- Email: [email protected]
P3-413: Altered MicroRNA expression in Alzheimer blood mononuclear cells
T642
Poster Presentations P3:
differences in the MuLV expression in SAMR1 and SAMP8 astrocytes; the cell lines we established in this study may provide a useful model to explore the functions and effects of murine endogenous retrovirus. P3-410
ALZHEIMER’S DISEASE–RELATED ENDOSOME DYSFUNCTION INDUCED BY APP OVEREXPRESSION IN NEURONS REQUIRES RAB5 OVERACTIVATION
Seonil Kim1, Takahiro Sasaki2, Ralph A. Nixon1,2, 1NYU School of Medicine, New York, NY, USA; 2Nathan Kline Institute, Orangeburg, NY, USA. Contact e-mail: [email protected] Background: The endocytic pathway is a target of converging genetic factors that increase the risk for Alzheimer’s disease and others that cause additional neurodegenerative disorders. Abnormalities of early endosomes are the earliest known cellular pathology in AD and have been linked to altered trophic signaling, synaptic deficits, and altered APP metabolism. Rab5 is a small GTPase that regulates homotypic fusion of early endosomes and its upregulation at the earliest stages of AD mediates the endosome enlargement characteristically seen in affected neurons. In addition, previous studies have linked APP triplication to development of this AD-related endosomal phenotype. Methods: We expressed GFP fused Rab5 proteins and mRFP-fused amyloid precursor protein (APP) in mouse primary neuronal cultures to test the idea that APP leads to endosomal dysfunction by activating Rab5. Results: Overexpression of wild type human APP695 increased the total area and number of Rab5-positive endosomes in neuronal cell bodies. In neurites of these cells, APP overexpression significantly increased both total area and average size of endosomes. In time-lapse videomicroscopy analyses, the trafficking of enlarged endosomes was markedly delayed along neurites. Expression of a dominant active Rab5 mutant (Q79L) mimicked the APP-related endosome enlargement and trafficking deficits in neurons in the absence of APP overexpression. Moreover, expression of a dominant-negative Rab5 (S34N) prevented APP-induced endosome enlargement. These results suggest that development of the APP-related endosomal phenotype requires Rab5 activation. To investigate this possibility biochemically, we analyzed Rab5 activation in mouse N2a neuroblasoma cells that stably express APP. Membrane bound Rab5, the major pool of activated Rab5, was increased in APP overexpressed N2a cells, consistent with an effect of APP in promoting Rab5 activation. Conclusions: Our findings indicate that Rab5 activation is a critical factor in the development of endosomal pathology induced by APP overexpression. APP-mediated effects on endosome morphology are associated with the retardation of endosome retrograde movements, which may impede the functions of endosomes in neurotrophin signaling, synaptic plasticity and receptor recycling, leading to neurodegeneration in AD. This study is supported by the National Institute on Aging. P3-411
AGE-RELATED CHANGES OF AXONAL TRANSPORT MOTOR PROTEINS: SIRNAINDUCED DOWN-REGULATION CAUSES ACCUMULATION OF TAU AND APP
Nobuyuki Kimura1, Osamu Imamura2, Fumiko Ono1, Keiji Terao1, 1 Tsukuba Primate Research Center, National Institute of Biomedical Innovation, Ibaraki, Japan; 2Faculty of Medical Science, Graduate School, Juntendo University, Tokyo, Japan. Contact e-mail: [email protected] Background: Impaired axonal transport may promote pathogenesis in neurodegenerative disorders, such as Alzheimer’s disease (AD). We previously showed that tau, amyloid precursor protein (APP), and intracellular amyloid -protein (A) accumulate in the nerve-ending fraction of aged monkey brains, perhaps due to impaired axonal transport. In the present study, we assessed age-related changes of axonal transport motor proteins in aged monkey brains. Methods: We inves-
tigated age-related changes of axonal transport motor proteins by using young and aged monkey brains. We also examined RNAi studies to investigate whether siRNA-induced down-regulation of motor protein causes intracellular accumulation of AD-related proteins in human neuroblastoma cells. Results: Western blotting showed that kinesin, dynein, and dynactin (DYN) localizations dramatically changed with aging, and dynein level in nerve-ending fractions increased significantly. Co-immunoprecipitation analyses showed that DYN-dynein intermediate chain (DIC) interactions decreased, suggesting that agerelated attenuation of this interaction may cause the impairment of dynein function. Moreover, siRNA-induced down-regulation of DIC in human neuroblastoma cells caused endogenous tau and APP to accumulate, and their subcellular localizations were also affected. Conclusions: Our findings suggest that aging attenuates DYN-DIC interaction, representing one of the risk factors for age-related impaired dynein function, and even for accumulation of disease proteins. P3-412
MELATONIN PROMOTES AXON OUTGROWTH AND RESCUES ALZHEIMER-LIKE AXONOPATHY
Dan Liu1,2, Ling-Qiang Zhu1, Jian-Zhi Wang1, 1Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China; 2Hubei Provincial Key Laboratory of Neurological Disease, Wuhan, China. Contact e-mail: [email protected] Background: Decreased melatonin in the plasma was seen in Alzheimer’s disease, which had been reported to be associated with many pathological changes in AD. Exogenous melatonin administration can improve the cognition of AD patients and rescue some AD-like pathological changes. However, the relationship of melatonin with axon outgrowth or melatonin can rescue axonopathy in AD or not had not been reported. Methods: Primary hippocampus neurons were cultured from E18 rats. Immunofluorescence with specific axon and dendrite markers was performed for detecting axon and dendrites number. FM4-64 releasing to evaluate the axon function. Wild type GSK-3 plasmid transfection and wortmannin treatment were used for mimic the axonopathy. Image J calculation were applied for analysis. Results: Melatonin administration promotes the axon outgrowth at both the establishment stage and the maintenance stage of neuronal polarity. The ratio of multiple-axons increased to about 60% and the “extra” axons also posses the normal vesicle recycling function. Additionally, melatonin can rescue the axon outgrowth inhibition induced by overexpressing wild type GSK-3 and wortmannin treatment. Conclusions: Melatonin can promote the axon outgrowth in hippocampus neurons and rescue AD like axonopathy. P3-413
ALTERED MicroRNA EXPRESSION IN ALZHEIMER BLOOD MONONUCLEAR CELLS
Olivier C. Maes1, Hyman M. Schipper1, Howard M. Chertkow1, Eugenia Wang2, 1Lady Davis Institute for Medical Research, Montreal, QC, Canada; 2University of Louisville, Louisville, KY, USA. Contact e-mail: [email protected] Background: The etiology of sporadic Alzheimer disease (AD) is multifactorial, and carriers of the apolipoprotein E ⑀4 (APOE⑀4) allele are at increased risk for developing this condition. AD is characterized by a systemic decline in various coding genes and proteins within multiple functional categories. By degrading transcripts or inhibiting translation, we hypothesized that non-coding microRNAs (miRNA) may play a role in this condition. Over 500 miRNAs, that have yet to be functionally characterized, have predicted target sites mostly in the 3’ untranslated region of the mRNA of many genes. Methods: As a paradigm for the study of systemic molecular alterations, we analyzed the expression of miRNAs in blood mononuclear cells (BMC) and correlated their predicted targets with microarray profiling data of BMC in sporadic AD (Gene Regulation and Systems Biology, 2007:1; 263-
Poster Presentations P3: 274). Thirty-two AD and normal elderly controls (NEC) subjects were matched for ethnicity, age, gender and education. Results: Various miRNAs were found to be significantly up-regulated (but none downregulated) in Alzheimer BMC, and patterns of miRNA expression in AD patients may be contingent on the presence or absence of the APOE⑀4 allele. Down-regulated genes that are targeted by the upregulated miRNAs were largely represented in the functional categories of Transcription/Translation and Synaptic Activity. Genes with multiple targeting miRNAs were within the functional category of Injury response/Redox homeostasis. Up-regulated miRNA miR-181b and -200a shared common targets in functional categories of Cell Cycle and DNA damage. Interestingly, miR-181b, which exhibited the greatest fold-increase expression in Alzheimer BMC, targeted the highest proportion of down-regulated genes in AD BMC (31.1 %), followed by miR-34a (21.7 %). Conclusions: The miRNA expression signatures exhibited good disease classification as judged by hierarchical clustering, and individual miRNAs may provide surrogate markers for AD progression. The current observations further support our model linking the development of AD pathology to systemic dysfunction in the cellular stress/antioxidant response and genomic maintenance. P3-414
ACTIVITY-REGULATED CYTOSKELETONASSOCIATED PROTEIN IN RODENT BRAIN IS DOWN-REGULATED BY HIGH FAT DIET IN VIVO AND BY 27-HYDROXYCHOLESTEROL IN VITRO
Laura Mateos, Susanne Akterin, Francisco-Javier Gil-Bea, Stefan Spulber, Atiqur Rahman, Ingemar Bjo¨rkhem, Marianne Schultzberg, Amilcar Flores-Morales, Angel Cedazo-Minguez, Karolinska Institutet, Stockholm, Sweden. Contact e-mail: [email protected] Background: Growing evidence strongly suggest that high fat diet (HFD) has an important role in some neurodegenerative disorders, including Alzheimer’s disease (AD). An unsolved paradox is how high serum cholesterol influences AD pathology, knowing that cholesterol metabolism in the central nervous system (CNS) is regulated independently and separately from blood cholesterol metabolism. 27-hydroxycholesterol (27-OH), a metabolite of cholesterol, can pass the bloodbrain barrier (BBB) and a large amount of 27-OH is taken up from the blood. However, the mechanisms by which hypercholesterolemia could contribute to neurodegenerative disorders are currently unknown. To identify new cellular pathways linking hypercholesterolemia and neurodegeneration, we analyzed the effects of HFD on gene expression in mouse brain. Methods: Using cDNA microarrays and RT-PCR, we found that HFD has a mild, but significant effect on the expression of several genes. The altered genes include molecules linked to AD pathology and others of potential interest for neurodegeneration. We further investigated the effect of HFD on the activity-regulated cytoskeleton-associated protein (Arc). Results: Expression of Arc was decreased in cerebral cortex and hippocampus of HFD fed animals. From the known regulatory mechanisms of Arc expression, HFD reduced N-methyl-D-aspartate receptor (NMDAR) activity, as seen by decreases in tyrosine phosphorylation of NMDAR2A and levels of NMDAR1. Additionally, we demonstrated that 27-hydroxycholesterol, a cholesterol metabolite that enters the brain from the blood, decreases Arc levels as well as NMDAR and Src kinase activities in rat primary hippocampal neurons. Finally, we showed that Arc levels are decreased in the hippocampus of AD brains. Conclusions: Our results suggest that high cholesterol diet may be a negative additive factor in several pathways involved in AD pathology, including the disruption of Arc. A feasible link between 27-OH and Arc down-regulation could be part of the mechanisms behind the association between hypercholesterolemia and AD.
P3-415
T643 THE PRESENCE OF ABETA DIMERS IN SOLUBLE BRAIN EXTRACT IS ASSOCIATED WITH DEMENTIA
Jessica M. McDonald1, Carol Brayne2, George Savva2, Fiona E. Mattews3, Alfred Welzel1, Ganesh M. Shankar4, Paul G. Ince5, Dennis J. Selkoe4, Dominic M. Walsh1, 1Laboratory for Neurodegenerative Research, Conway Institute, University College Dublin, Dublin, Ireland; 2Department of Public Health & Primary Care, Institute of Public Health, Cambridge University, Cambridge, United Kingdom; 3MRC Biostatistics Unit, Institute of Public Health, Cambridge University, Cambridge, United Kingdom; 4Center for Neurologic Diseases, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA, USA; 5Dept. Pathology, Royal Hallamshire Hospital, Sheffield University, Sheffield, United Kingdom. Contact e-mail: [email protected] Background: Burgeoning data indicate that soluble non-fibrillar forms of Abeta play a central role in Alzheimer’s disease pathogenesis, nonetheless the precise assemblies involved and mechanism(s) of their effect remain unclear. Consequently, there is an urgent need to fully describe the soluble, pre-fibrillar Abeta species actually present in human brain. Here we report preliminary data from a study designed to measure the relative concentrations of Abeta monomer and low-n oligomers in soluble extracts of human brain. Methods: Samples of frontal and temporal cortices of 45 brains were obtained from the Medical Research Council’s Cognitive Function and Ageing Study and used to prepare soluble extracts. Briefly, 0.2 g samples of frozen cortex were homogenized in ice-cold Tris-buffered saline (1 ml) and the homogenate spun at 125,000 g for 78 min. The supernate was then analyzed for Abeta content using an immunoprecipitation/Western blotting protocol that sensitively detects soluble oligomers in human cerebrospinal fluid (Walsh et al. 2000). Results: Abeta monomers and SDS-stable dimers and trimers were detected in the brain extracts of dementia patients, but were not detected in brain extracts from non-demented individuals. Abeta dimers and trimers were only detected in samples that contained detectable monomer; however, the level of dimer was not directly related to the level of monomer. Logistic regression for the effect of the presence of total soluble Abeta (monomer plus dimer) was conducted in both brain areas and for both types of Abeta simultaneously. Adjustment was made for the correlations within individuals, and the difference in levels between regions. The odds ratio for the relationship between detectable soluble amyloid and dementia is 3.47 (95% CI⫽1.25,9.63). In the temporal lobe there was an association between the presence and concentration of Abeta dimers and the severity of neurofibrillary tangle pathology. Conclusions: Total soluble Abeta concentrations predict dementia and are closely related to NFTs, diffuse and neuritic plaques, but the exact concentration of total Abeta does not appear to be associated with severity of dementia. Further analysis is required to delineate the relationship of Abeta dimer and severity of dementia. P3-416
A STIMULATION LEADS TO ELEVATED LEVELS OF HEPARAN SULFATE PROTEOGLYCANS IN GLIAL CELLS
Paul O’ Callaghan1, Elina Sandwall1, Jin-Ping Li1, Hong Yu2, Rivka Ravid3, Zhi-Zhong Guan4, Toin H. van Kuppevelt5, Lars N. G. Nilsson1, Martin Ingelsson1, Bradley T. Hyman6, Hannu Kalimo1, Ulf Lindahl1, Lars Lannfelt1, Xiao Zhang1, 1Uppsala University, Uppsala, Sweden; 2Astra Zeneca R&D, Stockholm, Sweden; 3 Netherlands Brain Bank, Amsterdam, Netherlands; 4Karolinska Institute, Stockhom, Sweden; 5Radboud University Nijmegen, Nijmegen, Netherlands; 6Alzheimer’s Disease Research Laboratory, Massachusetts, MA, USA. Contact e-mail: [email protected] Background: The presence of heparan sulfate (HS) and HS proteoglycans (HSPGs) in the -amyloid (A) plaques of Alzheimer’s disease (AD) is well documented. There is in vitro evidence that HS is capable of accelerating and stabilising A aggregation and it has been suggested that this interaction is
Poster Presentations P3:
differences in the MuLV expression in SAMR1 and SAMP8 astrocytes; the cell lines we established in this study may provide a useful model to explore the functions and effects of murine endogenous retrovirus. P3-410
ALZHEIMER’S DISEASE–RELATED ENDOSOME DYSFUNCTION INDUCED BY APP OVEREXPRESSION IN NEURONS REQUIRES RAB5 OVERACTIVATION
Seonil Kim1, Takahiro Sasaki2, Ralph A. Nixon1,2, 1NYU School of Medicine, New York, NY, USA; 2Nathan Kline Institute, Orangeburg, NY, USA. Contact e-mail: [email protected] Background: The endocytic pathway is a target of converging genetic factors that increase the risk for Alzheimer’s disease and others that cause additional neurodegenerative disorders. Abnormalities of early endosomes are the earliest known cellular pathology in AD and have been linked to altered trophic signaling, synaptic deficits, and altered APP metabolism. Rab5 is a small GTPase that regulates homotypic fusion of early endosomes and its upregulation at the earliest stages of AD mediates the endosome enlargement characteristically seen in affected neurons. In addition, previous studies have linked APP triplication to development of this AD-related endosomal phenotype. Methods: We expressed GFP fused Rab5 proteins and mRFP-fused amyloid precursor protein (APP) in mouse primary neuronal cultures to test the idea that APP leads to endosomal dysfunction by activating Rab5. Results: Overexpression of wild type human APP695 increased the total area and number of Rab5-positive endosomes in neuronal cell bodies. In neurites of these cells, APP overexpression significantly increased both total area and average size of endosomes. In time-lapse videomicroscopy analyses, the trafficking of enlarged endosomes was markedly delayed along neurites. Expression of a dominant active Rab5 mutant (Q79L) mimicked the APP-related endosome enlargement and trafficking deficits in neurons in the absence of APP overexpression. Moreover, expression of a dominant-negative Rab5 (S34N) prevented APP-induced endosome enlargement. These results suggest that development of the APP-related endosomal phenotype requires Rab5 activation. To investigate this possibility biochemically, we analyzed Rab5 activation in mouse N2a neuroblasoma cells that stably express APP. Membrane bound Rab5, the major pool of activated Rab5, was increased in APP overexpressed N2a cells, consistent with an effect of APP in promoting Rab5 activation. Conclusions: Our findings indicate that Rab5 activation is a critical factor in the development of endosomal pathology induced by APP overexpression. APP-mediated effects on endosome morphology are associated with the retardation of endosome retrograde movements, which may impede the functions of endosomes in neurotrophin signaling, synaptic plasticity and receptor recycling, leading to neurodegeneration in AD. This study is supported by the National Institute on Aging. P3-411
AGE-RELATED CHANGES OF AXONAL TRANSPORT MOTOR PROTEINS: SIRNAINDUCED DOWN-REGULATION CAUSES ACCUMULATION OF TAU AND APP
Nobuyuki Kimura1, Osamu Imamura2, Fumiko Ono1, Keiji Terao1, 1 Tsukuba Primate Research Center, National Institute of Biomedical Innovation, Ibaraki, Japan; 2Faculty of Medical Science, Graduate School, Juntendo University, Tokyo, Japan. Contact e-mail: [email protected] Background: Impaired axonal transport may promote pathogenesis in neurodegenerative disorders, such as Alzheimer’s disease (AD). We previously showed that tau, amyloid precursor protein (APP), and intracellular amyloid -protein (A) accumulate in the nerve-ending fraction of aged monkey brains, perhaps due to impaired axonal transport. In the present study, we assessed age-related changes of axonal transport motor proteins in aged monkey brains. Methods: We inves-
tigated age-related changes of axonal transport motor proteins by using young and aged monkey brains. We also examined RNAi studies to investigate whether siRNA-induced down-regulation of motor protein causes intracellular accumulation of AD-related proteins in human neuroblastoma cells. Results: Western blotting showed that kinesin, dynein, and dynactin (DYN) localizations dramatically changed with aging, and dynein level in nerve-ending fractions increased significantly. Co-immunoprecipitation analyses showed that DYN-dynein intermediate chain (DIC) interactions decreased, suggesting that agerelated attenuation of this interaction may cause the impairment of dynein function. Moreover, siRNA-induced down-regulation of DIC in human neuroblastoma cells caused endogenous tau and APP to accumulate, and their subcellular localizations were also affected. Conclusions: Our findings suggest that aging attenuates DYN-DIC interaction, representing one of the risk factors for age-related impaired dynein function, and even for accumulation of disease proteins. P3-412
MELATONIN PROMOTES AXON OUTGROWTH AND RESCUES ALZHEIMER-LIKE AXONOPATHY
Dan Liu1,2, Ling-Qiang Zhu1, Jian-Zhi Wang1, 1Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China; 2Hubei Provincial Key Laboratory of Neurological Disease, Wuhan, China. Contact e-mail: [email protected] Background: Decreased melatonin in the plasma was seen in Alzheimer’s disease, which had been reported to be associated with many pathological changes in AD. Exogenous melatonin administration can improve the cognition of AD patients and rescue some AD-like pathological changes. However, the relationship of melatonin with axon outgrowth or melatonin can rescue axonopathy in AD or not had not been reported. Methods: Primary hippocampus neurons were cultured from E18 rats. Immunofluorescence with specific axon and dendrite markers was performed for detecting axon and dendrites number. FM4-64 releasing to evaluate the axon function. Wild type GSK-3 plasmid transfection and wortmannin treatment were used for mimic the axonopathy. Image J calculation were applied for analysis. Results: Melatonin administration promotes the axon outgrowth at both the establishment stage and the maintenance stage of neuronal polarity. The ratio of multiple-axons increased to about 60% and the “extra” axons also posses the normal vesicle recycling function. Additionally, melatonin can rescue the axon outgrowth inhibition induced by overexpressing wild type GSK-3 and wortmannin treatment. Conclusions: Melatonin can promote the axon outgrowth in hippocampus neurons and rescue AD like axonopathy. P3-413
ALTERED MicroRNA EXPRESSION IN ALZHEIMER BLOOD MONONUCLEAR CELLS
Olivier C. Maes1, Hyman M. Schipper1, Howard M. Chertkow1, Eugenia Wang2, 1Lady Davis Institute for Medical Research, Montreal, QC, Canada; 2University of Louisville, Louisville, KY, USA. Contact e-mail: [email protected] Background: The etiology of sporadic Alzheimer disease (AD) is multifactorial, and carriers of the apolipoprotein E ⑀4 (APOE⑀4) allele are at increased risk for developing this condition. AD is characterized by a systemic decline in various coding genes and proteins within multiple functional categories. By degrading transcripts or inhibiting translation, we hypothesized that non-coding microRNAs (miRNA) may play a role in this condition. Over 500 miRNAs, that have yet to be functionally characterized, have predicted target sites mostly in the 3’ untranslated region of the mRNA of many genes. Methods: As a paradigm for the study of systemic molecular alterations, we analyzed the expression of miRNAs in blood mononuclear cells (BMC) and correlated their predicted targets with microarray profiling data of BMC in sporadic AD (Gene Regulation and Systems Biology, 2007:1; 263-
Poster Presentations P3: 274). Thirty-two AD and normal elderly controls (NEC) subjects were matched for ethnicity, age, gender and education. Results: Various miRNAs were found to be significantly up-regulated (but none downregulated) in Alzheimer BMC, and patterns of miRNA expression in AD patients may be contingent on the presence or absence of the APOE⑀4 allele. Down-regulated genes that are targeted by the upregulated miRNAs were largely represented in the functional categories of Transcription/Translation and Synaptic Activity. Genes with multiple targeting miRNAs were within the functional category of Injury response/Redox homeostasis. Up-regulated miRNA miR-181b and -200a shared common targets in functional categories of Cell Cycle and DNA damage. Interestingly, miR-181b, which exhibited the greatest fold-increase expression in Alzheimer BMC, targeted the highest proportion of down-regulated genes in AD BMC (31.1 %), followed by miR-34a (21.7 %). Conclusions: The miRNA expression signatures exhibited good disease classification as judged by hierarchical clustering, and individual miRNAs may provide surrogate markers for AD progression. The current observations further support our model linking the development of AD pathology to systemic dysfunction in the cellular stress/antioxidant response and genomic maintenance. P3-414
ACTIVITY-REGULATED CYTOSKELETONASSOCIATED PROTEIN IN RODENT BRAIN IS DOWN-REGULATED BY HIGH FAT DIET IN VIVO AND BY 27-HYDROXYCHOLESTEROL IN VITRO
Laura Mateos, Susanne Akterin, Francisco-Javier Gil-Bea, Stefan Spulber, Atiqur Rahman, Ingemar Bjo¨rkhem, Marianne Schultzberg, Amilcar Flores-Morales, Angel Cedazo-Minguez, Karolinska Institutet, Stockholm, Sweden. Contact e-mail: [email protected] Background: Growing evidence strongly suggest that high fat diet (HFD) has an important role in some neurodegenerative disorders, including Alzheimer’s disease (AD). An unsolved paradox is how high serum cholesterol influences AD pathology, knowing that cholesterol metabolism in the central nervous system (CNS) is regulated independently and separately from blood cholesterol metabolism. 27-hydroxycholesterol (27-OH), a metabolite of cholesterol, can pass the bloodbrain barrier (BBB) and a large amount of 27-OH is taken up from the blood. However, the mechanisms by which hypercholesterolemia could contribute to neurodegenerative disorders are currently unknown. To identify new cellular pathways linking hypercholesterolemia and neurodegeneration, we analyzed the effects of HFD on gene expression in mouse brain. Methods: Using cDNA microarrays and RT-PCR, we found that HFD has a mild, but significant effect on the expression of several genes. The altered genes include molecules linked to AD pathology and others of potential interest for neurodegeneration. We further investigated the effect of HFD on the activity-regulated cytoskeleton-associated protein (Arc). Results: Expression of Arc was decreased in cerebral cortex and hippocampus of HFD fed animals. From the known regulatory mechanisms of Arc expression, HFD reduced N-methyl-D-aspartate receptor (NMDAR) activity, as seen by decreases in tyrosine phosphorylation of NMDAR2A and levels of NMDAR1. Additionally, we demonstrated that 27-hydroxycholesterol, a cholesterol metabolite that enters the brain from the blood, decreases Arc levels as well as NMDAR and Src kinase activities in rat primary hippocampal neurons. Finally, we showed that Arc levels are decreased in the hippocampus of AD brains. Conclusions: Our results suggest that high cholesterol diet may be a negative additive factor in several pathways involved in AD pathology, including the disruption of Arc. A feasible link between 27-OH and Arc down-regulation could be part of the mechanisms behind the association between hypercholesterolemia and AD.
P3-415
T643 THE PRESENCE OF ABETA DIMERS IN SOLUBLE BRAIN EXTRACT IS ASSOCIATED WITH DEMENTIA
Jessica M. McDonald1, Carol Brayne2, George Savva2, Fiona E. Mattews3, Alfred Welzel1, Ganesh M. Shankar4, Paul G. Ince5, Dennis J. Selkoe4, Dominic M. Walsh1, 1Laboratory for Neurodegenerative Research, Conway Institute, University College Dublin, Dublin, Ireland; 2Department of Public Health & Primary Care, Institute of Public Health, Cambridge University, Cambridge, United Kingdom; 3MRC Biostatistics Unit, Institute of Public Health, Cambridge University, Cambridge, United Kingdom; 4Center for Neurologic Diseases, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA, USA; 5Dept. Pathology, Royal Hallamshire Hospital, Sheffield University, Sheffield, United Kingdom. Contact e-mail: [email protected] Background: Burgeoning data indicate that soluble non-fibrillar forms of Abeta play a central role in Alzheimer’s disease pathogenesis, nonetheless the precise assemblies involved and mechanism(s) of their effect remain unclear. Consequently, there is an urgent need to fully describe the soluble, pre-fibrillar Abeta species actually present in human brain. Here we report preliminary data from a study designed to measure the relative concentrations of Abeta monomer and low-n oligomers in soluble extracts of human brain. Methods: Samples of frontal and temporal cortices of 45 brains were obtained from the Medical Research Council’s Cognitive Function and Ageing Study and used to prepare soluble extracts. Briefly, 0.2 g samples of frozen cortex were homogenized in ice-cold Tris-buffered saline (1 ml) and the homogenate spun at 125,000 g for 78 min. The supernate was then analyzed for Abeta content using an immunoprecipitation/Western blotting protocol that sensitively detects soluble oligomers in human cerebrospinal fluid (Walsh et al. 2000). Results: Abeta monomers and SDS-stable dimers and trimers were detected in the brain extracts of dementia patients, but were not detected in brain extracts from non-demented individuals. Abeta dimers and trimers were only detected in samples that contained detectable monomer; however, the level of dimer was not directly related to the level of monomer. Logistic regression for the effect of the presence of total soluble Abeta (monomer plus dimer) was conducted in both brain areas and for both types of Abeta simultaneously. Adjustment was made for the correlations within individuals, and the difference in levels between regions. The odds ratio for the relationship between detectable soluble amyloid and dementia is 3.47 (95% CI⫽1.25,9.63). In the temporal lobe there was an association between the presence and concentration of Abeta dimers and the severity of neurofibrillary tangle pathology. Conclusions: Total soluble Abeta concentrations predict dementia and are closely related to NFTs, diffuse and neuritic plaques, but the exact concentration of total Abeta does not appear to be associated with severity of dementia. Further analysis is required to delineate the relationship of Abeta dimer and severity of dementia. P3-416
A STIMULATION LEADS TO ELEVATED LEVELS OF HEPARAN SULFATE PROTEOGLYCANS IN GLIAL CELLS
Paul O’ Callaghan1, Elina Sandwall1, Jin-Ping Li1, Hong Yu2, Rivka Ravid3, Zhi-Zhong Guan4, Toin H. van Kuppevelt5, Lars N. G. Nilsson1, Martin Ingelsson1, Bradley T. Hyman6, Hannu Kalimo1, Ulf Lindahl1, Lars Lannfelt1, Xiao Zhang1, 1Uppsala University, Uppsala, Sweden; 2Astra Zeneca R&D, Stockholm, Sweden; 3 Netherlands Brain Bank, Amsterdam, Netherlands; 4Karolinska Institute, Stockhom, Sweden; 5Radboud University Nijmegen, Nijmegen, Netherlands; 6Alzheimer’s Disease Research Laboratory, Massachusetts, MA, USA. Contact e-mail: [email protected] Background: The presence of heparan sulfate (HS) and HS proteoglycans (HSPGs) in the -amyloid (A) plaques of Alzheimer’s disease (AD) is well documented. There is in vitro evidence that HS is capable of accelerating and stabilising A aggregation and it has been suggested that this interaction is