- Email: [email protected]
THE ROLE OF TREM2 EXPRESSION ON MYELOID CELLS IN ALZHEIMER'S DISEASE
P876
Poster Presentations: P4
CN autoinhibitory domain located near the C terminus of the CN catalytic subunit. Commercially available antibodies that target the N terminus of the CN catalytic subunit reveal the presence of a highly-active 48 kDa proteolytic fragment in human brain tissue during early stages of cognitive decline and also in a variety of experimental models of neurodegeneration. While useful for determining the extent of CN proteolysis in Western blot applications, N terminus antibodies do not reveal the cellular location of the proteolysis. Knowing where CN is proteolyzed in nervous tissue seems critical to understanding the mechanistic basis of its many deleterious actions, particularly because CN is found in both neurons and glial cells where it is involved in different cellular functions. Methods: To address this gap in our understanding of CN regulation, we generated custom rabbit polyclonal antibodies to CN A based on previously identified calpain-dependent cleavage sites. One of these antibodies (referred to as “DCN48”) detects a 48 kDa fragment in Western blot assays, but does not detect full-length (60 kDa) CN. The DCN48 antibody was then used for DAB immunohistochemical labeling of human brain sections characterized by AD and mixed AD/vascular pathologies. Results: The anatomical features labeled by the DCN48 antibody included astrocyte clusters and vascular-associated elements and/or processes. We also observed numerous DCN48-positive astrocytes associated with microinfarcts. Surprisingly, we have seen very little neuronal labeling with this antibody. Conclusions: The results suggest that astrocytes and, perhaps astrocyte end-feet, are a primary locus for CPdependent CN proteolysis in injured or diseased nervous tissue. CN proteolysis appears to be especially pronounced in astrocytes associated with vascular pathology. This work may provide new mechanistic insights into the impact of Ca 2+ dysregulation on neurodegenerative diseases. P4-246
ACTIVATION OF THE SIGMA-1 RECEPTOR BY SPECIFIC LIGANDS INHIBITS HUMAN INFLAMMATORY DENDRITIC CELL FUNCTIONS AND EFFECTOR T-LYMPHOCYTE RESPONSES
Attila Szabo, Attila Kovacs, Ede Frecska, Eva Rajnavolgyi, Faculty of Medicine, University of Debrecen, Debrecen, Hungary. Contact e-mail: [email protected] Background: Neuropsychiatric diseases have recently been attributed to chronic inflammation in the central nervous system, and correlation between gene polymorphisms of innate immune receptors and the frequency of late onset of Alzheimer’s disease (AD) has also been shown. Ligation of murine maternal Toll-like receptors (TLRs) and RIG-I-like receptors (RLRs) by LPS or PolyI:C have been shown to cause decreased neurogenesis, cognitive deficits, and increased deposition of Ab aggregates in the brain of the offsprings. These data in line with the accumulation of monocyte-derived dendritic cells (moDCs) and macrophages during chronic inflammation suggest an activation-induced disease promoting mechanism. In contrast, the orphan receptor sigma-1 has been shown to mediate anti-inflammatory responses in rodent in vivo models, but the molecular background has not been elucidated. Methods: Western blot was used to monitor protein level expression of SIGMAR1 in human primary monocytes, macrophages and moDCs. Gene expression of sigma-1 receptor (SIGMAR1), and IL-1b, IL-6, TNFa, IL-8, IL-10 cytokines was assessed by Q-PCR. Concentration of secreted cytokines was measured by ELISA. ELISPOT was used to assess the numbers of moDC-primed autologous na€ıve Th1 and Th17 cells. Gene-specific RNA-interference was performed to silence sigmar-1 gene. N,N-dimethyltryptamine, 5-methoxy-N,N-dimethyltryptamine, and PRE084 hydrochloride were used to trigger SIGMAR1 in moDCs. To mimic different inflammatory conditions, we used TLR/RLR ligands (LPS, PolyI:C) and inactivated pathogens (E. coli and influenza virus). Results: In this study we used endogenous ligands (NN-DMT, 5-MeO-DMT) and high affinity synthetic PRE-084-hydrochloride to trigger sigma-1 in human moDCs and monitored their effects on LPS- and polyI:C-induced inflammatory responses. Co-administration of sigma-1 ligands with these activators inhibited the production of pro-inflammatory cytokines and chemokines (IL-1b, IL-6, TNFa, IL-8), while increased the secretion of anti-inflammatory IL-10. The antigen-presenting capacity of moDCs was also inhibited and co-administration of sigma-1 ligands with E. coli or influenza virus
decreased the differentiation of moDC-induced Th1 and Th17 inflammatory effector cells in a sigma-1 receptor specific manner confirmed by gene silencing. Conclusions: These results demonstrate the inhibitory potential of stimulated sigma-1 receptor in brain-resident moDCs that could be harnessed for the pharmacological treatment of AD and other chronic inflammatory conditions in the CNS. P4-247
IMMUNE-CHALLENGED TNF-DEFICIENT MICE SHOW A DECREASE IN HIPPOCAMPAL LEARNING
Christa Løth Myhre1, Alicia A. Babcock2, Kate Lambertsen2, Laura Ilkjær2, Morten Skovgaard Jensen3, Zengqiang Yuan4, Bente Finsen5, 1 Institute of Molecular Medicine, Odense, Denmark,; 2Institute of Molecular Medicine, Odense, Denmark; 3Department of Biomedicine, Aarhus, Denmark; 4Institute of Biophysics, CAS, Beijing, China; 5Institute of Molecular Medicine, Odense, Denmark. Contact e-mail: cmyhre@ health.sdu.dk ^ disease (AD) is activaBackground: A dominant feature of AlzheimerA’s tion of microglial cells, which can produce the pro-inflammatory cytokine TNF. TNF is deregulated in AD, and can be induced upon immune-challenge with endotoxin. TNF may also be involved in hippocampal neurogenesis, which is the basis for spatial learning. Spatial learning can be induced in mice by repeated exposure to the Barnes maze. Aim: The aim was to evaluate the involvement of TNF in hippocampal learning in mice during aging and after immune-challenge with endotoxin. Methods: To validate the Barnes maze paradigm, 3-month-old C57BL/6 mice and C57BL/6 mice injected with Scopolamine, a drug that interferes with learning, were exposed to the Barnes Maze with 4 trials/day for 6 days and killed on day 7. Threeand 9-month-old TNF-KO and C57BL/6 mice were then exposed to the Barnes Maze. To challenge the immune system of the mice, 9-month-old TNF-KO and C57BL/6 mice were twice injected with endotoxin, a strong inducer of TNF, and exposed to the Barnes maze 4 days later, after sickness behavior had ceased. Locomotor activity was assessed using Open Field. Results: The C57BL/6 mice injected with Scopolamine showed a significantly impaired learning ability compared with vehicle-injected C57BL/6 mice, as expected. Surprisingly, TNF deficiency did not affect the ability to learn in either 3- or 9-month-old mice. Interestingly, 9-month-old TNFKO mice injected with endotoxin showed a significantly impaired learning ability, whereas endotoxin treatment had no effect on learning in C57BL/6 mice. There was no difference in activity level between the two groups. Conclusions: We conclude that the TNF pathway is important for hippocampal learning when the immune system is activated, as takes place during systemic infection and pathological aging. Studies in progress focus on understanding the homeostatic role of TNF in hippocampal-dependent learning during normal and pathological aging. P4-248
THE ROLE OF TREM2 EXPRESSION ON MYELOID CELLS IN ALZHEIMER’S DISEASE
Bruce Lamb1, Crystal Miller1, Taylor Jay1, Leah Graham2, Shane Bemiller1, Guixiang Xu1, Daniel Margevicius1, Colleen Karlo3, Gregory Sousa2, Bunny Cotleur1, Lynn Bekris4, Susan Staugaitis1, James Leverenz5, Sanjay W. Pimplikar6, Gary Landreth3, Gareth R. Howell2, Richard Ransohoff1, 1The Cleveland Clinic, Cleveland, Ohio, United States; 2The Jackson Laboratory, Bar Harbor, Maine, United States; 3Case Western Reserve University, Cleveland, Ohio, United States; 4 The Cleveland Clinic, Seattle, Washington, United States; 5The Cleveland Clnic, Cleveland, Ohio, United States; 6Cleveland Clinic, Cleveland, Ohio, United States. Contact e-mail: [email protected] Background: Recent genetic studies implicate inflammatory genes and pathways in the etiology of Alzheimer’s disease (AD).Notably, mutations in TREM2, a gene expressed in myeloid cells, were recently demonstrated to confer high risk for developing AD. Mutations in TREM2 have also been identified as the genetic basis of Nasu-Hakola disease, and confer risk for frontotemporal dementia (FTD), Parkinson’s disease (PD) and amyotrophic lateral sclerosis (ALS). Since microglia are the primary myeloid cell type within brain, it has been proposed that TREM2 likely functions in AD
Poster Presentations: P4 and other neurodegenerative diseases by modifying microglial function. Methods: Here, we assess the cell-type specific expression of TREM2 during the course of AD pathology and in animals lacking TREM2. Results: TREM2 was highly expressed in human AD brains, localized mainly in cells surrounding b-amyloid (Ab) deposits. To define the expression and localization of TREM2 during the time course of Ab deposition, we examined three different AD mouse models. Quantitative RT-PCR documented significant age-related increases in TREM2 mRNA with corresponding elevations of TREM2 protein in Western blot analyses. Immunohistochemistry confirmed an age-dependent up-regulation of TREM2 around Ab plaques, as compared to non-transgenic controls. No staining was observed in a Trem2 -/- AD mouse model. Double fluorescent immunohistochemistry and confocal microscopy co-localized TREM2 immunoreactivity within Iba1+ cells surrounding Ab deposits in APPPS1 mice. These results that were confirmed via in-situ hybridization for TREM2 transcripts and via Xgal staining in a mouse line expressing lacZ under the endogenous Trem2 promoter. The identity of TREM2 expressing cells in AD mouse models was addressed using flow cytometric analysis of CD11b+/CD45 lo microglia and CD11b+/CD45 hi peripheral monocytes. Notably, there was an age-dependent increase in the percentage of TREM2 positive CD11b+/CD45 hi cells in the AD mouse models, while TREM2 expression by CD11b+/CD45 lo cells did not differ from that seen in wild-type animals. Conclusions: The current study demonstrates that TREM2 is highly-expressed by plaque-associated myeloid cells in human and animal model AD tissue, but suggests that these TREM2+ cells are macrophages derived from infiltrating peripheral monocytes and not microglia.
Background: Apolipoprotein E (APOE) genotype is a major genetic determinant for the risk of developing Alzheimer’s disease (AD), with APOE4 carriers more susceptible to developing AD compared to APOE2 and APOE3 carriers. APOE4 carriers have more fragmented ApoE, which contributes to the development of AD and the subsequent neurodegeneration. Mechanistically, this may be related to the metal dyshomeostasis that characterizes the AD brain. Therefore, it is important to determine whether metals like zinc, copper and iron, that are found in association with ApoE in the amyloid plaques that characterize the AD brain, can affect the proteolysis of ApoE in an isoform-dependent way. Methods: We investigated non-lipidated recombinant ApoE isoforms, lipidated ApoE isoforms and human plasma from healthy controls and AD patients. We incubated ApoE samples or human plasma with Tris-buffered saline (TBS) or different metal solutions (zinc chloride, copper chloride and ferrous chloride) at 37 C for 4 hours. Proteases (Kallikrein/Thrombin) were added to samples to digest ApoE at 37 C for 1 hour. Samples were then collected for western blot analysis of the amount of full length and fragmented ApoE. Results: Zinc promoted ApoE4 degradation induced by kallikrein and thrombin, leading to less full-length ApoE4 and more ApoE4 fragments. However, zinc had no effect on protease-induced degradation of ApoE3 or ApoE2. Neither copper nor iron had an effect on ApoE levels. Conclusions: Zinc may play a role in ApoE proteolysis, with protease-induced degradation of ApoE4 being promoted by zinc. These data might partially account for the observation that APOE4 carriers have higher risk for AD compared with APOE2 and APOE3 carriers. P4-251
P4-249
NOGO-A INHIBITS ADHESION AND MIGRATION OF MICROGLIA TO AB IN VITRO
Hong Liao, YinQuan Fang, National Center for Drug Screening, China Pharmaceutical University, Nanjing, China. Contact e-mail: fangyinquan@ gmail.com Background: Microglia plays a critical role in the brain aging process and in Alzheimer’s disease (AD). It has been reported the ability of microglia to clear Ab decreases with age and progression of AD pathology. Nogo-A/NgR signaling pathway not only arreste neurite outgrowth but also involve AD pathology. NgR expressed on the neuron also expresses on the microglia and Nogo-A/NgR signaling pathway mediates neuroinflammation via modulating microglia adhesion and migration. Therefore i n the present study we would like explore the role of Nogo-A/NgR signaling pathway on microglia adhesion and migration to A b deposition during aging in vitro. Methods: Adhesion assay, Boyden chamber migration assay and Western blot analysis were used for determinating ability of migration and adhesion of microglia isolated from different age to A b fibrils in vitro. Results: Our results show that microglia from aging mice decreased the adhesion and migration to A b fibrils compared with microglia from young mice in vitro. Nogo-A inhibited their adhesion and migration to Ab fibrils in vitro, which were mediated by NgR. We also found RhoA as the downstream signal transducers involved in the microglia adhesion and migration to A b fibrils in the presence of Nogo-A. Nogo-A also inhibited microglia polarization and membrane protrusion formation, thus might eventually contribute to the decreasing of microglia adhesion and migration to A b fibrils. Conclusions: Our data indicate that Nogo-A inhibits adhesion and migration of microglia to A b fibrils in the aging via NgR and Rho pathway. The NogoA/NgR pathway will maybe provide a potential target for modifying the course of AD and highlights the role of brain inflammation in the AD disease. P4-250
ZINC AFFECTS THE STABILITY OF APOLIPOPROTEIN E IN ALZHEIMER’S DISEASE
He Xu1, David Finkelstein2, Ashley I. Bush3, Paul Adlard2, 1The Florey Institute of Neuroscience and Mental Health, University of Melbourne, Melbourne, Australia; 2The Florey Institute of Neuroscience and Mental Health, Melbourne, Australia; 3The Florey Institute of Neuroscience and Mental Health, Parkville, Australia. Contact e-mail: hexu@student. unimelb.edu.au
P877
ALTERED METABOLISM OF BACE1 SUBSTRATES SEIZURE PROTEIN 6 AND SEIZURE 6-LIKE PROTEIN 1 IN NIEMANN-PICK TYPE C DISEASE
Stjepko Cermak1, Mirsada Causevic2, Peer-Hendrik Kuhn3, Jenny Gunnersen4, Birgit Hutter-Paier5, Manfred Windisch6, Stefan Lichtenthaler7, Silva Hecimovic2, 1Rudjer Boskovic Institute, Zagreb, Croatia; 2Rudjer Boskovic Institute, Zagreb, Croatia; 3German Center for Neurodegenerative Diseases (DZNE), Munich, Germany; 4 University of Melbourne, Victoria, Australia; 5QPS Austria GmbH, Grambach, Austria; 6NeuroScios GmbH - Neuroscience Optimized Solutions, St.Radegund/Graz, Austria; 7German Center for Neurodegenerative Diseases (DZNE), Technical University Munich Technische Universit€at M€unchen (TUM), Munich, Germany. Contact e-mail: [email protected] Background: Nieman-Pick type C (NPC) disease is a lysosomal storage disorder caused by mutations in NPC1 or NPC2 genes. It shares a number of similarities with Alzheimer’s disease, including disturbed cholesterol homeostasis and APP processing. Previously, in one of the models of NPC disease (CHO NPC1-null cells), we have shown a clear co-localization of APP and BACE1 within enlarged early/recycling endosomes leading to increased b-secretase processing of APP. In this study, in an effort to better characterize BACE1 (dys)function upon NPC1 loss, we investigated protein levels of BACE1 and several BACE1 substrates in two different experimental models of NPC disease. Methods: The levels of BACE1 and BACE1 substrates for which BACE1 is a major sheddase [seizure protein 6 (Sez6) and seizure 6-like protein 1 (Sez6L1)] were analyzed in the following models of NPC disease: 1) a cellular model of NPC disease, in which mouse, primary neurons were treated with the U18666A compound, that mimics the major phenotype of NPC disease - cholesterol accumulation and 2) in an animal model of NPC disease. Results: We observed a marked increase in BACE1 protein levels in both NPC disease models examined in this study. BACE1 increase was more pronounced in hippocampi vs. cerebella of 4week-old NPC1- mutant mice compared with wild-type mice. This increase paralleled the increase in Sez6 membrane-tethered precursor and its soluble ectodomain, suggesting full-length Sez6 accumulation and its enhanced shedding by BACE1 upon NPC1 dysfunction, respectively. Similar findings were observed for Sez6L1 and APP proteins. Currently, we are investigating other BACE1 substrates, including APP homologues APLP1 and APLP2. Conclusions: This study suggests the following: a) the NPC1 dysfunction has a strong effect on BACE1 as BACE1 protein levels are increased in
Poster Presentations: P4
CN autoinhibitory domain located near the C terminus of the CN catalytic subunit. Commercially available antibodies that target the N terminus of the CN catalytic subunit reveal the presence of a highly-active 48 kDa proteolytic fragment in human brain tissue during early stages of cognitive decline and also in a variety of experimental models of neurodegeneration. While useful for determining the extent of CN proteolysis in Western blot applications, N terminus antibodies do not reveal the cellular location of the proteolysis. Knowing where CN is proteolyzed in nervous tissue seems critical to understanding the mechanistic basis of its many deleterious actions, particularly because CN is found in both neurons and glial cells where it is involved in different cellular functions. Methods: To address this gap in our understanding of CN regulation, we generated custom rabbit polyclonal antibodies to CN A based on previously identified calpain-dependent cleavage sites. One of these antibodies (referred to as “DCN48”) detects a 48 kDa fragment in Western blot assays, but does not detect full-length (60 kDa) CN. The DCN48 antibody was then used for DAB immunohistochemical labeling of human brain sections characterized by AD and mixed AD/vascular pathologies. Results: The anatomical features labeled by the DCN48 antibody included astrocyte clusters and vascular-associated elements and/or processes. We also observed numerous DCN48-positive astrocytes associated with microinfarcts. Surprisingly, we have seen very little neuronal labeling with this antibody. Conclusions: The results suggest that astrocytes and, perhaps astrocyte end-feet, are a primary locus for CPdependent CN proteolysis in injured or diseased nervous tissue. CN proteolysis appears to be especially pronounced in astrocytes associated with vascular pathology. This work may provide new mechanistic insights into the impact of Ca 2+ dysregulation on neurodegenerative diseases. P4-246
ACTIVATION OF THE SIGMA-1 RECEPTOR BY SPECIFIC LIGANDS INHIBITS HUMAN INFLAMMATORY DENDRITIC CELL FUNCTIONS AND EFFECTOR T-LYMPHOCYTE RESPONSES
Attila Szabo, Attila Kovacs, Ede Frecska, Eva Rajnavolgyi, Faculty of Medicine, University of Debrecen, Debrecen, Hungary. Contact e-mail: [email protected] Background: Neuropsychiatric diseases have recently been attributed to chronic inflammation in the central nervous system, and correlation between gene polymorphisms of innate immune receptors and the frequency of late onset of Alzheimer’s disease (AD) has also been shown. Ligation of murine maternal Toll-like receptors (TLRs) and RIG-I-like receptors (RLRs) by LPS or PolyI:C have been shown to cause decreased neurogenesis, cognitive deficits, and increased deposition of Ab aggregates in the brain of the offsprings. These data in line with the accumulation of monocyte-derived dendritic cells (moDCs) and macrophages during chronic inflammation suggest an activation-induced disease promoting mechanism. In contrast, the orphan receptor sigma-1 has been shown to mediate anti-inflammatory responses in rodent in vivo models, but the molecular background has not been elucidated. Methods: Western blot was used to monitor protein level expression of SIGMAR1 in human primary monocytes, macrophages and moDCs. Gene expression of sigma-1 receptor (SIGMAR1), and IL-1b, IL-6, TNFa, IL-8, IL-10 cytokines was assessed by Q-PCR. Concentration of secreted cytokines was measured by ELISA. ELISPOT was used to assess the numbers of moDC-primed autologous na€ıve Th1 and Th17 cells. Gene-specific RNA-interference was performed to silence sigmar-1 gene. N,N-dimethyltryptamine, 5-methoxy-N,N-dimethyltryptamine, and PRE084 hydrochloride were used to trigger SIGMAR1 in moDCs. To mimic different inflammatory conditions, we used TLR/RLR ligands (LPS, PolyI:C) and inactivated pathogens (E. coli and influenza virus). Results: In this study we used endogenous ligands (NN-DMT, 5-MeO-DMT) and high affinity synthetic PRE-084-hydrochloride to trigger sigma-1 in human moDCs and monitored their effects on LPS- and polyI:C-induced inflammatory responses. Co-administration of sigma-1 ligands with these activators inhibited the production of pro-inflammatory cytokines and chemokines (IL-1b, IL-6, TNFa, IL-8), while increased the secretion of anti-inflammatory IL-10. The antigen-presenting capacity of moDCs was also inhibited and co-administration of sigma-1 ligands with E. coli or influenza virus
decreased the differentiation of moDC-induced Th1 and Th17 inflammatory effector cells in a sigma-1 receptor specific manner confirmed by gene silencing. Conclusions: These results demonstrate the inhibitory potential of stimulated sigma-1 receptor in brain-resident moDCs that could be harnessed for the pharmacological treatment of AD and other chronic inflammatory conditions in the CNS. P4-247
IMMUNE-CHALLENGED TNF-DEFICIENT MICE SHOW A DECREASE IN HIPPOCAMPAL LEARNING
Christa Løth Myhre1, Alicia A. Babcock2, Kate Lambertsen2, Laura Ilkjær2, Morten Skovgaard Jensen3, Zengqiang Yuan4, Bente Finsen5, 1 Institute of Molecular Medicine, Odense, Denmark,; 2Institute of Molecular Medicine, Odense, Denmark; 3Department of Biomedicine, Aarhus, Denmark; 4Institute of Biophysics, CAS, Beijing, China; 5Institute of Molecular Medicine, Odense, Denmark. Contact e-mail: cmyhre@ health.sdu.dk ^ disease (AD) is activaBackground: A dominant feature of AlzheimerA’s tion of microglial cells, which can produce the pro-inflammatory cytokine TNF. TNF is deregulated in AD, and can be induced upon immune-challenge with endotoxin. TNF may also be involved in hippocampal neurogenesis, which is the basis for spatial learning. Spatial learning can be induced in mice by repeated exposure to the Barnes maze. Aim: The aim was to evaluate the involvement of TNF in hippocampal learning in mice during aging and after immune-challenge with endotoxin. Methods: To validate the Barnes maze paradigm, 3-month-old C57BL/6 mice and C57BL/6 mice injected with Scopolamine, a drug that interferes with learning, were exposed to the Barnes Maze with 4 trials/day for 6 days and killed on day 7. Threeand 9-month-old TNF-KO and C57BL/6 mice were then exposed to the Barnes Maze. To challenge the immune system of the mice, 9-month-old TNF-KO and C57BL/6 mice were twice injected with endotoxin, a strong inducer of TNF, and exposed to the Barnes maze 4 days later, after sickness behavior had ceased. Locomotor activity was assessed using Open Field. Results: The C57BL/6 mice injected with Scopolamine showed a significantly impaired learning ability compared with vehicle-injected C57BL/6 mice, as expected. Surprisingly, TNF deficiency did not affect the ability to learn in either 3- or 9-month-old mice. Interestingly, 9-month-old TNFKO mice injected with endotoxin showed a significantly impaired learning ability, whereas endotoxin treatment had no effect on learning in C57BL/6 mice. There was no difference in activity level between the two groups. Conclusions: We conclude that the TNF pathway is important for hippocampal learning when the immune system is activated, as takes place during systemic infection and pathological aging. Studies in progress focus on understanding the homeostatic role of TNF in hippocampal-dependent learning during normal and pathological aging. P4-248
THE ROLE OF TREM2 EXPRESSION ON MYELOID CELLS IN ALZHEIMER’S DISEASE
Bruce Lamb1, Crystal Miller1, Taylor Jay1, Leah Graham2, Shane Bemiller1, Guixiang Xu1, Daniel Margevicius1, Colleen Karlo3, Gregory Sousa2, Bunny Cotleur1, Lynn Bekris4, Susan Staugaitis1, James Leverenz5, Sanjay W. Pimplikar6, Gary Landreth3, Gareth R. Howell2, Richard Ransohoff1, 1The Cleveland Clinic, Cleveland, Ohio, United States; 2The Jackson Laboratory, Bar Harbor, Maine, United States; 3Case Western Reserve University, Cleveland, Ohio, United States; 4 The Cleveland Clinic, Seattle, Washington, United States; 5The Cleveland Clnic, Cleveland, Ohio, United States; 6Cleveland Clinic, Cleveland, Ohio, United States. Contact e-mail: [email protected] Background: Recent genetic studies implicate inflammatory genes and pathways in the etiology of Alzheimer’s disease (AD).Notably, mutations in TREM2, a gene expressed in myeloid cells, were recently demonstrated to confer high risk for developing AD. Mutations in TREM2 have also been identified as the genetic basis of Nasu-Hakola disease, and confer risk for frontotemporal dementia (FTD), Parkinson’s disease (PD) and amyotrophic lateral sclerosis (ALS). Since microglia are the primary myeloid cell type within brain, it has been proposed that TREM2 likely functions in AD
Poster Presentations: P4 and other neurodegenerative diseases by modifying microglial function. Methods: Here, we assess the cell-type specific expression of TREM2 during the course of AD pathology and in animals lacking TREM2. Results: TREM2 was highly expressed in human AD brains, localized mainly in cells surrounding b-amyloid (Ab) deposits. To define the expression and localization of TREM2 during the time course of Ab deposition, we examined three different AD mouse models. Quantitative RT-PCR documented significant age-related increases in TREM2 mRNA with corresponding elevations of TREM2 protein in Western blot analyses. Immunohistochemistry confirmed an age-dependent up-regulation of TREM2 around Ab plaques, as compared to non-transgenic controls. No staining was observed in a Trem2 -/- AD mouse model. Double fluorescent immunohistochemistry and confocal microscopy co-localized TREM2 immunoreactivity within Iba1+ cells surrounding Ab deposits in APPPS1 mice. These results that were confirmed via in-situ hybridization for TREM2 transcripts and via Xgal staining in a mouse line expressing lacZ under the endogenous Trem2 promoter. The identity of TREM2 expressing cells in AD mouse models was addressed using flow cytometric analysis of CD11b+/CD45 lo microglia and CD11b+/CD45 hi peripheral monocytes. Notably, there was an age-dependent increase in the percentage of TREM2 positive CD11b+/CD45 hi cells in the AD mouse models, while TREM2 expression by CD11b+/CD45 lo cells did not differ from that seen in wild-type animals. Conclusions: The current study demonstrates that TREM2 is highly-expressed by plaque-associated myeloid cells in human and animal model AD tissue, but suggests that these TREM2+ cells are macrophages derived from infiltrating peripheral monocytes and not microglia.
Background: Apolipoprotein E (APOE) genotype is a major genetic determinant for the risk of developing Alzheimer’s disease (AD), with APOE4 carriers more susceptible to developing AD compared to APOE2 and APOE3 carriers. APOE4 carriers have more fragmented ApoE, which contributes to the development of AD and the subsequent neurodegeneration. Mechanistically, this may be related to the metal dyshomeostasis that characterizes the AD brain. Therefore, it is important to determine whether metals like zinc, copper and iron, that are found in association with ApoE in the amyloid plaques that characterize the AD brain, can affect the proteolysis of ApoE in an isoform-dependent way. Methods: We investigated non-lipidated recombinant ApoE isoforms, lipidated ApoE isoforms and human plasma from healthy controls and AD patients. We incubated ApoE samples or human plasma with Tris-buffered saline (TBS) or different metal solutions (zinc chloride, copper chloride and ferrous chloride) at 37 C for 4 hours. Proteases (Kallikrein/Thrombin) were added to samples to digest ApoE at 37 C for 1 hour. Samples were then collected for western blot analysis of the amount of full length and fragmented ApoE. Results: Zinc promoted ApoE4 degradation induced by kallikrein and thrombin, leading to less full-length ApoE4 and more ApoE4 fragments. However, zinc had no effect on protease-induced degradation of ApoE3 or ApoE2. Neither copper nor iron had an effect on ApoE levels. Conclusions: Zinc may play a role in ApoE proteolysis, with protease-induced degradation of ApoE4 being promoted by zinc. These data might partially account for the observation that APOE4 carriers have higher risk for AD compared with APOE2 and APOE3 carriers. P4-251
P4-249
NOGO-A INHIBITS ADHESION AND MIGRATION OF MICROGLIA TO AB IN VITRO
Hong Liao, YinQuan Fang, National Center for Drug Screening, China Pharmaceutical University, Nanjing, China. Contact e-mail: fangyinquan@ gmail.com Background: Microglia plays a critical role in the brain aging process and in Alzheimer’s disease (AD). It has been reported the ability of microglia to clear Ab decreases with age and progression of AD pathology. Nogo-A/NgR signaling pathway not only arreste neurite outgrowth but also involve AD pathology. NgR expressed on the neuron also expresses on the microglia and Nogo-A/NgR signaling pathway mediates neuroinflammation via modulating microglia adhesion and migration. Therefore i n the present study we would like explore the role of Nogo-A/NgR signaling pathway on microglia adhesion and migration to A b deposition during aging in vitro. Methods: Adhesion assay, Boyden chamber migration assay and Western blot analysis were used for determinating ability of migration and adhesion of microglia isolated from different age to A b fibrils in vitro. Results: Our results show that microglia from aging mice decreased the adhesion and migration to A b fibrils compared with microglia from young mice in vitro. Nogo-A inhibited their adhesion and migration to Ab fibrils in vitro, which were mediated by NgR. We also found RhoA as the downstream signal transducers involved in the microglia adhesion and migration to A b fibrils in the presence of Nogo-A. Nogo-A also inhibited microglia polarization and membrane protrusion formation, thus might eventually contribute to the decreasing of microglia adhesion and migration to A b fibrils. Conclusions: Our data indicate that Nogo-A inhibits adhesion and migration of microglia to A b fibrils in the aging via NgR and Rho pathway. The NogoA/NgR pathway will maybe provide a potential target for modifying the course of AD and highlights the role of brain inflammation in the AD disease. P4-250
ZINC AFFECTS THE STABILITY OF APOLIPOPROTEIN E IN ALZHEIMER’S DISEASE
He Xu1, David Finkelstein2, Ashley I. Bush3, Paul Adlard2, 1The Florey Institute of Neuroscience and Mental Health, University of Melbourne, Melbourne, Australia; 2The Florey Institute of Neuroscience and Mental Health, Melbourne, Australia; 3The Florey Institute of Neuroscience and Mental Health, Parkville, Australia. Contact e-mail: hexu@student. unimelb.edu.au
P877
ALTERED METABOLISM OF BACE1 SUBSTRATES SEIZURE PROTEIN 6 AND SEIZURE 6-LIKE PROTEIN 1 IN NIEMANN-PICK TYPE C DISEASE
Stjepko Cermak1, Mirsada Causevic2, Peer-Hendrik Kuhn3, Jenny Gunnersen4, Birgit Hutter-Paier5, Manfred Windisch6, Stefan Lichtenthaler7, Silva Hecimovic2, 1Rudjer Boskovic Institute, Zagreb, Croatia; 2Rudjer Boskovic Institute, Zagreb, Croatia; 3German Center for Neurodegenerative Diseases (DZNE), Munich, Germany; 4 University of Melbourne, Victoria, Australia; 5QPS Austria GmbH, Grambach, Austria; 6NeuroScios GmbH - Neuroscience Optimized Solutions, St.Radegund/Graz, Austria; 7German Center for Neurodegenerative Diseases (DZNE), Technical University Munich Technische Universit€at M€unchen (TUM), Munich, Germany. Contact e-mail: [email protected] Background: Nieman-Pick type C (NPC) disease is a lysosomal storage disorder caused by mutations in NPC1 or NPC2 genes. It shares a number of similarities with Alzheimer’s disease, including disturbed cholesterol homeostasis and APP processing. Previously, in one of the models of NPC disease (CHO NPC1-null cells), we have shown a clear co-localization of APP and BACE1 within enlarged early/recycling endosomes leading to increased b-secretase processing of APP. In this study, in an effort to better characterize BACE1 (dys)function upon NPC1 loss, we investigated protein levels of BACE1 and several BACE1 substrates in two different experimental models of NPC disease. Methods: The levels of BACE1 and BACE1 substrates for which BACE1 is a major sheddase [seizure protein 6 (Sez6) and seizure 6-like protein 1 (Sez6L1)] were analyzed in the following models of NPC disease: 1) a cellular model of NPC disease, in which mouse, primary neurons were treated with the U18666A compound, that mimics the major phenotype of NPC disease - cholesterol accumulation and 2) in an animal model of NPC disease. Results: We observed a marked increase in BACE1 protein levels in both NPC disease models examined in this study. BACE1 increase was more pronounced in hippocampi vs. cerebella of 4week-old NPC1- mutant mice compared with wild-type mice. This increase paralleled the increase in Sez6 membrane-tethered precursor and its soluble ectodomain, suggesting full-length Sez6 accumulation and its enhanced shedding by BACE1 upon NPC1 dysfunction, respectively. Similar findings were observed for Sez6L1 and APP proteins. Currently, we are investigating other BACE1 substrates, including APP homologues APLP1 and APLP2. Conclusions: This study suggests the following: a) the NPC1 dysfunction has a strong effect on BACE1 as BACE1 protein levels are increased in