast, the N-terminally truncated form of Abeta, known as "p3," formed by alpha-secretase cleavage, did not exhibit a rise. In addition to the Abeta rise, low betaCTF or C99 expression decreased gamma-secretase inhibitor potency. This "potency shift" may be explained by the relatively high enzyme to substrate ratio under conditions of low substrate because increased concentrations of inhibitor would be necessary to affect substrate turnover. Consistent with this hypothesis, gamma-secretase inhibitor radioligand occupancy studies showed that a high level of occupancy was correlated with inhibition of Abeta under conditions of low substrate expression. The Abeta rise was also observed in rat brain after dosing with the gamma-secretase inhibitor BMS-299897. The Abeta rise and potency shift are therefore relevant factors in the development of gamma-secretase inhibitors and can be evaluated using appropriate choices of animal and cell culture models. Hypothetical mechanisms for the Abeta rise, including the "incomplete processing" and endocytic models, are discussed.

    The Journal of biological chemistry 2008;283;34;22992-3003

  • Amyloid-beta protein dimers isolated directly from Alzheimer's brains impair synaptic plasticity and memory.

    Shankar GM, Li S, Mehta TH, Garcia-Munoz A, Shepardson NE, Smith I, Brett FM, Farrell MA, Rowan MJ, Lemere CA, Regan CM, Walsh DM, Sabatini BL and Selkoe DJ

    Center for Neurologic Diseases, Brigham and Women's Hospital and Harvard Medical School, 77 Avenue Louis Pasteur, Boston, Massachusetts 02115, USA.

    Alzheimer's disease constitutes a rising threat to public health. Despite extensive research in cellular and animal models, identifying the pathogenic agent present in the human brain and showing that it confers key features of Alzheimer's disease has not been achieved. We extracted soluble amyloid-beta protein (Abeta) oligomers directly from the cerebral cortex of subjects with Alzheimer's disease. The oligomers potently inhibited long-term potentiation (LTP), enhanced long-term depression (LTD) and reduced dendritic spine density in normal rodent hippocampus. Soluble Abeta from Alzheimer's disease brain also disrupted the memory of a learned behavior in normal rats. These various effects were specifically attributable to Abeta dimers. Mechanistically, metabotropic glutamate receptors were required for the LTD enhancement, and N-methyl D-aspartate receptors were required for the spine loss. Co-administering antibodies to the Abeta N-terminus prevented the LTP and LTD deficits, whereas antibodies to the midregion or C-terminus were less effective. Insoluble amyloid plaque cores from Alzheimer's disease cortex did not impair LTP unless they were first solubilized to release Abeta dimers, suggesting that plaque cores are largely inactive but sequester Abeta dimers that are synaptotoxic. We conclude that soluble Abeta oligomers extracted from Alzheimer's disease brains potently impair synapse structure and function and that dimers are the smallest synaptotoxic species.

    Funded by: NIA NIH HHS: AG R01 027443, R01 AG027443, R01 AG027443-01, R01 AG027443-02, R01 AG027443-03, R01 AG027443-04; NINDS NIH HHS: R01 NS046579, R01 NS046579-06A1; Wellcome Trust: 067660

    Nature medicine 2008;14;8;837-42

  • Embryonic stem cell-derived neurons as a cellular system to study gene function: lack of amyloid precursor proteins APP and APLP2 leads to defective synaptic transmission.

    Schrenk-Siemens K, Perez-Alcala S, Richter J, Lacroix E, Rahuel J, Korte M, Müller U, Barde YA and Bibel M

    Neurodegeneration Department, Neuroscience Research, Novartis Institutes for BioMedical Research, 4002 Basel, Switzerland.

    The in vitro generation of uniform populations of neurons from mouse embryonic stem cells (ESCs) provides a novel opportunity to study gene function in neurons. This is of particular interest when mutations lead to lethal in vivo phenotypes. Although the amyloid precursor protein (APP) and its proteolysis are regarded as key elements of the pathology of Alzheimer's disease, the physiological function of APP is not well understood and mice lacking App and the related gene Aplp2 die early postnatally without any obvious histopathological abnormalities. Here we show that glutamatergic neurons differentiated from ESCs lacking both genes reveal a decreased expression of the vesicular glutamate transporter 2 (VGLUT2) both at the mRNA and protein level, as well as a reduced uptake and/or release of glutamate. Blocking gamma-secretase cleavage of APP in wild-type neurons resulted in a similar decrease of VGLUT2 expression, whereas VGLUT2 levels could be restored in App-/-Aplp2-/- neurons by a construct encompassing the C-terminal intracellular domain of APP. Electrophysiological recordings of hippocampal organotypic slice cultures prepared from corresponding mutant mice corroborated these observations. Gene expression profiling and pathway analysis of the differentiated App-/-Aplp2-/- neurons identified dysregulation of additional genes involved in synaptic transmission pathways. Our results indicate a significant functional role of APP and amyloid precursor-like protein 2 (APLP2) in the development of synaptic function by the regulation of glutamatergic neurotransmission. Differentiation of ESCs into homogeneous populations thus represents a new opportunity to explore gene function and to dissect signaling pathways in neurons. Disclosure of potential conflicts of interest is found at the end of this article.

    Stem cells (Dayton, Ohio) 2008;26;8;2153-63

  • Evidence that CD147 modulation of beta-amyloid (Abeta) levels is mediated by extracellular degradation of secreted Abeta.

    Vetrivel KS, Zhang X, Meckler X, Cheng H, Lee S, Gong P, Lopes KO, Chen Y, Iwata N, Yin KJ, Lee JM, Parent AT, Saido TC, Li YM, Sisodia SS and Thinakaran G

    Department of Neurobiology and Neurology, The University of Chicago, Chicago, Illinois 60637, USA.

    Cerebral deposition of beta-amyloid (Abeta) peptides is a pathological hallmark of Alzheimer disease. Intramembranous proteolysis of amyloid precursor protein by a multiprotein gamma-secretase complex generates Abeta. Previously, it was reported that CD147, a glycoprotein that stimulates production of matrix metalloproteinases (MMPs), is a subunit of gamma-secretase and that the levels of secreted Abeta inversely correlate with CD147 expression. Here, we show that the levels and localization of CD147 in fibroblasts, as well as postnatal expression and distribution in brain, are distinct from those of integral gamma-secretase subunits. Notably, we show that although depletion of CD147 increased extracellular Abeta levels in intact cells, membranes isolated from CD147-depleted cells failed to elevate Abeta production in an in vitro gamma-secretase assay. Consistent with an extracellular source that modulates Abeta metabolism, synthetic Abeta was degraded more rapidly in the conditioned medium of cells overexpressing CD147. Moreover, modulation of CD147 expression had no effect on epsilon-site cleavage of amyloid precursor protein and Notch1 receptor. Collectively, our results demonstrate that CD147 modulates Abeta levels not by regulating gamma-secretase activity, but by stimulating extracellular degradation of Abeta. In view of the known function of CD147 in MMP production, we postulate that CD147 expression influences Abeta levels by an indirect mechanism involving MMPs that can degrade extracellular Abeta.

    Funded by: NIA NIH HHS: AG019070, AG021495, AG026660, R01 AG019070, R01 AG021495; NINDS NIH HHS: P01 NS032636, P01 NS032636-139001, R01 NS048283, R01 NS048283-03, R01 NS048283-04

    The Journal of biological chemistry 2008;283;28;19489-98

  • Regulation of FE65 nuclear translocation and function by amyloid beta-protein precursor in osmotically stressed cells.

    Nakaya T, Kawai T and Suzuki T

    Laboratory of Neuroscience, Graduate School of Pharmaceutical Sciences, Hokkaido University, Kita-12 Nishi-6, Kita-ku, Sapporo 060-0812, Japan.

    FE65, a neural adaptor protein, interacts with amyloid beta-protein precursor (APP) and is known to regulate amyloid beta generation from APP. FE65 also associates with nuclear proteins; however, its physiological function in the nucleus remains unclear. A fixed population of cytoplasmic FE65 is tethered to membranes by binding APP. This membrane-tethered FE65 is liberated from membranes by APP phosphorylation, which is facilitated by a stress-activated protein kinase in sorbitol-treated cells. Here we show that liberated FE65, which is distinct from "virgin" FE65 in the cytoplasm, translocates into the nucleus and accumulates in the nuclear matrix forming a patched structure. Targeting of FE65 into the nuclear matrix was suppressed by the APP intracellular domain fragment, which is generated by consecutive cleavages of APP. Thus, nuclear translocation of FE65 is under the regulation of APP. In the nucleus, FE65 induced gammaH2AX, which plays an important role in DNA repair as a cellular response by stress-damaged cells. These observations suggest that APP-regulated FE65 plays an important role in the early stress response of cells and that FE65 deregulated from APP induces apoptosis.

    The Journal of biological chemistry 2008;283;27;19119-31

  • Genes contributing to prion pathogenesis.

    Tamgüney G, Giles K, Glidden DV, Lessard P, Wille H, Tremblay P, Groth DF, Yehiely F, Korth C, Moore RC, Tatzelt J, Rubinstein E, Boucheix C, Yang X, Stanley P, Lisanti MP, Dwek RA, Rudd PM, Moskovitz J, Epstein CJ, Cruz TD, Kuziel WA, Maeda N, Sap J, Ashe KH, Carlson GA, Tesseur I, Wyss-Coray T, Mucke L, Weisgraber KH, Mahley RW, Cohen FE and Prusiner SB

    Institute for Neurodegenerative Diseases, University of California, San Francisco, CA, USA.

    Prion diseases are caused by conversion of a normally folded, non-pathogenic isoform of the prion protein (PrP(C)) to a misfolded, pathogenic isoform (PrP(Sc)). Prion inoculation experiments in mice expressing homologous PrP(C) molecules on different genetic backgrounds displayed different incubation times, indicating that the conversion reaction may be influenced by other gene products. To identify genes that contribute to prion pathogenesis, we analysed incubation times of prions in mice in which the gene product was inactivated, knocked out or overexpressed. We tested 20 candidate genes, because their products either colocalize with PrP, are associated with Alzheimer's disease, are elevated during prion disease, or function in PrP-mediated signalling, PrP glycosylation, or protein maintenance. Whereas some of the candidates tested may have a role in the normal function of PrP(C), our data show that many genes previously implicated in prion replication have no discernible effect on the pathogenesis of prion disease. While most genes tested did not significantly affect survival times, ablation of the amyloid beta (A4) precursor protein (App) or interleukin-1 receptor, type I (Il1r1), and transgenic overexpression of human superoxide dismutase 1 (SOD1) prolonged incubation times by 13, 16 and 19 %, respectively.

    Funded by: NIA NIH HHS: AG02132, AG021601, AG10770, P01 AG002132-26, P01 AG010770-14, P01 AG021601-03

    The Journal of general virology 2008;89;Pt 7;1777-88

  • High cholesterol-induced neuroinflammation and amyloid precursor protein processing correlate with loss of working memory in mice.

    Thirumangalakudi L, Prakasam A, Zhang R, Bimonte-Nelson H, Sambamurti K, Kindy MS and Bhat NR

    Department of Neurosciences, Medical University of South Carolina, Charleston, South Carolina 29425, USA.

    Recent findings suggest that hypercholesterolemia may contribute to the onset of Alzheimer's disease-like dementia but the underlying mechanisms remain unknown. In this study, we evaluated the cognitive performance in rodent models of hypercholesterolemia in relation to neuroinflammatory changes and amyloid precursor protein (APP) processing, the two key parameters of Alzheimer's disease pathogenesis. Groups of normal C57BL/6 and low density lipoprotein receptor (LDLR)-deficient mice were fed a high fat/cholesterol diet for an 8-week period and tested for memory in a radial arm maze. It was found that the C57BL/6 mice receiving a high fat diet were deficient in handling an increasing working memory load compared with counterparts receiving a control diet while the hypercholesterolemic LDLR-/- mice showed impaired working memory regardless of diet. Immunohistochemical analysis revealed the presence of activated microglia and astrocytes in the hippocampi from high fat-fed C57BL/6 mice and LDLR-/- mice. Consistent with a neuroinflammatory response, the hyperlipidemic mice showed increased expression of cytokines/mediators including tumor necrosis factor-alpha, interleukin-1beta and -6, nitric oxide synthase 2, and cycloxygenase 2. There was also an induced expression of the key APP processing enzyme i.e. beta-site APP cleaving enzyme 1 in both high fat/cholesterol-fed C57BL/6 and LDLR-/- mice accompanied by an increased generation of C-terminal fragments of APP. Although ELISA for beta-amyloid failed to record significant changes in the non-transgenic mice, a threefold increase in beta-amyloid 40 accumulation was apparent in a strain of transgenic mice expressing wild-type human APP on high fat/cholesterol diet. The findings link hypercholesterolemia with cognitive dysfunction potentially mediated by increased neuroinflammation and APP processing in a non-transgenic mouse model.

    Funded by: NCRR NIH HHS: C06 RR015455; NIA NIH HHS: R01 AG023055, R01 AG023055-01A1, R01 AG023055-02, R01 AG023055-03, R01 AG023055-04, R01 AG023055-05, R01AG023055; NINDS NIH HHS: R01 NS051575, R01NS051575

    Journal of neurochemistry 2008;106;1;475-85

  • SERCA pump activity is physiologically regulated by presenilin and regulates amyloid beta production.

    Green KN, Demuro A, Akbari Y, Hitt BD, Smith IF, Parker I and LaFerla FM

    Department of Neurobiology and Behavior, University of California, Irvine, Irvine, CA 92697, USA.

    In addition to disrupting the regulated intramembraneous proteolysis of key substrates, mutations in the presenilins also alter calcium homeostasis, but the mechanism linking presenilins and calcium regulation is unresolved. At rest, cytosolic Ca(2+) is maintained at low levels by pumping Ca(2+) into stores in the endoplasmic reticulum (ER) via the sarco ER Ca(2+)-ATPase (SERCA) pumps. We show that SERCA activity is diminished in fibroblasts lacking both PS1 and PS2 genes, despite elevated SERCA2b steady-state levels, and we show that presenilins and SERCA physically interact. Enhancing presenilin levels in Xenopus laevis oocytes accelerates clearance of cytosolic Ca(2+), whereas higher levels of SERCA2b phenocopy PS1 overexpression, accelerating Ca(2+) clearance and exaggerating inositol 1,4,5-trisphosphate-mediated Ca(2+) liberation. The critical role that SERCA2b plays in the pathogenesis of Alzheimer's disease is underscored by our findings that modulating SERCA activity alters amyloid beta production. Our results point to a physiological role for the presenilins in Ca(2+) signaling via regulation of the SERCA pump.

    Funded by: NIA NIH HHS: AG16573, AG17968, P50 AG016573, R01 AG017968; NIGMS NIH HHS: GM48071, R01 GM048071, R37 GM048071

    The Journal of cell biology 2008;181;7;1107-16

  • Evaluation of the APP23-model for Alzheimer's disease in the odour paired-associate test for hippocampus-dependent memory.

    Van Dijck A, Vloeberghs E, Van Dam D, Staufenbiel M and De Deyn PP

    Laboratory of Neurochemistry & Behaviour, Institute Born-Bunge, University of Antwerp, Department of Biomedical Sciences, Universiteitsplein 1, Wilrijk, Belgium.

    The APP23 model is a transgenic mouse model for Alzheimer's disease. Cognitive performance in the APP23-model was assessed by Morris Water Maze (MWM) and passive avoidance learning, but the latter failed to show any difference between the genotypes. In search of a non-spatial alternative for assessment of hippocampus-dependent memory, we evaluated an odour paired-associate test, which is based on learning an association between two sets of odours. The protocol includes a shaping phase, in which the animals learn to dig up a reward, a preliminary training phase and a training phase, where the actual association is learned. Subsequently, mice are tested for transitive inference and subjected to a symmetry test. Impairment was seen in the APP23 mice, in comparison with wild type mice, in training; however, both groups failed the transitivity and symmetry test. Possible explanations for this discrepancy with earlier published results are the advanced age of the mice or the C57Bl/6J background, in which the model was established.

    Behavioural brain research 2008;190;1;147-51

  • Females exhibit more extensive amyloid, but not tau, pathology in an Alzheimer transgenic model.

    Hirata-Fukae C, Li HF, Hoe HS, Gray AJ, Minami SS, Hamada K, Niikura T, Hua F, Tsukagoshi-Nagai H, Horikoshi-Sakuraba Y, Mughal M, Rebeck GW, LaFerla FM, Mattson MP, Iwata N, Saido TC, Klein WL, Duff KE, Aisen PS and Matsuoka Y

    Department of Neurology, Georgetown University Medical Center, Washington, DC 20057, USA.

    Epidemiological studies indicate that women have a higher risk of Alzheimer's disease (AD) even after adjustment for age. Though transgenic mouse models of AD develop AD-related amyloid beta (Abeta) and/or tau pathology, gender differences have not been well documented in these models. In this study, we found that female 3xTg-AD transgenic mice expressing mutant APP, presenilin-1 and tau have significantly more aggressive Abeta pathology. We also found an increase in beta-secretase activity and a reduction of neprilysin in female mice compared to males; this suggests that a combination of increased Abeta production and decreased Abeta degradation may contribute to higher risk of AD in females. In contrast to significantly more aggressive Abeta pathology in females, gender did not affect the levels of phosphorylated tau in 3xTg-AD mice. These results point to the involvement of Abeta pathways in the higher risk of AD in women. In addition to comparison of pathology between genders at 9, 16 and 23 months of age, we examined the progression of Abeta pathology at additional age points; i.e., brain Abeta load, intraneuronal oligomeric Abeta distribution and plaque load, in male 3xTg-AD mice at 3, 6, 9, 12, 16, 20 and 23 months of age. These findings confirm progressive Abeta pathology in 3xTg-AD transgenic mice, and provide guidance for their use in therapeutic research.

    Funded by: Intramural NIH HHS; NIA NIH HHS: AG022455, AG026478

    Brain research 2008;1216;92-103

  • Secreted APP regulates the function of full-length APP in neurite outgrowth through interaction with integrin beta1.

    Young-Pearse TL, Chen AC, Chang R, Marquez C and Selkoe DJ

    Center for Neurologic Diseases, Brigham and Women's Hospital and Harvard Medical School, Boston, MA 02115, USA. tyoung@rics.bwh.harvard.edu

    Background: Beta-amyloid precursor protein (APP) has been reported to play a role in the outgrowth of neurites from cultured neurons. Both cell-surface APP and its soluble, ectodomain cleavage product (APPs-alpha) have been implicated in regulating the length and branching of neurites in a variety of assays, but the mechanism by which APP performs this function is not understood.

    Results: Here, we report that APP is required for proper neurite outgrowth in a cell autonomous manner, both in vitro and in vivo. Neurons that lack APP undergo elongation of their longest neurite. Deletion of APLP1 or APLP2, homologues of APP, likewise stimulates neurite lengthening. Intriguingly, wild-type neurons exposed to APPs-alpha, the principal cleavage product of APP, also undergo neurite elongation. However, APPs-alpha is unable to stimulate neurite elongation in the absence of cellular APP expression. The outgrowth-enhancing effects of both APPs-alpha and the deletion of APP are inhibited by blocking antibodies to Integrin beta1 (Itgbeta1). Moreover, full length APP interacts biochemically with Itgbeta1, and APPs-alpha can interfere with this binding.

    Conclusion: Our findings indicate that APPs-alpha regulates the function of APP in neurite outgrowth via the novel mechanism of competing with the binding of APP to Itgbeta1.

    Funded by: NIA NIH HHS: R01 AG006173, R0I AG06173; NINDS NIH HHS: F32 NS053320, F32 NS053320-01A1

    Neural development 2008;3;15

  • Identification of Phox2b-regulated genes by expression profiling of cranial motoneuron precursors.

    Pla P, Hirsch MR, Le Crom S, Reiprich S, Harley VR and Goridis C

    Ecole normale supérieure, Département de Biologie, 75005 Paris, France. Patrick.Pla@curie.u-psud.fr

    Background: Branchiomotor neurons comprise an important class of cranial motor neurons that innervate the branchial-arch-derived muscles of the face, jaw and neck. They arise in the ventralmost progenitor domain of the rhombencephalon characterized by expression of the homeodomain transcription factors Nkx2.2 and Phox2b. Phox2b in particular plays a key role in the specification of branchiomotor neurons. In its absence, generic neuronal differentiation is defective in the progenitor domain and no branchiomotor neurons are produced. Conversely, ectopic expression of Phox2b in spinal regions of the neural tube promotes cell cycle exit and neuronal differentiation and, at the same time, induces genes and an axonal phenotype characteristic for branchiomotor neurons. How Phox2b exerts its pleiotropic functions, both as a proneural gene and a neuronal subtype determinant, has remained unknown.

    Results: To gain further insights into the genetic program downstream of Phox2b, we searched for novel Phox2b-regulated genes by cDNA microarray analysis of facial branchiomotor neuron precursors from heterozygous and homozygous Phox2b mutant embryos. We selected for functional studies the genes encoding the axonal growth promoter Gap43, the Wnt antagonist Sfrp1 and the transcriptional regulator Sox13, which were not previously suspected to play roles downstream of Phox2b and whose expression was affected by Phox2b misexpression in the spinal cord. While Gap43 did not produce an obvious phenotype when overexpressed in the neural tube, Sfrp1 induced the interneuron marker Lhx1,5 and Sox13 inhibited neuronal differentiation. We then tested whether Sfrp1 and Sox13, which are down-regulated by Phox2b in the facial neuron precursors, would antagonize some aspects of Phox2b activity. Co-expression of Sfrp1 prevented Phox2b from repressing Lhx1,5 and alleviated the commissural axonal phenotype. When expressed together with Sox13, Phox2b was still able to promote cell cycle exit and neuronal differentiation, but the cells failed to relocate to the mantle layer and to extinguish the neural stem cell marker Sox2.

    Conclusion: Our results suggest novel roles for Sfrp1 and Sox13 in neuronal subtype specification and generic neuronal differentiation, respectively, and indicate that down-regulation of Sfrp1 and Sox13 are essential aspects of the genetic program controlled by Phox2b in cranial motoneurons.

    Neural development 2008;3;14

  • Enzymatic characteristics of I213T mutant presenilin-1/gamma-secretase in cell models and knock-in mouse brains: familial Alzheimer disease-linked mutation impairs gamma-site cleavage of amyloid precursor protein C-terminal fragment beta.

    Shimojo M, Sahara N, Mizoroki T, Funamoto S, Morishima-Kawashima M, Kudo T, Takeda M, Ihara Y, Ichinose H and Takashima A

    Laboratory for Alzheimer's Disease, RIKEN Brain Science Institute, Wako-shi, Saitama 351-0198, Japan.

    Presenilin (PS)/gamma-secretase-mediated intramembranous proteolysis of amyloid precursor protein produces amyloid beta (Abeta) peptides in which Abeta species of different lengths are generated through multiple cleavages at the gamma-, zeta-, and epsilon-sites. An increased Abeta42/Abeta40 ratio is a common characteristic of most cases of familial Alzheimer disease (FAD)-linked PS mutations. However, the molecular mechanisms underlying amyloid precursor protein proteolysis leading to increased Abeta42/Abeta40 ratios still remain unclear. Here, we report our findings on the enzymatic analysis of gamma-secretase derived from I213T mutant PS1-expressing PS1/PS2-deficient (PS(-/-)) cells and from the brains of I213T mutant PS1 knock-in mice. Kinetics analyses revealed that the FAD mutation reduced de novo Abeta generation, suggesting that mutation impairs the total catalytic rate of gamma-secretase. Analysis of each Abeta species revealed that the FAD mutation specifically reduced Abeta40 levels more drastically than Abeta42 levels, leading to an increased Abeta42/Abeta40 ratio. By contrast, the FAD mutation increased the generation of longer Abeta species such as Abeta43, Abeta45, and >Abeta46. These results were confirmed by analyses of gamma-secretase derived from I213T knock-in mouse brains, in which the reduction of de novo Abeta generation was mutant allele dose-dependent. Our findings clearly indicate that the mechanism underlying the increased Abeta42/Abeta40 ratio observed in cases of FAD mutations is related to the differential inhibition of gamma-site cleavage reactions, in which the reaction producing Abeta40 is subject to more inhibition than that producing Abeta42. Our results also provide novel insight into how enhancing the generation of longer Abetas may contribute to Alzheimer disease onset.

    The Journal of biological chemistry 2008;283;24;16488-96

  • Transglutaminase induces protofibril-like amyloid beta-protein assemblies that are protease-resistant and inhibit long-term potentiation.

    Hartley DM, Zhao C, Speier AC, Woodard GA, Li S, Li Z and Walz T

    Department of Neurological Sciences, Rush University Medical Center, Chicago, Illinois 60612, USA. dean_hartley@rush.edu

    An increasing body of evidence suggests that soluble assemblies of amyloid beta-protein (Abeta) play an important role in the initiation of Alzheimer disease (AD). In vitro studies have found that synthetic Abeta can form soluble aggregates through self-assembly, but this process requires Abeta concentrations 100- to 1000-fold greater than physiological levels. Tissue transglutaminase (TGase) has been implicated in neurodegeneration and can cross-link Abeta. Here we show that TGase induces rapid aggregation of Abeta within 0.5-30 min, which was not observed with chemical cross-linkers. Both Abeta40 and Abeta42 are good substrates for TGase but show different aggregation patterns. Guinea pig and human TGase induced similar Abeta aggregation patterns, and oligomerization was observed with Abeta40 concentrations as low as 50 nm. The formed Abeta40 species range from 5 to 6 nm spheres to curvilinear structures of the same width, but up to 100 nm in length, that resemble the previously described self-assembled Abeta protofibrils. TGase-induced Abeta40 assemblies are resistant to a 1-h incubation with either neprilysin or insulin degrading enzyme, whereas the monomer is rapidly degraded by both proteases. In support of these species being pathological, TGase-induced Abeta40 assemblies (100 nm) inhibited long term potentiation recorded in the CA1 region of mouse hippocampus slices. Our data suggest that TGase can contribute to AD by initiating Abeta oligomerization and aggregation at physiological levels, by reducing the clearance of Abeta due to the generation of protease-resistant Abeta species, and by forming Abeta assemblies that inhibit processes involved in memory and learning. Our data suggest that TGase might constitute a specific therapeutic target for slowing or blocking the progression of AD.

    Funded by: NIA NIH HHS: AG19770; NIGMS NIH HHS: P01 GM062580

    The Journal of biological chemistry 2008;283;24;16790-800

  • The autophagy-related protein beclin 1 shows reduced expression in early Alzheimer disease and regulates amyloid beta accumulation in mice.

    Pickford F, Masliah E, Britschgi M, Lucin K, Narasimhan R, Jaeger PA, Small S, Spencer B, Rockenstein E, Levine B and Wyss-Coray T

    Department of Neurology and Neurological Sciences, Stanford University School of Medicine, Stanford, California, USA.

    Autophagy is the principal cellular pathway for degradation of long-lived proteins and organelles and regulates cell fate in response to stress. Recently, autophagy has been implicated in neurodegeneration, but whether it is detrimental or protective remains unclear. Here we report that beclin 1, a protein with a key role in autophagy, was decreased in affected brain regions of patients with Alzheimer disease (AD) early in the disease process. Heterozygous deletion of beclin 1 (Becn1) in mice decreased neuronal autophagy and resulted in neurodegeneration and disruption of lysosomes. In transgenic mice that express human amyloid precursor protein (APP), a model for AD, genetic reduction of Becn1 expression increased intraneuronal amyloid beta (Abeta) accumulation, extracellular Abeta deposition, and neurodegeneration and caused microglial changes and profound neuronal ultrastructural abnormalities. Administration of a lentiviral vector expressing beclin 1 reduced both intracellular and extracellular amyloid pathology in APP transgenic mice. We conclude that beclin 1 deficiency disrupts neuronal autophagy, modulates APP metabolism, and promotes neurodegeneration in mice and that increasing beclin 1 levels may have therapeutic potential in AD.

    Funded by: NCI NIH HHS: CA84254, R01 CA084254; NIA NIH HHS: AG02270, AG10435, AG18440, AG20603, AG22074, AG5131, P01 AG010435, P01 AG022074, P50 AG005131, R01 AG018440, R01 AG020603, R01 AG030144, R37 AG018440

    The Journal of clinical investigation 2008;118;6;2190-9

  • Identification of the Alzheimer's disease amyloid precursor protein (APP) and its homologue APLP2 as essential modulators of glucose and insulin homeostasis and growth.

    Needham BE, Wlodek ME, Ciccotosto GD, Fam BC, Masters CL, Proietto J, Andrikopoulos S and Cappai R

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

    The amyloid precursor protein (APP), the source of the neurotoxic amyloid beta (A beta) peptide involved in Alzheimer's disease (AD), belongs to a conserved family of related proteins. In mammals, the APP family contains amyloid precursor-like protein 1 (APLP1) and amyloid precursor-like protein 2 (APLP2). Whilst a number of activities have been attributed to the APP family, an overall function has not been definitively established. While ablating either the APP or APLP2 gene in mice produces minimal phenotypic change, the combined knockout of these genes in mice causes postnatal mortality. Postnatal survival therefore requires a shared but unknown function of APP and APLP2. To investigate the biochemical basis for the postnatal lethality, plasma was analysed from double knockout mice (APP-/- APLP2-/-) 2 days before birth, at gestational day E17, and from mice at 12-16 h after birth. The postnatal double knockouts had 66% lower plasma glucose levels than their wild-type controls and 50% lower than their single knockout counterparts. Interestingly, the postnatal double knockouts displayed hyperinsulinaemia, as shown by inappropriate plasma insulin levels, given their degree of hypoglycaemia. The single knockout mice also showed hyperinsulinaemia and had 31% lower plasma glucose than the wild-types. While the double knockouts did not survive more than 24 h after birth, the single knockouts reached adulthood and their hypoglycaemia continued. Therefore, APP and APLP2 expression modulates plasma insulin and glucose concentrations. Plasma calcium, magnesium and phosphate were also significantly reduced in the double knockouts compared to the wild-types, and they showed distinctive growth restriction, suggesting the involvement of a metabolic impairment. These results link the expression of the APP and APLP2 genes with glucose homeostasis and growth and therefore identify a novel function for the APP family.

    The Journal of pathology 2008;215;2;155-63

  • Neurogenesis and alterations of neural stem cells in mouse models of cerebral amyloidosis.

    Ermini FV, Grathwohl S, Radde R, Yamaguchi M, Staufenbiel M, Palmer TD and Jucker M

    Department of Cellular Neurology, Hertie Institute for Clinical Brain Research, University of Tübingen, Tübingen, Germany.

    The hippocampus in Alzheimer's disease is burdened with amyloid plaques and is one of the few locations where neurogenesis continues throughout adult life. To evaluate the impact of amyloid-beta deposition on neural stem cells, hippocampal neurogenesis was assessed using bromodeoxyuridine incorporation and doublecortin staining in two amyloid precursor protein (APP) transgenic mouse models. In 5-month-old APP23 mice prior to amyloid deposition, neurogenesis showed no robust difference relative to wild-type control mice, but 25-month-old amyloid-depositing APP23 mice showed significant increases in neurogenesis compared to controls. In contrast, 8-month-old amyloid-depositing APPPS1 mice revealed decreases in neurogenesis compared to controls. To study whether alterations in neurogenesis are the result of amyloid-induced changes at the level of neural stem cells, APPPS1 mice were crossed with mice expressing green fluorescence protein (GFP) under a central nervous system-specific nestin promoter. Eight-month-old nestin-GFP x APPPS1 mice exhibited decreases in quiescent nestin-positive astrocyte-like stem cells, while transient amplifying progenitor cells did not change in number. Strikingly, both astrocyte-like and transient-amplifying progenitor cells revealed an aberrant morphologic reaction toward congophilic amyloid-deposits. A similar reaction toward the amyloid was no longer observed in doublecortin-positive immature neurons. Results provide evidence for a disruption of neural stem cell biology in an amyloidogenic environment and support findings that neurogenesis is differently affected among various transgenic mouse models of Alzheimer's disease.

    The American journal of pathology 2008;172;6;1520-8

  • Regulation of Abeta pathology by beclin 1: a protective role for autophagy?

    Lee JA and Gao FB

    Gladstone Institute of Neurological Disease and Department of Neurology, UCSF, San Francisco, California 94158, USA.

    The amyloid beta (Abeta) peptide is thought to be a major culprit in Alzheimer disease (AD), and its production and degradation have been intensely investigated. Nevertheless, it remains largely unknown how Abeta pathology is modulated by the autophagy pathway. The study by Pickford and colleagues in this issue of the JCI shows that beclin 1, a multifunctional protein that also plays an important role in the autophagy pathway, affects some aspects of Abeta pathology in aged but not young transgenic mice expressing amyloid precursor protein (APP) (see the related article beginning on page 2190). These findings further support the notion that modulation of autophagy, in this case through beclin 1, may represent a novel therapeutic strategy for AD.

    The Journal of clinical investigation 2008;118;6;2015-8

  • Modulation of synaptic plasticity and Tau phosphorylation by wild-type and mutant presenilin1.

    Dewachter I, Ris L, Croes S, Borghgraef P, Devijver H, Voets T, Nilius B, Godaux E and Van Leuven F

    Experimental Genetics Group, K.U.Leuven, 3000 Leuven, Belgium.

    The function of presenilin1 (PS1) in intra-membrane proteolysis is undisputed, as is its role in neurodegeneration in FAD, in contrast to its exact function in normal conditions. In this study, we analyzed synaptic plasticity and its underlying mechanisms biochemically in brain of mice with a neuron-specific deficiency in PS1 (PS1(n-/-)) and compared them to mice that expressed human mutant PS1[A246E] or wild-type PS1. PS1(n-/-) mice displayed a subtle impairment in Schaffer collateral hippocampal long-term potentiation (LTP) as opposed to normal LTP in wild-type PS1 mice, and a facilitated LTP in mutant PS1[A246E] mice. This finding correlated with, respectively, increased and reduced NMDA receptor responses in PS1[A246E] mice and PS1(n-/-) mice in hippocampal slices. Postsynaptically, levels of NR1/NR2B NMDA-receptor subunits and activated alpha-CaMKII were reduced in PS1(n-/-) mice, while increased in PS1[A246E] mice. In addition, PS1(n-/-) mice, displayed reduced paired pulse facilitation, increased synaptic fatigue and lower number of total and docked synaptic vesicles, implying a presynaptic function for wild-type presenilin1, unaffected by the mutation in PS1[A246E] mice. In contrast to the deficiency in PS1, mutant PS1 activated GSK-3beta by decreasing phosphorylation on Ser-9, which correlated with increased phosphorylation of protein tau at Ser-396-Ser-404 (PHF1/AD2 epitope). The synaptic functions of PS1, exerted on presynaptic vesicles and on postsynaptic NMDA-receptor activity, were concluded to be independent of alterations in GSK-3beta activity and phosphorylation of protein tau.

    Neurobiology of aging 2008;29;5;639-52

  • Adaptor protein sorting nexin 17 regulates amyloid precursor protein trafficking and processing in the early endosomes.

    Lee J, Retamal C, Cuitiño L, Caruano-Yzermans A, Shin JE, van Kerkhof P, Marzolo MP and Bu G

    Department of Pediatrics and Cell Biology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.

    Accumulation of extracellular amyloid beta peptide (Abeta), generated from amyloid precursor protein (APP) processing by beta- and gamma-secretases, is toxic to neurons and is central to the pathogenesis of Alzheimer disease. Production of Abeta from APP is greatly affected by the subcellular localization and trafficking of APP. Here we have identified a novel intracellular adaptor protein, sorting nexin 17 (SNX17), that binds specifically to the APP cytoplasmic domain via the YXNPXY motif that has been shown previously to bind several cell surface adaptors, including Fe65 and X11. Overexpression of a dominant-negative mutant of SNX17 and RNA interference knockdown of endogenous SNX17 expression both reduced steady-state levels of APP with a concomitant increase in Abeta production. RNA interference knockdown of SNX17 also decreased APP half-life, which led to the decreased steady-state levels of APP. Immunofluorescence staining confirmed a colocalization of SNX17 and APP in the early endosomes. We also showed that a cell surface adaptor protein, Dab2, binds to the same YXNPXY motif and regulates APP endocytosis at the cell surface. Our results thus provide strong evidence that both cell surface and intracellular adaptor proteins regulate APP endocytic trafficking and processing to Abeta. The identification of SNX17 as a novel APP intracellular adaptor protein highly expressed in neurons should facilitate the understanding of the relationship between APP intracellular trafficking and processing to Abeta.

    Funded by: FIC NIH HHS: TW006456; NIA NIH HHS: R01 AG027924

    The Journal of biological chemistry 2008;283;17;11501-8

  • A neuronal model of Alzheimer's disease: an insight into the mechanisms of oxidative stress-mediated mitochondrial injury.

    Sompol P, Ittarat W, Tangpong J, Chen Y, Doubinskaia I, Batinic-Haberle I, Abdul HM, Butterfield DA and St Clair DK

    Graduate Center for Toxicology, University of Kentucky, Lexington, KY 40536, USA.

    Alzheimer's disease (AD) is associated with beta-amyloid accumulation, oxidative stress and mitochondrial dysfunction. However, the effects of genetic mutation of AD on oxidative status and mitochondrial manganese superoxide dismutase (MnSOD) production during neuronal development are unclear. To investigate the consequences of genetic mutation of AD on oxidative damages and production of MnSOD during neuronal development, we used primary neurons from new born wild-type (WT/WT) and amyloid precursor protein (APP) (NLh/NLh) and presenilin 1 (PS1) (P264L) knock-in mice (APP/PS1) which incorporated humanized mutations in the genome. Increasing levels of oxidative damages, including protein carbonyl, 4-hydroxynonenal (4-HNE) and 3-nitrotyrosine (3-NT), were accompanied by a reduction in mitochondrial membrane potential in both developing and mature APP/PS1 neurons compared with WT/WT neurons suggesting mitochondrial dysfunction under oxidative stress. Interestingly, developing APP/PS1 neurons were significantly more resistant to beta-amyloid 1-42 treatment, whereas mature APP/PS1 neurons were more vulnerable than WT/WT neurons of the same age. Consistent with the protective function of MnSOD, developing APP/PS1 neurons have increased MnSOD protein and activity, indicating an adaptive response to oxidative stress in developing neurons. In contrast, mature APP/PS1 neurons exhibited lower MnSOD levels compared with mature WT/WT neurons indicating that mature APP/PS1 neurons lost the adaptive response. Moreover, mature APP/PS1 neurons had more co-localization of MnSOD with nitrotyrosine indicating a greater inhibition of MnSOD by nitrotyrosine. Overexpression of MnSOD or addition of MnTE-2-PyP(5+) (SOD mimetic) protected against beta-amyloid-induced neuronal death and improved mitochondrial respiratory function. Together, the results demonstrate that compensatory induction of MnSOD in response to an early increase in oxidative stress protects developing neurons against beta-amyloid toxicity. However, continuing development of neurons under oxidative damage conditions may suppress the expression of MnSOD and enhance cell death in mature neurons.

    Funded by: NCI NIH HHS: R01 CA 49797, R01 CA049797, R01 CA049797-17; NIA NIH HHS: P01 AG005119, P01 AG005119-150007, P01AG 05119

    Neuroscience 2008;153;1;120-30

  • NFkappaB-dependent control of BACE1 promoter transactivation by Abeta42.

    Buggia-Prevot V, Sevalle J, Rossner S and Checler F

    Institut de Pharmacologie Moléculaire et Cellulaire, UMR 6097 CNRS/UNSA, Equipe Labellisée Fondation pour la Recherche Médicale, 06560 Valbonne, France.

    Beta-amyloid (Abeta) peptides that accumulate in Alzheimer disease are generated from the beta-amyloid precursor protein (betaAPP) by cleavages by beta-secretase BACE1 and by presenilin-dependent gamma-secretase activities. Very few data document a putative cross-talk between these proteases and the regulatory mechanisms underlying such interaction. We show that presenilin deficiency lowers BACE1 maturation and affects both BACE1 activity and promoter transactivation. The specific gamma-secretase inhibitor DFK167 triggers the decrease of BACE1 activity in wild-type but not in presenilin-deficient fibroblasts. This decrease is also elicited by catalytically inactive gamma-secretase. The overexpression of APP intracellular domain (AICD), the gamma/epsilon-secretase-derived C-terminal product of beta-amyloid precursor protein, does not modulate BACE1 activity or promoter transactivation in fibroblasts and does not alter BACE1 expression in AICD transgenic brains of mice. A DFK167-sensitive increase of BACE1 activity is observed in cells overexpressing APPepsilon (the N-terminal product of betaAPP generated by epsilon-secretase cleavage harboring the Abeta domain but lacking the AICD sequence), suggesting that the production of Abeta could account for the modulation of BACE1. Accordingly, we show that HEK293 cells overexpressing wild-type betaAPP exhibit a DFK167-sensitive increase in BACE1 promoter transactivation that is increased by the Abeta-potentiating Swedish mutation. This effect was mimicked by exogenous application of Abeta42 but not Abeta40 or by transient transfection of cDNA encoding Abeta42 sequence. The IkappaB kinase inhibitor BMS345541 prevents Abeta-induced BACE1 promoter transactivation suggesting that NFkappaB could mediate this Abeta-associated phenotype. Accordingly, the overexpression of wild-type or Swedish mutated betaAPP does not modify the transactivation of BACE1 promoter constructs lacking NFkappaB-responsive element. Furthermore, APP/beta-amyloid precursor protein-like protein deficiency does not affect BACE1 activity and expression. Overall, these data suggest that physiological levels of endogenous Abeta are not sufficient per se to modulate BACE1 promoter transactivation but that exacerbated Abeta production linked to wild-type or Swedish mutated betaAPP overexpression modulates BACE1 promoter transactivation and activity via an NFkappaB-dependent pathway.

    The Journal of biological chemistry 2008;283;15;10037-47

  • Aluminium exposure induces Alzheimer's disease-like histopathological alterations in mouse brain.

    Rodella LF, Ricci F, Borsani E, Stacchiotti A, Foglio E, Favero G, Rezzani R, Mariani C and Bianchi R

    Unit of Human Anatomy, Department of Biomedical Sciences and Biotechnology, University of Brescia, Brescia, Italy. rodella@med.unibs.it

    Aluminium (Al) is a neurotoxic metal and Al exposure may be a factor in the aetiology of various neurodegenerative diseases such as Alzheimer's disease (AD). The major pathohistological findings in the AD brain are the presence of neuritic plaques containing beta-amyloid (Abeta) which may interfere with neuronal communication. Moreover, it has been observed that GRP78, a stress-response protein induced by conditions that adversely affect endoplasmic reticulum (ER) function, is reduced in the brain of AD patients. In this study, we investigated the correlation between the expression of Abeta and GRP78 in the brain cortex of mice chronically treated with aluminium sulphate. Chronic exposure over 12 months to aluminium sulphate in drinking water resulted in deposition of Abeta similar to that seen in congophilic amyloid angiopathy (CAA) in humans and a reduction in neuronal expression of GRP78 similar to what has previously been observed in Alzheimer's disease. So, we hypothesise that chronic Al administration is responsible for oxidative cell damage that interferes with ER functions inducing Abeta accumulation and neurodegenerative damage.

    Histology and histopathology 2008;23;4;433-9

  • Amyloid precursor protein cytoplasmic domain antagonizes reelin neurite outgrowth inhibition of hippocampal neurons.

    Hoareau C, Borrell V, Soriano E, Krebs MO, Prochiantz A and Allinquant B

    INSERM U796, Centre Paul Broca, 2 ter rue d'Alésia, 75014 Paris, France.

    The function of the amyloid precursor protein (APP), a key molecule in Alzheimer's disease (AD) remains unknown. Among the proteins that interact with the APP cytoplasmic domain in vitro and in heterologous systems is Disabled-1, a signaling molecule of the reelin pathway. The physiological consequence of this interaction is unknown. Here we used an in vitro model of hippocampal neurons grown on a reelin substrate that inhibits neurite outgrowth. Our results show that an excess of APP cytoplasmic domain internalized by a cell permeable peptide, is able to antagonize the neurite outgrowth inhibition of reelin. The APP cytoplasmic domain binds Disabled-1 and retains it in the cytoplasm, preventing it from reaching the plasma membrane and sequesters tyrosine phosphorylated Disabled-1, both of which disrupt reelin signaling. In the context of AD, increased formation of APP cytoplasmic domain in the cytosol released after cleavage of the A beta peptide, could then inhibit reelin signaling pathway in the hippocampus and thus influence synaptic plasticity.

    Neurobiology of aging 2008;29;4;542-53

  • Acquisition of conditioned taste aversion is impaired in the amyloid precursor protein/presenilin 1 mouse model of Alzheimer's disease.

    Pistell PJ, Zhu M and Ingram DK

    Behavioral Neuroscience Section, Laboratory of Experimental Gerontology, National Institute on Aging, National Institutes of Health, Gerontology Research Center, 5600 Nathan Shock Drive, Baltimore, MD 21224, USA. Paul.Pistell@pbrc.edu

    Research into the underlying mechanisms of cognitive dysfunction in Alzheimer's disease (AD) has relied traditionally on tasks such as the water maze which evaluate spatial learning and memory. Since non-spatial forms of memory are also disrupted by AD, it is critical to establish other paradigms capable of investigating these deficits. Utilizing a non-spatial learning task, acquisition of conditioned taste aversion (CTA) was evaluated in a mouse model of AD. This line of transgenic mice encode a mutated allele of the human amyloid precursor protein (APP) and presenilin 1 (PS1) genes and exhibit extensive amyloid plaque deposition in the brain by 6-7 mo of age. Compared with wild-type mice, 10-17 month old APP/PS1 mice failed to acquire CTA to saccharin. Mice that only possessed one of the two mutations were able to acquire CTA to the saccharin. In 2-5 month old APP/PS1 mice acquisition of CTA was disrupted despite the lack of extensive plaque deposition. However, further analysis indicated a potential gender difference in both the CTA deficit and onset of plaque deposition with females showing greater conditioned aversion.

    Funded by: Intramural NIH HHS; NCRR NIH HHS: 1P20RR02-1945, P20 RR021945, P20 RR021945-01; NIDDK NIH HHS: 1P30 DK072476, P30 DK072476, P30 DK072476-01

    Neuroscience 2008;152;3;594-600

  • Fyn modulation of Dab1 effects on amyloid precursor protein and ApoE receptor 2 processing.

    Hoe HS, Minami SS, Makarova A, Lee J, Hyman BT, Matsuoka Y and Rebeck GW

    Department of Neuroscience, Georgetown University Medical Center, 3970 Reservoir Road NW, Washington, DC 20057, USA.

    Dab1 is an intracellular adaptor protein that interacts with amyloid precursor protein (APP) and apoE receptor 2 (apoEr2), increases their levels on the cell surface, and increases their cleavage by alpha-secretases. To investigate the mechanism underlying these alterations in processing and trafficking of APP and apoEr2, we examined the effect of Fyn, an Src family-tyrosine kinase known to interact with and phosphorylate Dab1. Co-immunoprecipitation, co-immunostaining, and fluorescence lifetime imaging demonstrated an association between Fyn and APP. Fyn induced phosphorylation of APP at Tyr-757 of the (757)YENPTY(762) motif and increased cell surface expression of APP. Overexpression of Fyn alone did not alter levels of sAPPalpha or cytoplasmic C-terminal fragments, although it significantly decreased production of Abeta. However, in the presence of Dab1, Fyn significantly increased sAPPalpha and C-terminal fragments. Fyn-induced APP phosphorylation and cell surface levels of APP were potentiated in the presence of Dab1. Fyn also induced phosphorylation of apoEr2 and increased its cell surface levels and, in the presence of Dab1, affected processing of its C-terminal fragment. In vivo studies showed that sAPPalpha was decreased in the Fyn knock-out, supporting a role for Fyn in APP processing. These data demonstrate that Fyn, due in part to its effects on Dab1, regulates the phosphorylation, trafficking, and processing of APP and apoEr2.

    Funded by: NIA NIH HHS: AG 022455, AG 14473, P01 AG030128

    The Journal of biological chemistry 2008;283;10;6288-99

  • A TAG1-APP signalling pathway through Fe65 negatively modulates neurogenesis.

    Ma QH, Futagawa T, Yang WL, Jiang XD, Zeng L, Takeda Y, Xu RX, Bagnard D, Schachner M, Furley AJ, Karagogeos D, Watanabe K, Dawe GS and Xiao ZC

    Institute of Molecular and Cell Biology, 61 Biopolis Drive, Proteos, Singapore 138673.

    The release of amyloid precursor protein (APP) intracellular domain (AICD) may be triggered by extracellular cues through gamma-secretase-dependent cleavage. AICD binds to Fe65, which may have a role in AICD-dependent signalling; however, the functional ligand has not been characterized. In this study, we have identified TAG1 as a functional ligand of APP. We found that, through an extracellular interaction with APP, TAG1 increased AICD release and triggered Fe65-dependent activity in a gamma-secretase-dependent manner. TAG1, APP and Fe65 colocalized in the neural stem cell niche of the fetal ventricular zone. Neural precursor cells from TAG1-/-, APP-/- and TAG1-/-;APP-/- mice had aberrantly enhanced neurogenesis, which was significantly reversed in TAG1-/- mice by TAG1 or AICD but not by AICD mutated at the Fe65 binding site. Notably, TAG1 reduced normal neurogenesis in Fe65+/+ mice. Abnormally enhanced neurogenesis also occurred in Fe65-/- mice but could not be reversed by TAG1. These results describe a TAG1-APP signalling pathway that negatively modulates neurogenesis through Fe65.

    Nature cell biology 2008;10;3;283-94

  • Cognitive evaluation of disease-modifying efficacy of donepezil in the APP23 mouse model for Alzheimer's disease.

    Van Dam D, Coen K and De Deyn PP

    Department of Biomedical Sciences, Laboratory of Neurochemistry and Behaviour, Institute Born-Bunge, University of Antwerp, Universiteitsplein 1, 2610, Wilrijk, Belgium.

    Rationale: The interest for acetylcholinesterase inhibitors in the treatment of Alzheimer's disease has been greatly renewed owing to the discovery of a broad range of additional cholinergic and non-cholinergic effects, exploitable to maximize the efficacy of these drugs beyond merely improving intellectual functions at the symptomatic level.

    Objectives: The age-dependent cognitive decline in the valid APP23 transgenic mouse model for Alzheimer's disease was employed to evaluate disease-modifying efficacy of chronic treatment with donepezil.

    At age 6 weeks, heterozygous APP23 mice and control littermates were subcutaneously implanted with osmotic pumps delivering saline or donepezil (0.27 or 0.58 mg/kg per day). After 2 months of treatment, a 3-week wash-out period was allowed to prevent bias from sustained symptomatic effects before cognitive evaluation in the Morris water maze commenced.

    Results: Donepezil (0.27 mg/kg per day)-treated APP23 mice performed significantly better than their sham-treated counterparts during the Morris water maze acquisition phase and the subsequent probe or retention trial. Chronic donepezil (0.27 mg/kg per day) treatment improved spatial accuracy in APP23 mice as to reach the same level of performance as wild-type control animals on this complex visual-spatial learning task.

    Conclusion: This is the first study reporting disease-modifying efficacy of donepezil at the level of cognitive performance in transgenic mice modeling Alzheimer's disease.

    Psychopharmacology 2008;197;1;37-43

  • Interaction of amyloid precursor protein with contactins and NgCAM in the retinotectal system.

    Osterfield M, Egelund R, Young LM and Flanagan JG

    Department of Cell Biology and Program in Neuroscience, Harvard Medical School, Boston, MA 02115, USA.

    The amyloid precursor protein (APP) plays a central role in Alzheimer's disease, but its actions in normal development are not well understood. Here, a tagged APP ectodomain was used to identify extracellular binding partners in developing chick brain. Prominent binding sites were seen in the olfactory bulb and on retinal axons growing into the optic tectum. Co-precipitation from these tissues and tandem mass spectrometry led to the identification of two associated proteins: contactin 4 and NgCAM. In vitro binding studies revealed direct interactions among multiple members of the APP and contactin protein families. Levels of the APP processing fragment, CTFalpha, were modulated by both contactin 4 and NgCAM. In the developing retinotectal system, APP, contactin 4 and NgCAM are expressed in the retina and tectum in suitable locations to interact. Functional assays revealed regulatory effects of both APP and contactin 4 on NgCAM-dependent growth of cultured retinal axons, demonstrating specific functional interactions among these proteins. These studies identify novel binding and functional interactions among proteins of the APP, contactin and L1CAM families, with general implications for mechanisms of APP action in neural development and disease.

    Funded by: NEI NIH HHS: EY11559; NICHD NIH HHS: HD29417

    Development (Cambridge, England) 2008;135;6;1189-99

  • Molecular dynamics study to investigate the effect of chemical substitutions of methionine 35 on the secondary structure of the amyloid beta (Abeta(1-42)) monomer in aqueous solution.

    Triguero L, Singh R and Prabhakar R

    Department of Chemistry, University of Miami, 1301 Memorial Drive, Coral Gables, Florida 33146, USA.

    In this study, all-atom molecular dynamics simulations in the explicit water solvent are performed to investigate conformational changes in the secondary structure of the Abeta(1-42) monomer associated with the substitution of the Cgamma-methylene position of the Met35 amino acid residue by sulfoxide (Met35(O)), sulfone (Met35(O2)), and norleucine (Met35(CH2)). The effects of these substitutions on the structural changes that occur in three distinct regions (the central hydrophobic core (CHC) region 17-21 (LVFFA), stable turn segment 24-27 (VGSN), and second hydrophobic region 29-35 (GAIIGLM)) of all monomers have been analyzed in detail, and results are compared with experiments. Our 20 ns simulations indicate that the most significant changes take place in the second hydrophobic region of the Met35(O) and Met35(O2) monomers. However, for the Met35(CH2) monomer, this region does not exhibit significant deviations. In comparison to the wild-type (WT)-Abeta(1-42) monomer, for Met35(O) the second hydrophobic region is characterized by the formation of internal beta-sheets separated by stable turns, whereas for Met35(O2) it exhibits a more helical conformation. These substantial changes in the secondary structure can be explained in terms of an increase in the computed dipole moment and solvent accessible surface area (SASA) per residue of these substituents. These structural modifications can affect interaction between monomers, which in turn may influence the oligomerization process involved in Alzheimer's disease (AD).

    The journal of physical chemistry. B 2008;112;7;2159-67

  • Development of homogeneous 384-well high-throughput screening assays for Abeta1-40 and Abeta1-42 using AlphaScreen technology.

    Szekeres PG, Leong K, Day TA, Kingston AE and Karran EH

    Eli Lilly and Co. Ltd, Lilly Research Centre, Windlesham, Surrey, UK. szekeresp@lilly.com

    Amyloid beta (Abeta) peptides are the major constituent of amyloid plaques, one of the hallmark pathologies of Alzheimer's disease. Accurate and precise quantitation of these peptides in biological fluids is a critical component of Alzheimer's disease research. The current most established assay for analysis of Abeta peptides in preclinical research is enzyme-linked immunosorbent assay (ELISA), which, although sensitive and of proven utility, is a multistep, labor-intensive assay that is difficult to automate completely. To overcome these limitations, the authors have developed and optimized simple, sensitive, homogeneous 384-well assays for Abeta1-42 and Abeta1-40 using AlphaScreen technology. The assays are capable of detecting Abeta peptides at concentrations <2 pg/mL and, using a final assay volume of 20 microL, routinely generate Z' values >0.85. The AlphaScreen format has the following key advantages: substantially less hands-on time to run, easier to automate, higher throughput, and less expensive to run than the traditional ELISA. The results presented here show that AlphaScreen technology permits robust, efficient, and cost-effective quantitation of Abeta peptides.

    Journal of biomolecular screening 2008;13;2;101-11

  • Intraneuronal amyloid beta and reduced brain volume in a novel APP T714I mouse model for Alzheimer's disease.

    Van Broeck B, Vanhoutte G, Pirici D, Van Dam D, Wils H, Cuijt I, Vennekens K, Zabielski M, Michalik A, Theuns J, De Deyn PP, Van der Linden A, Van Broeckhoven C and Kumar-Singh S

    Neurodegenerative Brain Diseases Group, Department of Molecular Genetics, VIB, Universiteitsplein 1, BE-2610 Antwerpen, Belgium.

    Transgenic mouse models of Alzheimer's disease (AD) expressing high levels of amyloid precursor protein (APP) with familial AD (FAD) mutations have proven to be extremely useful in understanding pathogenic processes of AD especially those that involve amyloidogenesis. We earlier described Austrian APP T714I pathology that leads to one of the earliest AD age-at-onsets with abundant intracellular and extracellular amyloid deposits in brain. The latter strikingly was non-fibrillar diffuse amyloid, composed of N-truncated A beta 42 in absence of A beta 40. In vitro, this mutation leads to one of the highest A beta 42/A beta 40 ratios among all FAD mutations. We generated an APP T714I transgenic mouse model that despite having 10 times lower transgene than endogenous murine APP deposited intraneuronal A beta in brain by 6 months of age. Accumulations increased with age, and this was paralleled by decreased brain sizes on volumetric MRI, compared to age-matched and similar transgene-expressing APP wild-type mice, although, with these levels of transgenic expression we did not detect neuronal loss or significant memory impairment. Immunohistochemical studies revealed that the majority of the intraneuronal A beta deposits colocalized with late endosomal markers, although some A beta inclusions were also positive for lysosomal and Golgi markers. These data support earlier observations of A beta accumulation in the endosomal-lysosomal pathway and the hypothesis that intraneuronal accumulation of A beta could be an important factor in the AD pathogenesis.

    Neurobiology of aging 2008;29;2;241-52

  • Amyloid precursor protein knockout mice show age-dependent deficits in passive avoidance learning.

    Senechal Y, Kelly PH and Dev KK

    Neuroscience Research, Novartis Institutes for BioMedical Research, Novartis Pharma AG, Lichtstrasse 35, CH-4056 Basel, Switzerland.

    Amyloid precursor protein (APP) is involved in the pathogenesis of Alzheimer's disease (AD), but its role in cognition has been relatively little studied. APP knockout (KO) animals have been described previously and show deficits in grip strength, reduced locomotor activity and impaired learning and memory in a conditioned avoidance test and the Morris water-maze. In order to further investigate the in vivo function of APP and its proteolytic derivatives, we tested APP KO mice and age-matched wild type controls at two different ages, 3 and 8 months, in a range of behavioural tests measuring neuromuscular, locomotor and cognitive functions. These tests included the acquisition of a passive avoidance response as a measure of long-term memory of an aversive experience, and spontaneous alternation in a Y-maze, regarded as a measure of spatial short-term memory. The absence of APP expression in APP KO mice was confirmed at the protein level using hippocampal tissue in Western blotting. APP KO mice displayed deficits in forelimb grip strength and locomotor activity in agreement with previous studies. In the Y-maze test used for spontaneous alternation behaviour, APP KO animals did not exhibit reduced alternation rates. On the other hand, in the passive avoidance test, APP KO mice showed an age-related deficit in retention of memory for an aversive experience. The results suggest that APP and/or its proteolytic derivatives may play a role in long-term memory in adult brain and/or may be required during the development and maintenance of neuronal networks involved in this type of memory.

    Behavioural brain research 2008;186;1;126-32

  • A genetic interaction between the APP and Dab1 genes influences brain development.

    Pramatarova A, Chen K and Howell BW

    Neurogenetics Branch, National Institute of Neurological Disorders and Stroke, NIH, 35 Convent Dr., Bethesda, MD 20892, USA.

    The Dab1 docking protein is required for the proper organization of brain laminae and for a signal transduction pathway initiated by Reelin binding to the ApoER2 and VLDLR receptors on the cell surface of neurons. Dab1 physically interacts with APP; however, it is not known whether the APP gene influences Dab1 function. Here we demonstrate a genetic interaction between Dab1 and APP. Dab1-hypomorphic animals have neuronal ectopias in the neocortex and reduced cerebellar volume, possibly a consequence of Purkinje cell misplacement. These phenotypes are exacerbated in transgenic animals overexpressing a mutant form of APP, APP(swe), which is characterized by increased processing at the beta-secretase site. The Dab1-hypomorphic phenotype is improved in the cerebellum of animals that are deficient for APP. Together this suggests that APP expression constrains the consequences of Dab1 activity during brain development.

    Funded by: Intramural NIH HHS: Z01 NS002987-08

    Molecular and cellular neurosciences 2008;37;1;178-86

  • The in vivo brain interactome of the amyloid precursor protein.

    Bai Y, Markham K, Chen F, Weerasekera R, Watts J, Horne P, Wakutani Y, Bagshaw R, Mathews PM, Fraser PE, Westaway D, St George-Hyslop P and Schmitt-Ulms G

    Centre for Research in Neurodegenerative Diseases and Departments of Laboratory Medicine and Pathobiology, University of Toronto, Ontario, Canada.

    Despite intense research efforts, the physiological function and molecular environment of the amyloid precursor protein has remained enigmatic. Here we describe the application of time-controlled transcardiac perfusion cross-linking, a method for the in vivo mapping of protein interactions in intact tissue, to study the interactome of the amyloid precursor protein (APP). To gain insights into the specificity of reported protein interactions the study was extended to the mammalian amyloid precursor-like proteins (APLP1 and APLP2). To rule out sampling bias as an explanation for differences in the individual datasets, a small scale quantitative iTRAQ (isobaric tags for relative and absolute quantitation)-based comparison of APP, APLP1, and APLP2 interactomes was carried out. An interactome map was derived that confirmed eight previously reported interactions of APP and revealed the identity of more than 30 additional proteins that reside in spatial proximity to APP in the brain. Subsequent validation studies confirmed a physiological interaction between APP and leucine-rich repeat and Ig domain-containing protein 1, demonstrated a strong influence of Ig domain-containing protein 1 on the proteolytic processing of APP, and consolidated similarities in the biology of APP and p75.

    Funded by: NIGMS NIH HHS: GM61898; Wellcome Trust: 081864

    Molecular & cellular proteomics : MCP 2008;7;1;15-34

  • Abeta deposition and related pathology in an APP x PS1 transgenic mouse model of Alzheimer's disease.

    Howlett DR, Bowler K, Soden PE, Riddell D, Davis JB, Richardson JC, Burbidge SA, Gonzalez MI, Irving EA, Lawman A, Miglio G, Dawson EL, Howlett ER and Hussain I

    Neurology & GI CEDD, GlaxoSmithKline, Harlow, Essex, UK. david.r.howlett@gsk.com

    A transgenic mouse bearing mutant transgenes linked to familial forms of Alzheimer's disease (AD) for the amyloid precursor protein and presenilin-1 (TASTPM) showed Abeta plaque deposition and age-related histological changes in associated brain pathology. The Abeta present was of multiple forms, including species with a C-terminus at position 40 or 42, as well as an N-terminus at position 1 or truncated in a pyro-3-glutamate form. Endogenous rodent Abeta was also present in the deposits. Laser capture microdissection extracts showed that multimeric forms of Abeta were present in both plaque and tissue surrounding plaques. Associated with the Abeta deposits was evidence of an inflammatory response characterised by the presence of astrocytes. Also present in close association with the deposits was phosphorylated tau and cathepsin D immunolabelling. The incidence of astrocytes and of phosphorylated tau and cathepsin D load showed that both of these potential disease markers increased in parallel to the age of the mice and with Abeta deposition. Immunohistochemical labelling of neurons in the cortex and hippocampus of TASTPM mice suggested that the areas of Abeta deposition were associated with the loss of neurons. TASTPM mice, therefore, exhibit a number of the pathological characteristics of disease progression in AD and may provide a means for assessment of novel therapeutic agents directed towards modifying or halting disease progression.

    Histology and histopathology 2008;23;1;67-76

  • Effects of progesterone administration and APPswe+PSEN1Deltae9 mutation for cognitive performance of mid-aged mice.

    Frye CA and Walf AA

    Department of Psychology, The University at Albany-SUNY, Life Sciences Research Building 01058, 1400 Washington Avenue, Albany, NY 12222, USA. cafrye@albany.edu

    Progesterone (P(4)) and its metabolite, 5alpha-pregnan-3alpha-ol-20-one (3alpha,5alpha-THP) have trophic effects and may improve cognitive function. We investigated the role of progestins in a murine model of Alzheimer's Disease (AD) in which transgenic mice co-overexpress a mutant form of amyloid precursor protein (APPswe) and a deletion in presenilin 1 Delta exon 9 (APPswe+PSEN1Deltae9). We hypothesized that: (1) mice with the APPswe+PSEN1Deltae9 mutation would have performance deficits compared to wildtype mice and (2) long-term administration of P(4) would enhance cognitive performance and increase brain progestin levels over placebo. Mice were ovariectomized at 6 months of age and administered placebo or P(4) via subcutaneously implanted pellets. Mice were tested between 9 and 12 months of age for cognitive performance in the object placement, water maze, object recognition, and T-maze tasks and for motor behavior in an activity monitor and then tissues were collected for steroid measurement. P(4) administration increased progestin levels in cortex, diencephalon, midbrain, and cerebellum of wildtype and mutant mice, but increases in 3alpha,5alpha-THP levels in the hippocampus of APPswe+PSEN1Deltae9 mutant mice were attenuated compared to that observed in wildtype mice. APPswe+PSEN1Deltae9 mice showed poorer performance in hippocampus measures (object placement and water maze tasks). In the object recognition and T-maze task, which are mediated by the cortex and hippocampus, P(4) administration improved performance in both wildtype and APPswe+PSEN1Deltae9 mutant mice compared to placebo administration. Thus, APPswe+PSEN1Delta9 mice have deficits in hippocampal performance and capacity to form 3alpha,5alpha-THP in the hippocampus and both wildtype and APPswe+PSEN1Delta9 mice show beneficial effects of P(4) in cortical function and similar capacity to form 3alpha,5alpha-THP in the cortex.

    Neurobiology of learning and memory 2008;89;1;17-26

  • A critical function for beta-amyloid precursor protein in neuronal migration revealed by in utero RNA interference.

    Young-Pearse TL, Bai J, Chang R, Zheng JB, LoTurco JJ and Selkoe DJ

    Center for Neurologic Diseases, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115, USA.

    Physiological processing of the beta-amyloid precursor protein (APP) generates amyloid beta-protein, which can assemble into oligomers that mediate synaptic failure in Alzheimer's disease. Two decades of research have led to human trials of compounds that chronically target this processing, and yet the normal function of APP in vivo remains unclear. We used the method of in utero electroporation of shRNA constructs into the developing cortex to acutely knock down APP in rodents. This approach revealed that neuronal precursor cells in embryonic cortex require APP to migrate correctly into the nascent cortical plate. cDNAs encoding human APP or its homologues, amyloid precursor-like protein 1 (APLP1) or APLP2, fully rescued the shRNA-mediated migration defect. Analysis of an array of mutations and deletions in APP revealed that both the extracellular and cytoplasmic domains of APP are required for efficient rescue. Whereas knock-down of APP inhibited cortical plate entry, overexpression of APP caused accelerated migration of cells past the cortical plate boundary, confirming that normal APP levels are required for correct neuronal migration. In addition, we found that Disabled-1 (Dab1), an adaptor protein with a well established role in cortical cell migration, acts downstream of APP for this function in cortical plate entry. We conclude that full-length APP functions as an important factor for proper migration of neuronal precursors into the cortical plate during the development of the mammalian brain.

    Funded by: NIA NIH HHS: R0I AG06173; NINDS NIH HHS: F32 NS053320-01A1

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2007;27;52;14459-69

  • Chemical and morphological alterations of spines within the hippocampus and entorhinal cortex precede the onset of Alzheimer's disease pathology in double knock-in mice.

    Aoki C, Mahadomrongkul V, Fujisawa S, Habersat R and Shirao T

    Center for Neural Science, New York University, New York, NY 10003, USA. chiye@cns.nyu.edu

    Mice with knock-in of two mutations that affect beta amyloid processing and levels (2xKI) exhibit impaired spatial memory by 9-12 months of age, together with synaptic plasticity dysfunction in the hippocampus. The goal of this study was to identify changes in the molecular and structural characteristics of synapses that precede and thus could exert constraints upon cellular mechanisms underlying synaptic plasticity. Drebrin A is one protein reported to modulate spine sizes and trafficking of proteins to and from excitatory synapses. Thus, we examined levels of drebrin A within postsynaptic spines in the hippocampus and entorhinal cortex. Our electron microscopic immunocytochemical analyses reveal that, by 6 months, the proportion of hippocampal spines containing drebrin A is reduced and this change is accompanied by an increase in the mean size of spines and decreased density of spines. In the entorhinal cortex of 2xKI brains, we detected no decrement in the proportion of spines labeled for drebrin A and no significant change in spine density at 6 months, but rather a highly significant reduction in the level of drebrin A immunoreactivity within each spine. These changes are unlike those observed for the somatosensory cortex of 2xKI mice, in which synapse density and drebrin A immunoreactivity levels remain unchanged at 6 months and older. These results indicate that brains of 2xKI mice, like those of humans, exhibit regional differences of vulnerability, with the hippocampus exhibiting the first signatures of structural changes that, in turn, may underlie the emergent inability to update spatial memory in later months.

    Funded by: NEI NIH HHS: 1P30 EY 13079, P30 EY013079, P30 EY013079-109003, R01 EY 13145, R01 EY013145, R01 EY013145-04; NINDS NIH HHS: R01 NS 41091, R01 NS041091, R01 NS041091-04

    The Journal of comparative neurology 2007;505;4;352-62

  • Increased asynchronous release and aberrant calcium channel activation in amyloid precursor protein deficient neuromuscular synapses.

    Yang L, Wang B, Long C, Wu G and Zheng H

    Huffington Center on Aging, Baylor College of Medicine, One Baylor Plaza, MS230, Houston, TX 77030, USA.

    Despite the critical roles of the amyloid precursor protein (APP) in Alzheimer's disease pathogenesis, its physiological function remains poorly established. Our previous studies implicated a structural and functional activity of the APP family of proteins in the developing neuromuscular junction (NMJ). Here we performed comprehensive analyses of neurotransmission in mature neuromuscular synapse of APP deficient mice. We found that APP deletion led to reduced paired-pulse facilitation and increased depression of synaptic transmission with repetitive stimulation. Readily releasable pool size and total releasable vesicles were not affected, but probability of release was significantly increased. Strikingly, the amount of asynchronous release, a measure sensitive to presynaptic calcium concentration, was dramatically increased, and pharmacological studies revealed that it was attributed to aberrant activation of N- and L-type Ca(2+) channels. We propose that APP modulates synaptic transmission at the NMJ by ensuring proper Ca(2+) channel function.

    Funded by: NIA NIH HHS: AG20670, R01 AG020670-05, R01 AG032051-05, R01 AG033467, R01 AG033467-04; NINDS NIH HHS: NS40039, R01 NS040039-07, R01 NS040039-08

    Neuroscience 2007;149;4;768-78

  • Amyloid beta deposition is related to decreased glucose transporter-1 levels and hippocampal atrophy in brains of aged APP/PS1 mice.

    Hooijmans CR, Graven C, Dederen PJ, Tanila H, van Groen T and Kiliaan AJ

    Radboud University Nijmegen Medical Centre, Department of Anatomy, Geert Grooteplein noord 21 6500 HB Nijmegen, The Netherlands.

    Unlabelled: The amount of the glucose transporter type-1 (GLUT-1) is decreased in the hippocampus and cerebral cortex of AD patients. In this study we therefore wanted to investigate the causal relationship between beta-amyloid (Abeta), GLUT-1 and hippocampal atrophy in the brains of young (8 months) and old (18 months) APP/PS1 mice.

    Methods: Abeta and GLUT-1 were visualized immunohistochemically. Abeta load, GLUT-1 amount, capillary density and GLUT-1 amount per capillary density were determined in cortical and hippocampal areas using computer-assisted analysis systems. Hippocampal atrophy was determined by calculating the width of the outer molecular layer of the dentate gyrus (DG).

    Results: In 18-month-old APP/PS1 mice we found a reduced GLUT-1 amount in the hippocampus but no differences in capillary density. The DG of these mice contained the highest level of Abeta in combination with hippocampal atrophy, and a reduced GLUT-1 amount per capillary density. At 8 months, no differences were observed. The highest Abeta deposition was found in the DG, although fourfold less compared to 18-month-old mice.

    Conclusions: We conclude that the GLUT-1 amount and capillary density in both wild type and transgenic mice decrease due to ageing. Further, a decreased amount of GLUT-1 is caused by decreased GLUT-1 amount/capillary density and not due to a reduced capillary density. We suggest that Abeta load in the hippocampus precedes the reduction of GLUT-1. A certain level of Abeta must be reached in the hippocampus, before it affects GLUT-1 amount/capillary density leading to further impairment of energy metabolism and hippocampal atrophy.

    Brain research 2007;1181;93-103

  • Fe65 stimulates proteolytic liberation of the beta-amyloid precursor protein intracellular domain.

    Wiley JC, Smith EA, Hudson MP, Ladiges WC and Bothwell M

    Department of Physiology and Biophysics, University of Washington, Seattle, Washington 98195, USA.

    The beta-amyloid precursor protein (APP)-binding protein Fe65 is involved in APP nuclear signaling and several steps in APP proteolytic processing. In this study, we show that Fe65 stimulates gamma-secretase-mediated liberation of the APP intracellular domain (AICD). The mechanism of Fe65-mediated stimulation of AICD formation appears to be through enhanced production of the carboxyl-terminal fragment substrates of gamma-secretase and direct stimulation of processing by gamma-secretase. The stimulatory capacity of Fe65 is isoform-dependent, as the non-neuronal and a2 isoforms promote APP processing more effectively than the exon 9 inclusive neuronal form of Fe65. Intriguingly, Fe65 stimulation of AICD production appears to be inversely related to pathogenic beta-amyloid production as the Fe65 isoforms profoundly stimulate AICD production and simultaneously decrease Abeta42 production. Despite the capacity of Fe65 to stimulate gamma-secretase-mediated APP proteolysis, it does not rescue the loss of proteolytic function associated with the presenilin-1 familial Alzheimer disease mutations. These data suggest that Fe65 regulation of APP proteolysis may be integrally associated with its nuclear signaling function, as all antecedent proteolytic steps prior to release of Fe65 from the membrane are fostered by the APP-Fe65 interaction.

    Funded by: NIA NIH HHS: AG21127, AG26476

    The Journal of biological chemistry 2007;282;46;33313-25

  • Soluble Abeta inhibits specific signal transduction cascades common to the insulin receptor pathway.

    Townsend M, Mehta T and Selkoe DJ

    Department of Neurology, Harvard Medical School and Center for Neurologic Diseases, Brigham and Women's Hospital, Boston, Massachusetts 02115, USA.

    Numerous studies have now shown that the amyloid beta-protein (Abeta), the principal component of cerebral plaques in Alzheimer disease, rapidly and potently inhibits certain forms of synaptic plasticity. The amyloid (or Abeta) hypothesis proposes that the continuous disruption of normal synaptic physiology by Abeta contributes to the development of Alzheimer disease. However, there is little consensus about how Abeta mediates this inhibition at the molecular level. Using mouse primary hippocampal neurons, we observed that a brief treatment with cell-derived, soluble, human Abeta disrupted the activation of three kinases (Erk/MAPK, CaMKII, and the phosphatidylinositol 3-kinase-activated protein Akt/protein kinase B) that are required for long term potentiation, whereas two other kinases (protein kinase A and protein kinase C) were stimulated normally. An antagonist of the insulin receptor family of tyrosine kinases was found to mimic the pattern of Abeta-mediated kinase inhibition. We then found that soluble Abeta binds to the insulin receptor and interferes with its insulin-induced autophosphorylation. Taken together, these data demonstrate that physiologically relevant levels of naturally secreted Abeta interfere with insulin receptor function in hippocampal neurons and prevent the rapid activation of specific kinases required for long term potentiation.

    Funded by: NIA NIH HHS: AG027443; NINDS NIH HHS: T32 NS07484-04

    The Journal of biological chemistry 2007;282;46;33305-12

  • Characterization of the adaptor protein ARH expression in the brain and ARH molecular interactions.

    Mameza MG, Lockard JM, Zamora E, Hillefors M, Lavina ZS and Kaplan BB

    Laboratory of Molecular Biology, National Institute of Mental Health, National Institutes of Health, Bethesda, Maryland 20892, USA.

    Previously, pA134 was identified as one of the mRNAs present in the squid giant axon. Comparative sequence analyses revealed that the pA134 gene product manifested significant similarity to the mammalian lipoprotein receptor adaptor protein also known as ARH (autosomal recessive hypercholesterolemia). ARH mRNA and protein displayed very similar pattern of expression throughout the mouse brain. Significant levels of expression were observed in cells with a predominantly neuronal profile in the cerebellum, brainstem, olfactory bulb, hippocampus, and cortex. A yeast two hybrid screen for ARH protein interactions in mouse brain identified the following binders: amyloid precursor-like protein 1, low density lipoprotein receptor-related protein (LRP) 1, LRP8, and GABA receptor-associated protein-like 1. The interactions of ARH with LRP1 and GABA receptor-associated protein-like 1 were subsequently verified by co-immunoprecipitation of the protein complexes from transfected human embryonic kidney cells. The presence of ARH mRNA in axon of primary sympathetic neurons was established by RT-PCR analyses and confirmed by in situ hybridization. Taken together, our data suggest that ARH is a multifunctional protein whose spectrum of function in the brain goes beyond the traditionally known metabolism of lipoproteins, and that ARH may be locally synthesized in the axon.

    Funded by: Intramural NIH HHS

    Journal of neurochemistry 2007;103;3;927-41

  • Valsartan lowers brain beta-amyloid protein levels and improves spatial learning in a mouse model of Alzheimer disease.

    Wang J, Ho L, Chen L, Zhao Z, Zhao W, Qian X, Humala N, Seror I, Bartholomew S, Rosendorff C and Pasinetti GM

    Department of Psychiatry, Mount Sinai School of Medicine, New York, New York 10029, USA.

    Recent epidemiological evidence suggests that some antihypertensive medications may reduce the risk for Alzheimer disease (AD). We screened 55 clinically prescribed antihypertensive medications for AD-modifying activity using primary cortico-hippocampal neuron cultures generated from the Tg2576 AD mouse model. These agents represent all drug classes used for hypertension pharmacotherapy. We identified 7 candidate antihypertensive agents that significantly reduced AD-type beta-amyloid protein (Abeta) accumulation. Through in vitro studies, we found that only 1 of the candidate drugs, valsartan, was capable of attenuating oligomerization of Abeta peptides into high-molecular-weight (HMW) oligomeric peptides, known to be involved in cognitive deterioration. We found that preventive treatment of Tg2576 mice with valsartan significantly reduced AD-type neuropathology and the content of soluble HMW extracellular oligomeric Abeta peptides in the brain. Most importantly, valsartan administration also attenuated the development of Abeta-mediated cognitive deterioration, even when delivered at a dose about 2-fold lower than that used for hypertension treatment in humans. These preclinical studies suggest that certain antihypertensive drugs may have AD-modifying activity and may protect against progressive Abeta-related memory deficits in subjects with AD or in those at high risk of developing AD.

    Funded by: NIA NIH HHS: AG02219, P01 AG002219

    The Journal of clinical investigation 2007;117;11;3393-402

  • Downregulation of myosin II-B by siRNA alters the subcellular localization of the amyloid precursor protein and increases amyloid-beta deposition in N2a cells.

    Massone S, Argellati F, Passalacqua M, Armirotti A, Melone L, d'Abramo C, Marinari UM, Domenicotti C, Pronzato MA and Ricciarelli R

    Department of Experimental Medicine, University of Genoa, Via L.B. Alberti 2, 16132 Genoa, Italy.

    The Alzheimer's disease (AD) brain pathology is characterized by extracellular deposits of amyloid-beta (Abeta) peptides and intraneuronal fibrillar structures. These pathological features may be functionally linked, but the mechanism by which Abeta accumulation relates to neuronal degeneration is still poorly understood. Abeta peptides are fragments cleaved from the amyloid precursor protein (APP), a transmembrane protein ubiquitously expressed in the nervous system. Although the proteolytic processing of APP has been implicated in AD, the physiological function of APP and the subcellular site of APP cleavages remain unknown. The overall structure of the protein and its fast anterograde transport along the axon support the idea that APP functions as a vesicular receptor for cytoskeletal motor proteins. In the current study, we test the hypothesis that myosin II, important contributor to the cytoskeleton of neuronal cells, may influence the trafficking and/or the processing of APP. Our results demonstrate that downregulation of myosin II-B, the major myosin isoform in neurons, is able to increase Abeta deposition, concomitantly altering the subcellular localization of APP. These new insights might be important for the understanding of the function of APP and provide a novel conceptual framework in which to analyze its pathological role.

    Biochemical and biophysical research communications 2007;362;3;633-8

  • Amyloid precursor protein regulates brain apolipoprotein E and cholesterol metabolism through lipoprotein receptor LRP1.

    Liu Q, Zerbinatti CV, Zhang J, Hoe HS, Wang B, Cole SL, Herz J, Muglia L and Bu G

    Department of Pediatrics, Washington University School of Medicine, St Louis, MO 63110, USA.

    Mutations in the amyloid precursor protein (APP) cause early-onset Alzheimer's disease (AD), but the only genetic risk factor for late-onset AD is the varepsilon4 allele of apolipoprotein E (apoE), a major cholesterol carrier. Using Cre-lox conditional knockout mice, we demonstrate that lipoprotein receptor LRP1 expression regulates apoE and cholesterol levels within the CNS. We also found that deletion of APP and its homolog APLP2, or components of the gamma-secretase complex, significantly enhanced the expression and function of LRP1, which was reversed by forced expression of the APP intracellular domain (AICD). We further show that AICD, together with Fe65 and Tip60, interacts with the LRP1 promoter and suppresses its transcription. Together, our findings support that the gamma-secretase cleavage of APP plays a central role in regulating apoE and cholesterol metabolism in the CNS via LRP1 and establish a biological linkage between APP and apoE, the two major genetic determinants of AD.

    Funded by: NHLBI NIH HHS: R37 HL063762-12; NIA NIH HHS: R01 AG027924, R01 AG027924-01, R01 AG027924-02

    Neuron 2007;56;1;66-78

  • HtrA2 regulates beta-amyloid precursor protein (APP) metabolism through endoplasmic reticulum-associated degradation.

    Huttunen HJ, Guénette SY, Peach C, Greco C, Xia W, Kim DY, Barren C, Tanzi RE and Kovacs DM

    Neurobiology of Disease Laboratory, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA 02129, USA.

    Alzheimer disease-associated beta-amyloid peptide is generated from its precursor protein APP. By using the yeast two-hybrid assay, here we identified HtrA2/Omi, a stress-responsive chaperone-protease as a protein binding to the N-terminal cysteinerich region of APP. HtrA2 coimmunoprecipitates exclusively with immature APP from cell lysates as well as mouse brain extracts and degrades APP in vitro. A subpopulation of HtrA2 localizes to the cytosolic side of the endoplasmic reticulum (ER) membrane where it contributes to ER-associated degradation of APP together with the proteasome. Inhibition of the proteasome results in accumulation of retrotranslocated forms of APP and increased association of APP with HtrA2 and Derlin-1 in microsomal membranes. In cells lacking HtrA2, APP holoprotein is stabilized and accumulates in the early secretory pathway correlating with elevated levels of APP C-terminal fragments and increased Abeta secretion. Inhibition of ER-associated degradation (either HtrA2 or proteasome) promotes binding of APP to the COPII protein Sec23 suggesting enhanced trafficking of APP out of the ER. Based on these results we suggest a novel function for HtrA2 as a regulator of APP metabolism through ER-associated degradation.

    The Journal of biological chemistry 2007;282;38;28285-95

  • TgCRND8 amyloid precursor protein transgenic mice exhibit an altered gamma-secretase processing and an aggressive, additive amyloid pathology subject to immunotherapeutic modulation.

    Van Vickle GD, Esh CL, Kalback WM, Patton RL, Luehrs DC, Kokjohn TA, Fifield FG, Fraser PE, Westaway D, McLaurin J, Lopez J, Brune D, Newel AJ, Poston M, Beach TG and Roher AE

    The Longtine Center for Molecular Biology and Genetics, Sun Health Research Institute, Sun City, Arizona 85351, USA.

    We investigated the morphology and biochemistry of the amyloid-beta (Abeta) peptides produced in TgCRND8 Tg mice carrying combined amyloid precursor protein (APP) Swedish (K670M/N671L) and Indiana (V717F) mutations. Histological analyses employing amyloid-specific staining and electron microscopy revealed that the TgCRND8 Tg mice produce an aggressive pathology, evident as early as 3 months of age, that is a composite of core plaques and peculiar floccular diffuse parenchymal deposits. The Abeta peptides were purified using combined FPLC-HPLC, Western blots, and immunoprecipitation methods and characterized by MALDI-TOF/SELDI-TOF mass spectrometry. The C-terminal APP peptides, assessed by Western blot experiments and mass spectrometry, suggested an alteration in the order of secretase processing, yielding a C-terminal fragment pattern that is substantially different from that observed in sporadic Alzheimer's disease (AD). This modified processing pattern generated longer Abeta peptides, as well as those ending at residues 40/42/43, which may partially explain the early onset and destructive nature of familial AD caused by APP mutations. Despite an aggressive pathology that extended to the cerebellum and white matter, these animals tolerated the presence of an imposing amount of Abeta load. Abeta immunization resulted in an impressive 7-fold reduction in the number of amyloid core plaques and, as previously demonstrated, a significant memory recovery. However, given the phylogenetic distance and the differences in APP processing and Abeta chemistry between Tg mice and AD, caution should be applied in projecting mouse therapeutic interventions onto human subjects.

    Funded by: NIA NIH HHS: P30 AG-19610, R01 AG 19795, R01 AG019795, R01 AG019795-08

    Biochemistry 2007;46;36;10317-27

  • Abeta solubility and deposition during AD progression and in APPxPS-1 knock-in mice.

    Murphy MP, Beckett TL, Ding Q, Patel E, Markesbery WR, St Clair DK, LeVine H and Keller JN

    Department of Molecular and Cellular Biochemistry, Sanders-Brown Center on Aging, University of Kentucky, Lexington, Kentucky 40536-0230, USA. mpmurp3@email.uky.edu

    Amnestic mild cognitive impairment (MCI) appears to be a very early stage of Alzheimer's disease (AD). The amyloid-beta peptide (Abeta) is believed to be a possible substrate for AD, but little is currently known about Abeta alterations in MCI and how these changes compare to later stages of disease. In the present study Abeta was differentially extracted from the brains of age-matched control, MCI, and AD cases and compared with plaque counts. For comparison, APPxPS-1 knock-in mice were processed in parallel. We observed that Abeta42 was significantly elevated in MCI subjects, even though there was no significant alteration in the total amount of Abeta. Relative Abeta solubility within the different extractable pools was identical between AD and MCI subjects, with both significantly altered relative to controls. Temporal analysis of Abeta levels and solubility in a knock-in mouse model of Abeta pathogenesis recapitulated many of the salient features observed in AD. Characterization of the SDS fraction showed some similarities between aged knock-in mice and AD subjects. These data suggest that distinct changes in Abeta occur throughout the progression of AD, and that elevations in Abeta42 occur at an early, clinically defined stage.

    Funded by: NCRR NIH HHS: RR020171; NIA NIH HHS: AG05119, P01 AG005119

    Neurobiology of disease 2007;27;3;301-11

  • Advancing age produces sex differences in vasomotor kinetics during and after skeletal muscle contraction.

    Bearden SE

    Department of Biological Sciences, Idaho State University, Pocatello, ID 83209, USA. bearshaw@isu.edu

    Little is known of the vasomotor responses of skeletal muscle arterioles during and following muscle contraction. We hypothesized that aging leads to impaired arteriolar responses to muscle contraction and recovery. Nitric oxide (NO) availability, which is age dependent, has been implicated in components of these kinetics. Therefore, we also hypothesized that changes in the kinetics of vascular responses are associated with the NO pathway. Groups were young (3 mo), old (24 mo), endothelial NO synthase knockout (eNOS-/-), and N(G)-nitro-L-arginine (L-NA)-treated male and female C57BL/6 mice. The kinetics of vasodilation during and following 1 min of contractions of the gluteus maximus muscle were recorded in second-order (regional distribution) and third-order (local control) arterioles. Baseline, peak (during contraction), and maximal diameters (pharmacological) were not affected by age or sex. The kinetics of dilation and recovery were not different between males and females at the young age. There was a significant slowing of vasodilation at the onset of contractions (approximately 2-fold; P < 0.05) and a significant speeding of recovery ( approximately 5-fold; P < 0.05) in old males vs. old females and vs. young eNOS-/-, and L-NA did not affect the kinetics at the onset of muscle contraction. eNOS-/- mimicked the rapid recovery of old males in second-order arterioles; acute NO production (L-NA) explained approximately 50% of this effect. These data demonstrate fundamental age-related differences between the sexes in the dynamic function of skeletal muscle arterioles. Understanding how youthful function persists in females but not males may provide therapeutic insight into clinical interventions to maintain dynamic microvascular control of nutrient supply with age.

    Funded by: NCRR NIH HHS: P20 RR 016454

    American journal of physiology. Regulatory, integrative and comparative physiology 2007;293;3;R1274-9

  • Amyloid precursor protein knockdown by siRNA impairs spontaneous alternation in adult mice.

    Senechal Y, Kelly PH, Cryan JF, Natt F and Dev KK

    Department of Neuroscience Research, Novartis Institutes for BioMedical Research, Novartis Pharma AG, Lichtstrasse, Basel, SwitzerlandDepartment of Functional Genomics, Novartis Institutes for BioMedical Research, Novartis Pharma AG, Lichtstrasse, Basel, Switzerland.

    The cleavage-product of amyloid precursor protein (APP) constitutes the core component of plaques found in the brains of Alzheimer's disease (AD) patients. APP is ubiquitously expressed and its precise physiological functions remain unclear. This protein has been proposed to regulate synaptic function and processes underlying learning and memory. While APP knockout mice show behavioral impairments, these may occur due to early changes during development and/or due to abolition of APP function in adult. To investigate the acute effects of APP knockdown without involving developmental processes, APP expression was reduced using RNA interference in adult mouse brain. Small interfering RNAs (siRNAs) that down-regulated mouse APP protein levels (APP-siRNA) were identified using an APP plasmid-siRNA co-transfection assay in mouse NIH/3T3 fibroblast cells. Infusion of APP-siRNAs into the ventricular system for 2 weeks also down-regulated APP mRNA in mouse brain. Highest knockdown of APP mRNA levels was found in the CA2-CA3 regions of the hippocampus. Mice treated with the most active APP-siRNA showed a significant reduction in spontaneous alternation rate in the Y-maze, without effects on forelimb grip strength or locomotor activity. These data suggest that acute knockdown of APP in adult mouse brain impairs hippocampus-dependent spatial working memory.

    Journal of neurochemistry 2007;102;6;1928-1940

  • Increased severity of hemorrhage in transgenic mice expressing cerebral protease nexin-2/amyloid beta-protein precursor.

    Xu F, Previti ML and Van Nostrand WE

    Secreted isoforms of amyloid beta-protein precursor (AbetaPP) that contain the Kunitz proteinase inhibitor domain, also known as protease nexin-2 (PN2), are enriched in brain. Although little is known of its physiological function, the potent inhibition of certain prothrombotic proteinases by PN2/AbetaPP suggests that it may function to regulate cerebral thrombosis during vascular injury events.

    Methods: To examine the antithrombotic function of cerebral PN2/AbetaPP in vivo, we performed measurements of carotid artery thrombosis and experimental intracerebral hemorrhage in transgenic mice with specific and modest overexpression of PN2/AbetaPP in brain. Comparisons were made with wild-type mice and Tg-rPF4/APP mice, a model that possesses specific and modest overexpression of PN2/AbetaPP in platelets and exhibits reduced thrombosis in vivo.

    Results: Modest overexpression of PN2/AbetaPP in transgenic mouse brain had no effect on intraluminal carotid arterial thrombosis but resulted in larger hematoma volumes and hemoglobin levels (23.1+/-2.7 mm(3) [n=6; P<0.01] and 1411+/-202 microg/hemisphere [n=12; P<0.01], respectively), compared with wild-type mice (15.9+/-2.2 mm(3) [n=6] and 935+/-418 microg/hemisphere [n=12], respectively).

    Conclusions: These findings indicate that cerebral PN2/AbetaPP plays a significant role in regulating thrombosis in brain and that modest age-related increases in the cerebral levels of this protein could markedly enhance the extent of cerebral hemorrhage.

    Funded by: NHLBI NIH HHS: R01-HL72533; NINDS NIH HHS: R01-NS36645, R01-NS52533

    Stroke 2007;38;9;2598-601

  • Amyolid precursor protein mediates presynaptic localization and activity of the high-affinity choline transporter.

    Wang B, Yang L, Wang Z and Zheng H

    Huffington Center on Aging and Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA.

    The key pathological features of Alzheimer's disease include synaptic dysfunction, profound changes in the cholinergic system, and deposition of beta-amyloid peptides generated by proteolytic processing of the amyloid-beta precursor protein (APP). However, the pathways linking APP with synaptic activity and cholinergic neuronal function are poorly understood. We report here that APP is essential in regulating the presynaptic expression and activity of the high-affinity choline transporter (CHT), a molecule that mediates the rate-limiting step of cholinergic synaptic transmission in both the neuromuscular junction and central cholinergic neurons. Loss of APP leads to aberrant localization of CHT at the neuromuscular synapses and reduced CHT activity at cholinergic projections. At the cellular level, we show that APP and CHT can be found in Rab5-positive endosomal compartments and that APP affects CHT endocytosis. Furthermore, we demonstrate that APP interacts with CHT through the C-terminal domain, providing support for a specific and direct regulation of CHT by APP through protein-protein interactions. These results identify a physiological activity of APP in cholinergic neurons, and our data indicate that deregulation of APP function may contribute to cholinergic impairment and AD pathogenesis.

    Funded by: NIA NIH HHS: AG20670, AG21141, T32 AG000183; NINDS NIH HHS: NS40039

    Proceedings of the National Academy of Sciences of the United States of America 2007;104;35;14140-5

  • Deletion of tumor necrosis factor death receptor inhibits amyloid beta generation and prevents learning and memory deficits in Alzheimer's mice.

    He P, Zhong Z, Lindholm K, Berning L, Lee W, Lemere C, Staufenbiel M, Li R and Shen Y

    Haldeman Laboratory of Molecular and Cellular Neurobiology, Sun Health Research Institute, Sun City, AZ 85351, USA.

    The tumor necrosis factor type 1 death receptor (TNFR1) contributes to apoptosis. TNFR1, a subgroup of the TNFR superfamily, contains a cytoplasmic death domain. We recently demonstrated that the TNFR1 cascade is required for amyloid beta protein (Abeta)-induced neuronal death. However, the function of TNFR1 in Abeta plaque pathology and amyloid precursor protein (APP) processing in Alzheimer's disease (AD) remains unclear. We report that the deletion of the TNFR1 gene in APP23 transgenic mice (APP23/TNFR1(-/-)) inhibits Abeta generation and diminishes Abeta plaque formation in the brain. Genetic deletion of TNFR1 leads to reduced beta-secretase 1 (BACE1) levels and activity. TNFR1 regulates BACE1 promoter activity via the nuclear factor-kappaB pathway, and the deletion of TNFR1 in APP23 transgenic mice prevents learning and memory deficits. These findings suggest that TNFR1 not only contributes to neurodegeneration but also that it is involved in APP processing and Abeta plaque formation. Thus, TNFR1 is a novel therapeutic target for AD.

    Funded by: NIA NIH HHS: AG025888

    The Journal of cell biology 2007;178;5;829-41

  • Insensitivity to Abeta42-lowering nonsteroidal anti-inflammatory drugs and gamma-secretase inhibitors is common among aggressive presenilin-1 mutations.

    Czirr E, Leuchtenberger S, Dorner-Ciossek C, Schneider A, Jucker M, Koo EH, Pietrzik CU, Baumann K and Weggen S

    Emmy Noether Research Group, Mainz, Germany.

    Abeta42-lowering nonsteroidal anti-inflammatory drugs (NSAIDs) constitute the founding members of a new class of gamma-secretase modulators that avoid side effects of pan-gamma-secretase inhibitors on NOTCH processing and function, holding promise as potential disease-modifying agents for Alzheimer disease (AD). These modulators are active in cell-free gamma-secretase assays indicating that they directly target the gamma-secretase complex. Additional support for this hypothesis was provided by the observation that certain mutations in presenilin-1 (PS1) associated with early-onset familial AD (FAD) change the cellular drug response to Abeta42-lowering NSAIDs. Of particular interest is the PS1-DeltaExon9 mutation, which provokes a pathogenic increase in the Abeta42/Abeta40 ratio and dramatically reduces the cellular response to the Abeta42-lowering NSAID sulindac sulfide. This FAD PS1 mutant is unusual as a splice-site mutation results in deletion of amino acids Thr(291)-Ser(319) including the endoproteolytic cleavage site of PS1, and an additional amino acid exchange (S290C) at the exon 8/10 splice junction. By genetic dissection of the PS1-DeltaExon9 mutation, we now demonstrate that a synergistic effect of the S290C mutation and the lack of endoproteolytic cleavage is sufficient to elevate the Abeta42/Abeta40 ratio and that the attenuated response to sulindac sulfide results partially from the deficiency in endoproteolysis. Importantly, a wider screen revealed that a diminished response to Abeta42-lowering NSAIDs is common among aggressive FAD PS1 mutations. Surprisingly, these mutations were also partially unresponsive to gamma-secretase inhibitors of different structural classes. This was confirmed in a mouse model with transgenic expression of the PS1-L166P mutation, in which the potent gamma-secretase inhibitor LY-411575 failed to reduce brain levels of soluble Abeta42. In summary, these findings highlight the importance of genetic background in drug discovery efforts aimed at gamma-secretase, suggesting that certain AD mouse models harboring aggressive PS mutations may not be informative in assessing in vivo effects of gamma-secretase modulators and inhibitors.

    The Journal of biological chemistry 2007;282;34;24504-13

  • {gamma}-Secretase Substrate Concentration Modulates the Abeta42/Abeta40 Ratio: IMPLICATIONS FOR ALZHEIMER DISEASE.

    Yin YI, Bassit B, Zhu L, Yang X, Wang C and Li YM

    Molecular Pharmacology and Chemistry Program, Memorial Sloan-Kettering Cancer Center, New York, New York 10021 and Department of Biology, Rensselaer Polytechnic Institute, Troy, New York 12180.

    Mutation of the amyloid precursor protein (APP), presenilin-1, or presenilin-2 results in the development of early onset autosomal dominant forms of Alzheimer disease (AD). These mutations lead to an increased Abeta42/Abeta40 ratio that correlates with the onset of disease. However, it remains unknown how these mutations affect gamma-secretase, a protease that generates the termini of Abeta40 and Abeta42. Here we have determined the reaction mechanism of gamma-secretase with wild type and three mutated APP substrates. Our findings indicate that despite the overall outcome of an increased Abeta42/Abeta40 ratio, these mutations each display rather distinct reactivity to gamma-secretase. Intriguingly, we found that the ratio of Abeta42/Abeta40 is variable with substrate concentration; increased substrate concentrations result in higher ratios of Abeta42/Abeta40. Moreover, we demonstrated that reduction of gamma-secretase substrate concentration by BACE1 inhibition in cells decreased the Abeta42/Abeta40 ratio. This study indicates that biological factors affecting targets such as BACE1 and APP, which ultimately cause an increased concentration of gamma-secretase substrate, can augment the Abeta42/Abeta40 ratio and may play a causative role in sporadic AD. Therefore, strategies lowering the Abeta42/Abeta40 ratio through partial reduction of gamma-secretase substrate production may introduce a practical therapeutic modality for treatment of AD.

    The Journal of biological chemistry 2007;282;32;23639-44

  • Rodent A beta modulates the solubility and distribution of amyloid deposits in transgenic mice.

    Jankowsky JL, Younkin LH, Gonzales V, Fadale DJ, Slunt HH, Lester HA, Younkin SG and Borchelt DR

    Division of Biology, California Institute of Technology, Pasadena, California 91125, USA.

    The amino acid sequence of amyloid precursor protein (APP) is highly conserved, and age-related A beta aggregates have been described in a variety of vertebrate animals, with the notable exception of mice and rats. Three amino acid substitutions distinguish mouse and human A beta that might contribute to their differing properties in vivo. To examine the amyloidogenic potential of mouse A beta, we studied several lines of transgenic mice overexpressing wild-type mouse amyloid precursor protein (moAPP) either alone or in conjunction with mutant PS1 (PS1dE9). Neither overexpression of moAPP alone nor co-expression with PS1dE9 caused mice to develop Alzheimer-type amyloid pathology by 24 months of age. We further tested whether mouse A beta could accelerate the deposition of human A beta by crossing the moAPP transgenic mice to a bigenic line expressing human APPswe with PS1dE9. The triple transgenic animals (moAPP x APPswe/PS1dE9) produced 20% more A beta but formed amyloid deposits no faster and to no greater extent than APPswe/PS1dE9 siblings. Instead, the additional mouse A beta increased the detergent solubility of accumulated amyloid and exacerbated amyloid deposition in the vasculature. These findings suggest that, although mouse A beta does not influence the rate of amyloid formation, the incorporation of A beta peptides with differing sequences alters the solubility and localization of the resulting aggregates.

    Funded by: NIA NIH HHS: 1 P01 AG-98-003, 1 P50 AG-14-248, AG-06-656, K01 AG-26144-01, K01 AG026144, K01 AG026144-01, K01 AG026144-02, K01 AG026144-03, K01 AG026144-04, K01 AG026144-05

    The Journal of biological chemistry 2007;282;31;22707-20

  • Alzheimer's presenilin 1 modulates sorting of APP and its carboxyl-terminal fragments in cerebral neurons in vivo.

    Gandy S, Zhang YW, Ikin A, Schmidt SD, Bogush A, Levy E, Sheffield R, Nixon RA, Liao FF, Mathews PM, Xu H and Ehrlich ME

    Farber Institute for Neurosciences and the Department of Neurology, Thomas Jefferson University, Philadelphia, Pennsylvania, USA. samgandy@earthlink.net

    Studies in continuously cultured cells have established that familial Alzheimer's disease (FAD) mutant presenilin 1 (PS1) delays exit of the amyloid precursor protein (APP) from the trans-Golgi network (TGN). Here we report the first description of PS1-regulated APP trafficking in cerebral neurons in culture and in vivo. Using neurons from transgenic mice or a cell-free APP transport vesicle biogenesis system derived from the TGN of those neurons, we demonstrated that knocking-in an FAD-associated mutant PS1 transgene was associated with delayed kinetics of APP arrival at the cell surface. Apparently, this delay was at least partially attributable to impaired exit of APP from the TGN, which was documented in the cell-free APP transport vesicle biogenesis assay. To extend the study to APP and carboxyl terminal fragment (CTF) trafficking to cerebral neurons in vivo, we performed subcellular fractionation of brains from APP transgenic mice, some of which carried a second transgene encoding an FAD-associated mutant form of PS1. The presence of the FAD mutant PS1 was associated with a slight shift in the subcellular localization of both holoAPP and APP CTFs toward iodixanol density gradient fractions that were enriched in a marker for the TGN. In a parallel set of experiments, we used an APP : furin chimeric protein strategy to test the effect of artificially forcing TGN concentration of an APP : furin chimera that could be a substrate for beta- and gamma-cleavage. This chimeric substrate generated excess Abeta42 when compared with wildtype APP. These data indicate that the presence of an FAD-associated mutant human PS1 transgene is associated with redistribution of the APP and APP CTFs in brain neurons toward TGN-enriched fractions. The chimera experiment suggests that TGN-enrichment of a beta-/gamma-secretase substrate may play an integral role in the action of mutant PS1 to elevate brain levels of Abeta42.

    Funded by: NIA NIH HHS: AG017617, AG021173, AG024895, AG08206, AG10491, AG18237, AG23611, P01 AG010491, P01 AG010491-14; NINDS NIH HHS: NS41017, NS46673

    Journal of neurochemistry 2007;102;3;619-26

  • Chronic administration of R-flurbiprofen attenuates learning impairments in transgenic amyloid precursor protein mice.

    Kukar T, Prescott S, Eriksen JL, Holloway V, Murphy MP, Koo EH, Golde TE and Nicolle MM

    Department of Neuroscience, Mayo Clinic College of Medicine, Jacksonville, FL 32224, USA. kukar.thomas@mayo.edu <kukar.thomas@mayo.edu&gt;

    Background: Long-term use of non-steroidal anti-inflammatory drugs (NSAIDs) is associated with a reduced incidence of Alzheimer's disease (AD). We and others have shown that certain NSAIDs reduce secretion of Abeta42 in cell culture and animal models, and that the effect of NSAIDs on Abeta42 is independent of the inhibition of cyclooxygenase by these compounds. Since Abeta42 is hypothesized to be the initiating pathologic molecule in AD, the ability of these compounds to lower Abeta42 selectively may be associated with their protective effect. We have previously identified R-flurbiprofen (tarenflurbil) as a selective Abeta42 lowering agent with greatly reduced cyclooxygenase activity that shows promise for testing this hypothesis. In this study we report the effect of chronic R-flurbiprofen treatment on cognition and Abeta loads in Tg2576 APP mice.

    Results: A four-month preventative treatment regimen with R-flurbiprofen (10 mg/kg/day) was administered to young Tg2576 mice prior to robust plaque or Abeta pathology. This treatment regimen improved spatial learning as assessed by the Morris water maze, indicated by an increased spatial bias during the third probe trial and an increased utilization of a place strategy to solve the water maze. These results are consistent with an improvement in hippocampal- and medial temporal lobe-dependent memory function. A modest, though not statistically significant, reduction in formic acid-soluble levels of Abeta was also observed. To determine if R-flurbiprofen could reverse cognitive deficits in Tg2576 mice where plaque pathology was already robust, a two-week therapeutic treatment was given to older Tg2576 mice with the same dose of R-flurbiprofen. This approach resulted in a significant decrease in Abeta plaque burden but no significant improvement in spatial learning.

    Conclusion: We have found that chronic administration of R-flurbiprofen is able to attenuate spatial learning deficits if given prior to plaque deposition in Tg2576 mice. Given its ability to selectively target Abeta42 production and improve cognitive impairments in transgenic APP mice, as well as promising data from a phase 2 human clinical trial, future studies are needed to investigate the utility of R-flurbiprofen as an AD therapeutic and its possible mechanisms of action.

    Funded by: NIA NIH HHS: P01 AG003949, P01 AG017216, P01 AG020206, P01 AG20206, P01-AG03949, P01-AG17216, P50 AG016574, P50 AG025711, P50-AG16574, P50-AG25711; NINDS NIH HHS: P50 NS040256, P50-NS40256

    BMC neuroscience 2007;8;54

  • The secreted beta-amyloid precursor protein ectodomain APPs alpha is sufficient to rescue the anatomical, behavioral, and electrophysiological abnormalities of APP-deficient mice.

    Ring S, Weyer SW, Kilian SB, Waldron E, Pietrzik CU, Filippov MA, Herms J, Buchholz C, Eckman CB, Korte M, Wolfer DP and Müller UC

    Department of Bioinformatics and Functional Genomics, Institute for Pharmacy and Molecular Biotechnology, University of Heidelberg, D-69120 Heidelberg, Germany.

    It is well established that the proteolytic processing of the beta-amyloid precursor protein (APP) generates beta-amyloid (Abeta), which plays a central role in the pathogenesis of Alzheimer's disease (AD). In contrast, the physiological role of APP and of its numerous proteolytic fragments and the question of whether a loss of these functions contributes to AD are still unknown. To address this question, we replaced the endogenous APP locus by gene-targeted alleles and generated two lines of knock-in mice that exclusively express APP deletion variants corresponding either to the secreted APP ectodomain (APPs alpha) or to a C-terminal (CT) truncation lacking the YENPTY interaction motif (APPdeltaCT15). Interestingly, the deltaCT15 deletion resulted in reduced turnover of holoAPP, increased cell surface expression, and strongly reduced Abeta levels in brain, likely because of reduced processing in the endocytic pathway. Most importantly, we demonstrate that in both APP knock-in lines the expression of APP N-terminal domains either grossly attenuated or completely rescued the prominent deficits of APP knock-out mice, such as reductions in brain and body weight, grip strength deficits, alterations in circadian locomotor activity, exploratory activity, and the impairment in spatial learning and long-term potentiation. Together, our data suggest that the APP C terminus is dispensable and that APPs alpha is sufficient to mediate the physiological functions of APP assessed by these tests.

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2007;27;29;7817-26

  • Cellular prion protein regulates beta-secretase cleavage of the Alzheimer's amyloid precursor protein.

    Parkin ET, Watt NT, Hussain I, Eckman EA, Eckman CB, Manson JC, Baybutt HN, Turner AJ and Hooper NM

    Proteolysis Research Group, Institute of Molecular and Cellular Biology, Faculty of Biological Sciences, and Leeds Institute of Genetics, Health and Therapeutics, University of Leeds, Leeds LS2 9JT, United Kingdom.

    Proteolytic processing of the amyloid precursor protein (APP) by beta-secretase, beta-site APP cleaving enzyme (BACE1), is the initial step in the production of the amyloid beta (Abeta) peptide, which is involved in the pathogenesis of Alzheimer's disease. The normal cellular function of the prion protein (PrP(C)), the causative agent of the transmissible spongiform encephalopathies such as Creutzfeldt-Jakob disease in humans, remains enigmatic. Because both APP and PrP(C) are subject to proteolytic processing by the same zinc metalloproteases, we tested the involvement of PrP(C) in the proteolytic processing of APP. Cellular overexpression of PrP(C) inhibited the beta-secretase cleavage of APP and reduced Abeta formation. Conversely, depletion of PrP(C) in mouse N2a cells by siRNA led to an increase in Abeta peptides secreted into the medium. In the brains of PrP knockout mice and in the brains from two strains of scrapie-infected mice, Abeta levels were significantly increased. Two mutants of PrP, PG14 and A116V, that are associated with familial human prion diseases failed to inhibit the beta-secretase cleavage of APP. Using constructs of PrP, we show that this regulatory effect of PrP(C) on the beta-secretase cleavage of APP required the localization of PrP(C) to cholesterol-rich lipid rafts and was mediated by the N-terminal polybasic region of PrP(C) via interaction with glycosaminoglycans. In conclusion, this is a mechanism by which the cellular production of the neurotoxic Abeta is regulated by PrP(C) and may have implications for both Alzheimer's and prion diseases.

    Funded by: Medical Research Council: G9824728; NINDS NIH HHS: NS042192, NS048554-01, R01 NS042192, R01 NS048554

    Proceedings of the National Academy of Sciences of the United States of America 2007;104;26;11062-7

  • Presenilin/gamma-secretase-dependent processing of beta-amyloid precursor protein regulates EGF receptor expression.

    Zhang YW, Wang R, Liu Q, Zhang H, Liao FF and Xu H

    Center for Neuroscience and Aging, Burnham Institute for Medical Research, La Jolla, CA 92037, USA. yunzhang@xmu.edu.cn

    Presenilins (PS, PS1/PS2) are necessary for the proteolytic activity of gamma-secretase, which cleaves multiple type I transmembrane proteins including Alzheimer's beta-amyloid precursor protein (APP), Notch, ErbB4, etc. Cleavage by PS/gamma-secretase releases the intracellular domain (ICD) of its substrates. Notch ICD translocates into the nucleus to regulate expression of genes important for development. However, the patho/physiological role of other ICDs, especially APP ICD (AICD), in regulating gene expression remains controversial because evidence supporting this functionality stems mainly from studies performed under supraphysiological conditions. EGF receptor (EGFR) is up-regulated in a wide variety of tumors and hence is a target for cancer therapeutics. Abnormal expression/activation of EGFR contributes to keratinocytic carcinomas, and mice with reduced PS dosages have been shown to develop skin tumors. Here we demonstrate that the levels of PS and EGFR in the skin tumors of PS1(+/-)/ PS2(-/-) mice and the brains of PS1/2 conditional double knockout mice are inversely correlated. Deficiency in PS/gamma-secretase activity or APP expression results in a significant increase of EGFR in fibroblasts. Importantly, we show that AICD mediates transcriptional regulation of EGFR. Furthermore, we provide in vivo evidence demonstrating direct binding of endogenous AICD to the EGFR promoter. Our results indicate an important role of PS/gamma-secretase-generated APP metabolite AICD in gene transcription and in EGFR-mediated tumorigenesis.

    Funded by: NIA NIH HHS: F32 AG024895, R01 AG021173, R01 AG030197; NINDS NIH HHS: R01 NS046673, R01 NS054880

    Proceedings of the National Academy of Sciences of the United States of America 2007;104;25;10613-8

  • Notch1 intracellular domain suppresses APP intracellular domain-Tip60-Fe65 complex mediated signaling through physical interaction.

    Kim SY, Kim MY, Mo JS and Park HS

    Hormone Research Center, School of Biological Sciences and Technology, Chonnam National University, Yongbong-dong, Buk-ku, Gwangju, 500-757, Republic of Korea.

    The amyloid beta-precursor protein (APP) and the Notch receptor are both type 1 integral transmembrane proteins, and both are cleaved by presenilin-dependent gamma-secretase activity. In this study, we have demonstrated that the Notch intracellular domain (Notch1-IC) suppresses APP-intracellular domain (AICD)-mediated ROS generation and cell death after being processed by gamma secretase. Notch1-IC physically interacts with AICD, Fe65, and Tip60, thereby disrupting the association of the AICD-Fe65-Tip60 trimeric transcription activator complex in AICD signaling. AICD-Fe65-Tip60 mediated reactive oxygen species generation was found to be suppressed by Notch1-IC. Furthermore, AICD-Fe65-Tip60 was shown to mediate cell death in human neuroblastoma cells, and the overexpression of Notch1-IC inhibited cell death induced by AICD-Fe65-Tip60. Collectively, our findings indicate that Notch1-IC plays the role of a negative regulator in AICD signaling via the disruption of the AICD-Fe65-Tip60 trimeric complex.

    Biochimica et biophysica acta 2007;1773;6;736-46

  • A macromolecular complex involving the amyloid precursor protein (APP) and the cytosolic adapter FE65 is a negative regulator of axon branching.

    Ikin AF, Sabo SL, Lanier LM and Buxbaum JD

    Laboratory of Molecular Neuropsychiatry, Departments of Psychiatry and Neurobiology, Mount Sinai School of Medicine, New York, NY 10029, USA. annatikin@gmail.com

    Several studies suggest a role for the amyloid precursor protein (APP) in neurite outgrowth and synaptogenesis, but the downstream interactions that mediate the function of APP during neuron development are unknown. By introducing interaction-deficient FE65 into cultured hippocampal neurons using adenovirus, we show that a complex including APP, FE65 and an additional protein is involved in neurite outgrowth at early stages of neuronal development. Both FE65 that is unable to interact with APP (PID2 mutants) or a WW mutant increased axon branching. Although the FE65 mutants did not affect total neurite output, both mutants decreased axon segment length, consistent with an overall slowing of axonal growth cones. FE65 mutants did not alter the localization of either APP or FE65 in axonal growth cones, suggesting that the effects on neurite outgrowth are achieved by alterations in local complex formation within the axonal growth cone.

    Funded by: NIA NIH HHS: R01 AG021792, R01 AG021792-04

    Molecular and cellular neurosciences 2007;35;1;57-63

  • Regulated intramembrane proteolysis of the interleukin-1 receptor II by alpha-, beta-, and gamma-secretase.

    Kuhn PH, Marjaux E, Imhof A, De Strooper B, Haass C and Lichtenthaler SF

    Adolf-Butenandt-Institut, Ludwig-Maximilians-University, Schillerstrasse 44, 80336 Munich, Germany.

    Ectodomain shedding and intramembrane proteolysis of the amyloid precursor protein (APP) by alpha-, beta- and gamma-secretase are involved in the pathogenesis of Alzheimer disease (AD). Increased proteolytic processing and secretion of another membrane protein, the interleukin-1 receptor II (IL-1R2), have also been linked to the pathogenesis of AD. IL-1R2 is a decoy receptor that may limit detrimental effects of IL-1 in the brain. At present, the proteolytic processing of IL-1R2 remains little understood. Here we show that IL-1R2 can be proteolytically processed in a manner similar to APP. IL-1R2 expressed in human embryonic kidney 293 cells first undergoes ectodomain shedding in an alpha-secretase-like manner, resulting in secretion of the IL-1R2 ectodomain and the generation of an IL-1R2 C-terminal fragment. This fragment undergoes further intramembrane proteolysis by gamma-secretase, leading to the generation of the soluble intracellular domain of IL-1R2. Intramembrane cleavage of IL-1R2 was abolished by a highly specific inhibitor of gamma-secretase and was absent in mouse embryonic fibroblasts deficient in gamma-secretase activity. Surprisingly, the beta-secretase BACE1 and its homolog BACE2 increased IL-1R2 secretion resulting in C-terminal fragments nearly identical to the ones generated by the alpha-secretase-like cleavage. This suggests that both proteases may act as alternative alpha-secretase-like proteases. Importantly, BACE1 and BACE2 did not cleave several other membrane proteins, demonstrating that both proteases do not contribute to general membrane protein turnover but only cleave specific proteins. This study reveals a similar proteolytic processing of IL-1R2 and APP and may provide an explanation for the increased IL-1R2 secretion observed in AD.

    The Journal of biological chemistry 2007;282;16;11982-95

  • Increased KPI containing amyloid precursor protein in experimental autoimmune encephalomyelitis brains.

    Beilin O, Karussis DM, Korczyn AD, Gurwitz D, Aronovich R, Mizrachi-Kol R and Chapman J

    Department of Physiology and Pharmacology, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv , Israel.

    Amyloid precursor protein can be translated from three alternatively spliced mRNAs. We measured levels of amyloid precursor protein isoforms containing the Kunitz protease inhibitor domain (KPIAPP), and amyloid precursor protein without the Kunitz protease inhibitor domain (KPIAPP) in brain homogenates of acute experimental autoimmune encephalomyelitis mice. At the preclinical phase of the disease, both KPIAPP and KPIAPP levels were significantly higher in homogenates from brains of autoimmune encephalomyelitis mice, whereas at the acute phase of the disease only KPIAPP remained significantly elevated compared with controls. At the recovery phase, no differences were observed between the groups. The early and isoform-specific elevation of KPIAPP in autoimmune encephalomyelitis mice suggests a possible role for amyloid precursor protein in the immune response mediating the disease.

    Neuroreport 2007;18;6;581-4

  • Physiologically regulated transgenic ABCA1 does not reduce amyloid burden or amyloid-beta peptide levels in vivo.

    Hirsch-Reinshagen V, Chan JY, Wilkinson A, Tanaka T, Fan J, Ou G, Maia LF, Singaraja RR, Hayden MR and Wellington CL

    Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, Canada.

    ABCA1-deficient mice have low levels of poorly lipidated apolipoprotein E (apoE) and exhibit increased amyloid load. To test whether excess ABCA1 protects from amyloid deposition, we crossed APP/PS1 mice to ABCA1 bacterial artificial chromosome (BAC) transgenic mice. Compared with wild-type animals, the ABCA1 BAC led to a 50% increase in cortical ABCA1 protein and a 15% increase in apoE abundance, demonstrating that this BAC supports modest ABCA1 overexpression in brain. However, this was observed only in animals that do not deposit amyloid. Comparison of ABCA1/APP/PS1 mice with APP/PS1 controls revealed no differences in levels of brain ABCA1 protein, amyloid, Abeta, or apoE, despite clear retention of ABCA1 overexpression in the livers of these animals. To further investigate ABCA1 expression in the amyloid-containing brain, we then compared ABCA1 mRNA and protein levels in young and aged cortex and cerebellum of APP/PS1 and ABCA1/APP/PS1 animals. Compared with APP/PS1 controls, aged ABCA1/APP/PS1 mice exhibited increased ABCA1 mRNA, but not protein, selectively in cortex. Additionally, ABCA1 mRNA levels were not increased before amyloid deposition but were induced only in the presence of extensive Abeta and amyloid levels. These data suggest that an induction of ABCA1 expression may be associated with late-stage Alzheimer's neuropathology.

    Journal of lipid research 2007;48;4;914-23

  • Endoplasmic reticulum chaperones inhibit the production of amyloid-beta peptides.

    Hoshino T, Nakaya T, Araki W, Suzuki K, Suzuki T and Mizushima T

    Graduate School of Medical and Pharmaceutical Sciences, Kumamoto University, Kumamoto 862-0973, Japan.

    Abeta (amyloid-beta peptides) generated by proteolysis of APP (beta-amyloid precursor protein), play an important role in the pathogenesis of AD (Alzheimer's disease). ER (endoplasmic reticulum) chaperones, such as GRP78 (glucose-regulated protein 78), make a major contribution to protein quality control in the ER. In the present study, we examined the effect of overexpression of various ER chaperones on the production of Abeta in cultured cells, which produce a mutant type of APP (APPsw). Overexpression of GRP78 or inhibition of its basal expression, decreased and increased respectively the level of Abeta40 and Abeta42 in conditioned medium. Co-expression of GRP78's co-chaperones ERdj3 or ERdj4 stimulated this inhibitory effect of GRP78. In the case of the other ER chaperones, overexpression of some (150 kDa oxygen-regulated protein and calnexin) but not others (GRP94 and calreticulin) suppressed the production of Abeta. These results indicate that certain ER chaperones are effective suppressors of Abeta production and that non-toxic inducers of ER chaperones may be therapeutically beneficial for AD treatment. GRP78 was co-immunoprecipitated with APP and overexpression of GRP78 inhibited the maturation of APP, suggesting that GRP78 binds directly to APP and inhibits its maturation, resulting in suppression of the proteolysis of APP. On the other hand, overproduction of APPsw or addition of synthetic Abeta42 caused up-regulation of the mRNA of various ER chaperones in cells. Furthermore, in the cortex and hippocampus of transgenic mice expressing APPsw, the mRNA of some ER chaperones was up-regulated in comparison with wild-type mice. We consider that this up-regulation is a cellular protective response against Abeta.

    The Biochemical journal 2007;402;3;581-9

  • Conditional neuronal simian virus 40 T antigen expression induces Alzheimer-like tau and amyloid pathology in mice.

    Park KH, Hallows JL, Chakrabarty P, Davies P and Vincent I

    Centre for Molecular Medicine and Therapeutics, Department of Pediatrics, Child and Family Research Institute, University of British Columbia, Vancouver, British Columbia, Canada, V5Z 4H4.

    A large body of evidence has shown the activation of a cohort of cell cycle regulators and the duplication of DNA in degenerating neurons of Alzheimer's disease (AD) brain. Activation of these regulators and duplication of chromosomes precede neurodegeneration and formation of neurofibrillary tangles (NFTs), one of the diagnostic lesions of AD. These findings, in combination with evidence for cell cycle regulation of amyloid precursor protein and tau, has led to the hypothesis that reentry into the cell cycle underlies AD pathogenesis. To test this hypothesis directly, we have created transgenic mice with forced cell cycle activation in postmitotic neurons via conditional expression of the simian virus 40 large T antigen (TAg) oncogene. We show that TAg mice recapitulate the cell cycle changes seen in AD and display a neurodegenerative phenotype accompanied by tau pathology and NFT-like profiles. Moreover, plaque-like amyloid deposits, similar to those seen in AD, are also observed in the brains of TAg mice. These data provide support for an essential role of ectopic cell cycle activation in the generation of the characteristic pathological hallmarks of AD. Furthermore, our TAg mice are the first model to develop NFTs and amyloid pathology simultaneously and in the absence of any human transgenes. These mice will be useful for further defining the nongenetic mechanisms in AD pathogenesis and for the development of cell cycle-based therapies for AD.

    Funded by: NIA NIH HHS: AG12721, P50 AG05136; PHS HHS: T32 00057

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2007;27;11;2969-78

  • Regulation of Alzheimer's disease amyloid-beta formation by casein kinase I.

    Flajolet M, He G, Heiman M, Lin A, Nairn AC and Greengard P

    Laboratory of Molecular and Cellular Neuroscience, The Rockefeller University, 1230 York Avenue, New York, NY 10021, USA.

    Alzheimer's disease (AD) is associated with accumulation of the neurotoxic peptide amyloid-beta (Abeta), which is produced by sequential cleavage of amyloid precursor protein (APP) by the aspartyl protease beta-secretase and the presenilin-dependent protease gamma-secretase. An increase of casein kinase 1 (CK1) expression has been described in the human AD brain. We show, by using in silico analysis, that APP, beta-secretase, and gamma-secretase subunits contain, in their intracellular regions, multiple CK1 consensus phosphorylation sites, many of which are conserved among human, rat, and mouse species. Overexpression of constitutively active CK1epsilon, one of the CK1 isoforms expressed in brain, leads to an increase in Abeta peptide production. Conversely, three structurally dissimilar CK1-specific inhibitors significantly reduced endogenous Abeta peptide production. By using mammalian cells expressing the beta C-terminal fragment of APP, it was possible to demonstrate that CK1 inhibitors act at the level of gamma-secretase cleavage. Importantly, Notch cleavage was not affected. Our results indicate that CK1 represents a therapeutic target for prevention of Abeta formation in AD.

    Funded by: NIA NIH HHS: AG 09464, P01 AG009464

    Proceedings of the National Academy of Sciences of the United States of America 2007;104;10;4159-64

  • Long-lasting impairment in hippocampal neurogenesis associated with amyloid deposition in a knock-in mouse model of familial Alzheimer's disease.

    Zhang C, McNeil E, Dressler L and Siman R

    Laboratory for Neurodegeneration, Center for Brain Injury and Repair, University of Pennsylvania School of Medicine, 105B Hayden Hall, 3320 Smith Walk, Philadelphia, PA 19104, USA.

    Neurogenesis in the adult hippocampus has been implicated in regulating long-term memory and mood, but its integrity in Alzheimer's disease (AD) is uncertain. Studies of neurogenesis in transgenic mouse models of familial AD are complicated by ectopic overexpression restricted to terminally differentiated neurons, while AD cases have been studied only at the pre-senile or end-stage of disease. To investigate further the fidelity of adult neurogenesis, we examined mice carrying targeted mutations in amyloid precursor protein (APP), presenilin-1 (PS-1), or both APP and PS-1, in which FAD-causing mutations have been inserted into their endogenous genes. The latter "double knock-in" mice developed aging- and region-dependent amyloid deposition starting around 6 months, and by 9 months exhibited microglial activation associated with the amyloid. In the 9-month-old dentate gyrus, the double knock-in mutations reduced the numbers of MCM2-positive neural stem and progenitor cells by 3-fold and doublecortin-positive neuroblasts by 2-fold. The reduction in dentate neuroblasts persisted at 18 months of age. The impairment in neurogenesis was confirmed by quantitative Western blot analysis of doublecortin content and was restricted to the hippocampal but not the olfactory bulb neurogenic system. In contrast, neither mutant PS-1 nor APP alone led to amyloid deposition or significant alterations in the two markers. These results demonstrate long-lasting and selective impairment in adult hippocampal neurogenesis in a knock-in mutant mouse model of FAD and suggest a novel mechanism by which amyloid and its attendant microglia-mediated neuroinflammation could contribute to the cognitive and behavioral abnormalities of AD.

    Funded by: NIA NIH HHS: AG17138, R01 AG017138, R01 AG017138-06A2, R56 AG017138

    Experimental neurology 2007;204;1;77-87

  • Suppression of beta-amyloid precursor protein signaling into the nucleus by estrogens mediated through complex formation between the estrogen receptor and Fe65.

    Bao J, Cao C, Zhang X, Jiang F, Nicosia SV and Bai W

    Department of Pathology, University of South Florida College of Medicine, 12901 Bruce B. Downs Blvd., Tampa, FL 33612-4799, USA.

    The C-terminal fragment of the beta-amyloid precursor protein produced after cleavage by gamma-secretase, namely, APPct or AICD, has been shown to form a multimeric complex with the adaptor protein Fe65 and to regulate transcription through the recruitment of the histone acetyltransferase Tip60. The present study shows that 17beta-estradiol inhibits the transcriptional and apoptotic activities of the APPct complex by a process involving the interaction of estrogen receptor alpha (ERalpha) with Fe65. ERalpha-Fe65 complexes were detected both in vitro and in the mouse brain, and recruitment of ERalpha to the promoter of an APPct target gene (KAI1) was demonstrated. Our studies reveal a novel mechanism of estrogen action, which may explain the well-known neuroprotective functions of estrogens as well as the complex role of this female hormone in the pathogenesis of neuronal degeneration diseases.

    Funded by: NCI NIH HHS: CA 79530

    Molecular and cellular biology 2007;27;4;1321-33

  • Essential roles for Fe65, Alzheimer amyloid precursor-binding protein, in the cellular response to DNA damage.

    Minopoli G, Stante M, Napolitano F, Telese F, Aloia L, De Felice M, Di Lauro R, Pacelli R, Brunetti A, Zambrano N and Russo T

    CEINGE Biotecnologie Avanzate, 80145 Napoli, Italy.

    Fe65 interacts with the cytosolic domain of the Alzheimer amyloid precursor protein (APP). The functions of the Fe65 are still unknown. To address this point we generated Fe65 knockout (KO) mice. These mice do not show any obvious phenotype; however, when fibroblasts (mouse embryonic fibroblasts), isolated from Fe65 KO embryos, were exposed to low doses of DNA damaging agents, such as etoposide or H2O2, an increased sensitivity to genotoxic stress, compared with wild type animals, clearly emerged. Accordingly, brain extracts from Fe65 KO mice, exposed to non-lethal doses of ionizing radiations, showed high levels of gamma-H2AX and p53, thus demonstrating a higher sensitivity to X-rays than wild type mice. Nuclear Fe65 is necessary to rescue the observed phenotype, and few minutes after the exposure of MEFs to DNA damaging agents, Fe65 undergoes phosphorylation in the nucleus. With a similar timing, the proteolytic processing of APP is rapidly affected by the genotoxic stress: in fact, the cleavage of the APP COOH-terminal fragments by gamma-secretase is induced soon after the exposure of cells to etoposide, in a Fe65-dependent manner. These results demonstrate that Fe65 plays an essential role in the response of the cells to DNA damage.

    The Journal of biological chemistry 2007;282;2;831-5

  • Modulation of gene expression and cytoskeletal dynamics by the amyloid precursor protein intracellular domain (AICD).

    Müller T, Concannon CG, Ward MW, Walsh CM, Tirniceriu AL, Tribl F, Kögel D, Prehn JH and Egensperger R

    Institute of Pathology and Neuropathology, University Hospital Essen, D-45122 Essen, Germany. thorsten.t.mueller@rub.de

    Amyloidogenic processing of the amyloid precursor protein (APP) results in the generation of beta-amyloid, the main constituent of Alzheimer plaques, and the APP intracellular domain (AICD). Recently, it has been demonstrated that AICD has transactivation potential; however, the targets of AICD-dependent gene regulation and hence the physiological role of AICD remain largely unknown. We analyzed transcriptome changes during AICD-dependent gene regulation by using a human neural cell culture system inducible for expression of AICD, its coactivator FE65, or the combination of both. Induction of AICD was associated with increased expression of genes with known function in the organization and dynamics of the actin cytoskeleton, including alpha2-Actin and Transgelin (SM22). AICD target genes were also found to be differentially regulated in the frontal cortex of Alzheimer's disease patients compared with controls as well as in AICD/FE65 transiently transfected murine cortical neurons. Confocal image analysis of neural cells and cortical neurons expressing both AICD and FE65 confirmed pronounced changes in the organization of the actin cytoskeleton, including the destabilization of actin fibers and clumping of actin at the sites of cellular outgrowth. Our data point to a role of AICD in developmental and injury-related cytoskeletal dynamics in the nervous system.

    Molecular biology of the cell 2007;18;1;201-10

  • Intranasal NAP administration reduces accumulation of amyloid peptide and tau hyperphosphorylation in a transgenic mouse model of Alzheimer's disease at early pathological stage.

    Matsuoka Y, Gray AJ, Hirata-Fukae C, Minami SS, Waterhouse EG, Mattson MP, LaFerla FM, Gozes I and Aisen PS

    Department of Neurology, Georgetown University Medical Center, Washington, DC 20057, USA.

    Accumulation of beta-amyloid (Abeta) peptide and hyperphosphorylation of tau in the brain are pathological hallmarks of Alzheimer's disease (AD). Agents altering these pathological events might modify clinical disease progression. NAP (Asn-Ala-Pro-Val-Ser-Ile-Pro-Gln) is an octapeptide that has shown neuroprotective effects in various in vitro and in vivo neurodegenerative models. Previous studies showed that NAP protected against Abeta-induced neurotoxicity, inhibited Abeta aggregation, and, by binding to tubulin, prevented disruption of microtubules. In this study, we investigated the effect of NAP on Abeta and tau pathology using a transgenic mouse model that recapitulates both aspects of AD. We administered NAP intranasally (0.5 microg/mouse per day, daily from Monday through Friday) for 3 mo, starting from 9 mo of age, which is a prepathological stage in these mice. NAP treatment significantly lowered levels of Abeta 1-40 and 1-42 in brain. In addition, NAP significantly reduced levels of hyperphosphorylated tau. Of particular interest, hyperphosphorylation at the threonine 231 site was reduced; phosphorylation at this site influences microtubule binding. Our results indicate that NAP treatment of transgenic mice initiated at an early stage reduced both Abeta and tau pathology, suggesting that NAP might be a potential therapeutic agent for AD.

    Funded by: Intramural NIH HHS; NIA NIH HHS: AG022455

    Journal of molecular neuroscience : MN 2007;31;2;165-70

  • p35/Cyclin-dependent kinase 5 is required for protection against beta-amyloid-induced cell death but not tau phosphorylation by ceramide.

    Seyb KI, Ansar S, Li G, Bean J, Michaelis ML and Dobrowsky RT

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

    Ceramide is a bioactive sphingolipid that can prevent calpain activation and beta-amyloid (A beta) neurotoxicity in cortical neurons. Recent evidence supports A beta induction of a calpain-dependent cleavage of the cyclin-dependent kinase 5 (cdk5) regulatory protein p35 that contributes to tau hyperphosphorylation and neuronal death. Using cortical neurons isolated from wild-type and p35 knockout mice, we investigated whether ceramide required p35/cdk5 to protect against A beta-induced cell death and tau phosphorylation. Ceramide inhibited A beta-induced calpain activation and cdk5 activity in wild-type neurons and protected against neuronal death and tau hyperphosphorylation. Interestingly, A beta also increased cdk5 activity in p35-/- neurons, suggesting that the alternate cdk5 regulatory protein, p39, might mediate this effect. In p35 null neurons, ceramide blocked A beta-induced calpain activation but did not inhibit cdk5 activity or cell death. However, ceramide blocked tau hyperphosphorylation potentially via inhibition of glycogen synthase kinase-3beta. These data suggest that ceramide can regulate A beta cell toxicity in a p35/cdk5-dependent manner.

    Journal of molecular neuroscience : MN 2007;31;1;23-35

  • Reduction of soluble Abeta and tau, but not soluble Abeta alone, ameliorates cognitive decline in transgenic mice with plaques and tangles.

    Oddo S, Vasilevko V, Caccamo A, Kitazawa M, Cribbs DH and LaFerla FM

    Departments of Neurobiology and Behavior and Neurology, and Institute for Brain Aging and Dementia, University of California, Irvine, California 92697, USA.

    Increasing evidence points to soluble assemblies of aggregating proteins as a major mediator of neuronal and synaptic dysfunction. In Alzheimer disease (AD), soluble amyloid-beta (Abeta) appears to be a key factor in inducing synaptic and cognitive abnormalities. Here we report the novel finding that soluble tau also plays a role in the cognitive decline in the presence of concomitant Abeta pathology. We describe improved cognitive function following a reduction in both soluble Abeta and tau levels after active or passive immunization in advanced aged 3xTg-AD mice that contain both amyloid plaques and neurofibrillary tangles (NFTs). Notably, reducing soluble Abeta alone did not improve the cognitive phenotype in mice with plaques and NFTs. Our results show that Abeta immunotherapy reduces soluble tau and ameliorates behavioral deficit in old transgenic mice.

    Funded by: NIA NIH HHS: AG0212982, AG20241

    The Journal of biological chemistry 2006;281;51;39413-23

  • Physiological mouse brain Abeta levels are not related to the phosphorylation state of threonine-668 of Alzheimer's APP.

    Sano Y, Nakaya T, Pedrini S, Takeda S, Iijima-Ando K, Iijima K, Mathews PM, Itohara S, Gandy S and Suzuki T

    RIKEN, Brain Science Institute, Behavioral Genetics, Wako, Japan.

    Background: Amyloid-beta peptide species ending at positions 40 and 42 (Abeta40, Abeta42) are generated by the proteolytic processing of the Alzheimer's amyloid precursor protein (APP). Abeta peptides accumulate in the brain early in the course of Alzheimer's disease (AD), especially Abeta42. The cytoplasmic domain of APP regulates intracellular trafficking and metabolism of APP and its carboxyl-terminal fragments (CTFalpha, CTFbeta). The role of protein phosphorylation in general, and that of the phosphorylation state of APP at threonine-668 (Thr668) in particular, has been investigated in detail by several laboratories (including our own). Some investigators have recently proposed that the phosphorylation state of Thr668 plays a pivotal role in governing brain Abeta levels, prompting the current study.

    Methodology: In order to evaluate whether the phosphorylation state of Thr668 controlled brain Abeta levels, we studied the levels and subcellular distributions of holoAPP, sAPPalpha, sAPPbeta, CTFalpha, CTFbeta, Abeta40 and Abeta42 in brains from "knock-in" mice in which a non-phosphorylatable alanyl residue had been substituted at position 668, replacing the threonyl residue present in the wild-type protein.

    Conclusions: The levels and subcellular distributions of holoAPP, sAPPalpha, sAPPbeta, CTFalpha, CTFbeta, Abeta40 and Abeta42 in the brains of Thr668Ala mutant mice were identical to those observed in wild-type mice. These results indicate that, despite speculation to the contrary, the phosphorylation state of APP at Thr668 does not play an obvious role in governing the physiological levels of brain Abeta40 or Abeta42 in vivo.

    Funded by: NIA NIH HHS: AG010491, AG023611, P01 AG010491, P01 AG010491-14, R01 AG023611; NINDS NIH HHS: NS41017, NS45357, R01 NS041017, R21 NS045357

    PloS one 2006;1;e51

  • Enhanced amyloidogenic metabolism of the amyloid beta-protein precursor in the X11L-deficient mouse brain.

    Sano Y, Syuzo-Takabatake A, Nakaya T, Saito Y, Tomita S, Itohara S and Suzuki T

    Laboratory of Neuroscience, Graduate School of Pharmaceutical Sciences, Hokkaido University, Kita-12 Nishi-6, Kita-ku, Sapporo 060-0812, Japan.

    X11L, a neuronal adaptor protein, associates with the cytoplasmic domain of APP and suppresses APP cellular metabolism. APP is the precursor of Abeta, whose metabolism is strongly implicated in Alzheimer disease pathogenesis. To examine the roles of X11L function in APP metabolism, including the generation of Abeta in the brain, we produced X11L-deficient mutant mice on the C57BL/6 background. The mutant mice did not exhibit histopathological alterations or compensatory changes in the expression of other X11 family proteins, X11 and X11L2. The expression level and distribution of APP in the brain of mutant mice were also identical to those in wild-type mice. However, in the hippocampus, where substantial levels of X11L and APP are expressed, the mutant mice exhibited a significant increase in the level of the C-terminal fragments of APP produced by cleavage with beta-secretase but not alpha-secretase. The levels of Abeta were increased in the hippocampus of aged mutant mice as compared with age-matched controls. These observations clearly indicate that X11L suppresses the amyloidogenic but not amyloidolytic processing of APP in regions of the brain such as the hippocampus, which express significant levels of X11L.

    The Journal of biological chemistry 2006;281;49;37853-60

  • Apolipoprotein E and low density lipoprotein receptor-related protein facilitate intraneuronal Abeta42 accumulation in amyloid model mice.

    Zerbinatti CV, Wahrle SE, Kim H, Cam JA, Bales K, Paul SM, Holtzman DM and Bu G

    Department of Pediatrics, Washington University School of Medicine, St. Louis, Missouri 63110, USA.

    The low density lipoprotein receptor-related protein (LRP) is highly expressed in the brain and has been shown to alter the metabolism of amyloid precursor protein and amyloid-beta peptide (Abeta) in vitro. Previously we developed mice that overexpress a functional LRP minireceptor (mLRP2) in their brains and crossed them to the PDAPP mouse model of Alzheimer disease. Overexpression of mLRP2 in 22-month-old PDAPP mice with amyloid plaques increased a pool of carbonate-soluble Abeta in the brain and worsened memory-related behavior. In the current study, we examined the effects of mLRP2 overexpression on 3-month-old PDAPP mice that had not yet developed amyloid plaques. We found significantly higher levels of membrane-associated Abeta42 in the hippocampus of mice that overexpressed mLRP2. Using immunohistochemical methods, we observed significant intraneuronal Abeta42 in the hippocampus and frontal cortex of PDAPP mice, which frequently co-localized with the lysosomal marker LAMP-1. Interestingly, PDAPP mice lacking apolipoprotein E (apoE) had much less intraneuronal Abeta42. We also found that PC12 cells overexpressing mLRP2 cleared Abeta42 and Abeta40 more rapidly from media than PC12 cells transfected with the vector only. Preincubation of apoE3 or apoE4 with Abeta42 increased the rate of Abeta clearance, and this effect was partially blocked by receptor-associated protein. Our results support the hypothesis that LRP binds and endocytoses Abeta42 both directly and via apoE but that endocytosed Abeta42 is not completely degraded and accumulates in intraneuronal lysosomes.

    Funded by: NIA NIH HHS: R01-AG027924; NINDS NIH HHS: F32-NS41872

    The Journal of biological chemistry 2006;281;47;36180-6

  • Gene silencing analyses against amyloid precursor protein (APP) gene family by RNA interference.

    Sakai T and Hohjoh H

    National Institute of Neuroscience, National Center of Neurology and Psychiatry, 4-1-1 Ogawahigashi, Kodaira, Tokyo 187-8502, Japan.

    Amyloid precursor protein (APP) and amyloid precursor-like proteins 1 and 2 (APLP1 and APLP2) are members of a large gene family. Although APP is known to be the source of the beta-amyloid peptides involved in the development of Alzheimer's disease, the normal functions of APP, APLP1 and APLP2 in cells are poorly understood. In this study, we carried out gene silencing analysis by means of RNA interference with synthetic small interfering RNA duplexes targeting the App, Aplp1 and Aplp2 genes in Neuro2a (N2a) cells, a mouse neuroblastoma cell line. The results demonstrated that cell viability and neurite outgrowth of N2a cells undergoing knockdown of Aplp1 were significantly reduced, compared with N2a cells undergoing knockdown of either App or Aplp2.

    Cell biology international 2006;30;11;952-6

  • Regulation of steady-state beta-amyloid levels in the brain by neprilysin and endothelin-converting enzyme but not angiotensin-converting enzyme.

    Eckman EA, Adams SK, Troendle FJ, Stodola BA, Kahn MA, Fauq AH, Xiao HD, Bernstein KE and Eckman CB

    Department of Neuroscience, Mayo Clinic College of Medicine, Jacksonville, Florida 32224, USA.

    The deposition of beta-amyloid in the brain is a pathological hallmark of Alzheimer disease (AD). Normally, the accumulation of beta-amyloid is prevented in part by the activities of several degradative enzymes, including the endothelin-converting enzymes, neprilysin, insulin-degrading enzyme, and plasmin. Recent reports indicate that another metalloprotease, angiotensin-converting enzyme (ACE), can degrade beta-amyloid in vitro and in cellular overexpression experiments. In addition, ACE gene variants are linked to AD risk in several populations. Angiotensin-converting enzyme, neprilysin and endothelin-converting enzyme function as vasopeptidases and are the targets of drugs designed to treat cardiovascular disorders, and ACE inhibitors are commonly prescribed. We investigated the potential physiological role of ACE in regulating endogenous brain beta-amyloid levels for two reasons: first, to determine whether beta-amyloid degradation might be the mechanism by which ACE is associated with AD, and second, to determine whether ACE inhibitor drugs might block beta-amyloid degradation in the brain and potentially increase the risk for AD. We analyzed beta-amyloid accumulation in brains from ACE-deficient mice and in mice treated with ACE inhibitors and found that ACE deficiency did not alter steady-state beta-amyloid concentration. In contrast, beta-amyloid levels are significantly elevated in endothelin-converting enzyme and neprilysin knock-out mice, and inhibitors of these enzymes cause a rapid increase in beta-amyloid concentration in the brain. The results of these studies do not support a physiological role for ACE in the degradation of beta-amyloid in the brain but confirm roles for endothelin-converting enzyme and neprilysin and indicate that reductions in these enzymes result in additive increases in brain amyloid beta-peptide levels.

    Funded by: NINDS NIH HHS: R01NS042192, R01NS048554

    The Journal of biological chemistry 2006;281;41;30471-8

  • Elevated levels of amyloid precursor protein in muscle of patients with amyotrophic lateral sclerosis and a mouse model of the disease.

    Koistinen H, Prinjha R, Soden P, Harper A, Banner SJ, Pradat PF, Loeffler JP and Dingwall C

    Neurodegeneration Research Department, GlaxoSmithKline Research & Development Ltd., New Frontiers Science Park, Third Avenue, Harlow, Essex, UK.

    Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease defined by motor neuron loss. Transgenic mouse models show features that closely mimic those seen in the clinical situation, reflected in the molecular changes observed in mouse models and in tissues from patients. We report a dramatic increase in the expression of amyloid precursor protein (APP) in the hindlimb muscles, but not the spinal cord of the G93A transgenic mouse model, significantly before the appearance of clinical abnormalities. APP levels were unchanged in nontransgenic mice and in mice overexpressing human wild-type Cu/Zn-dependent superoxide dismutase 1 (SOD1). Preliminary results indicate a similar change in APP expression in human deltoid muscle samples from ALS patients compared with age-matched controls. The inhibitory role of APP in innervation at the neuromuscular junction and increased expression in inclusion-body myositis suggest that presymptomatic upregulation of APP may be consistent with a potential role for APP in ALS pathology.

    Muscle & nerve 2006;34;4;444-50

  • Fibrillar beta-amyloid-stimulated intracellular signaling cascades require Vav for induction of respiratory burst and phagocytosis in monocytes and microglia.

    Wilkinson B, Koenigsknecht-Talboo J, Grommes C, Lee CY and Landreth G

    Alzheimer Research Laboratory, Department of Neuroscience, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106, USA.

    Microglial interaction with extracellular beta-amyloid fibrils (fAbeta) is mediated through an ensemble of cell surface receptors, including the B-class scavenger receptor CD36, the alpha(6)beta(1)-integrin, and the integrin-associated protein/CD47. The binding of fAbeta to this receptor complex has been shown to drive a tyrosine kinase-based signaling cascade leading to production of reactive oxygen species and stimulation of phagocytic activity; however, little is known about the intracellular signaling cascades governing the microglial response to fAbeta. This study reports a direct mechanistic link between the fAbeta cell surface receptor complex and downstream signaling events responsible for NADPH oxidase activation and phagosome formation. The Vav guanine nucleotide exchange factor is tyrosine-phosphorylated in response to fAbeta peptides as a result of the engagement of the microglia fAbeta cell surface receptor complex. Co-immunoprecipitation studies demonstrate an Abeta-dependent association between Vav and both Lyn and Syk kinases. The downstream target of Vav, the small GTPase Rac1, is GTP-loaded in an Abeta-dependent manner. Rac1 is both an essential component of the NADPH oxidase and a critical regulator of microglial phagocytosis. The direct role of Vav in fAbeta-stimulated intracellular signaling cascades was established using primary microglia obtained from Vav(-/-) mice. Stimulation of Vav(-/-) microglia with fAbeta failed to generate NADPH oxidase-derived reactive oxygen species and displayed a dramatically attenuated phagocytic response. These findings directly link Vav phosphorylation to the Abeta-receptor complex and demonstrate that Vav activity is required for fAbeta-stimulated intracellular signaling events upstream of reactive oxygen species production and phagosome formation.

    Funded by: NIA NIH HHS: AG16740, F32 AG24031

    The Journal of biological chemistry 2006;281;30;20842-50

  • Alzheimer's disease beta-amyloid peptides are released in association with exosomes.

    Rajendran L, Honsho M, Zahn TR, Keller P, Geiger KD, Verkade P and Simons K

    Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, 01307 Dresden, Germany.

    Although the exact etiology of Alzheimer's disease (AD) is a topic of debate, the consensus is that the accumulation of beta-amyloid (Abeta) peptides in the senile plaques is one of the hallmarks of the progression of the disease. The Abeta peptide is formed by the amyloidogenic cleavage of the amyloid precursor protein (APP) by beta- and gamma-secretases. The endocytic system has been implicated in the cleavages leading to the formation of Abeta. However, the identity of the intracellular compartment where the amyloidogenic secretases cleave and the mechanism by which the intracellularly generated Abeta is released into the extracellular milieu are not clear. Here, we show that beta-cleavage occurs in early endosomes followed by routing of Abeta to multivesicular bodies (MVBs) in HeLa and N2a cells. Subsequently, a minute fraction of Abeta peptides can be secreted from the cells in association with exosomes, intraluminal vesicles of MVBs that are released into the extracellular space as a result of fusion of MVBs with the plasma membrane. Exosomal proteins were found to accumulate in the plaques of AD patient brains, suggesting a role in the pathogenesis of AD.

    Proceedings of the National Academy of Sciences of the United States of America 2006;103;30;11172-7

  • Increased App expression in a mouse model of Down's syndrome disrupts NGF transport and causes cholinergic neuron degeneration.

    Salehi A, Delcroix JD, Belichenko PV, Zhan K, Wu C, Valletta JS, Takimoto-Kimura R, Kleschevnikov AM, Sambamurti K, Chung PP, Xia W, Villar A, Campbell WA, Kulnane LS, Nixon RA, Lamb BT, Epstein CJ, Stokin GB, Goldstein LS and Mobley WC

    Department of Neurology and Neurological Sciences, Stanford University, Stanford, California 94305, USA. asalehi@stanford.edu

    Degeneration of basal forebrain cholinergic neurons (BFCNs) contributes to cognitive dysfunction in Alzheimer's disease (AD) and Down's syndrome (DS). We used Ts65Dn and Ts1Cje mouse models of DS to show that the increased dose of the amyloid precursor protein gene, App, acts to markedly decrease NGF retrograde transport and cause degeneration of BFCNs. NGF transport was also decreased in mice expressing wild-type human APP or a familial AD-linked mutant APP; while significant, the decreases were less marked and there was no evident degeneration of BFCNs. Because of evidence suggesting that the NGF transport defect was intra-axonal, we explored within cholinergic axons the status of early endosomes (EEs). NGF-containing EEs were enlarged in Ts65Dn mice and their App content was increased. Our study thus provides evidence for a pathogenic mechanism for DS in which increased expression of App, in the context of trisomy, causes abnormal transport of NGF and cholinergic neurodegeneration.

    Funded by: NIA NIH HHS: AG16999; NINDS NIH HHS: NS38869; PHS HHS: 31498

    Neuron 2006;51;1;29-42

  • Synapse formation and function is modulated by the amyloid precursor protein.

    Priller C, Bauer T, Mitteregger G, Krebs B, Kretzschmar HA and Herms J

    Zentrum für Neuropathologie und Prion Forschung, Ludwig Maximilians Universität, 81377 Munich, Germany.

    The amyloid precursor protein (APP) is critical in the pathogenesis of Alzheimer's disease. The question of its normal biological function in neurons, in which it is predominantly located at synapses, is still unclear. Using autaptic cultures of hippocampal neurons, we demonstrate that hippocampal neurons lacking APP show significantly enhanced amplitudes of evoked AMPA- and NMDA-receptor-mediated EPSCs. The size of the readily releasable synaptic vesicle pool was also increased in neurons lacking APP, whereas the release probability was not affected. In addition, the analysis of spontaneous miniature synaptic currents revealed an augmented frequency in neurons lacking APP, whereas the amplitude of miniature synaptic currents was not found to be altered. Together, these findings strongly indicate that lack of APP increases the number of functional synapses. This hypothesis is further supported by morphometric immunohistochemical analysis revealing an increase of synaptophysin-positive puncta per cultured APP knock-out neuron. In conclusion, lack of APP affects synapse formation and transmission in cultured hippocampal neurons.

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2006;26;27;7212-21

  • Keratinocytes from APP/APLP2-deficient mice are impaired in proliferation, adhesion and migration in vitro.

    Siemes C, Quast T, Kummer C, Wehner S, Kirfel G, Müller U and Herzog V

    Institute of Cell Biology and Bonner Forum Biomedizin, University of Bonn, Ulrich-Haberlandstr. 61A, 53121 Bonn, Germany.

    Growing evidence shows that the soluble N-terminal form (sAPPalpha) of the amyloid precursor protein (APP) represents an epidermal growth factor fostering keratinocyte proliferation, migration and adhesion. APP is a member of a protein family including the two mammalian amyloid precursor-like proteins APLP1 and APLP2. In the mammalian epidermis, only APP and APLP2 are expressed. APP and APLP2-deficient mice die shortly after birth but do not display a specific epidermal phenotype. In this report, we investigated the epidermis of APP and/or APLP2 knockout mice. Basal keratinocytes showed reduced proliferation in vivo by about 40%. Likewise, isolated keratinocytes exhibited reduced proliferation rates in vitro, which could be completely rescued by either exogenously added recombinant sAPPalpha, or by co-culture with dermal fibroblasts derived from APP knockout mice. Moreover, APP-knockout keratinocytes revealed reduced migration velocity resulting from severely compromised cell substrate adhesion. Keratinocytes from double knockout mice died within the first week of culture, indicating essential functions of APP-family members for survival in vitro. Our data indicate that sAPPalpha has to be considered as an essential epidermal growth factor which, however, in vivo can be functionally compensated to a certain extent by other growth factors, e.g., factors released from dermal fibroblasts.

    Experimental cell research 2006;312;11;1939-49

  • Neprilysin-sensitive synapse-associated amyloid-beta peptide oligomers impair neuronal plasticity and cognitive function.

    Huang SM, Mouri A, Kokubo H, Nakajima R, Suemoto T, Higuchi M, Staufenbiel M, Noda Y, Yamaguchi H, Nabeshima T, Saido TC and Iwata N

    Laboratory for Proteolytic Neuroscience, RIKEN Brain Science Institute, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan.

    A subtle but chronic alteration in metabolic balance between amyloid-beta peptide (Abeta) anabolic and catabolic activities is thought to cause Abeta accumulation, leading to a decade-long pathological cascade of Alzheimer disease. However, it is still unclear whether a reduction of the catabolic activity of Abeta in the brain causes neuronal dysfunction in vivo. In the present study, to clarify a possible connection between a reduction in neprilysin activity and impairment of synaptic and cognitive functions, we cross-bred amyloid precursor protein (APP) transgenic mice (APP23) with neprilysin-deficient mice and biochemically and immunoelectron-microscopically analyzed Abeta accumulation in the brain. We also examined hippocampal synaptic plasticity using an in vivo recording technique and cognitive function using a battery of learning and memory behavior tests, including Y-maze, novel-object recognition, Morris water maze, and contextual fear conditioning tests at the age of 13-16 weeks. We present direct experimental evidence that reduced activity of neprilysin, the major Abeta-degrading enzyme, in the brain elevates oligomeric forms of Abeta at the synapses and leads to impaired hippocampal synaptic plasticity and cognitive function before the appearance of amyloid plaque load. Thus, reduced neprilysin activity appears to be a causative event that is at least partly responsible for the memory-associated symptoms of Alzheimer disease. This supports the idea that a strategy to reduce Abeta oligomers in the brain by up-regulating neprilysin activity would contribute to alleviation of these symptoms.

    The Journal of biological chemistry 2006;281;26;17941-51

  • ADAM10 activation is required for green tea (-)-epigallocatechin-3-gallate-induced alpha-secretase cleavage of amyloid precursor protein.

    Obregon DF, Rezai-Zadeh K, Bai Y, Sun N, Hou H, Ehrhart J, Zeng J, Mori T, Arendash GW, Shytle D, Town T and Tan J

    Neuroimmunology Laboratory, Silver Child Development Center, Department of Psychiatry and Behavioral Medicine, The Byrd Alzheimer's Center and Research Institute, 3515 E. Fletcher Avenue, Tampa, FL 33647, USA.

    Recently, we have shown that green tea polyphenol (-)-epigallocatechin-3-gallate (EGCG) exerts a beneficial role on reducing brain Abeta levels, resulting in mitigation of cerebral amyloidosis in a mouse model of Alzheimer disease. EGCG seems to accomplish this by modulating amyloid precursor protein (APP) processing, resulting in enhanced cleavage of the alpha-COOH-terminal fragment (alpha-CTF) of APP and corresponding elevation of the NH(2)-terminal APP product, soluble APP-alpha (sAPP-alpha). These beneficial effects were associated with increased alpha-secretase cleavage activity, but no significant alteration in beta-or gamma-secretase activities. To gain insight into the molecular mechanism whereby EGCG modulates APP processing, we evaluated the involvement of three candidate alpha-secretase enzymes, a-disintegrin and metalloprotease (ADAM) 9, 10, or 17, in EGCG-induced non-amyloidogenic APP metabolism. Results show that EGCG treatment of N2a cells stably transfected with "Swedish" mutant human APP (SweAPP N2a cells) leads to markedly elevated active ( approximately 60 kDa mature form) ADAM10 protein. Elevation of active ADAM10 correlates with increased alpha-CTF cleavage, and elevated sAPP-alpha. To specifically test the contribution of ADAM10 to non-amyloidogenic APP metabolism, small interfering RNA knockdown of ADAM9, -10, or -17 mRNA was employed. Results show that ADAM10 (but not ADAM9 or -17) is critical for EGCG-mediated alpha-secretase cleavage activity. In summary, ADAM10 activation is necessary for EGCG promotion of non-amyloidogenic (alpha-secretase cleavage) APP processing. Thus, ADAM10 represents an important pharmacotherapeutic target for the treatment of cerebral amyloidosis in Alzheimer disease.

    The Journal of biological chemistry 2006;281;24;16419-27

  • Genetically augmenting Abeta42 levels in skeletal muscle exacerbates inclusion body myositis-like pathology and motor deficits in transgenic mice.

    Kitazawa M, Green KN, Caccamo A and LaFerla FM

    Department of Neurobiology and Behavior, 1109 Gillespie Neuroscience Facility, University of California, Irvine, Irvine, CA 92697-4545, USA.

    The pathogenic basis of inclusion body myositis (IBM), the leading muscle degenerative disease afflicting the elderly, is unknown, although the histopathological features are remarkably similar to those observed in Alzheimer's disease. One leading hypothesis is that the buildup of amyloid-beta (Abeta) peptide within selective skeletal muscle fibers contributes to the degenerative phenotype. Abeta is a small peptide derived via endoproteolysis of the amyloid precursor protein (APP). To determine the pathogenic effect of augmenting Abeta42 levels in skeletal muscle, we used a genetic approach to replace the endogenous wild-type presenilin-1 (PS1) allele with the PS1(M146V) allele in MCK-APP mice. Although APP transgene expression was unaltered, Abeta levels, particularly Abeta42, were elevated in skeletal muscle of the double transgenic (MCK-APP/PS1) mice compared to the parental MCK-APP line. Elevated phospho-tau accumulation was found in the MCK-APP/PS1 mice, and the greater activation of GSK-3beta and cdk5 were observed. Other IBM-like pathological features, such as inclusion bodies and inflammatory infiltrates, were more severe and prominent in the MCK-APP/PS1 mice. Motor coordination and balance were more adversely affected and manifested at an earlier age in the MCK-APP/PS1 mice. The data presented here provide experimental evidence that Abeta42 plays a proximal and critical role in the muscle degenerative process.

    Funded by: NIA NIH HHS: AG 20335, R01 AG020335

    The American journal of pathology 2006;168;6;1986-97

  • Role of APP phosphorylation in FE65-dependent gene transactivation mediated by AICD.

    Nakaya T and Suzuki T

    Laboratory of Neuroscience, Graduate School of Pharmaceutical Sciences, Hokkaido University, Kita-12 Nishi-6, Kita-ku, Sapporo 060-0812, Japan.

    Consecutive cleavages of Alzheimer's amyloid beta-protein precursor (APP) generate intracellular domain fragment (AICD). Interaction of APP and/or AICD with the adaptor protein FE65 is thought to modulate the metabolism of APP and the function of AICD. Phosphorylation or amino acid substitution of APP and AICD at threonine 668 (Thr668) suppresses their association with FE65. Here, we analyzed the function of APP and AICD phosphorylation in the nuclear translocation of FE65. In brain, AICD was present as phosphorylated and non-phosphorylated forms with non-phosphorylated AICD being dominantly detected in the nucleus. However, a mutant AICD (AICDa), in which Thr668 of AICD was replaced with Ala, was also mostly localized to the nucleus. These observations indicate that phosphorylation of AICD does not regulate the translocation of FE65 and that FE65 does not accompany AICD into the nucleus. APP was known to tether FE65 to the membrane. We found that phosphorylation of APP liberated membrane-bound FE65, which was then translocated into the nucleus where it up-regulated gene transactivation mediated by AICD, which was translocated into the nucleus independently of FE65. Therefore, phosphorylation of APP but not AICD modulates FE65-dependent gene transactivation mediated by AICD through the regulation of FE65 intracellular localization.

    Genes to cells : devoted to molecular & cellular mechanisms 2006;11;6;633-45

  • Nonprogressive transgene-related callosal and hippocampal changes in PDAPP mice.

    Valla J, Schneider LE, Gonzalez-Lima F and Reiman EM

    Barrow Neurological Institute, St Joseph's Hospital & Medical Center, Phoenix, Arizona 85013, USA. jvalla@chw.edu

    We have previously shown that homozygous PDAPP mice, a transgenic model of Alzheimer's-like amyloidosis, have abnormal corpus callosi and anterior hippocampi. Now, we investigated the extent to which these morphological abnormalities are correlated with mutant gene dose in a larger, independent, and substantially younger cohort. Homozygous and heterozygous PDAPP mice had significantly smaller callosal commissure length and anterior hippocampal area than controls. Reductions correlated with mutant APP gene dose, with homozygotes showing the greatest reduction, and were present at 2 months of age. These findings and previous work with APP knockouts suggest that PDAPP mice have impaired white matter development due to interference with native murine APP.

    Funded by: NIA NIH HHS: P30 AG019610

    Neuroreport 2006;17;8;829-32

  • PKCepsilon increases endothelin converting enzyme activity and reduces amyloid plaque pathology in transgenic mice.

    Choi DS, Wang D, Yu GQ, Zhu G, Kharazia VN, Paredes JP, Chang WS, Deitchman JK, Mucke L and Messing RO

    Ernest Gallo Clinic and Research Center, Emeryville, CA 94608, USA.

    Deposition of plaques containing amyloid beta (Abeta) peptides is a neuropathological hallmark of Alzheimer's disease (AD). Here we demonstrate that neuronal overexpression of the epsilon isozyme of PKC decreases Abeta levels, plaque burden, and plaque-associated neuritic dystrophy and reactive astrocytosis in transgenic mice expressing familial AD-mutant forms of the human amyloid precursor protein (APP). Compared with APP singly transgenic mice, APP/PKCepsilon doubly transgenic mice had decreased Abeta levels but showed no evidence for altered cleavage of APP. Instead, PKCepsilon overexpression selectively increased the activity of endothelin-converting enzyme, which degrades Abeta. The activities of other Abeta-degrading enzymes, insulin degrading enzyme and neprilysin, were unchanged. These results indicate that increased neuronal PKCepsilon activity can promote Abeta clearance and reduce AD neuropathology through increased endothelin-converting enzyme activity.

    Funded by: NIA NIH HHS: AG022074, AG05834, AG11385, F32 AG005834, P01 AG022074, R01 AG011385, R37 AG011385; NIAAA NIH HHS: AA013588, R01 AA013588, R37 AA013588; NINDS NIH HHS: NS41787, R01 NS041787

    Proceedings of the National Academy of Sciences of the United States of America 2006;103;21;8215-20

  • Parkin protects against mitochondrial toxins and beta-amyloid accumulation in skeletal muscle cells.

    Rosen KM, Veereshwarayya V, Moussa CE, Fu Q, Goldberg MS, Schlossmacher MG, Shen J and Querfurth HW

    Department of Neurology, Caritas St. Elizabeth's Medical Center, Tufts University School of Medicine, Boston, Massachusetts 02135, USA. kenneth_rosen@cchcs.org

    Mutations in the ubiquitin ligase-encoding Parkin gene have been implicated in the pathogenesis of autosomal recessive Parkinson disease. Outside of the central nervous system, Parkin is prominently expressed in skeletal muscle. We have found accumulations of Parkin protein in skeletal muscle biopsies taken from patients with inclusion body myositis, a degenerative disorder in which intramyofiber accumulations of the beta-amyloid peptide are pathognomonic. In comparing primary cultures of skeletal muscle derived from parkin knock-out and wild-type mice, we have found the absence of parkin to result in greater sensitivity to mitochondrial stressors rotenone and carbonyl cyanide 3-chlorophenylhydrazone, without any alteration in sensitivity to calcium ionophore or hydrogen peroxide. Utilizing viral expression constructs coding for the Alzheimer disease and inclusion body myositis-linked beta-amyloid precursor protein and for its metabolic byproducts A beta42 and C100, we found that parkin knock-out muscle cells are also more sensitive to the toxic effects of intracellular A beta. We also constructed a lentiviral system to overexpress wild-type Parkin and have shown that boosting the levels of parkin expression in normal skeletal muscle cultures provides substantial protection against both mitochondrial toxins and overexpressed beta-amyloid. Correspondingly, exogenous Parkin significantly lowered A beta levels. These data support the hypothesis that in myocytes parkin has dual properties in the maintenance of skeletal muscle mitochondrial homeostasis and in the regulation of A beta levels.

    Funded by: NINDS NIH HHS: NS041373

    The Journal of biological chemistry 2006;281;18;12809-16

  • Reversal of Alzheimer's-like pathology and behavior in human APP transgenic mice by mutation of Asp664.

    Galvan V, Gorostiza OF, Banwait S, Ataie M, Logvinova AV, Sitaraman S, Carlson E, Sagi SA, Chevallier N, Jin K, Greenberg DA and Bredesen DE

    Buck Institute for Age Research, 8001 Redwood Boulevard, Novato, CA 94945, USA.

    The deficits characteristic of Alzheimer's disease (AD) are believed to result, at least in part, from the neurotoxic effects of beta-amyloid peptides, a set of 39-43 amino acid fragments derived proteolytically from beta-amyloid precursor protein (APP). APP also is cleaved intracytoplasmically at Asp-664 to generate a second cytotoxic peptide, APP-C31, but whether this C-terminal processing of APP plays a role in the pathogenesis of AD is unknown. Therefore, we compared elements of the Alzheimer's phenotype in transgenic mice modeling AD with vs. without a functional Asp-664 caspase cleavage site. Surprisingly, whereas beta-amyloid production and plaque formation were unaltered, synaptic loss, astrogliosis, dentate gyral atrophy, increased neuronal precursor proliferation, and behavioral abnormalities were completely prevented by a mutation at Asp-664. These results suggest that Asp-664 plays a critical role in the generation of Alzheimer-related pathophysiological and behavioral changes in human APP transgenic mice, possibly as a cleavage site or via protein-protein interactions.

    Funded by: NIA NIH HHS: AG05131, P50 AG005131; NINDS NIH HHS: NS45093, R01 NS045093

    Proceedings of the National Academy of Sciences of the United States of America 2006;103;18;7130-5

  • The endogenous estrogen status regulates microglia reactivity in animal models of neuroinflammation.

    Vegeto E, Belcredito S, Ghisletti S, Meda C, Etteri S and Maggi A

    Center of Excellence on Neurodegenerative Diseases and Department of Pharmacological Sciences, University of Milan, Italy. elisabetta.vegeto@unimi.it

    It has been previously demonstrated that 17beta-estradiol (E(2)) inhibits the response of microglia, the resident brain macrophages, to acute injuries in specific brain regions. We here show that the effect of E(2) in acute brain inflammation is widespread and that the hormone reduces the expression of inflammatory mediators, such as monocyte chemoattractant protein-1, macrophage inflammatory protein-2, and TNF-alpha, induced by lipopolysaccharide, demonstrating that microglia are a direct target of estrogen action in brain. Using the APP23 mice, an animal model of Alzheimer's disease reproducing chronic neuroinflammation, we demonstrate that ovary ablation increases microglia activation at beta-amyloid (Abeta) deposits and facilitates the progression of these cells toward a highly reactive state. Long-term administration of E(2) reverts the effects of ovariectomy and decreases microglia reactivity compared with control animals. In this animal model, these events do not correlate with a reduced number of Abeta deposits. Finally, we show that E(2) inhibits Abeta-induced expression of scavenger receptor-A in macrophage cells, providing a mechanism for the effect of E(2) on Abeta signaling observed in the APP23 mice. Altogether, our observations reveal a substantial involvement of endogenous estrogen in neuroinflammatory processes and provide novel mechanisms for hormone action in the brain.

    Funded by: Telethon: GP0127Y01

    Endocrinology 2006;147;5;2263-72

  • Beta-amyloid mediated nitration of manganese superoxide dismutase: implication for oxidative stress in a APPNLH/NLH X PS-1P264L/P264L double knock-in mouse model of Alzheimer's disease.

    Anantharaman M, Tangpong J, Keller JN, Murphy MP, Markesbery WR, Kiningham KK and St Clair DK

    Graduate Center for Toxicology, University of Kentucky, Lexington 40536-0305, USA.

    Alzheimer's disease is a multifactorial, progressive, age-related neurodegenerative disease. In familial Alzheimer's disease, Abeta is excessively produced and deposited because of mutations in the amyloid precursor protein, presenilin-1, and presenilin-2 genes. Here, we generated a double homozygous knock-in mouse model that incorporates the Swedish familial Alzheimer's disease mutations and converts mouse Abeta to the human sequence in amyloid precursor protein and had the P264L familial Alzheimer's disease mutation in presenilin-1. We observed Abeta deposition in double knock-in mice beginning at 6 months as well as an increase in the levels of insoluble Abeta1-40/1-42. Brain homogenates from 3-, 6-, 9-, 12-, and 14-month-old mice showed that protein levels of manganese superoxide dismutase (MnSOD) were unchanged in the double knock-in mice compared to controls. Genotype-associated increases in nitrotyrosine levels were observed. Protein immunoprecipitation revealed MnSOD as a target of this nitration. Although the levels of MnSOD protein did not change, MnSOD activity and mitochondrial respiration decreased in knock-in mice, suggesting compromised mitochondrial function. The compromised activity of MnSOD, a primary antioxidant enzyme protecting mitochondria, may explain mitochondrial dysfunction and provide the missing link between Abeta-induced oxidative stress and Alzheimer's disease.

    Funded by: NIA NIH HHS: AG-05119, P01 AG005119

    The American journal of pathology 2006;168;5;1608-18

  • Overexpression of Abeta is associated with acceleration of onset of motor impairment and superoxide dismutase 1 aggregation in an amyotrophic lateral sclerosis mouse model.

    Li QX, Mok SS, Laughton KM, McLean CA, Volitakis I, Cherny RA, Cheung NS, White AR and Masters CL

    Department of Pathology, The University of Melbourne, and The Mental Health Research Institute of Victoria, Parkville, Vic. 3010, Australia. q.li@unimelb.edu.au

    Transgenic mice carrying mutant Cu/Zn superoxide dismutase (SOD1) recapitulate the motor impairment of human amyotrophic lateral sclerosis (ALS). The amyloid-beta (Abeta) peptide associated with Alzheimer's disease is neurotoxic. To investigate the potential role of Abeta in ALS development, we generated a double transgenic mouse line that overexpresses SOD1(G93A) and amyloid precursor protein (APP)-C100. The transgenic mouse C100.SOD1(G93A) overexpresses Abeta and shows earlier onset of motor impairment but has the same lifespan as the single transgenic SOD1(G93A) mouse. To determine the mechanism associated with this early-onset phenotype, we measured copper and zinc levels in brain and spinal cord and found both significantly elevated in the single and double transgenic mice compared with their littermate control mice. Increased glial fibrillary acidic protein and decreased APP levels in the spinal cord of C100.SOD1(G93A) mice compared with the SOD1(G93A) mice agree with the neuronal damage observed by immunohistochemical analysis. In the spinal cords of C100.SOD1(G93A) double transgenic mice, soluble Abeta was elevated in mice at end-stage disease compared with the pre-symptomatic stage. Buffer-insoluble SOD1 aggregates were significantly elevated in the pre-symptomatic mice of C100.SOD1(G93A) compared with the age-matched SOD1(G93A) mice, correlating with the earlier onset of motor impairment in the C100.SOD1(G93A) mice. This study supports abnormal SOD1 protein aggregation as the pathogenic mechanism in ALS, and implicates a potential role for Abeta in the development of ALS by exacerbating SOD1(G93A) aggregation.

    Aging cell 2006;5;2;153-65

  • Progressive neuronal loss and behavioral impairments of transgenic C57BL/6 inbred mice expressing the carboxy terminus of amyloid precursor protein.

    Lee KW, Im JY, Song JS, Lee SH, Lee HJ, Ha HY, Koh JY, Gwag BJ, Yang SD, Paik SG and Han PL

    Department of Neuroscience, Neuroscience Research Center and Medical Research Institute, Ewha Womans University School of Medicine, 911-1 Mok-6-Dong, Yangchun-Gu, Seoul 158-710, South Korea.

    The beta-secretase cleaved Abeta-bearing carboxy-terminal fragments (betaCTFs) of amyloid precursor protein (APP) in neural cells have been suggested to be cytotoxic. However, the functional significance of betaCTFs in vivo remains elusive. We created a transgenic mouse line Tg-betaCTF99/B6 expressing the human betaCTF99 in the brain of inbred C57BL/6 strain. Tg-betaCTF99/B6 mouse brain at 12-16 months showed severely down-regulated calbindin, phospho-CREB, and Bcl-xL expression and up-regulated phospho-JNK, Bcl-2, and Bax expression. Neuronal cell density in the Tg-betaCTF99/B6 cerebral cortex at 16-18 months was lower than that of the non-transgenic control, but not at 5 months. At 11-14 months, Tg-betaCTF99/B6 mice displayed cognitive impairments and increased anxiety, which were not observed at 5 months. These results suggest that increased betaCTF99 expression is highly detrimental to the aging brain and that it produces a progressive and age-dependent AD-like pathogenesis.

    Neurobiology of disease 2006;22;1;10-24

  • Abeta-induced meningoencephalitis is IFN-gamma-dependent and is associated with T cell-dependent clearance of Abeta in a mouse model of Alzheimer's disease.

    Monsonego A, Imitola J, Petrovic S, Zota V, Nemirovsky A, Baron R, Fisher Y, Owens T and Weiner HL

    National Institute of Biotechnology and Department of Microbiology and Immunology, Faculty of Health Sciences, Ben-Gurion University, Beer-Sheva 84105, Israel.

    Vaccination against amyloid beta-peptide (Abeta) has been shown to be successful in reducing Abeta burden and neurotoxicity in mouse models of Alzheimer's disease (AD). However, although Abeta immunization did not show T cell infiltrates in the brain of these mice, an Abeta vaccination trial resulted in meningoencephalitis in 6% of patients with AD. Here, we explore the characteristics and specificity of Abeta-induced, T cell-mediated encephalitis in a mouse model of the disease. We demonstrate that a strong Abeta-specific T cell response is critically dependent on the immunizing T cell epitope and that epitopes differ depending on MHC genetic background. Moreover, we show that a single immunization with the dominant T cell epitope Abeta10-24 induced transient meningoencephalitis only in amyloid precursor protein (APP)-transgenic (Tg) mice expressing limited amounts of IFN-gamma under an myelin basic protein (MBP) promoter. Furthermore, immune infiltrates were targeted primarily to sites of Abeta plaques in the brain and were associated with clearance of Abeta. Immune infiltrates were not targeted to the spinal cord, consistent with what was observed in AD patients vaccinated with Abeta. Using primary cultures of microglia, we show that IFN-gamma enhanced clearance of Abeta, microglia, and T cell motility, and microglia-T cell immunological synapse formation. Our study demonstrates that limited expression of IFN-gamma in the brain, as observed during normal brain aging, is essential to promote T cell-mediated immune infiltrates after Abeta immunization and provides a model to investigate both the beneficial and detrimental effects of Abeta-specific T cells.

    Proceedings of the National Academy of Sciences of the United States of America 2006;103;13;5048-53

  • Secretion of the Notch-1 Abeta-like peptide during Notch signaling.

    Okochi M, Fukumori A, Jiang J, Itoh N, Kimura R, Steiner H, Haass C, Tagami S and Takeda M

    Department of Post-Genomics and Diseases, Division of Psychiatry and Behavioral Proteomics, Osaka University Graduate School of Medicine, Osaka 565-0871, Japan. mokochi@psy.med.osaka-u.ac.jp

    The canonical pathway of Notch signaling is mediated by regulated intramembrane proteolysis (RIP). In the pathway, ligand binding results in sequential proteolysis of the Notch receptor, and presenilin (PS)-dependent intramembrane proteolysis at the interface between the membrane and cytosol liberates the Notch-1 intracellular domain (NICD), a transcription modifier. Because the degradation of the Notch-1 transmembrane domain is thought to require an additional cleavage near the middle of the transmembrane domain, extracellular small peptides (Notch-1 Abeta-like peptide (Nbeta)) should be produced. Here we showed that Nbeta species are indeed secreted during the process of Notch signaling. We identified mainly two distinct molecular species of novel Nbeta, Nbeta21 and C-terminally elongated Nbeta25, which were produced in an approximately 5:1 ratio. This process is reminiscent of the production of Alzheimer disease-associated Abeta. PS pathogenic mutants increased the production of the longer species of Abeta (Abeta42) from beta-amyloid protein precursor. We revealed that several Alzheimer disease mutants also cause a parallel increase in the secretion of the longer form of Nbeta. Strikingly, chemicals that modify the Abeta42 level caused parallel changes in the Nbeta25 level. These results demonstrated that the characteristics of C-terminal elongation of Nbeta and Abeta are almost identical. In addition, because many other type 1 membrane-bound receptors release intracellular domains by PS-dependent intramembrane proteolysis, we suspect that the release of Abeta- or Nbeta-like peptides is a common feature of the proteolysis during RIP signaling. We anticipate that this study will open the door to searches for markers of RIP signaling and surrogate markers for Abeta42 production.

    The Journal of biological chemistry 2006;281;12;7890-8

  • The prolyl isomerase Pin1 regulates amyloid precursor protein processing and amyloid-beta production.

    Pastorino L, Sun A, Lu PJ, Zhou XZ, Balastik M, Finn G, Wulf G, Lim J, Li SH, Li X, Xia W, Nicholson LK and Lu KP

    Cancer Biology Program, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215, USA.

    Neuropathological hallmarks of Alzheimer's disease are neurofibrillary tangles composed of tau and neuritic plaques comprising amyloid-beta peptides (Abeta) derived from amyloid precursor protein (APP), but their exact relationship remains elusive. Phosphorylation of tau and APP on certain serine or threonine residues preceding proline affects tangle formation and Abeta production in vitro. Phosphorylated Ser/Thr-Pro motifs in peptides can exist in cis or trans conformations, the conversion of which is catalysed by the Pin1 prolyl isomerase. Pin1 has been proposed to regulate protein function by accelerating conformational changes, but such activity has never been visualized and the biological and pathological significance of Pin1 substrate conformations is unknown. Notably, Pin1 is downregulated and/or inhibited by oxidation in Alzheimer's disease neurons, Pin1 knockout causes tauopathy and neurodegeneration, and Pin1 promoter polymorphisms appear to associate with reduced Pin1 levels and increased risk for late-onset Alzheimer's disease. However, the role of Pin1 in APP processing and Abeta production is unknown. Here we show that Pin1 has profound effects on APP processing and Abeta production. We find that Pin1 binds to the phosphorylated Thr 668-Pro motif in APP and accelerates its isomerization by over 1,000-fold, regulating the APP intracellular domain between two conformations, as visualized by NMR. Whereas Pin1 overexpression reduces Abeta secretion from cell cultures, knockout of Pin1 increases its secretion. Pin1 knockout alone or in combination with overexpression of mutant APP in mice increases amyloidogenic APP processing and selectively elevates insoluble Abeta42 (a major toxic species) in brains in an age-dependent manner, with Abeta42 being prominently localized to multivesicular bodies of neurons, as shown in Alzheimer's disease before plaque pathology. Thus, Pin1-catalysed prolyl isomerization is a novel mechanism to regulate APP processing and Abeta production, and its deregulation may link both tangle and plaque pathologies. These findings provide new insight into the pathogenesis and treatment of Alzheimer's disease.

    Funded by: NIA NIH HHS: R01 AG029385

    Nature 2006;440;7083;528-34

  • Comparative analysis of cortical gene expression in mouse models of Alzheimer's disease.

    Wu ZL, Ciallella JR, Flood DG, O'Kane TM, Bozyczko-Coyne D and Savage MJ

    Cephalon, Inc., Neurobiology, 145 Brandywine Parkway, West Chester, PA 19380-4245, USA. zwu@cephalon.com

    Three mouse models of Alzheimer's disease (AD) were used to assess changes in gene expression potentially critical to amyloid beta-peptide (Abeta)-induced neuronal dysfunction. One mouse model harbored homozygous familial AD (FAD) knock-in mutations in both, amyloid precursor protein (APP) and presenilin 1 (PS-1) genes (APP(NLh/NLh)/PS-1(P264L/P264L)), the other two models harbored APP over-expression of FAD mutations (Tg2576) with the PS-1 knock-in mutation at either one or two alleles. These mouse models of AD had varying levels of Abeta40 and Abeta42 and different latencies and rates of Abeta deposition in brain. To assess changes in gene expression associated with Abeta accumulation, the Affymetrix murine genome array U74A was used to survey gene expression in the cortex of these three models both prior to and following Abeta deposition. Altered genes were identified by comparing the AD models with age-matched control littermates. Thirty-four gene changes were identified in common among the three models in mice with Abeta deposition. Among the up-regulated genes, three major classes were identified that encoded for proteins involved in immune responses, carbohydrate metabolism, and proteolysis. Down-regulated genes of note included pituitary adenylate cyclase-activating peptide (PACAP), brain-derived neurotrophic factor (BDNF), and insulin-like growth factor I receptor (IGF-IR). In young mice without detectable Abeta deposition, there were no regulated genes common among the three models, although 40 genes were similarly altered between the two Tg2576 models with the PS-1 FAD knock-in. Finally, changes in gene expression among the three mouse models of AD were compared with those reported in human AD samples. Sixty-nine up-regulated and 147 down-regulated genes were found in common with human AD brain. These comparisons across different genetic mouse models of AD and human AD brain provide greater support for the involvement of identified gene expression changes in the neuronal dysfunction and cognitive deficits accompanying amyloid deposition in mammalian brain.

    Neurobiology of aging 2006;27;3;377-86

  • Mutations in amyloid precursor protein and presenilin-1 genes increase the basal oxidative stress in murine neuronal cells and lead to increased sensitivity to oxidative stress mediated by amyloid beta-peptide (1-42), HO and kainic acid: implications for Alzheimer's disease.

    Mohmmad Abdul H, Sultana R, Keller JN, St Clair DK, Markesbery WR and Butterfield DA

    Department of Chemistry and Center of Membrane Sciences, University of Kentucky, Lexington, Kentucky 40506, USA.

    Oxidative stress is observed in Alzheimer's disease (AD) brain, including protein oxidation and lipid peroxidation. One of the major pathological hallmarks of AD is the brain deposition of amyloid beta-peptide (Abeta). This 42-mer peptide is derived from the beta-amyloid precursor protein (APP) and is associated with oxidative stress in vitro and in vivo. Mutations in the PS-1 and APP genes, which increase production of the highly amyloidogenic amyloid beta-peptide (Abeta42), are the major causes of early onset familial AD. Several lines of evidence suggest that enhanced oxidative stress, inflammation, and apoptosis play important roles in the pathogenesis of AD. In the present study, primary neuronal cultures from knock-in mice expressing mutant human PS-1 and APP were compared with those from wild-type mice, in the presence or absence of various oxidizing agents, viz, Abeta(1-42), H2O2 and kainic acid (KA). APP/PS-1 double mutant neurons displayed a significant basal increase in oxidative stress as measured by protein oxidation, lipid peroxidation, and 3-nitrotyrosine when compared with the wild-type neurons (p < 0.0005). Elevated levels of human APP, PS-1 and Abeta(1-42) were found in APP/PS-1 cultures compared with wild-type neurons. APP/PS-1 double mutant neuron cultures exhibited increased vulnerability to oxidative stress, mitochondrial dysfunction and apoptosis induced by Abeta(1-42), H2O2 and KA compared with wild-type neuronal cultures. The results are consonant with the hypothesis that Abeta(1-42)-associated oxidative stress and increased vulnerability to oxidative stress may contribute significantly to neuronal apoptosis and death in familial early onset AD.

    Funded by: NIA NIH HHS: AG-05119, AG-10836

    Journal of neurochemistry 2006;96;5;1322-35

  • Pathogenic accumulation of APP in fast twitch muscle of IBM patients and a transgenic model.

    Sugarman MC, Kitazawa M, Baker M, Caiozzo VJ, Querfurth HW and LaFerla FM

    Department of Neurobiology and Behavior, University of California, 1109 Gillespie Neuroscience Facility, Irvine, CA 92697-4545, USA.

    Inclusion body myositis (IBM) is the most common age-related degenerative skeletal muscle disorder. The aberrant intracellular accumulation of the beta-amyloid (Abeta) peptide within skeletal muscle is a pathological hallmark of IBM. Skeletal muscle is comprised of both slow and fast twitch fibers, which are present in different proportions in various muscles. It remains unclear if fast and/or slow twitch fibers are differentially involved in IBM pathogenesis. To better understand the molecular pathogenesis of IBM, we analyzed human IBM muscle biopsies and muscle from a transgenic mouse model of IBM (MCK-betaAPP). Here we report that the majority of histopathologically-affected fibers in human IBM biopsies were type II fast fibers. Skeletal muscle from MCK-betaAPP mice exhibited higher transgene expression and steady-state levels of human betaAPP in fast type IIB fibers compared to slow type I fibers. These findings indicate that fast twitch fibers may selectively accumulate and be more vulnerable to betaAPP- and Abeta-mediated damage in IBM. These findings also highlight parallels between the MCK-betaAPP mice and the human IBM condition.

    Funded by: NIA NIH HHS: AG 26175; NIAMS NIH HHS: AR 46856; NINDS NIH HHS: NS 41373

    Neurobiology of aging 2006;27;3;423-32

  • AMPA receptor downscaling at the onset of Alzheimer's disease pathology in double knockin mice.

    Chang EH, Savage MJ, Flood DG, Thomas JM, Levy RB, Mahadomrongkul V, Shirao T, Aoki C and Huerta PT

    Center for Neural Science, New York University, NY 10003, USA.

    It is widely thought that Alzheimer's disease (AD) begins as a malfunction of synapses, eventually leading to cognitive impairment and dementia. Homeostatic synaptic scaling is a mechanism that could be crucial at the onset of AD but has not been examined experimentally. In this process, the synaptic strength of a neuron is modified so that the overall excitability of the cell is maintained. Here, we investigate whether synaptic scaling mediated by l-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) contributes to pathology in double knockin (2 x KI) mice carrying human mutations in the genes for amyloid precursor protein and presenilin-1. By using whole-cell recordings, we show that 2 x KI mice exhibit age-related downscaling of AMPAR-mediated evoked currents and spontaneous, miniature currents. Electron microscopic analysis further corroborates the synaptic AMPAR decrease. Additionally, 2 x KI mice show age-related deficits in bidirectional plasticity (long-term potentiation and long-term depression) and memory flexibility. These results suggest that AMPARs are important synaptic targets for AD and provide evidence that cognitive impairment may involve downscaling of postsynaptic AMPAR function.

    Funded by: NEI NIH HHS: P30 EY013079-069003, R01 EY008055-09, R01 EY013145-04; NINDS NIH HHS: R01 NS041091-04

    Proceedings of the National Academy of Sciences of the United States of America 2006;103;9;3410-5

  • Molecular dissection of the interaction between amyloid precursor protein and its neuronal trafficking receptor SorLA/LR11.

    Andersen OM, Schmidt V, Spoelgen R, Gliemann J, Behlke J, Galatis D, McKinstry WJ, Parker MW, Masters CL, Hyman BT, Cappai R and Willnow TE

    Max-Delbrueck-Center for Molecular Medicine, Berlin, 13125 Berlin, Germany. o.andersen@mdc-berlin.de

    SorLA/LR11 is a sorting receptor that regulates the intracellular transport and processing of the amyloid precursor protein (APP) in neurons. SorLA/LR11-mediated binding results in sequestration of APP in the Golgi and in protection from processing into the amyloid-beta peptide (Abeta), the principal component of senile plaques in Alzheimer's disease (AD). To gain insight into the molecular mechanisms governing sorLA and APP interaction, we have dissected the respective protein interacting domains. Using a fluorescence resonance energy transfer (FRET) based assay of protein proximity, we identified binding sites in the extracellular regions of both proteins. Fine mapping by surface plasmon resonance analysis and analytical ultracentrifugation of recombinant APP and sorLA fragments further narrowed down the binding domains to the cluster of complement-type repeats in sorLA that forms a 1:1 stoichiometric complex with the carbohydrate-linked domain of APP. These data shed new light on the molecular determinants of neuronal APP trafficking and processing and on possible targets for intervention with senile plaque formation in patients with AD.

    Biochemistry 2006;45;8;2618-28

  • Formation of tau inclusions in knock-in mice with familial Alzheimer disease (FAD) mutation of presenilin 1 (PS1).

    Tanemura K, Chui DH, Fukuda T, Murayama M, Park JM, Akagi T, Tatebayashi Y, Miyasaka T, Kimura T, Hashikawa T, Nakano Y, Kudo T, Takeda M and Takashima A

    Laboratory for Alzheimer Disease and Neural Architecture, Brain Science Institute, RIKEN, Wako, Saitama 351-0198, Japan.

    Mutations in the presenilin 1 (PS1) gene are responsible for the early onset of familial Alzheimer disease (FAD). Accumulating evidence shows that PS1 is involved in gamma-secretase activity and that FAD-associated mutations of PS1 commonly accelerate Abeta(1-42) production, which causes Alzheimer disease (AD). Recent studies suggest, however, that PS1 is involved not only in Abeta production but also in other processes that lead to neurodegeneration. To better understand the causes of neurodegeneration linked to the PS1 mutation, we analyzed the development of tau pathology, another key feature of AD, in PS1 knock-in mice. Hippocampal samples taken from FAD mutant (I213T) PS1 knock-in mice contained hyperphosphorylated tau that reacted with various phosphodependent tau antibodies and with Alz50, which recognizes the conformational change of PHF tau. Some neurons exhibited Congo red birefringence and Thioflavin T reactivity, both of which are histological criteria for neurofibrillary tangles (NFTs). Biochemical analysis of the samples revealed SDS-insoluble tau, which under electron microscopy examination, resembled tau fibrils. These results indicate that our mutant PS1 knock-in mice exhibited NFT-like tau pathology in the absence of Abeta deposition, suggesting that PS1 mutations contribute to the onset of AD not only by enhancing Abeta(1-42) production but by also accelerating the formation and accumulation of filamentous tau.

    The Journal of biological chemistry 2006;281;8;5037-41

  • Phospholipase D1 corrects impaired betaAPP trafficking and neurite outgrowth in familial Alzheimer's disease-linked presenilin-1 mutant neurons.

    Cai D, Zhong M, Wang R, Netzer WJ, Shields D, Zheng H, Sisodia SS, Foster DA, Gorelick FS, Xu H and Greengard P

    Laboratory of Molecular and Cellular Neuroscience, and Fisher Center for Research on Alzheimer Disease, The Rockefeller University, 1230 York Avenue, New York, NY 10021, USA.

    Presenilins (PS1/PS2) regulate proteolysis of beta-amyloid precursor protein (betaAPP) and affect its intracellular trafficking. Here, we demonstrate that a PS1-interacting protein, phospholipase D1 (PLD1), affects intracellular trafficking of betaAPP. Overexpression of PLD1 in PS1wt cells promotes generation of betaAPP-containing vesicles from the trans-Golgi network. Conversely, inhibition of PLD1 activity by 1-butanol decreases betaAPP trafficking in both wt and PS1-deficient cells. The subcellular localization of PLD1 is altered, and PLD enzymatic activity is reduced in cells expressing familial Alzheimer's disease (FAD) PS1 mutations compared with PS1wt cells. Overexpression of wt, but not catalytically inactive, PLD1 increases budding of betaAPP-containing vesicles from the trans-Golgi network in FAD mutant cells. Surface delivery of betaAPP is also increased by PLD1 in these cells. The impaired neurite outgrowth capacity in FAD mutant neurons was corrected by introducing PLD1 into these cells. The results indicate that PLD1 may represent a therapeutic target for rescuing compromised neuronal function in AD.

    Funded by: NCI NIH HHS: CA46677, R01 CA046677; NIA NIH HHS: 5F32AG023431, AG09464, F32 AG023431, P01 AG009464; NIDDK NIH HHS: DK21860, R01 DK021860; NINDS NIH HHS: NS046673, R01 NS046673

    Proceedings of the National Academy of Sciences of the United States of America 2006;103;6;1936-40

  • Stabilization of ubiquitous mitochondrial creatine kinase preprotein by APP family proteins.

    Li X, Bürklen T, Yuan X, Schlattner U, Desiderio DM, Wallimann T and Homayouni R

    Department of Anatomy and Neurobiology, University of Tennessee Health Science Center, Memphis, TN 38163, USA.

    Amyloid precursor protein (APP) is involved in the pathogenesis of Alzheimer's disease (AD). However, the physiological role of APP and its family members is still unclear. To gain insights into APP function, we used a proteomic approach to identify APP interacting proteins. We report here for the first time a direct interaction between the C-terminal region of APP family proteins and ubiquitous mitochondrial creatine kinase (uMtCK). This interaction was confirmed in vitro as well as in cultured cells and in brain. Interestingly, expression of full-length and C-terminal domain of APP family proteins stabilized uMtCK preprotein in cultured cells. Our data suggest that APP may regulate cellular energy levels and mitochondrial function via a direct interaction and stabilization of uMtCK.

    Funded by: NCRR NIH HHS: RR 10522

    Molecular and cellular neurosciences 2006;31;2;263-72

  • Transgenic mice over-expressing human beta-amyloid have functional nicotinic alpha 7 receptors.

    Spencer JP, Weil A, Hill K, Hussain I, Richardson JC, Cusdin FS, Chen YH and Randall AD

    Neurology and GI CEDD, GlaxoSmithKline, Harlow, Essex CM19 5AW, UK. jon_p_spencer@gsk.com

    A potentially major factor in the development of Alzheimer's disease is the enhanced production of soluble beta-amyloid peptide fragments amyloid beta peptide(1-40) and amyloid beta peptide(1-42). These amyloid peptides are generated by cleavage of the amyloid-precursor protein and aggregate spontaneously to form amyloid plaques, which are a classical pathological hallmark in Alzheimer's disease. Although the precise mechanisms are unknown, it is widely believed that amyloid peptides initiate the degenerative process, resulting in subsequent cognitive decline. One interaction of amyloid beta peptide that may contribute to an impairment of cognition is its high affinity binding to the alpha 7 nicotinic receptor; a receptor shown to be important for cognition in a number of studies. There is some controversy, however, whether amyloid beta peptide inhibits or activates this receptor. We have cloned and stably expressed the human alpha 7 receptor and investigated its interaction with amyloid beta peptide using patch clamp electrophysiology. Human alpha 7 was activated in a concentration-dependent fashion by nicotine, acetylcholine and choline and potently inhibited by methyllycaconitine citrate. The responses were inwardly rectifying and exhibited rapid activation, desensitization and deactivation. Amyloid beta peptide(1-42) antagonized human alpha7 responses in a partially reversible fashion; no agonist effects of amyloid beta peptide(1-42) were detected. A similar inhibition of mouse alpha 7 was also observed. In addition, we have assessed the function of native alpha 7 receptors in hippocampal slices prepared from transgenic mice that over-express human amyloid. Despite this clear inhibition of recombinant receptors, hippocampal GABAergic interneurones in slices from beta-amyloid over-expressing mice still possess alpha 7 receptor-mediated currents.

    Neuroscience 2006;137;3;795-805

  • An early specific cell-cell interaction occurs in the production of beta-amyloid in cell cultures.

    Dewji NN, Mukhopadhyay D and Singer SJ

    Department of Medicine and Stein Institute for Research on Aging, School of Medicine, University of California at San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA. ndewji@ucsd.edu

    We have earlier proposed that a cell-cell interaction, mediated by the specific binding of molecules of the beta-amyloid precursor protein (beta-APP) on one cell surface with molecules of presenilin (PS) on the other cell surface, is a required initial step in the ultimate production of beta-amyloid (Abeta) from beta-APP. Abeta is widely believed to be the neurotoxic agent in Alzheimer's disease. In this paper, we test this proposal by modifying cells to express surface beta-APP but no PS, and other cells to express surface PS but no beta-APP. Coculturing these two cell populations at appropriate cell densities produces substantial amounts of Abeta that appear both in cell extracts and culture media. Such Abeta production could occur only if the two cell types interacted with one another to provide the beta-APP and the PS required for the generation of Abeta. The addition to the coculture, from the start, of the soluble specific N-terminal domain of the appropriate PS significantly reduces the amount of Abeta produced. These and related experiments, therefore, suggest a very different mechanism for Abeta production than the one that is currently widely accepted.

    Funded by: NIA NIH HHS: 5R01 AG 17858; NINDS NIH HHS: 5R01 NS 044768, 5R01 NS 27580, P30 NS 047101, P30 NS047101, R01 NS044768

    Proceedings of the National Academy of Sciences of the United States of America 2006;103;5;1540-5

  • Essential roles for the FE65 amyloid precursor protein-interacting proteins in brain development.

    Guénette S, Chang Y, Hiesberger T, Richardson JA, Eckman CB, Eckman EA, Hammer RE and Herz J

    Genetics and Aging Research Unit, MassGeneral Institute for Neurodegenerative Disease, Charlestown, MA 02129-4404, USA. guenette@helix.mgh.harvard.edu

    Targeted deletion of two members of the FE65 family of adaptor proteins, FE65 and FE65L1, results in cortical dysplasia. Heterotopias resembling those found in cobblestone lissencephalies in which neuroepithelial cells migrate into superficial layers of the developing cortex, aberrant cortical projections and loss of infrapyramidal mossy fibers arise in FE65/FE65L1 compound null animals, but not in single gene knockouts. The disruption of pial basal membranes underlying the heterotopias and poor organization of fibrillar laminin by isolated meningeal fibroblasts from double knockouts suggests that FE65 proteins are involved in basement membrane assembly. A similar phenotype is observed in triple mutant mice lacking the APP family members APP, APLP1 and APLP2, all of which interact with FE65 proteins, suggesting that this phenotype may be caused by decreased transmission of an APP-dependent signal through the FE65 proteins. The defects observed in the double knockout may also involve the family of Ena/Vasp proteins, which participate in actin cytoskeleton remodeling and interact with the WW domains of FE65 proteins.

    Funded by: NHLBI NIH HHS: HL20948, HL63762, P01 HL020948, R01 HL063762, R37 HL063762; NIA NIH HHS: AG15903, R01 AG015903; NINDS NIH HHS: NS43408, R01 NS043408

    The EMBO journal 2006;25;2;420-31

  • Interaction of the cytosolic domains of sorLA/LR11 with the amyloid precursor protein (APP) and beta-secretase beta-site APP-cleaving enzyme.

    Spoelgen R, von Arnim CA, Thomas AV, Peltan ID, Koker M, Deng A, Irizarry MC, Andersen OM, Willnow TE and Hyman BT

    Alzheimer's Disease Research Laboratory, Massachusetts General Hospital, Harvard Medical School, Charlestown, Massachusetts 02129, USA.

    sorLA is a recently identified neuronal receptor for amyloid precursor protein (APP) that is known to interact with APP and affect its intracellular transport and processing. Decreased levels of sorLA in the brain of Alzheimer's disease (AD) patients and elevated levels of amyloid-beta peptide (Abeta) in sorLA-deficient mice point to the importance of the receptor in this neurodegenerative disorder. We analyzed APP cleavage in an APP-shedding assay and found that both sorLA and, surprisingly, a sorLA tail construct inhibited APP cleavage in a beta-site APP-cleaving enzyme (BACE)-dependent manner. In line with this finding, sorLA and the sorLA tail significantly reduced secreted Abeta levels when BACE was overexpressed, suggesting that sorLA influences beta-cleavage. To understand the effect of sorLA on APP cleavage by BACE, we analyzed whether sorLA interacts with APP and/or BACE. Because both full-length sorLA and sorLA C-terminal tail constructs were functionally relevant for APP processing, we analyzed sorLA-APP for a potential cytoplasmatic interaction domain. sorLA and C99 coimmunoprecipitated, pointing toward the existence of a new cytoplasmatic interaction site between sorLA and APP. Moreover, sorLA and BACE also coimmunoprecipitate. Thus, sorLA interacts both with BACE and APP and might therefore directly affect BACE-APP complex formation. To test whether sorLA impacts BACE-APP interactions, we used a fluorescence resonance energy transfer assay to evaluate BACE-APP interactions in cells. We discovered that sorLA significantly reduced BACE-APP interactions in Golgi. We postulate that sorLA acts as a trafficking receptor that prevents BACE-APP interactions and hence BACE cleavage of APP.

    Funded by: NIA NIH HHS: AG 12406

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2006;26;2;418-28

  • The Arctic Alzheimer mutation facilitates early intraneuronal Abeta aggregation and senile plaque formation in transgenic mice.

    Lord A, Kalimo H, Eckman C, Zhang XQ, Lannfelt L and Nilsson LN

    Department of Public Health and Caring Sciences, Geriatrics, Uppsala University, Dag Hammarskjölds Väg 20, SE-751 85 Uppsala, Sweden.

    The Arctic mutation (APP E693G) is unique, since it is located within the amyloid-beta (Abeta) sequence and leads to Alzheimer's disease (AD). Arctic Abeta peptides more easily form Abeta protofibrils in vitro, but little is known about the pathogenic mechanism of the Arctic mutation in vivo. Here, we analyzed APP transgenic mice with both the Swedish and Arctic mutations (tg-APPArcSwe) and transgenic mice with the Swedish mutation alone (tg-APPSwe). Intense intraneuronal Abeta-immunoreactive staining was present in young tg-APPArcSwe mice, but not in tg-APPSwe mice. Intracellular Abeta aggregates in tg-APPArcSwe were strongly stained by antibodies recognizing the N-terminus of Abeta, while those recognizing the C-terminus of Abeta stained weakly. The Abeta aggregates inside neurons increased with age and predated extracellular Abeta deposition in both tg-APPArcSwe and tg-APPSwe mice. Senile plaque deposition was markedly accelerated in tg-APPArcSwe mice, as compared to tg-APPSwe mice. We conclude that the Arctic mutation causes AD by facilitating amyloidosis through early accumulation of intracellular Abeta aggregates in association with a rapid onset of senile plaque deposition.

    Neurobiology of aging 2006;27;1;67-77

  • The absence of ABCA1 decreases soluble ApoE levels but does not diminish amyloid deposition in two murine models of Alzheimer disease.

    Hirsch-Reinshagen V, Maia LF, Burgess BL, Blain JF, Naus KE, McIsaac SA, Parkinson PF, Chan JY, Tansley GH, Hayden MR, Poirier J, Van Nostrand W and Wellington CL

    Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, British Columbia V4Z 5H5, Canada.

    ABCA1, a cholesterol transporter expressed in the brain, has been shown recently to be required to maintain normal apoE levels and lipidation in the central nervous system. In addition, ABCA1 has been reported to modulate beta-amyloid (Abeta) production in vitro. These observations raise the possibility that ABCA1 may play a role in the pathogenesis of Alzheimer disease. Here we report that the deficiency of ABCA1 does not affect soluble or guanidine-extractable Abeta levels in Tg-SwDI/B or amyloid precursor protein/presenilin 1 (APP/PS1) mice, but rather is associated with a dramatic reduction in soluble apoE levels in brain. Although this reduction in apoE was expected to reduce the amyloid burden in vivo, we observed that the parenchymal and vascular amyloid load was increased in Tg-SwDI/B animals and was not diminished in APP/PS1 mice. Furthermore, we observed an increase in the proportion of apoE retained in the insoluble fraction, particularly in the APP/PS1 model. These data suggested that ABCA1-mediated effects on apoE levels and lipidation influenced amyloidogenesis in vivo.

    Funded by: NINDS NIH HHS: NS36645

    The Journal of biological chemistry 2005;280;52;43243-56

  • Deletion of Abca1 increases Abeta deposition in the PDAPP transgenic mouse model of Alzheimer disease.

    Wahrle SE, Jiang H, Parsadanian M, Hartman RE, Bales KR, Paul SM and Holtzman DM

    Program in Neurosciences, Department of Neurology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.

    Apolipoprotein E (apoE) genotype has a major influence on the risk for Alzheimer disease (AD). Different apoE isoforms may alter AD pathogenesis via their interactions with the amyloid beta-peptide (Abeta). Mice lacking the lipid transporter ABCA1 were found to have markedly decreased levels and lipidation of apoE in the central nervous system. We hypothesized that if Abca1-/- mice were bred to the PDAPP mouse model of AD, PDAPP Abca1-/ mice would have a phenotype similar to that of PDAPP Apoe+/- and PDAPP Apoe-/- mice, which develop less amyloid deposition than PDAPP Apoe+/+ mice. In contrast to this prediction, 12-month-old PDAPP Abca -/- mice had significantly higher levels of hippocampal Abeta, and cerebral amyloid angiopathy was significantly more common compared with PDAPP Abca1+/+ mice. Amyloid precursor protein (APP) C-terminal fragments were not different between Abca1 genotypes prior to plaque deposition in 3-month-old PDAPP mice, suggesting that deletion of Abca1 did not affect APP processing or Abeta production. As expected, 3-month-old PDAPP Abca1-/- mice had decreased apoE levels, but they also had a higher percentage of carbonate-insoluble apoE, suggesting that poorly lipidated apoE is less soluble in vivo. We also found that 12-month-old PDAPP Abca1-/- mice had a higher percentage of carbonate-insoluble apoE and that apoE deposits co-localize with amyloid plaques, demonstrating that poorly lipidated apoE co-deposits with insoluble Abeta. Together, these data suggest that despite substantially lower apoE levels, poorly lipidated apoE produced in the absence of ABCA1 is strongly amyloidogenic in vivo.

    Funded by: NIA NIH HHS: AG11355, AG13956

    The Journal of biological chemistry 2005;280;52;43236-42

  • Brain estrogen deficiency accelerates Abeta plaque formation in an Alzheimer's disease animal model.

    Yue X, Lu M, Lancaster T, Cao P, Honda S, Staufenbiel M, Harada N, Zhong Z, Shen Y and Li R

    L. J. Roberts Center for Alzheimer's Research and Haldeman Laboratory for Molecular and Cellular Neurobiology, Sun Health Research Institute, Sun City, AZ 85351, USA.

    Much evidence indicates that women have a higher risk of developing Alzheimer's disease (AD) than do men. The reason for this gender difference is unclear. We hypothesize that estrogen deficiency in the brains of women with AD may be a key risk factor. In rapidly acquired postmortem brains from women with AD, we found greatly reduced estrogen levels compared with those from age- and gender-matched normal control subjects; AD and control subjects had comparably low levels of serum estrogen. We examined the onset and severity of AD pathology associated with estrogen depletion by using a gene-based approach, by crossing the estrogen-synthesizing enzyme aromatase gene knockout mice with APP23 transgenic mice, a mouse model of AD, to produce estrogen-deficient APP23 mice. Compared with APP23 transgenic control mice, estrogen-deficient APP23 mice exhibited greatly reduced brain estrogen and early-onset and increased beta amyloid peptide (Abeta) deposition. These mice also exhibited increased Abeta production, and microglia cultures prepared from the brains of these mice were impaired in Abeta clearance/degradation. In contrast, ovariectomized APP23 mice exhibited plaque pathology similar to that observed in the APP23 transgenic control mice. Our results indicate that estrogen depletion in the brain may be a significant risk factor for developing AD neuropathology.

    Proceedings of the National Academy of Sciences of the United States of America 2005;102;52;19198-203

  • BACE1, a major determinant of selective vulnerability of the brain to amyloid-beta amyloidogenesis, is essential for cognitive, emotional, and synaptic functions.

    Laird FM, Cai H, Savonenko AV, Farah MH, He K, Melnikova T, Wen H, Chiang HC, Xu G, Koliatsos VE, Borchelt DR, Price DL, Lee HK and Wong PC

    Department of Pathology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.

    A transmembrane aspartyl protease termed beta-site APP cleavage enzyme 1 (BACE1) that cleaves the amyloid-beta precursor protein (APP), which is abundant in neurons, is required for the generation of amyloid-beta (Abeta) peptides implicated in the pathogenesis of Alzheimer's disease (AD). We now demonstrate that BACE1, enriched in neurons of the CNS, is a major determinant that predisposes the brain to Abeta amyloidogenesis. The physiologically high levels of BACE1 activity coupled with low levels of BACE2 and alpha-secretase anti-amyloidogenic activities in neurons is a major contributor to the accumulation of Abeta in the CNS, whereas other organs are spared. Significantly, deletion of BACE1 in APPswe;PS1DeltaE9 mice prevents both Abeta deposition and age-associated cognitive abnormalities that occur in this model of Abeta amyloidosis. Moreover, Abeta deposits are sensitive to BACE1 dosage and can be efficiently cleared from the CNS when BACE1 is silenced. However, BACE1 null mice manifest alterations in hippocampal synaptic plasticity as well as in performance on tests of cognition and emotion. Importantly, memory deficits but not emotional alterations in BACE1(-/-) mice are prevented by coexpressing APPswe;PS1DeltaE9 transgenes, indicating that other potential substrates of BACE1 may affect neural circuits related to emotion. Our results establish BACE1 and APP processing pathways as critical for cognitive, emotional, and synaptic functions, and future studies should be alert to potential mechanism-based side effects that may occur with BACE1 inhibitors designed to ameliorate Abeta amyloidosis in AD.

    Funded by: Intramural NIH HHS: Z01 AG000959-04, Z99 AG999999; NIA NIH HHS: AG02556, P50 AG005146, P50 AG05146; NINDS NIH HHS: P01 NS047308, R01 NS041438, R01 NS045150, R01 NS41438, R01 NS45150

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2005;25;50;11693-709

  • Suppression of cyclin-dependent kinase 5 activation by amyloid precursor protein: a novel excitoprotective mechanism involving modulation of tau phosphorylation.

    Han P, Dou F, Li F, Zhang X, Zhang YW, Zheng H, Lipton SA, Xu H and Liao FF

    Center for Neuroscience and Aging, The Burnham Institute, La Jolla, California 92037, USA.

    Alzheimer's disease is cytopathologically characterized by loss of synapses and neurons, neuritic amyloid plaques consisting of beta-amyloid (Abeta) peptides, and neurofibrillary tangles consisting of hyperphosphorylated tau protein in susceptible brain regions. Abeta, which triggers a cascade of pathogenic events including tau phosphorylation and neuronal excitotoxicity, is proteolytically derived from beta-amyloid precursor protein (APP); the pathological and physiological functions of APP, however, remain undefined. Here we demonstrate that the level of tau phosphorylation in cells and brains deficient in APP is significantly higher than that in wild-type controls, resulting from activation of cyclin-dependent kinase 5 (CDK5) but not glycogen synthase kinase 3, the two major tau kinases. In addition, we show that overexpression of APP or its non-amyloidogenic homolog amyloid precursor-like protein 1 suppresses both basal and stress-induced CDK5 activation. The ectodomain of APP, sAPPalpha, is responsible for inhibiting CDK5 activation. Furthermore, neurons derived from APP-deficient mice exhibit reduced metabolism and survival rates and are more susceptible to excitotoxic glutamate-induced apoptosis. These neurons also manifest significant defects in neurite outgrowth compared with neurons from the wild-type littermates. The observed neuronal excitotoxicity/apoptosis is mediated through a mechanism involving CDK5 activation. Our study defines a novel neuroprotective function for APP in preventing tau hyperphosphorylation via suppressing overactivation of CDK5. We suggest that CDK5 activation, through a calcium/calpain/p25 pathway, plays a key role in neuronal excitotoxicity and represents an underlying mechanism for the physiological functions of APP.

    Funded by: NIA NIH HHS: R01 AG024895; NICHD NIH HHS: P01 HD29587; NINDS NIH HHS: R01 NS046673

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2005;25;50;11542-52

  • Protease nexin-2/amyloid beta-protein precursor limits cerebral thrombosis.

    Xu F, Davis J, Miao J, Previti ML, Romanov G, Ziegler K and Van Nostrand WE

    Department of Medicine, Stony Brook University, NY 11794, USA.

    The amyloid beta-protein precursor (AbetaPP) is best known as the parent molecule to the amyloid beta-peptide that accumulates in the brains of patients with Alzheimer's disease. Secreted isoforms of AbetaPP that contain the Kunitz proteinase inhibitor domain are analogous to the previously identified cell-secreted proteinase inhibitor known as protease nexin-2 (PN2). Although PN2/AbetaPP is enriched in brain and in circulating blood platelets, little is understood of its physiological function and potential role in disease processes outside of amyloid beta-peptide generation. We hypothesized that the potent inhibition of certain procoagulant proteinases by PN2/AbetaPP, coupled with its abundance in platelets and brain, indicate that it may function to regulate cerebral thrombosis. Here we show that specific and modest 2-fold overexpression of PN2/AbetaPP in circulating platelets of transgenic mice caused a marked inhibition of thrombosis in vivo. In contrast, deletion of PN2/AbetaPP in AbetaPP gene knockout mice resulted in a significant increase in thrombosis. Similarly, platelet PN2/AbetaPP transgenic mice developed larger hematomas in experimental intracerebral hemorrhage, whereas AbetaPP gene knockout mice exhibited reduced hemorrhage size. These findings indicate that PN2/AbetaPP plays a significant role in regulating cerebral thrombosis and that modest increases in this protein can profoundly enhance cerebral hemorrhage.

    Funded by: NHLBI NIH HHS: HL72553; NINDS NIH HHS: NS36645

    Proceedings of the National Academy of Sciences of the United States of America 2005;102;50;18135-40

  • Stability of the distribution of spines containing drebrin A in the sensory cortex layer I of mice expressing mutated APP and PS1 genes.

    Mahadomrongkul V, Huerta PT, Shirao T and Aoki C

    Center for Neural Science, New York University, 4 Washington Place, New York, NY 10003, USA.

    Post-mortem cortices from patients diagnosed with Alzheimer's disease (AD) exhibit reduced levels of drebrin, an F-actin binding protein of dendritic spines and shafts. We used a mouse model of familial AD (FAD) to determine whether the density of cortical spines engaged in asymmetric (presumably excitatory) synapses and containing drebrin A is reduced and if so, whether this occurs prior to the emergence of beta amyloid deposits, when only soluble beta amyloid (Abeta) is present. Quantitative electron microscopic immunocytochemistry revealed that by 6 months, the proportion of postsynaptic spines with drebrin A within somatosensory cortex layer I was smaller for the FAD model mice, when compared to the corresponding region of WT mice (P < 0.0005). However, the areal density of postsynaptic spines containing drebrin A was relatively constant from 3 to 18 months and beyond for both genotypes, suggesting that drebrin A confers stability to postsynaptic spines. Further measurements confirmed that the reduced proportion of drebrin A-containing spines in brains of FAD mice at 6 months is due to the greater size and areal density of spine profiles lacking drebrin A. Thus, soluble Abeta could affect spines lacking drebrin A more strongly than spines containing drebrin A. At 6 months and older, a larger fraction of spinous drebrin A in 2xKI mice was located near the synaptic membrane, as compared to those of WT mice. This pattern may reflect an altered trafficking of synaptic molecules within spines, a factor adding to the decline of synaptic function and plasticity.

    Funded by: NEI NIH HHS: P30 EY13079, R01-EY13145; NINDS NIH HHS: R01-NS41091

    Brain research 2005;1064;1-2;66-74

  • Inactivation of the peroxisomal ABCD2 transporter in the mouse leads to late-onset ataxia involving mitochondria, Golgi and endoplasmic reticulum damage.

    Ferrer I, Kapfhammer JP, Hindelang C, Kemp S, Troffer-Charlier N, Broccoli V, Callyzot N, Mooyer P, Selhorst J, Vreken P, Wanders RJ, Mandel JL and Pujol A

    Institut de Neuropatologia, Hospital Universitari de Bellvitge, Department de Biologia Cel.lular i Anatomia Patologica, Facultat de Medicina, Universitat de Barcelona, Spain.

    ATP-binding cassette (ABC) transporters facilitate unidirectional translocation of chemically diverse substances, ranging from peptides to lipids, across cell or organelle membranes. In peroxisomes, a subfamily of four ABC transporters (ABCD1 to ABCD4) has been related to fatty acid transport, because patients with mutations in ABCD1 (ALD gene) suffer from X-linked adrenoleukodystrophy (X-ALD), a disease characterized by an accumulation of very-long-chain fatty acids (VLCFAs). Inactivation in the mouse of the abcd1 gene leads to a late-onset neurodegenerative condition, comparable to the late-onset form of X-ALD [Pujol, A., Hindelang, C., Callizot, N., Bartsch, U., Schachner, M. and Mandel, J.L. (2002) Late onset neurological phenotype of the X-ALD gene inactivation in mice: a mouse model for adrenomyeloneuropathy. Hum. Mol. Genet., 11, 499-505.]. In the present work, we have generated and characterized a mouse deficient for abcd2, the closest paralog to abcd1. The main pathological feature in abcd2-/- mice is a late-onset cerebellar and sensory ataxia, with loss of cerebellar Purkinje cells and dorsal root ganglia cell degeneration, correlating with accumulation of VLCFAs in the latter cellular population. Axonal degeneration was present in dorsal and ventral columns in spinal cord. We have identified mitochondrial, Golgi and endoplasmic reticulum damage as the underlying pathological mechanism, thus providing evidence of a disturbed organelle cross-talk, which may be at the origin of the pathological cascade.

    Human molecular genetics 2005;14;23;3565-77

  • Amyloid-beta induces disulfide bonding and aggregation of GAPDH in Alzheimer's disease.

    Cumming RC and Schubert D

    Cellular Neurobiology Laboratory, The Salk Institute for Biological Studies, La Jolla, California 92037, USA.

    GAPDH is a redox-sensitive glycolytic enzyme that also promotes apoptosis when translocated to the nucleus and associates with aggregate-prone proteins involved in neurodegenerative disorders. Recent evidence indicates that polymorphic variation within GAPDH genes is associated with an elevated risk of developing Alzheimer's disease (AD). We previously demonstrated that GAPDH readily undergoes disulfide bonding following oxidant exposure, although the consequence of disulfide bonding on GAPDH activity or function is unknown. Here we show that increased GAPDH disulfide bonding is observed in detergent-insoluble extracts from AD patient and transgenic AD mouse brain tissue compared with age-matched controls. Exposure of primary rat cortical neurons to the pro-oxidant amyloid beta peptide promotes nuclear accumulation of a disulfide-linked form of GAPDH, which becomes detergent-insoluble. Disulfide bonding leads to a reduction in GAPDH enzymatic activity and correlates with the appearance of punctate aggregate-like GAPDH staining within the cytoplasm of both oxidant-treated HT22 cells and amyloid beta-treated primary cortical neurons. Our findings suggest that disulfide bonding of GAPDH and subsequent protein aggregate formation may have relevance to the pathophysiology of AD.

    FASEB journal : official publication of the Federation of American Societies for Experimental Biology 2005;19;14;2060-2

  • Amyloid peptide attenuates the proteasome activity in neuronal cells.

    Oh S, Hong HS, Hwang E, Sim HJ, Lee W, Shin SJ and Mook-Jung I

    Neuroscience Program, Ajou University, Suwon, Republic of Korea.

    Previous studies have suggested a possible relationship between the ubiquitin-proteasome pathway and some pathological manifestations of Alzheimer's disease (AD). This study investigated the possibility that the Abeta peptides interact with the ubiquitin-proteasome pathway inside neuronal cells. The ubiquitin-proteasome activity decreased with age in the brains of Tg2576 mice while the Abeta(1-42) levels increased. In cultured neuronal cells, an extracellular treatment of Abeta markedly decreased the proteasome activity and extracellular treated Abeta peptides were found in the cytoplasmic compartment. These results suggest that the extracellular Abeta peptides enter the cell and inhibit the proteasome activity, which might play a role in the pathogenesis of AD.

    Mechanisms of ageing and development 2005;126;12;1292-9

  • CpG-containing oligodeoxynucleotide promotes microglial cell uptake of amyloid beta 1-42 peptide by up-regulating the expression of the G-protein- coupled receptor mFPR2.

    Iribarren P, Chen K, Hu J, Gong W, Cho EH, Lockett S, Uranchimeg B and Wang JM

    Laboratory of Molecular Immunoregulation, Center for Cancer Research, National Cancer Institute at Frederick, Frederick, Maryland 21702-1201, USA.

    Human G protein-coupled formyl peptide receptor like 1 (FPRL1) and its mouse homologue murine formyl peptide receptor 2 (mFPR2) mediate the chemotactic activity of amyloid beta 1-42 (Abeta42), a key pathogenic peptide in Alzheimer's disease (AD). Since mFPR2 is up-regulated in mouse microglia by lipopolysaccharide (LPS), a Toll-like receptor 4 ligand, we investigated the capacity of CpG-containing oligodeoxynucleotide (ODN), a Toll-like receptor (TLR) 9 ligand, to regulate the expression of mFPR2 in mouse microglia. CpG ODN markedly enhanced the expression and function of mFPR2 in microglial cells, which exhibited increased chemotactic responses to mFPR2 agonists, including Abeta42. The effect of CpG ODN is dependent on activation of p38 MAPK. Further studies showed that CpG ODN-treated microglia increased their capacity to endocytose Abeta42 through mFPR2, as this process was abrogated by pertussis toxin, a Gi protein inhibitor, and W peptide, another potent mFPR2 agonist. Our results suggest that TLR9 may play an important role in promoting microglial recognition of Abeta42, thus affecting the pathogenic process of AD.

    Funded by: Intramural NIH HHS; NCI NIH HHS: N01-CO-12400

    FASEB journal : official publication of the Federation of American Societies for Experimental Biology 2005;19;14;2032-4

  • Mitochondrial Abeta: a potential focal point for neuronal metabolic dysfunction in Alzheimer's disease.

    Caspersen C, Wang N, Yao J, Sosunov A, Chen X, Lustbader JW, Xu HW, Stern D, McKhann G and Yan SD

    Department of Surgery, College of Physicians & Surgeons of Columbia University, New York, New York 10032, USA.

    Although amyloid-beta peptide (Abeta) is the neurotoxic species implicated in the pathogenesis of Alzheimer's disease (AD), mechanisms through which intracellular Abeta impairs cellular properties, resulting in neuronal dysfunction, remain to be clarified. Here we demonstrate that intracellular Abeta is present in mitochondria from brains of transgenic mice with targeted neuronal overexpression of mutant human amyloid precursor protein and AD patients. Abeta progressively accumulates in mitochondria and is associated with diminished enzymatic activity of respiratory chain complexes (III and IV) and a reduction in the rate of oxygen consumption. Importantly, mitochondria-associated Abeta, principally Abeta42, was detected as early as 4 months, before extensive extracellular Abeta deposits. Our studies delineate a new means through which Abeta potentially impairs neuronal energetics, contributing to cellular dysfunction in AD.

    Funded by: NIA NIH HHS: AG16736, P01 AG17490, P50 AG08702; NINDS NIH HHS: NS 042855

    FASEB journal : official publication of the Federation of American Societies for Experimental Biology 2005;19;14;2040-1

  • Amyloid precursor proteins anchor CPEB to membranes and promote polyadenylation-induced translation.

    Cao Q, Huang YS, Kan MC and Richter JD

    Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, 01605, USA

    The cytoplasmic polyadenylation element (CPE) binding factor, CPEB, is a sequence-specific RNA binding protein that controls polyadenylation-induced translation in germ cells and at postsynaptic sites of neurons. A yeast two-hybrid screen with a mouse brain cDNA library identified the transmembrane amyloid precursor-like protein 1 (APLP1) as a CPEB-interacting factor. CPEB binds the small intracellular domain (ICD) of APLP1 and the related proteins APLP2 and APP. These proteins promote polyadenylation and translation by stimulating Aurora A catalyzed CPEB serine 174 phosphorylation. Surprisingly, CPEB, Maskin, CPSF, and several other factors involved in polyadenylation and translation and CPE-containing RNA are all detected on membranes by cell fractionation and immunoelectron microscopy. Moreover, most of the RNA that undergoes polyadenylation does so in membrane-containing fractions. These data demonstrate a link between cytoplasmic polyadenylation and membrane association and implicate APP family member proteins as anchors for localized mRNA polyadenylation and translation.

    Funded by: NIDDK NIH HHS: DK32520, P30 DK032520

    Molecular and cellular biology 2005;25;24;10930-9

  • Coordinated transport of phosphorylated amyloid-beta precursor protein and c-Jun NH2-terminal kinase-interacting protein-1.

    Muresan Z and Muresan V

    Department of Physiology and Biophysics, Case School of Medicine, Case Western Reserve University, Cleveland, OH 44106, USA. zoia.muresan@case.edu

    The transmembrane protein amyloid-beta precursor protein (APP) and the vesicle-associated protein c-Jun NH(2)-terminal kinase-interacting protein-1 (JIP-1) are transported into axons by kinesin-1. Both proteins may bind to kinesin-1 directly and can be transported separately. Because JIP-1 and APP can interact, kinesin-1 may recruit them as a complex, enabling their cotransport. In this study, we tested whether APP and JIP-1 are transported together or separately on different vesicles. We found that, within the cellular context, JIP-1 preferentially interacts with Thr(668)-phosphorylated APP (pAPP), compared with nonphosphorylated APP. In neurons, JIP-1 colocalizes with vesicles containing pAPP and is excluded from those containing nonphosphorylated APP. The accumulation of JIP-1 and pAPP in neurites requires kinesin-1, and the expression of a phosphomimetic APP mutant increases JIP-1 transport. Down-regulation of JIP-1 by small interfering RNA specifically impairs transport of pAPP, with no effect on the trafficking of nonphosphorylated APP. These results indicate that the phosphorylation of APP regulates the formation of a pAPP-JIP-1 complex that accumulates in neurites independent of nonphosphorylated APP.

    Funded by: NIA NIH HHS: AG08012, P50 AG008012; NIGMS NIH HHS: 5R01GM068596-02, R01 GM068596

    The Journal of cell biology 2005;171;4;615-25

  • Alzheimer's disease-like neuropathology of gene-targeted APP-SLxPS1mut mice expressing the amyloid precursor protein at endogenous levels.

    Köhler C, Ebert U, Baumann K and Schröder H

    Institute II of Anatomy, Department of Neuroanatomy, University of Cologne, Josef Stelzmann-Strasse 9, D-50931 Cologne, Germany. c.koehler@uni-koeln.de

    Most transgenic mice used for preclinical evaluation of potential disease-modifying treatments of Alzheimer's disease develop major histopathological features of this disease by several-fold overexpression of the human amyloid precursor protein. We studied the phenotype of three different strains of gene-targeted mice which express the amyloid precursor protein at endogenous levels. Only further crossing with transgenic mice overexpressing mutant human presenilin1 led to the deposition of extracellular amyloid, accompanied by the deposition of apolipoprotein E, an astrocyte and microglia reaction, and the occurrence of dilated cholinergic terminals in the cortex. Features of neurodegeneration, however, were absent. The pattern of plaque development and deposition in these mice was similar to that of amyloid precursor protein overproducing strains if crossed to presenilin1-transgenics. However, plaque development started much later and developed slowly until the age of 18 months but then increased more rapidly.

    Neurobiology of disease 2005;20;2;528-40

  • Beta-amyloid accumulation in APP mutant neurons reduces PSD-95 and GluR1 in synapses.

    Almeida CG, Tampellini D, Takahashi RH, Greengard P, Lin MT, Snyder EM and Gouras GK

    Department of Neurology and Neuroscience, Laboratory of Alzheimer's Disease Neurobiology, Weill Medical College of Cornell University, 525 E 68th Street, NY 10021, USA.

    Synaptic dysfunction is increasingly viewed as an early manifestation of Alzheimer's disease (AD), but the cellular mechanism by which beta-amyloid (Abeta) may affect synapses remains unclear. Since cultured neurons derived from APP mutant transgenic mice secrete elevated levels of Abeta and parallel the subcellular Abeta accumulation seen in vivo, we asked whether alterations in synapses occur in this setting. We report that cultured Tg2576 APP mutant neurons have selective alterations in pre- and post-synaptic compartments compared to wild-type neurons. Post-synaptic compartments appear fewer in number and smaller, while active pre-synaptic compartments appear fewer in number and enlarged. Among the earliest changes in synaptic composition in APP mutant neurons were reductions in PSD-95, a protein involved in recruiting and anchoring glutamate receptor subunits to the post-synaptic density. In agreement, we observed early reductions in surface expression of glutamate receptor subunit GluR1 in APP mutant neurons. We provide evidence that Abeta is specifically involved in these alterations in synaptic biology, since alterations in PSD-95 and GluR1 are blocked by gamma-secretase inhibition, and since exogenous addition of synthetic Abeta to wild-type neurons parallels changes in synaptic PSD-95 and GluR1 observed in APP mutant neurons.

    Funded by: NIA NIH HHS: AG09464; NINDS NIH HHS: NS002037, NS045677

    Neurobiology of disease 2005;20;2;187-98

  • Proteomic determination of widespread detergent-insolubility including Abeta but not tau early in the pathogenesis of Alzheimer's disease.

    Woltjer RL, Cimino PJ, Boutté AM, Schantz AM, Montine KS, Larson EB, Bird T, Quinn JF, Zhang J and Montine TJ

    Department of Pathology, Division of Neuropathology, University of Washington, Seattle, WA, USA.

    Biochemical characterization of the major detergent-insoluble proteins that comprise hallmark histopathologic lesions initiated the molecular era of Alzheimer's disease (AD) research. Here, we reinvestigated detergent-insoluble proteins in AD using modern proteomic techniques. Using liquid chromatography (LC)-mass spectrometry (MS)-MS-based proteomics, we robustly identified 125 proteins in the detergent-insoluble fraction of late-onset AD (LOAD) temporal cortex that included several proteins critical to Abeta production, components of synaptic scaffolding, and products of genes linked to an increased risk of LOAD; we verified 15 of 15 of these proteins by Western blot. Following multiple analyses, we estimated that these represent ~80% of detergent-insoluble proteins in LOAD detectable by our method. Abeta, tau, and 7 of 8 other newly identified detergent-insoluble proteins were disproportionately increased in temporal cortex from patients with LOAD and AD derived from mutations in PSEN1 and PSEN2; all of these except tau were elevated in individuals with prodromal dementia, while none except Abeta were elevated in aged APPswe mice. These results are consistent with the amyloid hypothesis of AD and extend it to include widespread protein insolubility, not exclusively Abeta insolubility, early in AD pathogenesis even before the onset of clinical dementia.

    Funded by: NIA NIH HHS: AG05136, AG06781, AG22040, AG23801, AG24011

    FASEB journal : official publication of the Federation of American Societies for Experimental Biology 2005;19;13;1923-5

  • Regulation of lung injury and repair by Toll-like receptors and hyaluronan.

    Jiang D, Liang J, Fan J, Yu S, Chen S, Luo Y, Prestwich GD, Mascarenhas MM, Garg HG, Quinn DA, Homer RJ, Goldstein DR, Bucala R, Lee PJ, Medzhitov R and Noble PW

    Department of Medicine, Section of Pulmonary and Critical Care Medicine, Yale University School of Medicine, 333 Cedar Street, New Haven, Connecticut 06520, USA.

    Mechanisms that regulate inflammation and repair after acute lung injury are incompletely understood. The extracellular matrix glycosaminoglycan hyaluronan is produced after tissue injury and impaired clearance results in unremitting inflammation. Here we report that hyaluronan degradation products require MyD88 and both Toll-like receptor (TLR)4 and TLR2 in vitro and in vivo to initiate inflammatory responses in acute lung injury. Hyaluronan fragments isolated from serum of individuals with acute lung injury stimulated macrophage chemokine production in a TLR4- and TLR2-dependent manner. Myd88(-/-) and Tlr4(-/-)Tlr2(-/-) mice showed impaired transepithelial migration of inflammatory cells but decreased survival and enhanced epithelial cell apoptosis after lung injury. Lung epithelial cell-specific overexpression of high-molecular-mass hyaluronan was protective against acute lung injury. Furthermore, epithelial cell-surface hyaluronan was protective against apoptosis, in part, through TLR-dependent basal activation of NF-kappaB. Hyaluronan-TLR2 and hyaluronan-TLR4 interactions provide signals that initiate inflammatory responses, maintain epithelial cell integrity and promote recovery from acute lung injury.

    Funded by: NHLBI NIH HHS: HL57486; NIAID NIH HHS: AI52487

    Nature medicine 2005;11;11;1173-9

  • Transforming growth factor beta2 is a neuronal death-inducing ligand for amyloid-beta precursor protein.

    Hashimoto Y, Chiba T, Yamada M, Nawa M, Kanekura K, Suzuki H, Terashita K, Aiso S, Nishimoto I and Matsuoka M

    Department of Pharmacology, KEIO University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan.

    APP, amyloid beta precursor protein, is linked to the onset of Alzheimer's disease (AD). We have here found that transforming growth factor beta2 (TGFbeta2), but not TGFbeta1, binds to APP. The binding affinity of TGFbeta2 to APP is lower than the binding affinity of TGFbeta2 to the TGFbeta receptor. On binding to APP, TGFbeta2 activates an APP-mediated death pathway via heterotrimeric G protein G(o), c-Jun N-terminal kinase, NADPH oxidase, and caspase 3 and/or related caspases. Overall degrees of TGFbeta2-induced death are larger in cells expressing a familial AD-related mutant APP than in those expressing wild-type APP. Consequently, superphysiological concentrations of TGFbeta2 induce neuronal death in primary cortical neurons, whose one allele of the APP gene is knocked in with the V642I mutation. Combined with the finding indicated by several earlier reports that both neural and glial expression of TGFbeta2 was upregulated in AD brains, it is speculated that TGFbeta2 may contribute to the development of AD-related neuronal cell death.

    Molecular and cellular biology 2005;25;21;9304-17

  • Activation of GSK-3 and phosphorylation of CRMP2 in transgenic mice expressing APP intracellular domain.

    Ryan KA and Pimplikar SW

    Department of Pathology and Cell Biology Program, Case Western Reserve University, Cleveland, OH 44106, USA.

    Amyloid precursor protein (APP), implicated in Alzheimer's disease, is a trans-membrane protein of undetermined function. APP is cleaved by gamma-secretase that releases the APP intracellular domain (AICD) in the cytoplasm. In vitro studies have implicated AICD in cell signaling and transcriptional regulation, but its biologic relevance has been uncertain and its in vivo function has not been examined. To investigate its functional role, we generated AICD transgenic mice, and found that AICD causes significant biologic changes in vivo. AICD transgenic mice show activation of glycogen synthase kinase-3beta (GSK-3beta) and phosphorylation of CRMP2 protein, a GSK-3beta substrate that plays a crucial role in Semaphorin3a-mediated axonal guidance. Our data suggest that AICD is biologically relevant, causes significant alterations in cell signaling, and may play a role in axonal elongation or pathfinding.

    Funded by: NIA NIH HHS: P50 AG008012, P50-AG08012

    The Journal of cell biology 2005;171;2;327-35

  • Homo- and heterodimerization of APP family members promotes intercellular adhesion.

    Soba P, Eggert S, Wagner K, Zentgraf H, Siehl K, Kreger S, Löwer A, Langer A, Merdes G, Paro R, Masters CL, Müller U, Kins S and Beyreuther K

    ZMBH, University of Heidelberg, Heidelberg, Germany. p.soba@zmbh.uni-heidelberg.de

    The amyloid precursor protein (APP) plays a central role in Alzheimer's disease, but its physiological function and that of its mammalian paralogs, the amyloid precursor-like proteins 1 and 2 (APLPs), is still poorly understood. APP has been proposed to form dimers, a process that could promote cell adhesion via trans-dimerization. We investigated the dimerization and cell adhesion properties of APP/APLPs and provide evidence that all three paralogs are capable of forming homo- and heterocomplexes. Moreover, we show that trans-interaction of APP family proteins promotes cell-cell adhesion in a homo- and heterotypic fashion and that endogenous APLP2 is required for cell-cell adhesion in mouse embryonic fibroblasts. We further demonstrate interaction of all the three APP family members in mouse brain, genetic interdependence, and molecular interaction of APP and APLPs in synaptically enriched membrane compartments. Together, our results provide evidence that homo- and heterocomplexes of APP/APLPs promote trans-cellular adhesion in vivo.

    The EMBO journal 2005;24;20;3624-34

  • The ciliary rootlet interacts with kinesin light chains and may provide a scaffold for kinesin-1 vesicular cargos.

    Yang J and Li T

    The Berman-Gund Laboratory for the Study of Retinal Degenerations, Harvard Medical School, Massachusetts Eye and Ear Infirmary, 243 Charles Street, Boston, MA 02114, USA. jun_yang@meei.harvard.edu

    The ciliary rootlet is a large striated fibrous network originating from basal bodies in ciliated cells. To explore its postulated role in intracellular transport, we investigated the interaction between kinesin light chains (KLCs) and rootletin, the structural component of ciliary rootlets. We show here that KLCs directly interact with rootletin and are located along ciliary rootlets. Their interactions are mediated by the heptad repeats of KLCs. Further studies found that these interactions tethered kinesin heavy chains along ciliary rootlets. However, the ciliary rootlet-bound kinesin-1 did not recruit microtubules or move along ciliary rootlets. Additionally, amyloid precursor protein (APP; a kinesin-1 vesicular cargo receptor) and presenilin 1 (a presumed cargo of APP/kinesin-1) were found to be enriched along the rootletin fibers, suggesting that the interaction between ciliary rootlets and kinesin-1 recruits APP and presenilin 1 along ciliary rootlets. These findings indicate that ciliary rootlets may provide a scaffold for kinesin-1 vesicular cargos and, thus, play a role in the intracellular transport in ciliated cells.

    Funded by: NEI NIH HHS: EY14104, EY14426, P30 EY014104, R01 EY014226

    Experimental cell research 2005;309;2;379-89

  • Reduced synaptic vesicle density and active zone size in mice lacking amyloid precursor protein (APP) and APP-like protein 2.

    Yang G, Gong YD, Gong K, Jiang WL, Kwon E, Wang P, Zheng H, Zhang XF, Gan WB and Zhao NM

    State Key Lab of Biomembrane and Membrane Biotechnology, Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing, China.

    Although abnormal processing of amyloid precursor protein (APP) leads to early onset of Alzheimer's disease, the normal function of this protein is poorly understood. APP is widely expressed in axons, dendrites, and synapses in both central and peripheral nervous systems. Mice homozygous for APP or its homologue APP-like protein 2 (APLP2) null mutation (KO) are viable, but double mutants for APP and APLP2 deletions (DKO) are early postnatal lethal. To investigate the role of APP in synapse development, we compared the ultrastructure of submandibular ganglion synapses between DKO and littermate APLP2 KO mice at birth. Using serial electron microscopy, we found that the size of presynaptic boutons and the number of active zones per bouton were comparable in both strains of animals. However, the synaptic vesicle density, active zone size, and docked vesicle number per active zone were significantly reduced in DKO compared to those in APLP2 KO. These results indicate that the APP family of proteins plays an important role in regulating the formation and function of inter-neuronal synapses.

    Neuroscience letters 2005;384;1-2;66-71

  • Cerebral microvascular amyloid beta protein deposition induces vascular degeneration and neuroinflammation in transgenic mice expressing human vasculotropic mutant amyloid beta precursor protein.

    Miao J, Xu F, Davis J, Otte-Höller I, Verbeek MM and Van Nostrand WE

    Department of Medicine, HSC, Stony Brook University, Stony Brook, NY 11794-8153, USA.

    Cerebral vascular amyloid beta-protein (Abeta) deposition, also known as cerebral amyloid angiopathy, is a common pathological feature of Alzheimer's disease. Additionally, several familial forms of cerebral amyloid angiopathy exist including the Dutch (E22Q) and Iowa (D23N) mutations of Abeta. Increasing evidence has associated cerebral microvascular amyloid deposition with neuroinflammation and dementia in these disorders. We recently established a transgenic mouse model (Tg-SwDI) that expresses human vasculotropic Dutch/Iowa mutant amyloid beta-protein precursor in brain. Tg-SwDI mice were shown to develop early-onset deposition of Abeta exhibiting high association with cerebral microvessels. Here we present quantitative temporal analysis showing robust and progressive accumulation of cerebral microvascular fibrillar Abeta accompanied by decreased cerebral vascular densities, the presence of apoptotic cerebral vascular cells, and cerebral vascular cell loss in Tg-SwDI mice. Abundant neuroinflammatory reactive astrocytes and activated microglia strongly associated with the cerebral microvascular fibrillar Abeta deposits. In addition, Tg-SwDI mouse brain exhibited elevated levels of the inflammatory cytokines interleukin-1beta and -6. Together, these studies identify the Tg-SwDI mouse as a unique model to investigate selective accumulation of cerebral microvascular amyloid and the associated neuroinflammation.

    Funded by: NINDS NIH HHS: NS36645, R01 NS036645

    The American journal of pathology 2005;167;2;505-15

  • Regulation of NMDA receptor trafficking by amyloid-beta.

    Snyder EM, Nong Y, Almeida CG, Paul S, Moran T, Choi EY, Nairn AC, Salter MW, Lombroso PJ, Gouras GK and Greengard P

    Laboratory for Molecular and Cellular Neuroscience, Rockefeller University, 1230 York Avenue, New York, New York 10021, USA. ericmaltesnyder@gmail.com

    Amyloid-beta peptide is elevated in the brains of patients with Alzheimer disease and is believed to be causative in the disease process. Amyloid-beta reduces glutamatergic transmission and inhibits synaptic plasticity, although the underlying mechanisms are unknown. We found that application of amyloid-beta promoted endocytosis of NMDA receptors in cortical neurons. In addition, neurons from a genetic mouse model of Alzheimer disease expressed reduced amounts of surface NMDA receptors. Reducing amyloid-beta by treating neurons with a gamma-secretase inhibitor restored surface expression of NMDA receptors. Consistent with these data, amyloid-beta application produced a rapid and persistent depression of NMDA-evoked currents in cortical neurons. Amyloid-beta-dependent endocytosis of NMDA receptors required the alpha-7 nicotinic receptor, protein phosphatase 2B (PP2B) and the tyrosine phosphatase STEP. Dephosphorylation of the NMDA receptor subunit NR2B at Tyr1472 correlated with receptor endocytosis. These data indicate a new mechanism by which amyloid-beta can cause synaptic dysfunction and contribute to Alzheimer disease pathology.

    Funded by: NIA NIH HHS: AG09464; PHS HHS: 01527, 52711

    Nature neuroscience 2005;8;8;1051-8

  • Inhibition of glycosphingolipid biosynthesis reduces secretion of the beta-amyloid precursor protein and amyloid beta-peptide.

    Tamboli IY, Prager K, Barth E, Heneka M, Sandhoff K and Walter J

    Department of Neurology, University of Bonn, Sigmund-Freud-Strasse 25, 53127 Bonn, Germany.

    Alzheimer disease is associated with extracellular deposits of amyloid beta-peptides in the brain. Amyloid beta-peptides are generated by proteolytic processing of the beta-amyloid precursor protein by beta- and gamma-secretases. The cleavage by secretases occurs predominantly in post-Golgi secretory and endocytic compartments and is influenced by cholesterol, indicating a role of the membrane lipid composition in proteolytic processing of the beta-amyloid precursor protein. To analyze the role of glycosphingolipids in these processes we inhibited glycosyl ceramide synthase, which catalyzes the first step in glycosphingolipid biosynthesis. The depletion of glycosphingolipids markedly reduced the secretion of endogenous beta-amyloid precursor protein in different cell types, including human neuroblastoma SH-SY5Y cells. Importantly, secretion of amyloid beta-peptides was also strongly decreased by inhibition of glycosphingolipid biosynthesis. Conversely, the addition of exogenous brain gangliosides to cultured cells reversed these effects. Biochemical and cell biological experiments demonstrate that the pharmacological reduction of cellular glycosphingolipid levels inhibited maturation and cell surface transport of the beta-amyloid precursor protein. In the glycosphingolipid-deficient cell line GM95, cellular levels and maturation of beta-amyloid precursor protein were also significantly reduced as compared with normal B16 cells. Together, these data demonstrate that glycosphingolipids are implicated in the regulation of the subcellular transport of the beta-amyloid precursor protein in the secretory pathway and its proteolytic processing. Thus, enzymes involved in glycosphingolipid metabolism might represent targets to inhibit the production of amyloid beta-peptides.

    The Journal of biological chemistry 2005;280;30;28110-7

  • Disruption of the paternal necdin gene diminishes TrkA signaling for sensory neuron survival.

    Kuwako K, Hosokawa A, Nishimura I, Uetsuki T, Yamada M, Nada S, Okada M and Yoshikawa K

    Institute for Protein Research, Osaka University, Osaka 565-0871, Japan.

    Necdin is a multifunctional signaling protein that stabilizes terminal differentiation of postmitotic neurons. The human necdin gene in chromosome 15q11-q12 is maternally imprinted, paternally transcribed, and not expressed in Prader-Willi syndrome, a human genomic imprinting-associated neurodevelopmental disorder. Although necdin-deficient mice display several abnormal phenotypes reminiscent of this syndrome, little is known about molecular mechanisms that lead to the neurodevelopmental defects. Here, we demonstrate that paternally expressed necdin is required for physiological development of nerve growth factor (NGF)-dependent sensory neurons. Mouse embryos defective in the paternal necdin allele displayed absent necdin expression in the dorsal root ganglia, in which the tropomyosin-related kinase A (TrkA) receptor tyrosine kinase and the p75 neurotrophin receptor were expressed in a normal manner. Necdin interacted with both TrkA and p75 to facilitate the association between these receptors. NGF-induced phosphorylation of TrkA and mitogen-activated protein kinase was significantly diminished in the necdin-null sensory ganglia. Furthermore, the mice lacking the paternal necdin allele displayed augmented apoptosis in the sensory ganglia in vivo and had a reduced population of substance P-containing neurons. These mutant mice showed significantly high tolerance to thermal pain, which is often seen in individuals with Prader-Willi syndrome. These results suggest that paternally expressed necdin facilitates TrkA signaling to promote the survival of NGF-dependent nociceptive neurons.

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2005;25;30;7090-9

  • Spatial segregation of gamma-secretase and substrates in distinct membrane domains.

    Vetrivel KS, Cheng H, Kim SH, Chen Y, Barnes NY, Parent AT, Sisodia SS and Thinakaran G

    Department of Neurobiology, Pharmacology, and Physiology, the University of Chicago, Chicago, Illinois 60637, USA.

    Gamma-secretase facilitates the regulated intramembrane proteolysis of select type I membrane proteins that play diverse physiological roles in multiple cell types and tissue. In this study, we used biochemical approaches to examine the distribution of amyloid precursor protein (APP) and several additional gamma-secretase substrates in membrane microdomains. We report that APP C-terminal fragments (CTFs) and gamma-secretase reside in Lubrol WX detergent-insoluble membranes (DIM) of cultured cells and adult mouse brain. APP CTFs that accumulate in cells lacking gamma-secretase activity preferentially associate with DIM. Cholesterol depletion and magnetic immunoisolation studies indicate recruitment of APP CTFs into cholesterol- and sphingolipid-rich lipid rafts, and co-residence of APP CTFs, PS1, and syntaxin 6 in DIM patches derived from the trans-Golgi network. Photoaffinity cross-linking studies provided evidence for the preponderance of active gamma-secretase in lipid rafts of cultured cells and adult brain. Remarkably, unlike the case of APP, CTFs derived from Notch1, Jagged2, deleted in colorectal cancer (DCC), and N-cadherin remain largely detergent-soluble, indicative of their spatial segregation in non-raft domains. In embryonic brain, the majority of PS1 and nicastrin is present in Lubrol WX-soluble membranes, wherein the CTFs derived from APP, Notch1, DCC, and N-cadherin also reside. We suggest that gamma-secretase residence in non-raft membranes facilitates proteolysis of diverse substrates during embryonic development but that the translocation of gamma-secretase to lipid rafts in adults ensures processing of certain substrates, including APP CTFs, while limiting processing of other potential substrates.

    Funded by: NIA NIH HHS: AG019070, AG021494, AG021495, R01 AG019070, R01 AG021494, R01 AG021495

    The Journal of biological chemistry 2005;280;27;25892-900

  • Akt-dependent expression of NAIP-1 protects neurons against amyloid-{beta} toxicity.

    Lesné S, Gabriel C, Nelson DA, White E, Mackenzie ET, Vivien D and Buisson A

    UMR CNRS 6185, Université de Caen, Bd. H. Becquerel BP5229, 14074 Caen, France and Howard Hughes Medical Institute, Rutgers University, Piscataway, New Jersey 08854, USA.

    Neurotrophins are a family of growth factors that attenuate several forms of pathological neuronal cell death and may represent a putative therapeutic approach to neurodegenerative diseases. In Alzheimer disease, amyloid-beta (Abeta) is thought to play a central role in the neuronal death occurring in brains of patients. In the present study, we evaluate the ability of neurotrophin-3 (NT-3) to protect neurons against the toxicity induced by aggregated Abeta. We showed that in primary cultures of cortical neurons, NT-3 reduces Abeta-induced apoptosis by limiting caspase-8, caspase-9, and caspase-3 cleavage. This neuroprotective effect of NT-3 was concomitant to an increased level of Akt phosphorylation and was abolished by an inhibitor of the phosphatidylinositol-3 kinase (PI-3K), LY294002. In parallel, NT-3 treatment reduced Abeta induced caspase-3 processing to control levels. In an attempt to link PI-3K/Akt to caspase inhibition, we evaluated the influence of the PI-3K/Akt axis on the expression of a member of the inhibitors of apoptosis proteins (IAPs), the neuronal apoptosis inhibitory protein-1. We demonstrated that NT-3 induces an up-regulation of neuronal apoptosis inhibitory protein-1 expression in neurons that promotes the inhibition of Abeta-induced neuronal apoptosis. Together, these findings demonstrate that NT-3 signaling counters Abeta-dependent neuronal cell death and may represent an innovative therapeutic intervention to limit neuronal death in Alzheimer disease.

    The Journal of biological chemistry 2005;280;26;24941-7

  • mTOR/p70S6k signalling alteration by Abeta exposure as well as in APP-PS1 transgenic models and in patients with Alzheimer's disease.

    Lafay-Chebassier C, Paccalin M, Page G, Barc-Pain S, Perault-Pochat MC, Gil R, Pradier L and Hugon J

    Department of Pharmacology, Poitiers University Hospital, France.

    In Alzheimer's disease, neuropathological hallmarks include the accumulation of beta-amyloid peptides (Abeta) in senile plaques, phosphorylated tau in neurofibrillary tangles and neuronal death. Abeta is the major aetiological agent according to the amyloid cascade hypothesis. Translational control includes phosphorylation of the kinases mammalian target of rapamycin (mTOR) and p70S6k which modulate cell growth, proliferation and autophagy. It is mainly part of an anti-apoptotic cellular signalling. In this study, we analysed modifications of mTOR/p70S6k signalling in cellular and transgenic models of Alzheimer's disease, as well as in lymphocytes of patients and control individuals. Abeta 1-42 produced a rapid and persistent down-regulation of mTOR/p70S6k phosphorylation in murine neuroblastoma cells associated with caspase 3 activation. Using western blottings, we found that phosphorylated forms of mTOR and p70S6k are decreased in the cortex but not in the cerebellum (devoid of plaques) of double APP/PS1 transgenic mice compared with control mice. These results were confirmed by immunohistochemical methods. Finally, the expression of phosphorylated p70S6k was significantly reduced in lymphocytes of Alzheimer's patients, and levels of phosphorylated p70S6k were statistically correlated with Mini Mental Status Examination (MMSE) scores. Taken together, these findings demonstrate that the mainly anti-apoptotic mTOR/p70S6k signalling is altered in cellular and transgenic models of Alzheimer's disease and in peripheral cells of patients, and could contribute to the pathogenesis of the disease.

    Journal of neurochemistry 2005;94;1;215-25

  • Estradiol regulates APP mRNA alternative splicing in the mice brain cortex.

    Thakur MK and Mani ST

    Biochemistry & Molecular Biology Laboratory, Department of Zoology, Banaras Hindu University, Varanasi 221005, India. mkt_bhu@yahoo.com

    Alternative splicing of amyloid precursor protein (APP), one of the candidate genes for Alzheimer's disease, yields three major mRNAs, which give rise to APP770, APP751 and APP695 protein isoforms. Out of these three isoforms, APP695 is expressed most predominantly in the brain. The splicing and processing of APP are shown to be influenced by several factors including hormones. In this study, we report the effect of withdrawal and administration of sex steroid hormones on the alternative splicing of APP mRNA during aging in the brain cortex of mice of both sexes. The level of APP695 mRNA isoform was higher in intact adult as compared to old mice of both sexes. Gonadectomy upregulated the APP695 mRNA isoform levels in all groups except in adult female where the level was downregulated. Estradiol supplementation upregulated the level of APP695 mRNA isoform in all groups except in old male where the level was downregulated. Thus these results show that the level of APP695 mRNA changes with age and estradiol may play a key role in the development of Alzheimer's disease by modulating the level of APP mRNA isoforms.

    Neuroscience letters 2005;381;1-2;154-7

  • Presenilin-dependent transcriptional control of the Abeta-degrading enzyme neprilysin by intracellular domains of betaAPP and APLP.

    Pardossi-Piquard R, Petit A, Kawarai T, Sunyach C, Alves da Costa C, Vincent B, Ring S, D'Adamio L, Shen J, Müller U, St George Hyslop P and Checler F

    Institut de Pharmacologie Moléculaire et Cellulaire, Centre National de la Recherche Scientifique, UMR6097 CNRS/UNSA, Valbonne 06560, France.

    Amyloid beta-peptide (Abeta), which plays a central role in Alzheimer's disease, is generated by presenilin-dependent gamma-secretase cleavage of beta-amyloid precursor protein (betaAPP). We report that the presenilins (PS1 and PS2) also regulate Abeta degradation. Presenilin-deficient cells fail to degrade Abeta and have drastic reductions in the transcription, expression, and activity of neprilysin, a key Abeta-degrading enzyme. Neprilysin activity and expression are also lowered by gamma-secretase inhibitors and by PS1/PS2 deficiency in mouse brain. Neprilysin activity is restored by transient expression of PS1 or PS2 and by expression of the amyloid intracellular domain (AICD), which is cogenerated with Abeta, during gamma-secretase cleavage of betaAPP. Neprilysin gene promoters are transactivated by AICDs from APP-like proteins (APP, APLP1, and APLP2), but not by Abeta or by the gamma-secretase cleavage products of Notch, N- or E- cadherins. The presenilin-dependent regulation of neprilysin, mediated by AICDs, provides a physiological means to modulate Abeta levels with varying levels of gamma-secretase activity.

    Neuron 2005;46;4;541-54

  • Neocortical synaptic bouton number is maintained despite robust amyloid deposition in APP23 transgenic mice.

    Boncristiano S, Calhoun ME, Howard V, Bondolfi L, Kaeser SA, Wiederhold KH, Staufenbiel M and Jucker M

    Department of Neuropathology, Institute of Pathology, University of Basel, Switzerland.

    Major pathological findings in Alzheimer's disease (AD) brain include the deposition of amyloid-beta and synapse loss. Synaptic loss has been shown to correlate with the cognitive decline in AD patients, but the relationship between cerebral amyloidosis and synapse loss is complicated by the presence of neurofibrillary tangles and other lesions in AD brain. With the use of the APP23 transgenic mouse model that overexpresses human amyloid precursor protein (APP) with the Swedish double mutation, we investigated whether the development of cortical amyloid deposition was accompanied by synaptic bouton loss. With stereological methods, we show that despite robust age-related cortical amyloid deposition with associated synaptic degeneration, the total number of cortical synaptophysin-positive presynaptic terminals is not changed in 24-month-old animals compared with 3-, 8-, and 15-month-old APP23 mice. Wild-type mice also do not show an age-related loss of presynaptic boutons in the neocortex and are not significantly different from APP23 mice. Synaptophysin Western blotting revealed no significant difference between APP23 mice and wild-type controls at 3 and 25 months of age. Our results suggest that cerebral amyloidosis is not sufficient to account for the global synapse loss in AD. Alternatively, a putative trophic effect of APP may prevent, compensate, or delay a loss of synapses in this mouse model.

    Neurobiology of aging 2005;26;5;607-13

  • The amyloid precursor protein (APP) of Alzheimer disease and its paralog, APLP2, modulate the Cu/Zn-Nitric Oxide-catalyzed degradation of glypican-1 heparan sulfate in vivo.

    Cappai R, Cheng F, Ciccotosto GD, Needham BE, Masters CL, Multhaup G, Fransson LA and Mani K

    Department of Pathology and Center for Neuroscience, The University of Melbourne, Victoria 3010, Australia.

    Processing of the recycling proteoglycan glypican-1 involves the release of its heparan sulfate chains by copper ion- and nitric oxide-catalyzed ascorbate-triggered autodegradation. The Alzheimer disease amyloid precursor protein (APP) and its paralogue, the amyloid precursor-like protein 2 (APLP2), contain copper ion-, zinc ion-, and heparan sulfate-binding domains. We have investigated the possibility that APP and APLP2 regulate glypican-1 processing during endocytosis and recycling. By using cell-free biochemical experiments, confocal laser immunofluorescence microscopy, and flow cytometry of tissues and cells from wild-type and knock-out mice, we find that (a) APP and glypican-1 colocalize in perinuclear compartments of neuroblastoma cells, (b) ascorbate-triggered nitric oxidecatalyzed glypican-1 autodegradation is zinc ion-dependent in the same cells, (c) in cell-free experiments, APP but not APLP2 stimulates glypican-1 autodegradation in the presence of both Cu(II) and Zn(II) ions, whereas the Cu(I) form of APP and the Cu(II) and Cu(I) forms of APLP2 inhibit autodegradation, (d) in primary cortical neurons from APP or APLP2 knock-out mice, there is an increased nitric oxide-catalyzed degradation of heparan sulfate compared with brain tissue and neurons from wild-type mice, and (e) in growth-quiescent fibroblasts from APLP2 knock-out mice, but not from APP knock-out mice, there is also an increased heparan sulfate degradation. We propose that the rate of autoprocessing of glypican-1 is modulated by APP and APLP2 in neurons and by APLP2 in fibroblasts. These observation identify a functional relationship between the heparan sulfate and copper ion binding activities of APP/APLP2 in their modulation of the nitroxyl anion-catalyzed heparan sulfate degradation in glypican-1.

    The Journal of biological chemistry 2005;280;14;13913-20

  • Endoproteolytic cleavage of FE65 converts the adaptor protein to a potent suppressor of the sAPPalpha pathway in primates.

    Hu Q, Wang L, Yang Z, Cool BH, Zitnik G and Martin GM

    Department of Pathology, University of Washington, Seattle, Washington, 98195, USA. qhu@u.washington.edu

    Adaptor protein FE65 (APBB1) specifically binds to the intracellular tail of the type I transmembrane protein, beta-amyloid precursor protein (APP). The formation of this complex may be important for modulation of the processing and function of APP. APP is proteolytically cleaved at multiple sites. The cleavages and their regulation are of central importance in the pathogenesis of dementias of the Alzheimer type. In cell cultures and perhaps in vivo, secretion of the alpha-cleaved APP ectodomain (sAPPalpha) is the major pathway in the most cells. Regulation of the process may require extracellular/intracellular cues. Neither extracellular ligands nor intracellular mediators have been identified, however. Here, we show novel evidence that the major isoform of FE65 (97-kDa FE65, p97FE65) can be converted to a 65-kDa N-terminally truncated C-terminal fragment (p65FE65) via endoproteolysis. The cleavage region locates immediately after an acidic residue cluster but before the three major protein-protein binding domains. The cleavage activity is particularly high in human and non-human primate cells and low in rodent cells; the activity appears to be triggered/enhanced by high cell density, presumably via cell-cell/cell-substrate contact cues. As a result, p65FE65 exhibits extraordinarily high affinity for APP (up to 40-fold higher than p97FE65) and potent suppression (up to 90%) of secretion of sAPPalpha. Strong p65FE65-APP binding is required for the suppression. The results suggest that p65FE65 may be an intracellular mediator in a signaling cascade regulating alpha-secretion of APP, particularly in primates.

    Funded by: NIA NIH HHS: AG05136-19, AG19711

    The Journal of biological chemistry 2005;280;13;12548-58

  • Analysis of cholinergic markers, biogenic amines, and amino acids in the CNS of two APP overexpression mouse models.

    Van Dam D, Marescau B, Engelborghs S, Cremers T, Mulder J, Staufenbiel M and De Deyn PP

    Laboratory of Neurochemistry and Behaviour, Born-Bunge Institute, Department of Biomedical Sciences, University of Antwerp, Universiteitsplein 1, B-2610 Wilrijk, Belgium.

    Two transgenic mouse models expressing mutated human amyloid precursor protein and previously found to display cognitive and behavioural alterations, reminiscent of Alzheimer patients' symptomatology, were scrutinised for putative brain region-specific changes in neurochemical parameters. Brains of NSE-hAPP751m-57, APP23 and wild-type mice were microdissected to perform brain region-specific neurochemical analyses. Impairment of cholinergic transmission, the prominent neurochemical deficit in Alzheimer brain, was examined; acetylcholinesterase and choline acetyltransferase activity levels were determined as markers of the cholinergic system. Since Alzheimer neurodegeneration is not restricted to the cholinergic system, brain levels of biogenic amines and metabolites, and amino acidergic neurotransmitters and systemic amino acids were analysed as well. Cholinergic dysfunction, reflected in reduced enzymatic activity in the basal forebrain nuclei, was restricted to the APP23 model, which also exhibited more outspoken and more widespread changes in other neurotransmitter systems. Significant changes in compounds of the noradrenergic and serotonergic system were observed, as well as alterations in levels of the inhibitory neurotransmitter glycine and systemic amino acids. These observations were clearly in occurrence with the more pronounced histopathological and behavioural phenotype of the APP23 model. As transgenic models often do not represent an end-stage of the disease, some discrepancies with results from post-mortem human Alzheimer brain analyses were apparent; in particular, no significant alterations in excitatory amino acid levels were detected. Our findings of brain region-specific alterations in compound levels indicate disturbed neurotransmission pathways, and greatly add to the validity of APP23 mice as a model for Alzheimer's disease. Transgenic mouse models may be employed as a tool to study early-stage neurochemical changes, which are often not accessible in Alzheimer brain.

    Neurochemistry international 2005;46;5;409-22

  • Somatostatin regulates brain amyloid beta peptide Abeta42 through modulation of proteolytic degradation.

    Saito T, Iwata N, Tsubuki S, Takaki Y, Takano J, Huang SM, Suemoto T, Higuchi M and Saido TC

    Laboratory for Proteolytic Neuroscience, RIKEN Brain Science Institute, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan.

    Expression of somatostatin in the brain declines during aging in various mammals including apes and humans. A prominent decrease in this neuropeptide also represents a pathological characteristic of Alzheimer disease. Using in vitro and in vivo paradigms, we show that somatostatin regulates the metabolism of amyloid beta peptide (Abeta), the primary pathogenic agent of Alzheimer disease, in the brain through modulating proteolytic degradation catalyzed by neprilysin. Among various effector candidates, only somatostatin upregulated neprilysin activity in primary cortical neurons. A genetic deficiency of somatostatin altered hippocampal neprilysin activity and localization, and increased the quantity of a hydrophobic 42-mer form of Abeta, Abeta(42), in a manner similar to presenilin gene mutations that cause familial Alzheimer disease. These results indicate that the aging-induced downregulation of somatostatin expression may be a trigger for Abeta accumulation leading to late-onset sporadic Alzheimer disease, and suggest that somatostatin receptors may be pharmacological-target candidates for prevention and treatment of Alzheimer disease.

    Nature medicine 2005;11;4;434-9

  • Axonal transport, amyloid precursor protein, kinesin-1, and the processing apparatus: revisited.

    Lazarov O, Morfini GA, Lee EB, Farah MH, Szodorai A, DeBoer SR, Koliatsos VE, Kins S, Lee VM, Wong PC, Price DL, Brady ST and Sisodia SS

    Department of Neurobiology, Pharmacology, and Physiology, The University of Chicago, Chicago, Illinois 60637, USA.

    The sequential enzymatic actions of beta-APP cleaving enzyme 1 (BACE1), presenilins (PS), and other proteins of the gamma-secretase complex liberate beta-amyloid (Abeta) peptides from larger integral membrane proteins, termed beta-amyloid precursor proteins (APPs). Relatively little is known about the normal function(s) of APP or the neuronal compartment(s) in which APP undergoes proteolytic processing. Recent studies have been interpreted as consistent with the idea that APP serves as a kinesin-1 cargo receptor and that PS and BACE1 are associated with the APP-resident membranous cargos that undergo rapid axonal transport. In this report, derived from a collaboration among several independent laboratories, we examined the potential associations of APP and kinesin-1 using glutathione S-transferase pull-down and coimmunoprecipitation assays. In addition, we assessed the trafficking of membrane proteins in the sciatic nerves of transgenic mice with heterozygous or homozygous deletions of APP. In contrast to previous reports, we were unable to find evidence for direct interactions between APP and kinesin-1. Furthermore, the transport of kinesin-1 and tyrosine kinase receptors, previously reported to require APP, was unchanged in axons of APP-deficient mice. Finally, we show that two components of the APP proteolytic machinery, i.e., PS1 and BACE1, are not cotransported with APP in the sciatic nerves of mice. These findings suggest that the hypothesis that APP serves as a kinesin-1 receptor and that the proteolytic processing machinery responsible for generating Abeta is transported in the same vesicular compartment in axons of peripheral nerves requires revision.

    Funded by: NIA NIH HHS: AG-021494, AG05146, AG11542; NINDS NIH HHS: NS23320, NS23868, NS41170, NS41438, NS43408, NS45150

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2005;25;9;2386-95

  • Axonopathy and transport deficits early in the pathogenesis of Alzheimer's disease.

    Stokin GB, Lillo C, Falzone TL, Brusch RG, Rockenstein E, Mount SL, Raman R, Davies P, Masliah E, Williams DS and Goldstein LS

    Howard Hughes Medical Institute and Department of Cellular and Molecular Medicine, School of Medicine, University of California San Diego (UCSD), 9500 Gilman Drive, La Jolla, CA 92093, USA.

    We identified axonal defects in mouse models of Alzheimer's disease that preceded known disease-related pathology by more than a year; we observed similar axonal defects in the early stages of Alzheimer's disease in humans. Axonal defects consisted of swellings that accumulated abnormal amounts of microtubule-associated and molecular motor proteins, organelles, and vesicles. Impairing axonal transport by reducing the dosage of a kinesin molecular motor protein enhanced the frequency of axonal defects and increased amyloid-beta peptide levels and amyloid deposition. Reductions in microtubule-dependent transport may stimulate proteolytic processing of beta-amyloid precursor protein, resulting in the development of senile plaques and Alzheimer's disease.

    Funded by: NEI NIH HHS: EY12598, EY13408, R01 EY007042, R01 EY007042-19, R01 EY013408, R01 EY013408-02; NIA NIH HHS: P50 AG05131

    Science (New York, N.Y.) 2005;307;5713;1282-8

  • The liver X receptor ligand T0901317 decreases amyloid beta production in vitro and in a mouse model of Alzheimer's disease.

    Koldamova RP, Lefterov IM, Staufenbiel M, Wolfe D, Huang S, Glorioso JC, Walter M, Roth MG and Lazo JS

    Department of Pharmacology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261, USA. radak@pitt.edu

    Recent studies indicate that oxysterols, which are ligands for the nuclear hormone liver X receptors (LXR), decrease amyloid beta (Abeta) secretion in vitro. The effect was attributed primarily to the ATP-binding cassette transporter A1 (ABCA1) transcriptionally up-regulated by ligand-activated LXRs. We now examined the effect of the synthetic LXR ligand T0901317, which can be used in vivo, on Abeta production in vitro and in APP23 transgenic mice. T0901317 applied to a variety of in vitro models, including immortalized fibroblasts from Tangier patients, and primary embryonic mouse neurons caused a concentration-dependent decrease in Abeta secretion, and this effect was increased by the addition of apolipoprotein A-I. The inhibition of Abeta production by T0901317 was cell-type specific, being more prominent in primary neurons than in non-neuronal cells. Tangier fibroblasts lacking a functional ABCA1 secreted more Abeta than control fibroblasts, thus demonstrating the role of ABCA1 in amyloid precursor protein (APP) processing and Abeta generation. T0901317 treatment of 11-week-old APP23 mice for 6 days showed a significant increase in ABCA1 expression and a decrease in the ratio of soluble APP (sAPP)beta- to sAPPalpha-cleavage products. Most importantly, the treatment caused a statistically significant reduction in the levels of soluble Abeta40 and of Abeta42 in the brain these mice. Our experiments demonstrate that T0901317 decreases amyloidogenic processing of APP in vitro and in vivo, thus supporting the search for potent and specific LXR ligands with properties allowing therapeutic application.

    Funded by: NIA NIH HHS: AG 18558, AG 23304

    The Journal of biological chemistry 2005;280;6;4079-88

  • Defective neuromuscular synapses in mice lacking amyloid precursor protein (APP) and APP-Like protein 2.

    Wang P, Yang G, Mosier DR, Chang P, Zaidi T, Gong YD, Zhao NM, Dominguez B, Lee KF, Gan WB and Zheng H

    Huffington Center on Aging, Baylor College of Medicine, Houston, Texas 77030, USA.

    Biochemical and genetic studies place the amyloid precursor protein (APP) at the center stage of Alzheimer's disease (AD) pathogenesis. Although mutations in the APP gene lead to dominant inheritance of familial AD, the normal function of APP remains elusive. Here, we report that the APP family of proteins plays an essential role in the development of neuromuscular synapses. Mice deficient in APP and its homolog APP-like protein 2 (APLP2) exhibit aberrant apposition of presynaptic marker proteins with postsynaptic acetylcholine receptors and excessive nerve terminal sprouting. The number of synaptic vesicles at presynaptic terminals is dramatically reduced. These structural abnormalities are accompanied by defective neurotransmitter release and a high incidence of synaptic failure. Our results identify APP/APLP2 as key regulators of structure and function of developing neuromuscular synapses.

    Funded by: NIA NIH HHS: AG20670, AG21141; NINDS NIH HHS: NS041846, NS40039

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2005;25;5;1219-25

  • Altered metabolic profile in the frontal cortex of PS2APP transgenic mice, monitored throughout their life span.

    von Kienlin M, Künnecke B, Metzger F, Steiner G, Richards JG, Ozmen L, Jacobsen H and Loetscher H

    F. Hoffmann-La Roche Pharmaceuticals Ltd., CH-4070 Basel, Switzerland. markus.von_kienlin@roche.com

    The transgenic mouse line PS2APP (PS2N141I x APP(swe)) develops an age-related cognitive decline associated with severe amyloidosis, mimicking the pathophysiologic processes in Alzheimer disease (AD). In the quest for biomarkers to monitor, noninvasively, the progression of the disease, we used magnetic resonance imaging and 1H-spectroscopy to characterize PS2APP mice throughout their life span. Morphometric measurements revealed only small size differences to controls. The metabolic profile, however, showed clear indicators of hypometabolism with age in the PS2APP mice: both N-acetyl-aspartate and glutamate were significantly reduced in the older animals. These spectroscopic measures in vivo correlated well with the plaque load in the frontal cortex. A diagnostic test, based on these measures, reached 92% sensitivity and 82% specificity at age 20 months. These noninvasive biomarkers can be exploited in preclinical pharmaceutical research to cope with the high variability in transgenic animal models and to enhance the power of drug efficacy studies.

    Neurobiology of disease 2005;18;1;32-9

  • Increased tau phosphorylation on mitogen-activated protein kinase consensus sites and cognitive decline in transgenic models for Alzheimer's disease and FTDP-17: evidence for distinct molecular processes underlying tau abnormalities.

    Lambourne SL, Sellers LA, Bush TG, Choudhury SK, Emson PC, Suh YH and Wilkinson LS

    Neurobiology Programme, The Babraham Institute, Babraham, Cambridge CB2 4AT, United Kingdom.

    Abnormal tau phosphorylation occurs in several neurodegenerative disorders, including Alzheimer's disease (AD) and frontotemporal dementia with Parkinsonism linked to chromosome 17 (FTDP-17). Here, we compare mechanisms of tau phosphorylation in mouse models of FTDP-17 and AD. Mice expressing a mutated form of human tau associated with FTDP-17 (tau(V337M)) showed age-related increases in exogenous tau phosphorylation in the absence of increased activation status of a number of kinases known to phosphorylate tau in vitro. In a "combined" model, expressing both tau(V337M) and the familial amyloid precursor protein AD mutation APP(V717I) in a CT100 fragment, age-dependent tau phosphorylation occurred at the same sites and was significantly augmented compared to "single" tau(V337M) mice. These effects were concomitant with increased activation status of mitogen-activated protein kinase (MAPK) family members (extracellular regulated kinases 1 and 2, p38, and c-Jun NH(2)-terminal kinase) but not glycogen synthase kinase-3alphabeta or cyclin-dependent kinase 5. The increase in MAPK activation was a discrete effect of APP(V717I)-CT100 transgene expression as near identical changes were observed in single APP(V717I)-CT100 mice. Age-dependent deficits in memory were also associated with tau(V337M) and APP(V717I)-CT100 expression. The data reveal distinct routes to abnormal tau phosphorylation in models of AD and FTDP-17 and suggest that in AD, tau irregularities may be linked to processing of APP C-terminal fragments via specific effects on MAPK activation status.

    Molecular and cellular biology 2005;25;1;278-93

  • Lengthening of G2/mitosis in cortical precursors from mice lacking beta-amyloid precursor protein.

    López-Sánchez N, Müller U and Frade JM

    Instituto Cajal, CSIC, Avda. Dr Arce, 37, E-28002 Madrid, Spain.

    The beta-amyloid precursor protein (APP) is expressed within the nervous system, even at the earliest stages of embryonic development when cell growth and proliferation is particularly important. In order to study the function of APP at these early developmental stages, we have studied the development of the cerebral cortex in both wild type and App-/- mutant mice. Here, we demonstrate that APP mRNA is expressed in cortical precursor cells and that APP protein is concentrated within their apical domains during interphase. However, during mitosis, APP re-localizes to the peripheral space surrounding the metaphase plate. In APP-deficient cortical precursors, the duration of mitosis is increased and a higher proportion of cortical precursor cells contained nuclei in late G2. We conclude that during cortical development APP plays a role in controlling cell cycle progression, particularly affecting G2 and mitosis. These observations may have important implications for our understanding of how APP influences the progression of Alzheimer's disease, since degenerating cortical neurons have been shown to up-regulate cell cycle markers and re-enter the mitotic cycle before dying.

    Neuroscience 2005;130;1;51-60

  • Gene expression profiling in chronic copper overload reveals upregulation of Prnp and App.

    Armendariz AD, Gonzalez M, Loguinov AV and Vulpe CD

    Department of Nutritional Science and Toxicology, University of California, Berkeley 94720, USA.

    The level at which copper becomes toxic is not clear. Several studies have indicated that copper causes oxidative stress; however, most have tested very high levels of copper exposure. We currently have only a limited understanding of the protective systems that operate in cells chronically exposed to copper. Additionally, the limits of homeostatic regulation are not known, making it difficult to define the milder effects of copper excess. Furthermore, a robust assay to facilitate the diagnosis of copper excess and to distinguish mild, moderate, and severe copper overload is needed. To address these issues, we have investigated the effects on steady-state gene expression of chronic copper overload in a cell culture model system using cDNA microarrays. For this study we utilized cells from genetic models of copper overload: fibroblast cells from two mouse mutants, C57BL/6-Atp7a(Mobr) and C57BL/6-Atp7a(Modap). These cell lines accumulate copper to abnormally high levels in normal culture media due to a defect in copper export from the cell. We identified 12 differentially expressed genes in common using our outlier identification methods. Surprisingly, our results show no evidence of oxidative stress in the copper-loaded cells. In addition, candidate components perhaps responsible for a copper-specific homeostatic response are identified. The genes that encode for the prion protein and the amyloid-beta precursor protein, two known copper-binding proteins, are upregulated in both cell lines.

    Physiological genomics 2004;20;1;45-54

  • Neuronal membrane cholesterol loss enhances amyloid peptide generation.

    Abad-Rodriguez J, Ledesma MD, Craessaerts K, Perga S, Medina M, Delacourte A, Dingwall C, De Strooper B and Dotti CG

    Cavalieri Ottolenghi Scientific Institute, Universita degli Studi di Torino, Orbassano, Italy.

    Recent experimental and clinical retrospective studies support the view that reduction of brain cholesterol protects against Alzheimer's disease (AD). However, genetic and pharmacological evidence indicates that low brain cholesterol leads to neurodegeneration. This apparent contradiction prompted us to analyze the role of neuronal cholesterol in amyloid peptide generation in experimental systems that closely resemble physiological and pathological situations. We show that, in the hippocampus of control human and transgenic mice, only a small pool of endogenous APP and its beta-secretase, BACE 1, are found in the same membrane environment. Much higher levels of BACE 1-APP colocalization is found in hippocampal membranes from AD patients or in rodent hippocampal neurons with a moderate reduction of membrane cholesterol. Their increased colocalization is associated with elevated production of amyloid peptide. These results suggest that loss of neuronal membrane cholesterol contributes to excessive amyloidogenesis in AD and pave the way for the identification of the cause of cholesterol loss and for the development of specific therapeutic strategies.

    The Journal of cell biology 2004;167;5;953-60

  • Interaction of human and mouse Abeta peptides.

    Fung J, Frost D, Chakrabartty A and McLaurin J

    Centre for Research in Neurodegenerative Diseases, Ontario Cancer Institute, Toronto, Ontario, Canada.

    Transgenic mice over-expressing mutant human amyloid precursor protein have become an important tool for research on Alzheimer's disease (AD) and, in particular, for therapeutic screening. Many models have reported formation of amyloid plaques with age as is detected in AD. However, the plaques generated in transgenic mice are more soluble than human plaques. Differences in solubility may occur for a number of reasons; one proposal is the presence of murine Abeta peptides within the CNS milieu. Here, we report the interaction of human and murine Abeta peptides, Abeta40 and Abeta42, utilizing a fluorescence assay to monitor formation of mixed pre-fibrillar aggregates, electron microscopy to examine morphological characteristics and detergent solubility to monitor stability. Our results demonstrate that interspecies Abeta aggregates and fibres are readily formed and are more stable than homogenous human fibres. Furthermore, these results suggest that the presence of endogenous murine Abeta in human APP transgenic mice does not account for the increased solubility of plaques.

    Journal of neurochemistry 2004;91;6;1398-403

  • Libraries enriched for alternatively spliced exons reveal splicing patterns in melanocytes and melanomas.

    Watahiki A, Waki K, Hayatsu N, Shiraki T, Kondo S, Nakamura M, Sasaki D, Arakawa T, Kawai J, Harbers M, Hayashizaki Y and Carninci P

    Genome Science Laboratory, RIKEN, Wako main campus, 2-1 Hirosawa, Wako, Saitama, 351-0198 Japan.

    It is becoming increasingly clear that alternative splicing enables the complex development and homeostasis of higher organisms. To gain a better understanding of how splicing contributes to regulatory pathways, we have developed an alternative splicing library approach for the identification of alternatively spliced exons and their flanking regions by alternative splicing sequence enriched tags sequencing. Here, we have applied our approach to mouse melan-c melanocyte and B16-F10Y melanoma cell lines, in which 5,401 genes were found to be alternatively spliced. These genes include those encoding important regulatory factors such as cyclin D2, Ilk, MAPK12, MAPK14, RAB4, melastatin 1 and previously unidentified splicing events for 436 genes. Real-time PCR further identified cell line-specific exons for Tmc6, Abi1, Sorbs1, Ndel1 and Snx16. Thus, the ASL approach proved effective in identifying splicing events, which suggest that alternative splicing is important in melanoma development.

    Nature methods 2004;1;3;233-9

  • The neuronal adaptor protein X11beta reduces amyloid beta-protein levels and amyloid plaque formation in the brains of transgenic mice.

    Lee JH, Lau KF, Perkinton MS, Standen CL, Rogelj B, Falinska A, McLoughlin DM and Miller CC

    Department of Neuroscience and Section of Old Age Psychiatry, The Institute of Psychiatry, King's College London SE5 8AF, United Kingdom.

    Accumulation of cerebral amyloid beta-protein (Abeta) is believed to be part of the pathogenic process in Alzheimer's disease. Abeta is derived by proteolytic cleavage from a precursor protein, the amyloid precursor protein (APP). APP is a type-1 membrane-spanning protein, and its carboxyl-terminal intracellular domain binds to X11beta, a neuronal adaptor protein. X11beta has been shown to inhibit the production of Abeta in transfected non-neuronal cells in culture. However, whether this is also the case in vivo in the brain and whether X11beta can also inhibit the deposition of Abeta as amyloid plaques is not known. Here we show that transgenic overexpression of X11beta in neurons leads to a decrease in cerebral Abeta levels in transgenic APPswe Tg2576 mice that are a model of the amyloid pathology of Alzheimer's disease. Moreover, overexpression of X11beta retards amyloid plaque formation in these APPswe mice. Our findings suggest that modulation of X11beta function may represent a novel therapeutic approach for preventing the amyloid pathology of Alzheimer's disease.

    Funded by: Medical Research Council: G0000749

    The Journal of biological chemistry 2004;279;47;49099-104

  • Reduced beta-amyloid production and increased inflammatory responses in presenilin conditional knock-out mice.

    Beglopoulos V, Sun X, Saura CA, Lemere CA, Kim RD and Shen J

    Center for Neurologic Diseases, Brigham and Women's Hospital, Program in Neuroscience, Harvard Medical School, Boston, Massachusetts 02115, USA.

    Mutations in presenilins (PS) 1 and 2 are the major cause of familial Alzheimer's disease. Conditional double knock-out mice lacking both presenilins in the postnatal forebrain (PS cDKO mice) exhibit memory and synaptic plasticity impairments followed by progressive neurodegeneration in the cerebral cortex. Here we further investigate the molecular events that may underlie the observed phenotypes and identify additional neuropathological markers in the PS cDKO brain. Enzyme-linked immunosorbent assay analysis showed reduced levels of the toxic beta-amyloid (Abeta) peptides in the cerebral cortex of PS cDKO mice. Interestingly, the reduction in Abeta40 and Abeta42 peptides is similar in PS1 conditional knock-out and PS cDKO mice. We further examined the gene expression profile by oligonucleotide microarrays in the PS cDKO cerebral cortex and found that a high number of genes are differentially expressed, most notably a group of up-regulated inflammatory genes. Quantitative real-time reverse transcription PCR and Western analyses confirmed the elevated levels of glial fibrillary acidic protein, complement component C1q, and cathepsin S, up-regulation of which has been associated with inflammatory responses in various neurodegenerative processes. Immunohistochemical analysis revealed that the increase in complement component C1q is confined to the hippocampal formation, whereas glial fibrillary acidic protein and cathepsin S are up-regulated throughout the entire neocortex and hippocampus. In addition, strong microglial activation occurs in the hippocampus and the deeper cortical layers of PS cDKO mice. These results indicate that the memory impairment and neurodegeneration in PS cDKO mice are not caused by Abeta accumulation and that loss of PS function leads to differential up-regulation of inflammatory markers in the cerebral cortex.

    Funded by: NINDS NIH HHS: NS41783

    The Journal of biological chemistry 2004;279;45;46907-14

  • Subcellular topography of neuronal Abeta peptide in APPxPS1 transgenic mice.

    Langui D, Girardot N, El Hachimi KH, Allinquant B, Blanchard V, Pradier L and Duyckaerts C

    Laboratoire de Neuropathologie Raymond Escourolle, Groupe hospitalier Pitié-Salpêtrière, 47, boulevard de l'Hôpital, 75013 Paris, France.

    In transgenic mice expressing human mutant beta-amyloid precursor protein (APP) and mutant presenilin-1 (PS1), Abeta antibodies labeled granules, about 1 microm in diameter, in the perikaryon of neurons clustered in the isocortex, hippocampus, amygdala, thalamus, and brainstem. The granules were present before the onset of Abeta deposits; their number increased up to 9 months and decreased in 15-month-old animals. They were immunostained by antibodies against Abeta 40, Abeta 42, and APP C-terminal region. In double immunofluorescence experiments, the intracellular Abeta co-localized with lysosome markers and less frequently with MG160, a Golgi marker. Abeta accumulation correlated with an increased volume of lysosomes and Golgi apparatus, while the volume of endoplasmic reticulum and early endosomes did not change. Some granules were immunolabeled with an antibody against flotillin-1, a raft marker. At electron microscopy, Abeta, APP-C terminal, cathepsin D, and flotillin-1 epitopes were found in the lumen of multivesicular bodies. This study shows that Abeta peptide and APP C-terminal region accumulate in multivesicular bodies containing lysosomal enzymes, while APP N-terminus is excluded from them. Multivesicular bodies could secondarily liberate their content in the extracellular space as suggested by the association of cathepsin D with Abeta peptide in the extracellular space.

    The American journal of pathology 2004;165;5;1465-77

  • Time-dependent reduction in Abeta levels after intracranial LPS administration in APP transgenic mice.

    Herber DL, Roth LM, Wilson D, Wilson N, Mason JE, Morgan D and Gordon MN

    Alzheimer Research Laboratory, Department of Pharmacology and Therapeutics, University of South Florida, Tampa, FL 33612-4799, USA.

    Inflammation has been argued to play a primary role in the pathogenesis of Alzheimer's disease (AD). Lipopolysaccharide (LPS) activates the innate immune system, triggering gliosis and inflammation when injected in the central nervous system. In studies described here, APP transgenic mice were injected intrahippocampally with 4 or 10 microg of LPS and evaluated 1, 3, 7, 14, or 28 days later. Abeta load was significantly reduced at 3, 7, and 14 days but surprisingly returned near baseline 28 days after the injection. No effects of LPS on congophilic amyloid deposits could be detected. LPS also activated both microglia and astrocytes in a time-dependent manner. The GFAP astrocyte reaction and the Fcgamma receptor microglial reaction peaked at 7 days after LPS injection, returning to baseline by 2 weeks postinjection. When stained for CD45, microglial activation was detected at all time points, although the morphology of these cells transitioned from an ameboid to a ramified and bushy appearance between 7 and 14 days postinjection. These results indicate that activation of brain glia can rapidly and transiently clear diffuse Abeta deposits but has no effect on compacted fibrillar amyloid.

    Funded by: NIA NIH HHS: AG 15490, AG 18478

    Experimental neurology 2004;190;1;245-53

  • Construction of a multi-functional cDNA library specific for mouse pancreatic islets and its application to microarray.

    Nishimura M, Yokoi N, Miki T, Horikawa Y, Yoshioka H, Takeda J, Ohara O and Seino S

    Department of Cellular and Molecular Medicine, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan.

    We have constructed a high-quality and multi-applicable cDNA library specific for mouse pancreatic islets. This is the first pancreatic islet cDNA library created using a recombination-based method, which can readily be converted into other applications including yeast two-hybrid and mammalian expression libraries. Based on sequence data of the library, we constructed a sequence database specific for mouse pancreatic islets. Among the 8882 non-redundant clones, 5799 were classified into specific functional categories using a classification system designed by the Gene Ontology Consortium, 10% of which were "molecular function unknown" genes. We also developed cDNA microarray membranes with 8108 non-redundant clones. Analyses of expression profiles of three different cell lines and of MIN6 cells with or without overexpression of transcription factor NeuroD1 established the usefulness and applicability of our microarrays. The mouse pancreatic islet cDNA library, sequence database, set of clones, and microarrays developed in this study should be useful resources for studies of pancreatic islets and related diseases including diabetes mellitus.

    DNA research : an international journal for rapid publication of reports on genes and genomes 2004;11;5;315-23

  • Synapsin and synaptic vesicle protein expression during embryonic and post-natal lens fiber cell differentiation.

    Frederikse PH, Yun E, Kao HT, Zigler JS, Sun Q and Qazi AS

    Department of Pharmacology & Physiology, UMDNJ-New Jersey Medical School, Newark, NJ 07103, USA. frederph@umdnj.edu

    Purpose: Reorganization of cytoskeleton and membrane biogenesis are dynamically coordinated during lens fiber cell differentiation and development to produce an organ with precise dimensions and optical properties. Cargo vesicle trafficking is fundamental to cell elongation and has also been implicated in degenerative disease mechanisms. Alzheimer precursor protein (AbetaPP) acts with kinesin, synapsin, and synaptic vesicle proteins to mediate cargo vesicle transport and membrane fusion in neurons. In our previous studies we demonstrated that AbetaPP is also a key element in lens fiber cell formation, and in early-onset cataract that occurs along with early-onset Alzheimer disease in Down syndrome. In the present study we examine lens expression and regulation of a complement of genes associated with cargo and synaptic vesicle transport in neurons.

    Methods: RT-PCR, immunoblot, and immunohistochemical methods were used to characterize expression of AbetaPP and kinesin associated motor proteins, synapsins, and synaptic vesicle proteins in mouse and rat embryonic, post-natal, and adult lenses. Phospho-specific anti-synapsin antibodies were used to determine the distributions of site-1 phosphorylated and dephosphorylated synapsin protein.

    Results: We demonstrate that a substantial complement of cargo and synaptic vesicle proteins involved in AbetaPP mediated vesicle transport are expressed in lenses along the anterior-posterior axis of fiber cells in embryonic and adult lenses, consistent with vesicles, actin filaments, and neuron-like arrangement of microtubules in lenses shown by others. We identify temporal regulation of synapsins I, II, and III during embryonic and post-natal lens development consistent with their roles in neurons. Regulation of vesicle cytoskeleton attachment, actin polymerization, and the capacity to stimulate cell differentiation by synapsins are governed in large part by phosphorylation at a conserved Ser9 residue (site-1). We demonstrate discrete distributions of Ser9 phospho- and dephospho-synapsins along the axial length of rapidly elongating embryonic lens fiber cells, and decreased levels of site-1 phosphorylated synapsins in adult lenses.

    Conclusions: The present findings demonstrate several fundamental parallels between lens and neuron vesicle trafficking cell biology and development, and suggest that more extensive AbetaPP related vesicle trafficking disease mechanisms may be shared by lens and brain.

    Molecular vision 2004;10;794-804

  • Cortical dysplasia resembling human type 2 lissencephaly in mice lacking all three APP family members.

    Herms J, Anliker B, Heber S, Ring S, Fuhrmann M, Kretzschmar H, Sisodia S and Müller U

    Zentrum für Neuropathologie und Prionforschung, Universität München, München, Germany.

    The Alzheimer's disease beta-amyloid precursor protein (APP) is a member of a larger gene family that includes the amyloid precursor-like proteins, termed APLP1 and APLP2. We previously documented that APLP2-/-APLP1-/- and APLP2-/-APP-/- mice die postnatally, while APLP1-/-APP-/- mice and single mutants were viable. We now report that mice lacking all three APP/APLP family members survive through embryonic development, and die shortly after birth. In contrast to double-mutant animals with perinatal lethality, 81% of triple mutants showed cranial abnormalities. In 68% of triple mutants, we observed cortical dysplasias characterized by focal ectopic neuroblasts that had migrated through the basal lamina and pial membrane, a phenotype that resembles human type II lissencephaly. Moreover, at E18.5 triple mutants showed a partial loss of cortical Cajal Retzius (CR) cells, suggesting that APP/APLPs play a crucial role in the survival of CR cells and neuronal adhesion. Collectively, our data reveal an essential role for APP family members in normal brain development and early postnatal survival.

    The EMBO journal 2004;23;20;4106-15

  • The status, quality, and expansion of the NIH full-length cDNA project: the Mammalian Gene Collection (MGC).

    Gerhard DS, Wagner L, Feingold EA, Shenmen CM, Grouse LH, Schuler G, Klein SL, Old S, Rasooly R, Good P, Guyer M, Peck AM, Derge JG, Lipman D, Collins FS, Jang W, Sherry S, Feolo M, Misquitta L, Lee E, Rotmistrovsky K, Greenhut SF, Schaefer CF, Buetow K, Bonner TI, Haussler D, Kent J, Kiekhaus M, Furey T, Brent M, Prange C, Schreiber K, Shapiro N, Bhat NK, Hopkins RF, Hsie F, Driscoll T, Soares MB, Casavant TL, Scheetz TE, Brown-stein MJ, Usdin TB, Toshiyuki S, Carninci P, Piao Y, Dudekula DB, Ko MS, Kawakami K, Suzuki Y, Sugano S, Gruber CE, Smith MR, Simmons B, Moore T, Waterman R, Johnson SL, Ruan Y, Wei CL, Mathavan S, Gunaratne PH, Wu J, Garcia AM, Hulyk SW, Fuh E, Yuan Y, Sneed A, Kowis C, Hodgson A, Muzny DM, McPherson J, Gibbs RA, Fahey J, Helton E, Ketteman M, Madan A, Rodrigues S, Sanchez A, Whiting M, Madari A, Young AC, Wetherby KD, Granite SJ, Kwong PN, Brinkley CP, Pearson RL, Bouffard GG, Blakesly RW, Green ED, Dickson MC, Rodriguez AC, Grimwood J, Schmutz J, Myers RM, Butterfield YS, Griffith M, Griffith OL, Krzywinski MI, Liao N, Morin R, Morrin R, Palmquist D, Petrescu AS, Skalska U, Smailus DE, Stott JM, Schnerch A, Schein JE, Jones SJ, Holt RA, Baross A, Marra MA, Clifton S, Makowski KA, Bosak S, Malek J and MGC Project Team

    The National Institutes of Health's Mammalian Gene Collection (MGC) project was designed to generate and sequence a publicly accessible cDNA resource containing a complete open reading frame (ORF) for every human and mouse gene. The project initially used a random strategy to select clones from a large number of cDNA libraries from diverse tissues. Candidate clones were chosen based on 5'-EST sequences, and then fully sequenced to high accuracy and analyzed by algorithms developed for this project. Currently, more than 11,000 human and 10,000 mouse genes are represented in MGC by at least one clone with a full ORF. The random selection approach is now reaching a saturation point, and a transition to protocols targeted at the missing transcripts is now required to complete the mouse and human collections. Comparison of the sequence of the MGC clones to reference genome sequences reveals that most cDNA clones are of very high sequence quality, although it is likely that some cDNAs may carry missense variants as a consequence of experimental artifact, such as PCR, cloning, or reverse transcriptase errors. Recently, a rat cDNA component was added to the project, and ongoing frog (Xenopus) and zebrafish (Danio) cDNA projects were expanded to take advantage of the high-throughput MGC pipeline.

    Funded by: PHS HHS: N01-C0-12400

    Genome research 2004;14;10B;2121-7

  • Gene knockout of amyloid precursor protein and amyloid precursor-like protein-2 increases cellular copper levels in primary mouse cortical neurons and embryonic fibroblasts.

    Bellingham SA, Ciccotosto GD, Needham BE, Fodero LR, White AR, Masters CL, Cappai R and Camakaris J

    Department of Genetics, The University of Melbourne, Victoria, Australia.

    Alzheimer's disease is characterised by the accumulation of amyloid-beta peptide, which is cleaved from the copper-binding amyloid-beta precursor protein. Recent in vivo and in vitro studies have illustrated the importance of copper in Alzheimer's disease neuropathogenesis and suggested a role for amyloid-beta precursor protein and amyloid-beta in copper homeostasis. Amyloid-beta precursor protein is a member of a multigene family, including amyloid precursor-like proteins-1 and -2. The copper-binding domain is similar among amyloid-beta precursor protein family members, suggesting an overall conservation in its function or activity. Here, we demonstrate that double knockout of amyloid-beta precursor protein and amyloid precursor-like protein-2 expression results in significant increases in copper accumulation in mouse primary cortical neurons and embryonic fibroblasts. In contrast, over-expression of amyloid-beta precursor protein in transgenic mice results in significantly reduced copper levels in primary cortical neurons. These findings provide cellular neuronal evidence for the role of amyloid-beta precursor protein in copper homeostasis and support the existing hypothesis that amyloid-beta precursor protein and amyloid precursor-like protein-2 are copper-binding proteins with functionally interchangeable roles in copper homeostasis.

    Journal of neurochemistry 2004;91;2;423-8

  • Antisense directed at the Abeta region of APP decreases brain oxidative markers in aged senescence accelerated mice.

    Poon HF, Joshi G, Sultana R, Farr SA, Banks WA, Morley JE, Calabrese V and Butterfield DA

    Department of Chemistry, Center of Membrane Sciences, University of Kentucky, Lexington 40506-0055, USA.

    Amyloid beta-peptide (Abeta) is known to induce free radical-mediated oxidative stress in the brain. Free radical-mediated damage to the neuronal membrane components has been implicated in the etiology of Alzheimer's disease (AD). Abeta is produced by proteolytic processing of the amyloid precursor protein (APP). The senescence accelerated mouse prone 8 (SAMP8) strain was developed by phenotypic selection from a common genetic pool. The SAMP8 strain exhibits age-related deterioration in memory and learning as well as Abeta accumulation, and it is considered an effective model for studying brain aging in accelerated senescence. Previous research has shown that a phosphorothiolated antisense oligonucleotide directed against the Abeta region of APP decreases the expression of APP and reverses deficits in learning and memory in aged SAMP8 mice. Consistent with other reports, our previous study showed that 12-month-old SAMP8 mice have increased levels of oxidative stress markers in the brain compared with that in brains from 4-month-old SAMP8 mice. In the current study, 12-month-old SAMP8 mice were treated with antisense oligonucleotide directed against the Abeta region of APP, and the oxidative markers in brain were decreased significantly. Therefore, we conclude that Abeta may contribute to the oxidative stress found in aged SAMP8 mice that have learning and memory impairments. These results are discussed in reference to AD.

    Funded by: NIA NIH HHS: AG-05119, AG-10836; NIAAA NIH HHS: R01 AA12743; NINDS NIH HHS: R01 NS41863

    Brain research 2004;1018;1;86-96

  • Cognitive correlates of Abeta deposition in male and female mice bearing amyloid precursor protein and presenilin-1 mutant transgenes.

    Howlett DR, Richardson JC, Austin A, Parsons AA, Bate ST, Davies DC and Gonzalez MI

    Neurology and GI CEDD, GlaxoSmithKline Research and Development Limited, New Frontiers Science Park, Third Avenue, Harlow, Essex, CM19 5AW, UK. david_howlett-1@gsk.com

    Several transgenic mouse models of Alzheimer's disease (AD) have been developed that exhibit beta-amyloid (Abeta) neuropathology and behavioural deficits. However, not all studies have investigated the relationship between the development of cognitive impairment and neuropathology. Therefore, temporal changes in cognition were investigated in male and female double-mutant APPswexPS1.M146V (TASTPM) transgenic mice using an object recognition test and correlated with the development of cerebral Abeta neuropathology. Both male and female TASTPM mice exhibited similar significant cognitive impairment at 6, 8 and 10 months of age in the object recognition test, compared to wild-type littermates. There was no such cognitive impairment at 3 or 4 months of age. Quantitative immunohistochemistry using a battery of Abeta antibodies demonstrated that cerebral Abeta deposition was first apparent in 3-month-old mice, and it increased with age. The early appearance of cerebral Abeta deposits in the double-transgenic TASTPM mice supports the evidence that mutations in the PS1 gene accelerate Abeta deposition. The cerebral Abeta load was greater in female than in male TASTPM mice at all ages investigated. In the electron microscope, mature Abeta plaques comprising a fibrillar core surrounded by degenerating neurites and reactive glia were first observed in the cortex of TASTPM mice at 6 months of age, the same age at which cognitive impairment became apparent. These results suggest that the cognitive impairment in TASTPM mice is related to the disruption of neural connectivity and not simply Abeta deposition, which first occurs 3 months earlier.

    Brain research 2004;1017;1-2;130-6

  • Presence of a "CAGA box" in the APP gene unique to amyloid plaque-forming species and absent in all APLP-1/2 genes: implications in Alzheimer's disease.

    Maloney B, Ge YW, Greig N and Lahiri DK

    Departments of Psychiatry, Institute of Psychiatric Research, Indiana University School of Medicine, Indianapolis, Indiana 46202, USA.

    Potentially toxic amyloid beta-peptide (Abeta) in Alzheimer's disease (AD) is generated from a family of Abeta-containing precursor proteins (APP), which is regulated via the 5'-untranslated region (5'-UTR) of its mRNA. We analyzed 5'-UTRs of the APP superfamily, including amyloid plaque-forming and non-amyloid plaque-forming species, and of prions (27 different DNA sequences). A "CAGA" sequence proximal to the "ATG" start codon was present in a location unique to APP genes of amyloid plaque-forming species and absent in all other genes surveyed. This CAGA box is immediately upstream of an interleukin-1-responsive element (acute box). In addition, the proximal CAGA box is predicted to appear on a stem-loop structure in both human and guinea pig APP mRNA. This stem-loop is part of a predicted bulge-loop that encompasses a known iron regulatory element (IRE). Electrophoretic mobility shift with segments of the APP 5'-UTR showed that a region with the proximal CAGA sequence binds nuclear proteins, and this UTR fragment is active in a reporter gene functional assay. Thus, the 5'-UTR in the human APP but not those of APP-like proteins contains a specific region that may participate in APP regulation and may determine a more general model for amyloid generation as seen in AD. The 5'-UTR of human APP contains several interesting control elements, such as an acute box element, a CAGA box, an IRE, and a transforming growth factor-beta-responsive element, that could control APP expression and provide suitable and specific drug targets for AD.

    Funded by: NIA NIH HHS: AG 18379, AG 18884

    FASEB journal : official publication of the Federation of American Societies for Experimental Biology 2004;18;11;1288-90

  • Regional cerebral blood volume reduction in transgenic mutant APP (V717F, K670N/M671L) mice.

    Wu EX, Tang H, Asai T and Yan SD

    Department of Radiology, Columbia University, 710 West 168th Street-Unit 63, New York, NY 10032, USA. xew1@columbia.edu

    Recent advance in nuclear magnetic resonance (NMR) microimaging has enabled in vivo cerebral blood volume (CBV) mapping with high spatial resolution. Using an intravascular susceptibility contrast agent and T(2)-weighted magnetic resonance imaging (MRI) on a 9.4T NMR microimager, the regional CBV was measured in mice as the transverse relaxation increase induced by the contrast agent. CBV maps in an Alzheimer's disease mouse model at resting state were obtained and examined. Four-month-old male transgenic mutant APP (V717F, K670N/M671L) mice (N = 10) and littermate wild-type controls (N = 12) were used. Regional analysis of the multi-slice CBV maps revealed statistically significant CBV reductions among the APP mice in cerebral cortex (-9.29%, P = 0.0002), hippocampus (-4.22%, P = 0.02), and thalamus (-5.21%, P = 0.03), indicating an early change of microvasculature in these selected regions. No significant difference was found in olfactory bulb, pons, midbrain, superior colliculus, medulla, and cerebellum.

    Funded by: NCI NIH HHS: CA 85594; NIA NIH HHS: AG 08702, AG 16223, AG 16736, AG 17490

    Neuroscience letters 2004;365;3;223-7

  • Munc13-1-mediated vesicle priming contributes to secretory amyloid precursor protein processing.

    Rossner S, Fuchsbrunner K, Lange-Dohna C, Hartlage-Rübsamen M, Bigl V, Betz A, Reim K and Brose N

    Paul Flechsig Institut für Hirnforschung, Abteilung Neurochemie, Universität Leipzig, Jahnallee 59, D-04109 Leipzig, Germany. rossn@medizin.uni-leipzig.de

    The amyloid precursor protein (APP) gives rise toc beta-amyloid peptides, which are the main constituents of senile plaques in brains of Alzheimer's disease patients. Non-amyloidogenic processing of the APP can be stimulated by phorbol esters (PEs) and by intracellular diacylglycerol (DAG) generation. This led to the hypothesis that classical and novel protein kinase Cs (PKCs), which are activated by DAG/PEs, regulate APP processing. However, in addition to PKCs, there are other DAG/PE receptors present in neurons that may participate in the modulation of APP processing. Munc13-1, a presynaptic protein with an essential role in synaptic vesicle priming, represents such an alternative target of the DAG second messenger pathway. Using Munc13-1 knock-out mice and knock-in mice expressing a Munc13-1(H567K) variant deficient in DAG/PE binding, we determined the relative contributions of PKCs and Munc13-1 to PE-stimulated secretory APP processing. We establish that, in addition to PKC, Munc13-1 significantly contributes to the regulation of secretory APP metabolism.

    The Journal of biological chemistry 2004;279;27;27841-4

  • Growth factor receptor-bound protein 2 interaction with the tyrosine-phosphorylated tail of amyloid beta precursor protein is mediated by its Src homology 2 domain.

    Zhou D, Noviello C, D'Ambrosio C, Scaloni A and D'Adamio L

    Albert Einstein College of Medicine, Department of Microbiology and Immunology, Bronx, New York 10461, USA.

    The sequential processing of the familial disease gene product amyloid beta precursor protein (AbetaPP) by beta- and gamma-secretases generates amyloid beta, which is considered to be the pathogenic factor of Alzheimer's disease, and the AID peptide (AbetaPP intracellular domain). The AID peptide acts as a positive regulator of apoptosis and modulates transcription and calcium release. To gain clues about the molecular mechanisms regulating the function of AbetaPP and AID, proteins interacting with the AID region of AbetaPP have been isolated using the yeast two-hybrid system. Recent evidence indicates that AbetaPP undergoes post-translational modification events in the AID region and that phosphorylation might regulate its affinity for interacting proteins. To test this possibility and to uncover AbetaPP-binding partners whose interaction depends on AbetaPP phosphorylation, we used a proteomic approach. Here we describe a protein, growth factor receptor-bound protein 2 (Grb2), that specifically binds AbetaPP, phosphorylated in Tyr(682). Furthermore, we show that this interaction is direct and that Grb2 binds to phospho-AbetaPP via its Src homology 2 region. Together with the evidence that Grb2 is in complex with AbetaPP in human brains and that these complexes are augmented in brains from Alzheimer's cases, our data indicate that Grb2 may mediate some biological and possibly pathological AbetaPP-AID function.

    Funded by: NIA NIH HHS: R01 AG21588-01, R01 AG22024-01; Telethon: GFP02006

    The Journal of biological chemistry 2004;279;24;25374-80

  • Early-onset and robust cerebral microvascular accumulation of amyloid beta-protein in transgenic mice expressing low levels of a vasculotropic Dutch/Iowa mutant form of amyloid beta-protein precursor.

    Davis J, Xu F, Deane R, Romanov G, Previti ML, Zeigler K, Zlokovic BV and Van Nostrand WE

    Department of Medicine, Health Sciences Center, Stony Brook University, Stony Brook, NY 11794-8153, USA.

    Cerebrovascular deposition of amyloid beta-protein (Abeta) is a common pathological feature of Alzheimer's disease and related disorders. In particular, the Dutch E22Q and Iowa D23N mutations in Abeta cause familial cerebrovascular amyloidosis with abundant diffuse amyloid plaque deposits. Both of these charge-altering mutations enhance the fibrillogenic and pathogenic properties of Abeta in vitro. Here, we describe the generation of several transgenic mouse lines (Tg-SwDI) expressing human neuronal Abeta precursor protein (AbetaPP) harboring the Swedish K670N/M671L and vasculotropic Dutch/Iowa E693Q/D694N mutations under the control of the mouse Thy1.2 promoter. Tg-SwDI mice expressed transgenic human AbetaPP only in the brain, but at levels below those of endogenous mouse AbetaPP. Despite the paucity of human AbetaPP expression, quantitative enzyme-linked immunosorbent assay measurements revealed that Tg-SwDI mice developed early-onset and robust accumulation of Abeta in the brain with high association with isolated cerebral microvessels. Tg-SwDI mice exhibited striking perivascular/vascular Abeta deposits that markedly increased with age. The vascular Abeta accumulations were fibrillar, exhibiting strong thioflavin S staining, and occasionally presented signs of microhemorrhage. In addition, numerous largely diffuse, plaque-like structures were observed starting at 3 months of age. In vivo transport studies demonstrated that Dutch/Iowa mutant Abeta was more readily retained in the brain compared with wild-type Abeta. These results with Tg-SwDI mice demonstrate that overexpression of human AbetaPP is not required for early-onset and robust accumulation of both vascular and parenchymal Abeta in mouse brain.

    Funded by: NIA NIH HHS: AG 16233; NINDS NIH HHS: NS 34467, NS 36645

    The Journal of biological chemistry 2004;279;19;20296-306

  • Luteinizing hormone, a reproductive regulator that modulates the processing of amyloid-beta precursor protein and amyloid-beta deposition.

    Bowen RL, Verdile G, Liu T, Parlow AF, Perry G, Smith MA, Martins RN and Atwood CS

    Voyager Pharmaceutical Corporation, Raleigh, NC 27615, USA.

    Hormonal changes associated with the dysregulation of the hypothalamic-pituitary-gonadal (HPG) axis following menopause/andropause have been implicated in the pathogenesis of Alzheimer's disease (AD). Experimental support for this has come from studies demonstrating an increase in amyloid-beta (Abeta) deposition following ovariectomy/castration. Because sex steroids and gonadotropins are both part of the HPG feedback loop, any loss in sex steroids results in a proportionate increase in gonadotropins. To assess whether Abeta generation was due to the loss of serum 17beta-estradiol or to the up-regulation of serum gonadotropins, we treated C57Bl/6J mice with the anti-gonadotropin leuprolide acetate, which suppresses both sex steroids and gonadotropins. Leuprolide acetate treatment resulted in a 3.5-fold (p < 0.0001) and a 1.5-fold (p < 0.024) reduction in total brain Abeta1-42 and Abeta1-40 concentrations, respectively, after 8 weeks of treatment. To further explore the role of gonadotropins in promoting amyloidogenesis, M17 neuroblastoma cells were treated with the gonadotropin luteinizing hormone (LH) at concentrations equivalent to early adulthood (10 mIU/ml) or post-menopause/andropause (30 mIU/ml). LH did not alter amyloid-beta precursor protein (AbetaPP) expression but did alter AbetaPP processing toward the amyloidogenic pathway as evidenced by increased secretion and insolubility of Abeta, decreased alphaAbetaPP secretion, and increased AbetaPP-C99 levels. These results suggest the marked increases in serum LH following menopause/andropause as a physiologically relevant signal that could promote Abeta secretion and deposition in the aging brain. Suppression of the age-related increase in serum gonadotropins using anti-gonadotropin agents may represent a novel therapeutic strategy for AD.

    Funded by: NIA NIH HHS: R01 AG 19356

    The Journal of biological chemistry 2004;279;19;20539-45

  • Amyloid-beta deposition is associated with decreased hippocampal glucose metabolism and spatial memory impairment in APP/PS1 mice.

    Sadowski M, Pankiewicz J, Scholtzova H, Ji Y, Quartermain D, Jensen CH, Duff K, Nixon RA, Gruen RJ and Wisniewski T

    Department of Neurology, New York University School of Medicine, New York, New York 10016, USA.

    In Alzheimer disease (AD) patients, early memory dysfunction is associated with glucose hypometabolism and neuronal loss in the hippocampus. Double transgenic (Tg) mice co-expressing the M146L presenilin 1 (PS1) and K670N/M671L, the double "Swedish" amyloid precursor protein (APP) mutations, are a model of AD amyloid-beta deposition (Abeta) that exhibits earlier and more profound impairments of working memory and learning than single APP mutant mice. In this study we compared performance on spatial memory tests, regional glucose metabolism, Abeta deposition, and neuronal loss in APP/PS1, PS1, and non-Tg (nTg) mice. At the age of 2 months no significant morphological and metabolic differences were detected between 3 studied genotypes. By 8 months, however, APP/PS1 mice developed selective impairment of spatial memory, which was significantly worse at 22 months and was accompanied by reduced glucose utilization in the hippocampus and a 35.8% dropout of neurons in the CA1 region. PS1 mice exhibited a similar degree of neuronal loss in CA1 but minimal memory deficit and no impairment of glucose utilization compared to nTg mice. Deficits in 22 month APP/PS1 mice were accompanied by a substantially elevated Abeta load, which rose from 2.5% +/- 0.4% at 8 months to 17.4% +/- 4.6%. These findings implicate Abeta or APP in the behavioral and metabolic impairments in APP/PS1 mice and the failure to compensate functionally for PS1-related hippocampal cell loss.

    Funded by: NIA NIH HHS: AG 17617, AG 20245, AG 20747; NIDA NIH HHS: DA00017

    Journal of neuropathology and experimental neurology 2004;63;5;418-28

  • Soluble form of amyloid precursor protein regulates proliferation of progenitors in the adult subventricular zone.

    Caillé I, Allinquant B, Dupont E, Bouillot C, Langer A, Müller U and Prochiantz A

    CNRS UMR 8542, Ecole Normale Supérieure, 46 rue d'Ulm, 75005 Paris, France.

    The amyloid precursor protein (APP) is a type I transmembrane protein of unknown physiological function. Its soluble secreted form (sAPP) shows similarities with growth factors and increases the in vitro proliferation of embryonic neural stem cells. As neurogenesis is an ongoing process in the adult mammalian brain, we have investigated a role for sAPP in adult neurogenesis. We show that the subventricular zone (SVZ) of the lateral ventricle, the largest neurogenic area of the adult brain, is a major sAPP binding site and that binding occurs on progenitor cells expressing the EGF receptor. These EGF-responsive cells can be cultured as neurospheres (NS). In vitro, EGF provokes soluble APP (sAPP) secretion by NS and anti-APP antibodies antagonize the EGF-induced NS proliferation. In vivo, sAPP infusions increase the number of EGF-responsive progenitors through their increased proliferation. Conversely, blocking sAPP secretion or downregulating APP synthesis decreases the proliferation of EGF-responsive cells, which leads to a reduction of the pool of progenitors. These results reveal a new function for sAPP as a regulator of SVZ progenitor proliferation in the adult central nervous system.

    Development (Cambridge, England) 2004;131;9;2173-81

  • Targeted introduction of V642I mutation in amyloid precursor protein gene causes functional abnormality resembling early stage of Alzheimer's disease in aged mice.

    Kawasumi M, Chiba T, Yamada M, Miyamae-Kaneko M, Matsuoka M, Nakahara J, Tomita T, Iwatsubo T, Kato S, Aiso S, Nishimoto I and Kouyama K

    Department of Pharmacology, Keio University School of Medicine, Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan.

    While the exact aetiology of Alzheimer's disease (AD) is unknown, distinct genetic mutations have been identified for the rare cases of familial AD (FAD). V642I mutation in amyloid precursor protein (APP) co-segregates with FAD with perfect penetration, and the clinicopathological characteristics of patients with this mutation resemble that of sporadic AD. To examine the pathogenic process of this FAD-linked trait in vivo, we produced a mouse with the corresponding point mutation in the APP gene using homologous recombination and Cre-loxP site-specific recombination ('knock-in' technique). Mice with the heterozygous V642I-APP allele most precisely reflected the genotype of humans bearing this mutation. For the observation period of 2.5 years the mutants stayed apparently indistinguishable from the wild-type littermates. However, behavioural analysis revealed significantly deteriorated long-term memory in mutants when examined for the retention of spatial attention. Interestingly, acquisition of spatial memory was slightly affected but short-term working memory was not deteriorated at all. Histological examination was negative for formation of neuritic plaques or neurofibrillary tangles, whereas the relative amount of longer form of beta-amyloid species A beta 42(43) was significantly increased against that of the shorter form (A beta 40) in the mutant brain homogenates. We conclude that a V642I-APP mutant allele in aged mice confers functional components, but not organic components, of the AD-related phenotype that are observed in the early stage of AD. This V642I-APP knock-in mutant line may serve as a model to study the early pathogenic processes of AD in vivo and to develop therapeutics for this stage.

    The European journal of neuroscience 2004;19;10;2826-38

  • Global expression analysis of gene regulatory pathways during endocrine pancreatic development.

    Gu G, Wells JM, Dombkowski D, Preffer F, Aronow B and Melton DA

    Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, USA.

    To define genetic pathways that regulate development of the endocrine pancreas, we generated transcriptional profiles of enriched cells isolated from four biologically significant stages of endocrine pancreas development: endoderm before pancreas specification, early pancreatic progenitor cells, endocrine progenitor cells and adult islets of Langerhans. These analyses implicate new signaling pathways in endocrine pancreas development, and identified sets of known and novel genes that are temporally regulated, as well as genes that spatially define developing endocrine cells from their neighbors. The differential expression of several genes from each time point was verified by RT-PCR and in situ hybridization. Moreover, we present preliminary functional evidence suggesting that one transcription factor encoding gene (Myt1), which was identified in our screen, is expressed in endocrine progenitors and may regulate alpha, beta and delta cell development. In addition to identifying new genes that regulate endocrine cell fate, this global gene expression analysis has uncovered informative biological trends that occur during endocrine differentiation.

    Funded by: NIDDK NIH HHS: F32 DK009832, R01 DK065949

    Development (Cambridge, England) 2004;131;1;165-79

  • The presence of the APP(swe) mutation in mice does not increase the vulnerability of cholinergic basal forebrain neurons to neuroinflammation.

    Wenk GL, McGann-Gramling K and Hauss-Wegrzyniak B

    Division of Neural Systems, Memory and Aging, University of Arizona, 350 Life Sciences North Building, Tucson, AZ 85724, USA. gary@nsma.arizona.edu

    Neuroinflammation, and elevated levels of inflammatory proteins, such as tumor necrosis factor-alpha, and the deposition of beta-amyloid may interact to contribute to the pathogenesis of Alzheimer's disease. We reproduced a component of the neuroinflammatory state within the basal forebrain cholinergic system, a region that is vulnerable to degeneration in Alzheimer's disease, of transgenic Tg2576 mice that express the Swedish double mutation of the human amyloid precursor protein (APPswe). We have previously shown that basal forebrain cholinergic neurons are selectively vulnerable to the consequences of neuroinflammation. In the current study, tumor necrosis factor-alpha was infused into the basal forebrain region of APPswe and nontransgenic control mice for 20 days with the expectation that the presence of the transgene would enhance the loss of cholinergic neurons. Chronic infusion of tumor necrosis factor-alpha significantly decreased cortical choline acetyltransferase activity, reduced the number of choline acetyltransferase-immunoreactive cells and increased the number of activated astrocytes and microglia within the basal forebrain. The presence of the APPswe gene did not enhance the vulnerability of forebrain cholinergic neurons to the chronic neuroinflammation. Furthermore, combined treatment of these mice with memantine demonstrated that the neurotoxic effects of tumor necrosis factor-alpha upon cholinergic cells did not require the activation of the N-methyl-d-aspartate receptors. In contrast, we have previously shown that memantine was able to provide neuroprotection to cholinergic forebrain neurons from the consequences of exposure to the inflammogen lipopolysaccharide. These results provide insight into the mechanism by which neuroinflammation may selectively target specific neural systems during the progression of Alzheimer's disease.

    Funded by: NIA NIH HHS: AG10546

    Neuroscience 2004;125;3;769-76

  • Central cholinergic functions in human amyloid precursor protein knock-in/presenilin-1 transgenic mice.

    Hartmann J, Erb C, Ebert U, Baumann KH, Popp A, König G and Klein J

    Alzheimer Research Group, Bayer Health Care AG, D-42096 Wuppertal, Germany.

    Alzheimer's disease is characterized by amyloid peptide formation and deposition, neurofibrillary tangles, central cholinergic dysfunction, and dementia; however, the relationship between these parameters is not well understood. We studied the effect of amyloid peptide formation and deposition on central cholinergic function in knock-in mice carrying the human amyloid precursor protein (APP) gene with the Swedish/London double mutation (APP-SL mice) which were crossbred with transgenic mice overexpressing normal (PS1wt) or mutated (M146L; PS1mut) human presenilin-1. APP-SLxPS1mut mice had increased levels of Abeta peptides at 10 months of age and amyloid plaques at 14 months of age while APP-SLxPS1wt mice did not have increased peptide levels and did not develop amyloid plaques. We used microdialysis in 15-27 months old mice to compare hippocampal acetylcholine (ACh) levels in the two mouse lines and found that extracellular ACh levels were slightly but significantly reduced in the APP-SLxPS1mut mice (-26%; P=0.044). Exploratory activity in the open field increased hippocampal ACh release by two-fold in both mouse lines; total and relative increases were not significantly different for the two strains under study. Similarly, infusion of scopolamine (1 microM) increased hippocampal ACh release to a similar extent (3-5-fold) in both groups. High-affinity choline uptake, a measure of the ACh turnover rate, was identical in both mouse lines. Neurons expressing choline acetyltransferase were increased in the septum of APP-SLxPS1mut mice (+26%; P=0.046). We conclude that amyloid peptide production causes a small decrease of extracellular ACh levels. The deposition of amyloid plaques, however, does not impair stimulated ACh release and proceeds without major changes of central cholinergic function.

    Neuroscience 2004;125;4;1009-17

  • Notch1 competes with the amyloid precursor protein for gamma-secretase and down-regulates presenilin-1 gene expression.

    Lleó A, Berezovska O, Ramdya P, Fukumoto H, Raju S, Shah T and Hyman BT

    Alzheimer Research Unit, Massachusetts General Hospital, Charlestown, Massachusetts 02114, USA.

    Presenilin 1 (PS1) is a critical component of the gamma-secretase complex, which is involved in the cleavage of several substrates including the amyloid precursor protein (APP) and Notch1. Based on the fact that APP and Notch are processed by the same gamma-secretase, we postulated that APP and Notch compete for the enzyme activity. In this report, we examined the interactions between APP, Notch, and PS1 using the direct gamma-secretase substrates, Notch 1 Delta extracellular domain (N1DeltaEC) and APP carboxyl-terminal fragment of 99 amino acids, and measured the effects on amyloid-beta protein production and Notch signaling, respectively. Additionally, we tested the hypothesis that downstream effects on PS1 expression may coexist with the competition phenomenon. We observed significant competition between Notch and APP for gamma-secretase activity; transfection with either of two direct substrates of gamma-secretase led to a reduction in the gamma-cleaved products, Notch intracellular domain or amyloid-beta protein. In addition, however, we found that activation of the Notch signaling pathway, by either N1 Delta EC or Notch intracellular domain, induced down-regulation of PS1 gene expression. This finding suggests that Notch activation directly engages gamma-secretase and subsequently leads to diminished PS1 expression, suggesting a complex set of feedback interactions following Notch activation.

    Funded by: NIA NIH HHS: AG15379

    The Journal of biological chemistry 2003;278;48;47370-5

  • Genetic background regulates beta-amyloid precursor protein processing and beta-amyloid deposition in the mouse.

    Lehman EJ, Kulnane LS, Gao Y, Petriello MC, Pimpis KM, Younkin L, Dolios G, Wang R, Younkin SG and Lamb BT

    Department of Genetics, Case Western Reserve University, and Center for Human Genetics, University Hospitals of Cleveland, OH 44106-4955, USA.

    Alzheimer's disease (AD) is a multigenic neurodegenerative disorder characterized by distinct neuropathological hallmarks including deposits of the beta-amyloid (A beta) peptide. A beta is a 39- to 43-amino acid peptide derived from the proteolytic processing of the amyloid precursor protein (APP). While increasing evidence suggests that altered APP processing and A beta metabolism is a common feature of AD, the relationship between the levels of A beta and various APP products and the onset of AD remains unclear. We have undertaken a screen to characterize genetic factors that modify APP processing, A beta metabolism and A beta deposition in a genomic-based yeast artificial chromosome (YAC) transgenic mouse model of AD. A mutant human APP YAC transgene was transferred to three inbred mouse strains. Despite similar levels of holo-APP expression in the congenic strains, the levels of APP C-terminal fragments as well as brain and plasma A beta in young animals varied by genetic background. Furthermore, we demonstrate that age-dependent A beta deposition in the APP YAC transgenic model is dramatically altered depending on the congenic strain examined. These studies demonstrate that APP processing, A beta metabolism and A beta deposition are regulated by genetic background and that analysis of these phenotypes in mice should provide new insights into the factors that regulate AD pathogenesis.

    Funded by: NCI NIH HHS: CA43703; NIA NIH HHS: AG08012, AG10491, AG14451; NIGMS NIH HHS: GM08613

    Human molecular genetics 2003;12;22;2949-56

  • Neurotoxic, redox-competent Alzheimer's beta-amyloid is released from lipid membrane by methionine oxidation.

    Barnham KJ, Ciccotosto GD, Tickler AK, Ali FE, Smith DG, Williamson NA, Lam YH, Carrington D, Tew D, Kocak G, Volitakis I, Separovic F, Barrow CJ, Wade JD, Masters CL, Cherny RA, Curtain CC, Bush AI and Cappai R

    Department of Pathology, The University of Melbourne and The Mental Health Research Institute of Victoria, Victoria 3010, Australia. kbarnham@unimelb.edu.au

    The amyloid beta peptide is toxic to neurons, and it is believed that this toxicity plays a central role in the progression of Alzheimer's disease. The mechanism of this toxicity is contentious. Here we report that an Abeta peptide with the sulfur atom of Met-35 oxidized to a sulfoxide (Met(O)Abeta) is toxic to neuronal cells, and this toxicity is attenuated by the metal chelator clioquinol and completely rescued by catalase implicating the same toxicity mechanism as reduced Abeta. However, unlike the unoxidized peptide, Met(O)Abeta is unable to penetrate lipid membranes to form ion channel-like structures, and beta-sheet formation is inhibited, phenomena that are central to some theories for Abeta toxicity. Our results show that, like the unoxidized peptide, Met(O)Abeta will coordinate Cu2+ and reduce the oxidation state of the metal and still produce H2O2. We hypothesize that Met(O)Abeta production contributes to the elevation of soluble Abeta seen in the brain in Alzheimer's disease.

    The Journal of biological chemistry 2003;278;44;42959-65

  • Analysis of neurons created from wild-type and Alzheimer's mutation knock-in embryonic stem cells by a highly efficient differentiation protocol.

    Abe Y, Kouyama K, Tomita T, Tomita Y, Ban N, Nawa M, Matsuoka M, Niikura T, Aiso S, Kita Y, Iwatsubo T and Nishimoto I

    Department of Pharmacology, KEIO University School of Medicine, Tokyo 160-8582, Japan.

    It is impossible to obtain and amplify live neurons from Alzheimer's disease (AD) patients. To establish the neurons harboring AD abnormality, we constructed mouse embryonic stem (ES) cells, in which the AD-causative V642I mutation was introduced to the endogenous amyloid precursor protein (APP) gene, in combination with a protocol to efficiently differentiate ES cells into postmitotic neurons without using a cell sorter. By this protocol, ES cells differentiated into >90% of the central type of adult postmitotic neurons. Neurons derived from V642I-APP knock-in ES cells were indistinguishable from wild-type ES-derived neurons, as determined by the expression of various markers for neuronal differentiation. Notably, V642I-APP knock-in ES cell-derived neurons exhibited significantly increased secretion of Abeta42 without AD-related hyperphosphorylation of tau, indicating that the direct output of the AD-causative mutation is increased Abeta42 secretion. In this study, we analyze created neurons with wild-type and AD genotypes and propose a new strategy for generating neurons for any dominantly inherited neurodegenerative diseases. The strategy can be applied to create human neurons with AD or any other neurodegenerative disease by using human ES cells.

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2003;23;24;8513-25

  • Both raft- and non-raft proteins associate with CHAPS-insoluble complexes: some APP in large complexes.

    Rouvinski A, Gahali-Sass I, Stav I, Metzer E, Atlan H and Taraboulos A

    Department of Molecular Biology, The Hebrew University-Hadassah Medical School, P.O. Box 12272, Jerusalem 91120, Israel.

    Components of caveolae and lipid rafts are characterized by their buoyancy after detergent extraction. Using flotations in density gradients, we now show that non-raft membrane molecules are also associated with detergent-insoluble, buoyant assemblies. When Triton X-100 cellular extracts were spun to equilibrium in Nycodenz, only components of classical rafts floated. In contrast, with the zwitterionic detergent CHAPS, non-raft residents such as calnexin and APP also buoyed. When CHAPS extracts were spun in non-equilibrium (velocity) conditions, some raft components rapidly exited the input fractions while other raft markers and non-raft molecules remained relatively immobile. This pointed to size heterogeneities of CHAPS-insoluble complexes. Combined velocity/equilibrium gradients broadly divided CHAPS-insoluble membrane complexes into three size categories, which all contained cholesterol and the glycosphingolipid GM1. Large complexes were enriched in caveolin and ESA. Medium size complexes were enriched in PrP, whereas small complexes contained non-raft proteins, PrP, and some ESA. While Alzheimer's APP was primarily confined to small assemblies, a portion of its glycosylated form did buoy with large complexes. Large CHAPS-insoluble complexes resemble, but are not equal to, classical rafts. These findings extend considerably the range of detergent-insoluble membranal domains.

    Biochemical and biophysical research communications 2003;308;4;750-8

  • App gene dosage modulates endosomal abnormalities of Alzheimer's disease in a segmental trisomy 16 mouse model of down syndrome.

    Cataldo AM, Petanceska S, Peterhoff CM, Terio NB, Epstein CJ, Villar A, Carlson EJ, Staufenbiel M and Nixon RA

    Mailman Research Center, McLean Hospital, Belmont, Massachusetts 02478, USA. acataldo@mclean.harvard.edu

    Altered neuronal endocytosis is the earliest known pathology in sporadic Alzheimer's disease (AD) and Down syndrome (DS) brain and has been linked to increased Abeta production. Here, we show that a genetic model of DS (trisomy 21), the segmental trisomy 16 mouse Ts65Dn, develops enlarged neuronal early endosomes, increased immunoreactivity for markers of endosome fusion (rab5, early endosomal antigen 1, and rabaptin5), and endosome recycling (rab4) similar to those in AD and DS individuals. These abnormalities are most prominent in neurons of the basal forebrain, which later develop aging-related atrophy and degenerative changes, as in AD and DS. We also show that App, one of the triplicated genes in Ts65Dn mice and human DS, is critical to the development of these endocytic abnormalities. Selectively deleting one copy of App or a small portion of the chromosome 16 segment containing App from Ts65Dn mice eliminated the endosomal phenotype. Overexpressing App at high levels in mice did not alter early endosomes, implying that one or more additional genes on the triplicated segment of chromosome 16 are also required for the Ts65Dn endosomal phenotype. These results identify an essential role for App gene triplication in causing AD-related endosomal abnormalities and further establish the pathogenic significance of endosomal dysfunction in AD.

    Funded by: NIA NIH HHS: AG17617; NICHD NIH HHS: HD31498

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2003;23;17;6788-92

  • Expression of liver X receptor target genes decreases cellular amyloid beta peptide secretion.

    Sun Y, Yao J, Kim TW and Tall AR

    Division of Molecular Medicine, Department of Medicine, College of Physicians and Surgeons, Columbia University, New York, NY 10032, USA.

    A hallmark of Alzheimer's disease is the deposition of plaques of amyloid beta peptide (Abeta) in the brain. Abeta is thought to be formed from the amyloid precursor protein (APP) in cholesterol-enriched membrane rafts, and cellular cholesterol depletion decreases Abeta formation. The liver X receptors (LXR) play a key role in regulating genes that control cellular cholesterol efflux and membrane composition and are widely expressed in cells of the central nervous system. We show that treatment of APP-expressing cells with LXR activators reduces the formation of Abeta. LXR activation resulted in increased levels of the ATP-binding cassette transporter A1 (ABCA1) and stearoyl CoA desaturase, and expression of these genes individually decreased formation of Abeta. Expression of ABCA1 led to both decreased beta-cleavage product of APPSw (i.e. C99 peptide) and reduced gamma-secretase-cleavage of C99 peptide. Remarkably, these effects of ABCA1 on APP processing were independent of cellular lipid efflux. LXR and ABCA1-induced changes in membrane lipid organization had favorable effects on processing of APP, suggesting a new approach to the treatment of Alzheimer's disease.

    Funded by: NHLBI NIH HHS: HL 22682, HL 54591; NIA NIH HHS: AG18026

    The Journal of biological chemistry 2003;278;30;27688-94

  • Presenilin redistribution associated with aberrant cholesterol transport enhances beta-amyloid production in vivo.

    Burns M, Gaynor K, Olm V, Mercken M, LaFrancois J, Wang L, Mathews PM, Noble W, Matsuoka Y and Duff K

    Center for Dementia Research, Nathan S. Kline Institute, Orangeburg, New York 10962, USA.

    Epidemiology, in vitro, and in vivo studies strongly implicate a role for cholesterol in the pathogenesis of Alzheimer's disease (AD). We have examined the impact of aberrant intracellular cholesterol transport on the processing of the amyloid precursor protein (APP) in a mouse model of Niemann-Pick type C (NPC) disease. In the NPC mouse brain, cholesterol accumulates in late endosomes/lysosomes. This was associated with the accumulation of beta-C-terminal fragments (CTFs) of APP, but the level of beta-secretase and its activity were not affected. Alpha-secretase activity and secreted APPalpha generation were also not affected, suggesting CTFs increased because of decreased clearance. The level of presenilin-1 (PS-1) was unchanged, but gamma-secretase activity was greatly enhanced, which correlated with an increase in Abeta40 and Abeta42 levels. These events were associated with abnormal distribution of PS-1 in the endosomal system. Our results show that aberrant cholesterol trafficking is associated with the potentiation of APP processing components in vivo, leading to an overall increase in Abeta levels.

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2003;23;13;5645-9

  • Learning and memory deficits in APP transgenic mouse models of amyloid deposition.

    Morgan D

    Alzheimer Research Laboratory, Department of Pharmacology, University of South Florida, Tampa, Florida 33612, USA. dmorgan@hsc.usf.edu

    Several different transgenic APP mice develop learning and memory deficits. In some cases the mice have deficits very early in life, while in other instances the mice exhibit deficits only after they have aged and amyloid deposits have accumulated. In many cases, there is a correlation in individual mice of the same age and genotype between the extent of learning and memory deficits and the amounts of deposited amyloid found in the central nervous system. While superficially this might imply that the deposited material is somehow toxic to cognition, it is likely that deposited amyloid is also an index of the overall rate of amyloid production in each mouse. Rate of production would be expected to modify not only the amounts of deposited amyloid, but also other amyloid pools, including soluble, oligomeric, conjugated (e.g. ADDLs) and intracellular. Thus, the deposited material may be an integrated reflection of total A beta production, in addition to indicating the amounts in fibrillar forms. As such, it is conceivable that other A beta pools may be more directly linked to memory deficits. Thus far, the one manipulation found to mitigate the learning and memory deficits in APP transgenic mice is immunotherapy for A beta, either using active or passive immunization against the peptide. These data together with other findings are leading to a conclusion that the fibrillar A beta deposits are not directly linked to the memory deficits in mice, and that some other A beta pool, more readily diminished by immunotherapy, is more directly linked to the mechanisms leading to poor performance in learning and memory tasks.

    Funded by: NIA NIH HHS: AG 15490, AG18478, AG20227

    Neurochemical research 2003;28;7;1029-34

  • Using advanced intercross lines for high-resolution mapping of HDL cholesterol quantitative trait loci.

    Wang X, Le Roy I, Nicodeme E, Li R, Wagner R, Petros C, Churchill GA, Harris S, Darvasi A, Kirilovsky J, Roubertoux PL and Paigen B

    The Jackson Laboratory, Bar Harbor, Maine 04609, USA.

    Mapping quantitative trait loci (QTLs) with high resolution facilitates identification and positional cloning of the underlying genes. The novel approach of advanced intercross lines (AILs) generates many more recombination events and thus can potentially narrow QTLs significantly more than do conventional backcrosses and F2 intercrosses. In this study, we carried out QTL analyses in (C57BL/6J x NZB/BlNJ) x C57BL/6J backcross progeny fed either chow or an atherogenic diet to detect QTLs that regulate high-density lipoprotein cholesterol (HDL)concentrations, and in (C57BL/6J x NZB/BlNJ) F11 AIL progeny to confirm and narrow those QTLs. QTLs for HDL concentrations were found on chromosomes 1, 5, and 16. AIL not only narrowed the QTLs significantly more than did a conventional backcross but also resolved a chromosome 5 QTL identified in the backcross into two QTLs, the peaks of both being outside the backcross QTL region. We tested 27 candidate genes and found significant mRNA expression differences for 12 (Nr1i3, Apoa2, Sap, Tgfb2, Fgfbp1, Prom, Ppargc1, Tcf1, Ncor2, Srb1, App, and Ifnar). Some of these underlay the same QTL, indicating that expression differences are common and not sufficient to identify QTL genes. All the major HDL QTLs in our study had homologous counterparts in humans, implying that their underlying genes regulate HDL in humans.

    Funded by: NHLBI NIH HHS: HL66611

    Genome research 2003;13;7;1654-64

  • Upregulation and antiapoptotic role of endogenous Alzheimer amyloid precursor protein in dorsal root ganglion neurons.

    Nishimura I, Takazaki R, Kuwako Ki, Enokido Y and Yoshikawa K

    Division of Regulation of Macromolecular Functions, Osaka University, Yamadaoka 3-2, Suita, Osaka 565-0871, Japan.

    The amyloid precursor protein (APP) is a transmembrane protein whose abnormal processing is associated with the pathogenesis of Alzheimer's disease. In this study, we examined the expression and role of cell-associated APP in primary dorsal root ganglion (DRG) neurons. When dissociated DRG cells prepared from mouse embryos were treated with nerve growth factor (NGF), neuronal APP levels were transiently elevated. DRG neurons treated with an antibody against cell surface APP failed to mature and underwent apoptosis. When NGF was withdrawn from the cultures after a 36-h NGF treatment, virtually all neurons underwent apoptosis by 48 h. During the course of apoptosis, some neurons with intact morphology contained increased levels of APP immunoreactivity, whereas the APP levels were greatly reduced in apoptotic neurons. Furthermore, affected neurons contained immunoreactivities for activated caspase-3, a caspase-cleaved APP fragment (APPDeltaC31), and Abeta. Downregulation of endogenous APP expression by treatment with an APP antisense oligodeoxynucleotide significantly increased the number of apoptotic neurons in NGF-deprived DRG cultures. Furthermore, overexpression of APP by adenovirus vector-mediated gene transfer reduced the number of apoptotic neurons deprived of NGF. These results suggest that endogenous APP is upregulated to exert an antiapoptotic effect on neurotrophin-deprived DRG neurons and subsequently undergoes caspase-dependent proteolysis.

    Experimental cell research 2003;286;2;241-51

  • Downregulation and increased turnover of beta-amyloid precursor protein in skeletal muscle cultures by neuregulin-1.

    Rosen KM, Ford BD and Querfurth HW

    Division of Neurology, St. Elizabeth's Medical Center, Tufts University School of Medicine, Boston, MA 02135, USA. Kenneth_Rosen@cchcs.org

    The beta-amyloid precursor protein (betaAPP) is found in skeletal muscle localized to the base of the postsynaptic folds of the neuromuscular junction; yet here, as well as in neurons, its function remains enigmatic. Here we report that the motor nerve-derived trophic factor neuregulin-1 (NRG1) regulates both steady-state betaAPP levels as well as the metabolism of the cell surface-associated protein in cultured muscle cells. These two effects occur over two discernible time scales. At short times (minutes to hours), NRG1 increases the rate of internalization and apparent degradation of cell surface betaAPP while reducing the release of soluble APP to the medium. At longer times (hours to days), NRG1 causes a decrease in mRNA for betaAPP with a concomitant reduction in steady-state protein levels. These are novel findings for this trophic factor originally identified as inducing the expression of nicotinic acetylcholine receptors and other important synaptic proteins in skeletal muscle. They suggest that betaAPP may play a receptor or signal transduction role at the neuromuscular junction since other receptor protein's actions are terminated in a similar fashion. The effects of NRG1 on betaAPP metabolism are overcome by inhibitors of both the phosphatidylinositol-3 (PI3) kinase and mitogen-activated protein (MAP) kinase pathways, yet are distinct from those activated during induction of nicotinic acetylcholine receptor biosynthesis. BetaAPP should be added to the list of specialized post-neuromuscular junction proteins that are regulated by cholinergic terminal-derived factors critical to synaptogenesis.

    Funded by: NINDS NIH HHS: NS34194

    Experimental neurology 2003;181;2;170-80

  • Transforming growth factor-beta 1 potentiates amyloid-beta generation in astrocytes and in transgenic mice.

    Lesné S, Docagne F, Gabriel C, Liot G, Lahiri DK, Buée L, Plawinski L, Delacourte A, MacKenzie ET, Buisson A and Vivien D

    Unité Mixte de Recherche (UMR) CNRS 6551, IFR47, Université de Caen, Cyceron, Caen Cedex 14074, France.

    Accumulation of the amyloid-beta peptide (Abeta) in the brain is crucial for development of Alzheimer's disease. Expression of transforming growth factor-beta1 (TGF-beta1), an immunosuppressive cytokine, has been correlated in vivo with Abeta accumulation in transgenic mice and recently with Abeta clearance by activated microglia. Here, we demonstrate that TGF-beta1 drives the production of Abeta40/42 by astrocytes leading to Abeta production in TGF-beta1 transgenic mice. First, TGF-beta1 induces the overexpression of the amyloid precursor protein (APP) in astrocytes but not in neurons, involving a highly conserved TGF-beta1-responsive element in the 5'-untranslated region (+54/+74) of the APP promoter. Second, we demonstrated an increased release of soluble APP-beta which led to TGF-beta1-induced Abeta generation in both murine and human astrocytes. These results demonstrate that TGF-beta1 potentiates Abeta production in human astrocytes and may enhance the formation of plaques burden in the brain of Alzheimer's disease patients.

    The Journal of biological chemistry 2003;278;20;18408-18

  • Amyloid-beta peptide levels in brain are inversely correlated with insulysin activity levels in vivo.

    Miller BC, Eckman EA, Sambamurti K, Dobbs N, Chow KM, Eckman CB, Hersh LB and Thiele DL

    Department of Internal Medicine, University of Texas Southwestern Medical School, Dallas 75390-9151, USA. bonnie.miller@utsouthwestern.edu

    Factors that elevate amyloid-beta (Abeta) peptide levels are associated with an increased risk for Alzheimer's disease. Insulysin has been identified as one of several proteases potentially involved in Abeta degradation based on its hydrolysis of Abeta peptides in vitro. In this study, in vivo levels of brain Abeta40 and Abeta42 peptides were found to be increased significantly (1.6- and 1.4-fold, respectively) in an insulysin-deficient gene-trap mouse model. A 6-fold increase in the level of the gamma-secretase-generated C-terminal fragment of the Abeta precursor protein in the insulysin-deficient mouse also was found. In mice heterozygous for the insulysin gene trap, in which insulysin activity levels were decreased approximately 50%, brain Abeta peptides were increased to levels intermediate between those in wild-type mice and homozygous insulysin gene-trap mice that had no detectable insulysin activity. These findings indicate that there is an inverse correlation between in vivo insulysin activity levels and brain Abeta peptide levels and suggest that modulation of insulysin activity may alter the risk for Alzheimer's disease.

    Funded by: NIDA NIH HHS: N01DA08801

    Proceedings of the National Academy of Sciences of the United States of America 2003;100;10;6221-6

  • Comparison of expression patterns between CREB family transcription factor OASIS and proteoglycan core protein genes during murine tooth development.

    Hikake T, Mori T, Iseki K, Hagino S, Zhang Y, Takagi H, Yokoya S and Wanaka A

    Department of Cell Science, Institute of Biomedical Sciences, Fukushima Medical University School of Medicine, 960-1295 Fukushima, Japan.

    The transcription factor OASIS gene, which encodes for a CREB/ATF family member, is specifically expressed in the salivary gland, the cartilage and the tooth germs of the mouse embryo. In the present study, the expression patterns were compared between OASIS mRNA and major vertebrate proteoglycans, which might be the downstream genes of OASIS in the tooth germs of mouse first mandibular molars, through in situ hybridization histochemistry. OASIS mRNA expression was observed in the inner enamel epithelium during the cap and bell stages (E14.5-E18.5) in the preodontoblasts during differentiation stage (E18.5-P0) and in the differentiating odontoblasts during the early secretory stage (P2.5-P4.5). Proteoglycans (versican, decorin, biglycan, glypican, syndecan-1, and syndecan-3) were expressed in the tooth germs in various patterns. Decorin, biglycan, syndecan-1 and syndecan-3 showed gene expressions overlapping with OASIS. Especially the expression pattern of decorin and syndecan-3 coincided temporally and spatially exactly with that of OASIS. These results suggest that the OASIS gene might be related to proteoglycan expression and may play an important role in the differentiation of the odontoblast and cells in inner enamel epithelium.

    Anatomy and embryology 2003;206;5;373-80

  • beta-Amyloid-specific upregulation of stearoyl coenzyme A desaturase-1 in macrophages.

    Uryu S, Tokuhiro S and Oda T

    Lead Discovery Research Laboratories, Sankyo, 2-58, Hiromachi 1-chome, Shinagawa-ku, Tokyo 140-8710, Japan. shigek@shina.sankyo.co.jp

    beta-Amyloid peptide (A beta), a major component of senile plaques, the formation of which is characteristic of Alzheimer's disease (AD), is believed to induce inflammation of the brain mediated by microglia, leading to neuronal cell loss. In this study, we performed an oligonucleotide microarray analysis to investigate the molecular events underlying the A beta-induced activation of macrophages and its specific suppression by the A beta-specific-macrophage-activation inhibitor, RS-1178. Of the approximately 36,000 genes and expressed sequence tags analyzed, eight genes were specifically and significantly upregulated by a treatment with interferon gamma (IFN gamma) and A beta compared to a treatment with IFN gamma alone (p<0.002). We found that the gene for a well-characterized lipogenetic enzyme, stearoyl coenzyme A desaturase-1 (SCD-1), was specifically upregulated by A beta treatment and was suppressed to basal levels by RS-1178. Although the underlying mechanisms remain unknown, our results suggest the presence of a link between AD and SCD-1.

    Biochemical and biophysical research communications 2003;303;1;302-5

  • Capacitive calcium entry is directly attenuated by mutant presenilin-1, independent of the expression of the amyloid precursor protein.

    Herms J, Schneider I, Dewachter I, Caluwaerts N, Kretzschmar H and Van Leuven F

    Department of Neuropathology, Ludwig-Maximilians-Universität, 81377 Munich, Germany.

    Mutant presenilin-1 (PS1) increases amyloid peptide production, attenuates capacitative calcium entry (CCE), and augments calcium release from the endoplasmatic reticulum (ER). Here we measured the intracellular free Ca(2+) concentration in hippocampal neurons from six different combinations of transgenic and gene-ablated mice to demonstrate that mutant PS1 attenuated CCE directly, independent of the expression of the amyloid precursor protein (APP). On the other hand, increased Ca(2+) release from the ER in mutant PS1 neurons, as induced by thapsigargin, was clearly dependent on the presence of APP and its processing by PS1, i.e. on the generation of the amyloid peptides and the APP C99 fragments. This observation was corroborated by the thapsigargin-induced increase in cytosolic [Ca(2+)](i) in PS1 deficient neurons, which accumulate C99 fragments due to deficient gamma-secretase activity. Moreover, co-expression of mutant APP[V717I] in PS1-deficient neurons further increased the apparent size of the ER calcium stores in parallel with increasing levels of the APP processing products. We conclude that mutant PS1 deregulates neuronal calcium homeostasis by two different actions: (i) direct attenuation of CCE at the cell-surface independent of APP; and (ii) indirect increase of ER-calcium stores via processing of APP and generation of amyloid peptides and C99 fragments.

    The Journal of biological chemistry 2003;278;4;2484-9

  • Amyloidogenic processing of the Alzheimer beta-amyloid precursor protein depends on lipid rafts.

    Ehehalt R, Keller P, Haass C, Thiele C and Simons K

    Max Planck Institute of Molecular Cell Biology and Genetics, D-01307 Dresden, Germany.

    Formation of senile plaques containing the beta-amyloid peptide (A beta) derived from the amyloid precursor protein (APP) is an invariant feature of Alzheimer's disease (AD). APP is cleaved either by beta-secretase or by alpha-secretase to initiate amyloidogenic (release of A beta) or nonamyloidogenic processing of APP, respectively. A key to understanding AD is to unravel how access of these enzymes to APP is regulated. Here, we demonstrate that lipid rafts are critically involved in regulating A beta generation. Reducing cholesterol levels in N2a cells decreased A beta production. APP and the beta-site APP cleavage enzyme (BACE1) could be induced to copatch at the plasma membrane upon cross-linking with antibodies and to segregate away from nonraft markers. Antibody cross-linking dramatically increased production of A beta in a cholesterol-dependent manner. A beta generation was dependent on