- Email: [email protected]
Role of the LRP-RanBP9 pathway in APP processing and Alzheimer's disease susceptibility
Oral O4-02: Disease Mechanisms: Other replicate association signals from our discovery dataset, a previously published LOAD GWAS on 492 LOAD cases and 496 controls. To validate associations and follow-up on a series of LOAD candidate genes, association analyses were performed under an additive model with corrections for population substructure. Results: Our analyses replicated a number of LOAD association signals, with unequivocal confirmation of APOE’s risk effect (rs2075650, P ¼ 1.9 3 1013). Several SNPs demonstrated strong associations (105-106) near previous signals on chromosomes 3q13.33 (near CD86), 6q25.1 (in MTHFD1L), and others. Additionally, several previously moderate signals were much stronger in the replication dataset, including signals on chromosomes 1q42, 3q26.31, and in the chromosome 20 gene PTPRT, which has several roles in the central nervous system. In the joint analysis comprising 891 LOAD cases and 853 controls, we observed notably stronger associations for SNPs in candidate genes, though none met genomewide significance (e.g., MTHFD1L, rs11754661, P ¼ 9.3 3 107; ELAVL4, rs9659092, P ¼ 1.4 3 106). ELAVL4 SNPs were more significantly associated in LOAD cases with Depression (rs11583200, P ¼ 4.6 3 108) (see Slifer et al., this conference). Among other candidate genes previously associated with LOAD, we observed nominal associations in genes for VDR, WWC1, and for SORL1. Conclusions: In this genome-wide association study of late onset Alzheimer disease, we replicated and validated previously observed associations, including several in multiple candidate genes including ELAVL4, VDR, SORL1, WWC1, and APOE, and several in candidate genes with potential biological roles, including PTRPT and MTHFD1L, meriting further investigation. O4-01-08
ANALYSIS OF ALS6 IN FRONTOTEMPORAL LOBAR DEGENERATION
Stuart M. Pickering-Brown1, Christopher E. Shaw2, 1Manchester University, Manchester, United Kingdom; 2King’s College London, London, United Kingdom. Contact e-mail: [email protected] Background: It has been proposed that Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Lobar Degeneration (FTLD) represent separate ends of the same clinicopathological disease spectrum because approximately 95% of ALS and 50% of FTLD cases have a common aggregated and ubiquitinated protein, TAR DNA-binding protein (TDP-43), in neuronal cytoplasmic inclusions. Furthermore, up to 30% of patients with FTLD develop sub-clinical features of ALS and 30% of patients with ALS develop cognitive verbal fluency and executive function deficits similar to those observed in FTLD. Mutations in TDP-43 have been shown to be a cause of familial ALS but other genes for this disease remain to be discovered. Recently, the gene in the ALS6 locus on chr16 has been identified as a cause of ALS (Vance et al. Science - In Press). That ALS and FTLD have common features makes the ALS6 gene a good candidate for FTLD. Methods: 310 DNA samples from cases with FTLD were available for ALS6 sequencing using conventional methods. Results: Currently, the 2 mutational hotspots of ALS6 have been sequenced in 195 cases of FTLD but no mutations have yet been identified. Conclusions: The complete open reading frame of ALS6 requires sequencing in all cases before conclusions can be made to the role, if any, that this gene plays in the aetiology of FTLD. Completed sequencing data in addition to a linkage dysequilibrium analysis of the ALS6 locus, to assess if common variation of this gene modifies disease risk of FTLD, will be presented. WEDNESDAY, JULY 15, 2009 ORAL O4-02 DISEASE MECHANISMS: OTHER O4-02-01
A NOVEL ROLE OF BETA-SECRETASE1 (BACE1) IN THE PATHOGENESIS OF CEREBRAL AMYLOID ANGIOPATHY (CAA)
Xin Cheng1, Qiang Dong1, Ping He2, Hikmet F. Nural2, Rena Li2, Yong Shen2,1, 1Huashan Hospital, Fudan University, Shanghai, China; 2 Sun Health Research Institute, Sun City, AZ, USA. Contact e-mail: [email protected]
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Background: Cerebral amyloid angiopathy (CAA) is an age-associated condition that is pathologically characterized by deposition of b-amyloid peptide (Ab) in small and medium sized arteries of the cerebral cortex and leptomeninges. While studies demonstrated that the pathologies of CAA are associated with Alzheimer’s disease (AD), mechanisms of Ab deposition and secondary vascular degeneration remain unclear. The generation of Ab from the amyloid precursor protein is initiated by beta-secretase 1 (b-site amyloid precursor protein cleaving enzyme 1 [BACE1]), which is increased in AD brains. Here, we propose the hypothesis that BACE1 may play an important role in CAA pathogenesis in vessel endothelial cells. Methods: To examine the relationship between CAA and AD, we examined BACE1 protein expression and enzymatic activities in the leptomeninges from AD, AD with CAA (AD þ CAA), and non AD control individuals. To investigate the role of BACE1 and Ab in CAA, we studied Ab and BACE1 expression in brains of a CAA mouse transgenic animal model, APP23 mice. Furthermore, to study whether BACE1 was related to blood vessel degeneration, we detected protein expression levels of tight junction proteins (TJPs) in cerebral microvessel endothelial cell line overexpressed with BACE1. Results: We found a significant elevation of BACE1 protein levels and the enzyme activity in cerebral vessels of AD þ CAA patients compared to that in AD or control individuals. Moreover, TJPs levels in cerebral vessels from the same cases are much lower than that in AD or control cases. Similar results were observed in APP23 mice. Importantly, we observed that overexpressing BACE1 in cerebral microvessel endothelial cells resulted in TJP degradation, which was prevented by BACE1 inhibitor. Conclusions: The results suggest that BACE1 contributes to the Ab accumulation in cerebral vessels and endothelial degeneration in CAA patients. O4-02-02
ROLE OF THE LRP-RANBP9 PATHWAY IN APP PROCESSING AND ALZHEIMER’S DISEASE SUSCEPTIBILITY
David E. Kang, Madepalli K. Lakshmana, Eileen Tak, Edward H. Koo, IlSang Yoon, Eunice Chen, University of California, San Diego, La Jolla, CA, USA. Contact e-mail: [email protected] Background: Accumulation of the amyloid b (Ab) peptide derived from the proteolytic processing of amyloid precursor protein (APP) is the defining pathological hallmark of Alzheimer’s disease (AD). We previously demonstrated that the C-terminal 37 amino acids of LRP robustly promoted Ab generation independent of FE65 and specifically interacted with Ran Binding Protein 9 (RanBP9). Methods: In this study, we employed non-neuronal and neuronal cell lines as well as primary neurons to study whether modulation of RanBP9 expression alters the trafficking and processing of APP and Ab generation. In addition, we utilized several genetic variations in the promoter/enhancer region of the RanBP9 gene to examine the potential genetic association of RanBP9 with the risk of developing AD. Results: We found that increased RanBP9 expression strongly promoted b-secretase cleavage of APP and Ab generation in HEK293 T, CHO, and Neuro-2a cells. This pro-amyloidogenic activity of RanBP9 did not depend on the KPI domain or the Swedish APP mutation. In addition, RanBP9 reduced cell surface APP and accelerated APP internalization, consistent with enhanced b-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, suggestive of a scaffolding mechanism to bridge these molecular interactions. Importantly, knockdown of endogenous RanBP9 significantly reduced Ab generation in CHO cells, Neuro-2a cells, and in primary neurons, demonstrating its physiological role in regulating b-secretase cleavage of APP. In vivo, RanBP9 protein expression was strongly elevated both in AD as well as in APP transgenic mice compared to their respective controls. Finally, genetic analysis of RanBP9 promoter polymorphisms revealed a strong and highly significant distortion of RanBP9 genotypes and alleles between AD and age-matched subjects. Conclusions: These findings taken together reveal a novel role of the
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Oral O4-02: Disease Mechanisms: Other
LRP-RanBP9 pathway in both the susceptibility to late-onset AD and in the critical regulation of APP processing. O4-02-03
PREVENTING COGNITIVE DECLINE IN ELDERLY DEMENTED/DEPRESSED PATIENTS AND IN APOE4 MOUSE AS A MODEL OF HUMAN ALZHEIMER’S DISEASE BY FEEDING ACETYL-L-CARNITINE AND LIPOIC ACID
Gjumrakch Aliev1,2, Jiankang Liu3, Amanda Lipsitt4, Kathryn Fischbach5, Mark E. Obrenovich6, Bruce N. Ames3, Eldar Qasimov7, Joseph C. LaManna8, Ludis Morales2, Valentin Bragin1, 1Stress Relief Center, Brooklyn, NY, USA; 2Departamento de Nutricio´n y Bioquı´mica, Facultad de Ciencias, Pontificia Universidad Javeriana, Bogota´, Colombia; 3 Children’s Hospital Oakland Research Institute, Oakland, CA, USA; 4Department of Infectious Diseases, Medicine University of Texas Health Science Center at San Antonio, San Antonio, TX, USA; 5The University of Texas at San Antonio, San Antonio, TX, USA; 6Department of Pathology, School of Medicine, Case Western Reserve University, Cleveland, OH, USA; 7Department of Cytology, Histology and Embryology, Baku Medical University, Baku, Azerbaijan; 8Department of Physiology, School of Medicine, Case Western Reserve University, Cleveland, OH, USA. Contact e-mail: aliev03@ gmail.com Background: The E4 isoform of apolipoprotein E (ApoE) is involved in cardiovascular and cerebrovascular disorders and is the most prevalent risk factor for late onset or sporadic AD. ApoE4 transgenic mice are appropriate models for studying the pathogenesis and preclinical treatment of ApoE-related cognitive deficits associated with late onset AD. Methods: This study uses the vascular dementia paradigm to analyze the effects of selective mitochondrial antioxidants, acetyl-Carnitine and R-Lipoic acid, and ApoE4 on CBF, neuropathology, brain and vessel ultrastructural abnormalities, and behavior in ApoE4 mice. We have also compared these results with our ongoing clinical study of clinically depressed demented seniors with cardiovascular diseases symptoms. The patients received Alpha-Lipoic Acid, Acetyl-L-Carnitine, Omega-3 and Coenzyme Q-10, along with diet changes, and our recently developed brain activation program. Results: ApoE4 associated factors reduce cerebral blood flow (CBF) gradually to create brain hypoperfusion when compared to wild-type (WT) and the differences in CBF are greatest as animals age from 6-weeks to 12-months. Transmission electron microscopy (TEM) with colloidal gold immunocytochemistry and in situ hybridization using human and mouse DNA probes shows structural damage and mitochondrial DNA overproliferation and/or deletion in young and aged microvessel endothelium of ApoE4 animals, extending to the cytoplasm of perivascular cells, perivascular nerve terminals, hippocampal neurons, and glial cells. Spatial memory and temporal memory tests show a trend in improving cognitive function in ApoE4 mice fed selective mitochondrial antioxidants acetyl-L-Carnitine and R-Lipoic acid. Our clinical results show that patients who receive mitochondrial antioxidants present the maximum significant cognitive improvement at the end of 24 months of the treatment. Conclusions: Further examination of ultrastructural degeneration caused by aging, especially under cardiovascular diseases complication, will likely contribute to our understanding of neurodegenerative etiology and indicates a new avenue of development for novel prophylactic and treatment strategies by ensuing selective mitochondrial antioxidants for the demented patients. O4-02-04
SELECTIVE TARGETING OF PERIVASCULAR MACROPHAGES FOR b-AMYLOID CLEARANCE IN CEREBRAL AMYLOID ANGIOPATHY
JoAnne McLaurin, Cheryl Hawkes, University of Toronto, Toronto, ON, Canada. Contact e-mail: [email protected] Background: The majority of Alzheimer’s disease (AD) cases present with cerebral amyloid angiopathy (CAA), the deposition of Ab in cortical blood vessels and leptomeninges. Although the pathogenesis of CAA remains unknown, it has been suggested to develop in part due to impaired clearance of
cerebral Ab via perivascular interstitial fluid (ISF) drainage pathways. Perivascular macrophages are a group of innate immune cells that contribute to the formation and function of the blood brain barrier by acting as scavenger phagocytes. However, the interaction of perivascular macrophages with Ab and the significance of this interaction in the development of CAA is unknown. The purpose of these experiments was to determine the effect of perivascular macrophage depletion and stimulation on CAA severity in the TgCRND8 animal model of AD. Methods: To deplete perivascular macrophages, 4 month-old mice received an i.c.v. injection of liposome-encapsulated clodronate into the left lateral ventricle. Perivascular macrophage turnover was induced in 5 month-old TgCRND8 mice following two i.c.v. injections of chitin. Mice were sacrificed 1 month after injection and brain tissues were processed for immunocytochemistry, immunoblotting and ELISA. Results: Depletion of perivascular macrophages significantly increased the number of thioflavin S-positive cortical and leptomeningeal blood vessels. ELISA analyses confirmed that this increase was underscored by elevations in total vascular Ab42 levels. Conversely, stimulation of perivascular macrophage turnover reduced cerebral CAA load, an effect that was specifically mediated by perivascular macrophages, not via clearance by microglia or astrocytes. Conclusions: These results highlight a function for the physiological role of perivascular macrophages in the regulation of CAA and suggest that selective targeting of perivascular macrophage activation might constitute a therapeutic strategy to clear vascular amyloid. O4-02-05
ABNORMAL MITOCHONDRIAL STRUCTURAL AND FUNCTIONAL CHANGES CAUSED BY AMYLOID BETA IN ALZHEIMER’S DISEASE
Peizhong Mao, Maria Manczak, Divya Shree, P. Hemachandra Reddy, Oregon Health and Science University, Beaverton, OR, USA. Contact e-mail: [email protected] Background: Oxidative stress and synaptic damage have recently been reported as early events in Alzheimer’s disease (AD) progression. The purpose of this study was to investigate the role of amyloid beta in mitochondrial structure and function. Methods: Our recent global gene expression studies have revealed an up-regulation of mitochondrial genes in AD transgenic mice (a Tg2576 mouse model), suggesting that mitochondrial metabolism is impaired by mutant APP/Ab and that the up-regulation of mitochondrial genes may be a compensatory response to mutant APP/Ab (Reddy et al. 2004). Recently, we (Manczak et al 2006) and others (Caspersen et al 2005, Devi et al 2006) found that Ab is associated with mitochondria and is responsible for generating free radicals and initiating mitochondrial dysfunction. However, the connection between mutant APP/Ab and mitochondrial structural abnormalities is still unclear. To determine the link between Ab and mitochondrial structural changes, we treated mouse neuroblastoma (N2a) cells with Ab peptide 25-35 (30uM) for 48 hrs and studied the mitochondrial structure and function. We also measured mRNA of mitochondrial fission (Fis1, Drp1) and fusion (Mfn1 m Mfn2, Opa1) genes using qRT-PCR. Further, using EM and qRT-PCR, we studied mitochondrial structure in the cerebral cortex of 13-month-old Tg2576 mice and non-transgenic littermates. In addition, we investigated to determine, if mitochondrial targeted antioxidants (MTAs), MitoQ and SS31 rescue abnormal mitochondrial structure and function in N2a cells treated with Ab. Results: Our EM studies of Ab and N2a cells revealed increased mitochondrial number in N2a cells treated with Ab compared to untreated cells. We found up-regulated Fis1 and Drp1 in N2a cells treated with Ab. Our mitochondrial functional studies revealed decreased cytochrome oxidase, cell viability and mitochondrial membrane potential, and increased H2O2 and lipid peroxidation. However, MitoQ and SS31 rescued abnormal mitochondrial structure and function induced by Ab. Our EM studies of Tg2576 mice revealed increased mitochondrial number in 13-month-old Tg576 mice compared to control mice. Our qRT-PCR revealed that increased Fis1 and decreased Mfn1 and Mfn2 in Tg2576 mice relative to non-transgenic littermates. Conclusions: Overall our mitochondrial structural and functional studies revealed that mutant APP and Ab damage mitochondrial structure and cause mitochondrial dysfunction and neuronal damage.
ANALYSIS OF ALS6 IN FRONTOTEMPORAL LOBAR DEGENERATION
Stuart M. Pickering-Brown1, Christopher E. Shaw2, 1Manchester University, Manchester, United Kingdom; 2King’s College London, London, United Kingdom. Contact e-mail: [email protected] Background: It has been proposed that Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Lobar Degeneration (FTLD) represent separate ends of the same clinicopathological disease spectrum because approximately 95% of ALS and 50% of FTLD cases have a common aggregated and ubiquitinated protein, TAR DNA-binding protein (TDP-43), in neuronal cytoplasmic inclusions. Furthermore, up to 30% of patients with FTLD develop sub-clinical features of ALS and 30% of patients with ALS develop cognitive verbal fluency and executive function deficits similar to those observed in FTLD. Mutations in TDP-43 have been shown to be a cause of familial ALS but other genes for this disease remain to be discovered. Recently, the gene in the ALS6 locus on chr16 has been identified as a cause of ALS (Vance et al. Science - In Press). That ALS and FTLD have common features makes the ALS6 gene a good candidate for FTLD. Methods: 310 DNA samples from cases with FTLD were available for ALS6 sequencing using conventional methods. Results: Currently, the 2 mutational hotspots of ALS6 have been sequenced in 195 cases of FTLD but no mutations have yet been identified. Conclusions: The complete open reading frame of ALS6 requires sequencing in all cases before conclusions can be made to the role, if any, that this gene plays in the aetiology of FTLD. Completed sequencing data in addition to a linkage dysequilibrium analysis of the ALS6 locus, to assess if common variation of this gene modifies disease risk of FTLD, will be presented. WEDNESDAY, JULY 15, 2009 ORAL O4-02 DISEASE MECHANISMS: OTHER O4-02-01
A NOVEL ROLE OF BETA-SECRETASE1 (BACE1) IN THE PATHOGENESIS OF CEREBRAL AMYLOID ANGIOPATHY (CAA)
Xin Cheng1, Qiang Dong1, Ping He2, Hikmet F. Nural2, Rena Li2, Yong Shen2,1, 1Huashan Hospital, Fudan University, Shanghai, China; 2 Sun Health Research Institute, Sun City, AZ, USA. Contact e-mail: [email protected]
P151
Background: Cerebral amyloid angiopathy (CAA) is an age-associated condition that is pathologically characterized by deposition of b-amyloid peptide (Ab) in small and medium sized arteries of the cerebral cortex and leptomeninges. While studies demonstrated that the pathologies of CAA are associated with Alzheimer’s disease (AD), mechanisms of Ab deposition and secondary vascular degeneration remain unclear. The generation of Ab from the amyloid precursor protein is initiated by beta-secretase 1 (b-site amyloid precursor protein cleaving enzyme 1 [BACE1]), which is increased in AD brains. Here, we propose the hypothesis that BACE1 may play an important role in CAA pathogenesis in vessel endothelial cells. Methods: To examine the relationship between CAA and AD, we examined BACE1 protein expression and enzymatic activities in the leptomeninges from AD, AD with CAA (AD þ CAA), and non AD control individuals. To investigate the role of BACE1 and Ab in CAA, we studied Ab and BACE1 expression in brains of a CAA mouse transgenic animal model, APP23 mice. Furthermore, to study whether BACE1 was related to blood vessel degeneration, we detected protein expression levels of tight junction proteins (TJPs) in cerebral microvessel endothelial cell line overexpressed with BACE1. Results: We found a significant elevation of BACE1 protein levels and the enzyme activity in cerebral vessels of AD þ CAA patients compared to that in AD or control individuals. Moreover, TJPs levels in cerebral vessels from the same cases are much lower than that in AD or control cases. Similar results were observed in APP23 mice. Importantly, we observed that overexpressing BACE1 in cerebral microvessel endothelial cells resulted in TJP degradation, which was prevented by BACE1 inhibitor. Conclusions: The results suggest that BACE1 contributes to the Ab accumulation in cerebral vessels and endothelial degeneration in CAA patients. O4-02-02
ROLE OF THE LRP-RANBP9 PATHWAY IN APP PROCESSING AND ALZHEIMER’S DISEASE SUSCEPTIBILITY
David E. Kang, Madepalli K. Lakshmana, Eileen Tak, Edward H. Koo, IlSang Yoon, Eunice Chen, University of California, San Diego, La Jolla, CA, USA. Contact e-mail: [email protected] Background: Accumulation of the amyloid b (Ab) peptide derived from the proteolytic processing of amyloid precursor protein (APP) is the defining pathological hallmark of Alzheimer’s disease (AD). We previously demonstrated that the C-terminal 37 amino acids of LRP robustly promoted Ab generation independent of FE65 and specifically interacted with Ran Binding Protein 9 (RanBP9). Methods: In this study, we employed non-neuronal and neuronal cell lines as well as primary neurons to study whether modulation of RanBP9 expression alters the trafficking and processing of APP and Ab generation. In addition, we utilized several genetic variations in the promoter/enhancer region of the RanBP9 gene to examine the potential genetic association of RanBP9 with the risk of developing AD. Results: We found that increased RanBP9 expression strongly promoted b-secretase cleavage of APP and Ab generation in HEK293 T, CHO, and Neuro-2a cells. This pro-amyloidogenic activity of RanBP9 did not depend on the KPI domain or the Swedish APP mutation. In addition, RanBP9 reduced cell surface APP and accelerated APP internalization, consistent with enhanced b-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, suggestive of a scaffolding mechanism to bridge these molecular interactions. Importantly, knockdown of endogenous RanBP9 significantly reduced Ab generation in CHO cells, Neuro-2a cells, and in primary neurons, demonstrating its physiological role in regulating b-secretase cleavage of APP. In vivo, RanBP9 protein expression was strongly elevated both in AD as well as in APP transgenic mice compared to their respective controls. Finally, genetic analysis of RanBP9 promoter polymorphisms revealed a strong and highly significant distortion of RanBP9 genotypes and alleles between AD and age-matched subjects. Conclusions: These findings taken together reveal a novel role of the
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Oral O4-02: Disease Mechanisms: Other
LRP-RanBP9 pathway in both the susceptibility to late-onset AD and in the critical regulation of APP processing. O4-02-03
PREVENTING COGNITIVE DECLINE IN ELDERLY DEMENTED/DEPRESSED PATIENTS AND IN APOE4 MOUSE AS A MODEL OF HUMAN ALZHEIMER’S DISEASE BY FEEDING ACETYL-L-CARNITINE AND LIPOIC ACID
Gjumrakch Aliev1,2, Jiankang Liu3, Amanda Lipsitt4, Kathryn Fischbach5, Mark E. Obrenovich6, Bruce N. Ames3, Eldar Qasimov7, Joseph C. LaManna8, Ludis Morales2, Valentin Bragin1, 1Stress Relief Center, Brooklyn, NY, USA; 2Departamento de Nutricio´n y Bioquı´mica, Facultad de Ciencias, Pontificia Universidad Javeriana, Bogota´, Colombia; 3 Children’s Hospital Oakland Research Institute, Oakland, CA, USA; 4Department of Infectious Diseases, Medicine University of Texas Health Science Center at San Antonio, San Antonio, TX, USA; 5The University of Texas at San Antonio, San Antonio, TX, USA; 6Department of Pathology, School of Medicine, Case Western Reserve University, Cleveland, OH, USA; 7Department of Cytology, Histology and Embryology, Baku Medical University, Baku, Azerbaijan; 8Department of Physiology, School of Medicine, Case Western Reserve University, Cleveland, OH, USA. Contact e-mail: aliev03@ gmail.com Background: The E4 isoform of apolipoprotein E (ApoE) is involved in cardiovascular and cerebrovascular disorders and is the most prevalent risk factor for late onset or sporadic AD. ApoE4 transgenic mice are appropriate models for studying the pathogenesis and preclinical treatment of ApoE-related cognitive deficits associated with late onset AD. Methods: This study uses the vascular dementia paradigm to analyze the effects of selective mitochondrial antioxidants, acetyl-Carnitine and R-Lipoic acid, and ApoE4 on CBF, neuropathology, brain and vessel ultrastructural abnormalities, and behavior in ApoE4 mice. We have also compared these results with our ongoing clinical study of clinically depressed demented seniors with cardiovascular diseases symptoms. The patients received Alpha-Lipoic Acid, Acetyl-L-Carnitine, Omega-3 and Coenzyme Q-10, along with diet changes, and our recently developed brain activation program. Results: ApoE4 associated factors reduce cerebral blood flow (CBF) gradually to create brain hypoperfusion when compared to wild-type (WT) and the differences in CBF are greatest as animals age from 6-weeks to 12-months. Transmission electron microscopy (TEM) with colloidal gold immunocytochemistry and in situ hybridization using human and mouse DNA probes shows structural damage and mitochondrial DNA overproliferation and/or deletion in young and aged microvessel endothelium of ApoE4 animals, extending to the cytoplasm of perivascular cells, perivascular nerve terminals, hippocampal neurons, and glial cells. Spatial memory and temporal memory tests show a trend in improving cognitive function in ApoE4 mice fed selective mitochondrial antioxidants acetyl-L-Carnitine and R-Lipoic acid. Our clinical results show that patients who receive mitochondrial antioxidants present the maximum significant cognitive improvement at the end of 24 months of the treatment. Conclusions: Further examination of ultrastructural degeneration caused by aging, especially under cardiovascular diseases complication, will likely contribute to our understanding of neurodegenerative etiology and indicates a new avenue of development for novel prophylactic and treatment strategies by ensuing selective mitochondrial antioxidants for the demented patients. O4-02-04
SELECTIVE TARGETING OF PERIVASCULAR MACROPHAGES FOR b-AMYLOID CLEARANCE IN CEREBRAL AMYLOID ANGIOPATHY
JoAnne McLaurin, Cheryl Hawkes, University of Toronto, Toronto, ON, Canada. Contact e-mail: [email protected] Background: The majority of Alzheimer’s disease (AD) cases present with cerebral amyloid angiopathy (CAA), the deposition of Ab in cortical blood vessels and leptomeninges. Although the pathogenesis of CAA remains unknown, it has been suggested to develop in part due to impaired clearance of
cerebral Ab via perivascular interstitial fluid (ISF) drainage pathways. Perivascular macrophages are a group of innate immune cells that contribute to the formation and function of the blood brain barrier by acting as scavenger phagocytes. However, the interaction of perivascular macrophages with Ab and the significance of this interaction in the development of CAA is unknown. The purpose of these experiments was to determine the effect of perivascular macrophage depletion and stimulation on CAA severity in the TgCRND8 animal model of AD. Methods: To deplete perivascular macrophages, 4 month-old mice received an i.c.v. injection of liposome-encapsulated clodronate into the left lateral ventricle. Perivascular macrophage turnover was induced in 5 month-old TgCRND8 mice following two i.c.v. injections of chitin. Mice were sacrificed 1 month after injection and brain tissues were processed for immunocytochemistry, immunoblotting and ELISA. Results: Depletion of perivascular macrophages significantly increased the number of thioflavin S-positive cortical and leptomeningeal blood vessels. ELISA analyses confirmed that this increase was underscored by elevations in total vascular Ab42 levels. Conversely, stimulation of perivascular macrophage turnover reduced cerebral CAA load, an effect that was specifically mediated by perivascular macrophages, not via clearance by microglia or astrocytes. Conclusions: These results highlight a function for the physiological role of perivascular macrophages in the regulation of CAA and suggest that selective targeting of perivascular macrophage activation might constitute a therapeutic strategy to clear vascular amyloid. O4-02-05
ABNORMAL MITOCHONDRIAL STRUCTURAL AND FUNCTIONAL CHANGES CAUSED BY AMYLOID BETA IN ALZHEIMER’S DISEASE
Peizhong Mao, Maria Manczak, Divya Shree, P. Hemachandra Reddy, Oregon Health and Science University, Beaverton, OR, USA. Contact e-mail: [email protected] Background: Oxidative stress and synaptic damage have recently been reported as early events in Alzheimer’s disease (AD) progression. The purpose of this study was to investigate the role of amyloid beta in mitochondrial structure and function. Methods: Our recent global gene expression studies have revealed an up-regulation of mitochondrial genes in AD transgenic mice (a Tg2576 mouse model), suggesting that mitochondrial metabolism is impaired by mutant APP/Ab and that the up-regulation of mitochondrial genes may be a compensatory response to mutant APP/Ab (Reddy et al. 2004). Recently, we (Manczak et al 2006) and others (Caspersen et al 2005, Devi et al 2006) found that Ab is associated with mitochondria and is responsible for generating free radicals and initiating mitochondrial dysfunction. However, the connection between mutant APP/Ab and mitochondrial structural abnormalities is still unclear. To determine the link between Ab and mitochondrial structural changes, we treated mouse neuroblastoma (N2a) cells with Ab peptide 25-35 (30uM) for 48 hrs and studied the mitochondrial structure and function. We also measured mRNA of mitochondrial fission (Fis1, Drp1) and fusion (Mfn1 m Mfn2, Opa1) genes using qRT-PCR. Further, using EM and qRT-PCR, we studied mitochondrial structure in the cerebral cortex of 13-month-old Tg2576 mice and non-transgenic littermates. In addition, we investigated to determine, if mitochondrial targeted antioxidants (MTAs), MitoQ and SS31 rescue abnormal mitochondrial structure and function in N2a cells treated with Ab. Results: Our EM studies of Ab and N2a cells revealed increased mitochondrial number in N2a cells treated with Ab compared to untreated cells. We found up-regulated Fis1 and Drp1 in N2a cells treated with Ab. Our mitochondrial functional studies revealed decreased cytochrome oxidase, cell viability and mitochondrial membrane potential, and increased H2O2 and lipid peroxidation. However, MitoQ and SS31 rescued abnormal mitochondrial structure and function induced by Ab. Our EM studies of Tg2576 mice revealed increased mitochondrial number in 13-month-old Tg576 mice compared to control mice. Our qRT-PCR revealed that increased Fis1 and decreased Mfn1 and Mfn2 in Tg2576 mice relative to non-transgenic littermates. Conclusions: Overall our mitochondrial structural and functional studies revealed that mutant APP and Ab damage mitochondrial structure and cause mitochondrial dysfunction and neuronal damage.