- Email: [email protected]
Dysregulation of brain ApoE and cholesterol metabolism in APP transgenic mice
Poster Presentations P4
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months (p<0.002), respectively. Likewise, IHC analysis revealed an 83% increase in PHF1 IR tau in the pyriform cortex at 8-9 months, (p¼0.02), and a comparable increase at 5-6 months and in AT8 IR (IHC and Western) that are being quantitated. Importantly, the htau/PS1/mtau-/- mice were more cognitively impaired than controls in the Radial Arm Maze (p<0.03). Analyses of other cortical and hippocampal regions with advanced tau pathology, with other tau antibodies as well as of insoluble tau is underway. Furthermore, older animals, tau-related pathology and the potential involvement of various signaling pathways are being assessed. Conclusions: The M146L mutation promotes age-related tau phosphorylation and aggregation, and impairs cognition compared to controls, suggesting that PS1/tau interaction may be important in the etiology and/or pathogenesis of AD. This novel model can be very useful for studying the onset and progression of AD as well as for therapeutic studies. Supported by AG032611. P4-050
DYSREGULATION OF BRAIN APOE AND CHOLESTEROL METABOLISM IN APP TRANSGENIC MICE
Helen K. Warwick, Julius Jefferson, Kevin Atchison, Hua Zhou, Yolanda Kirksey, Cathleen Gonzales, Suzan Aschmies, J. S. Jacobsen, Menelas N. Pangalos, Peter H. Reinhart, David R. Riddell, Wyeth Research, Monmouth Junction, NJ, USA. Contact e-mail: [email protected] Background: Recent evidence has suggested a potential role for APP in the regulation of brain Apolipoprotein E (ApoE) and cholesterol metabolism. In support of this function we have previously presented data from a transcriptional profiling study which demonstrates dysregulation of cholesterol-related genes in the Tg2576 APP transgenic mouse model of AD. We have now expanded these findings to investigate the effects of APP transgene expression on ApoE metabolism. Methods: ApoE protein levels in the cortex and hippocampus of wildtype and APP transgenic mice have been examined using western blotting and an ApoE-specific immunoassay. Results: In both brain regions ApoE protein levels were significantly increased in the transgenic animals compared to wildtype controls. This was not a result of increased ApoE transcription, as mRNA levels in the Tg2576 mice were unchanged. ApoE is one of a number of ligands for the low density lipoprotein-related receptor, LRP1. To investigate whether changes in ApoE protein levels arise as a result of decreased functionality of LRP1, we first investigated levels of other LRP1 ligands in APP transgenic mice. Immunohistochemical analysis demonstrated increased protein levels of two additional LRP1 ligands, tissue plasminogen activator (tPA) and plasminogen activator inhibitor-1 (PAI-1). Together these findings suggest that LRP1 function may be altered in the APP transgenic mice leading to a reduction in the catabolism of LRP1 ligands. Previously, LRP1 expression has been shown to be regulated at the transcriptional level by the APP intracellular domain (AICD). To investigate whether this mechanism underlies the differences in ApoE, tPA and PAI-1 protein levels observed in the Tg2576 mice we examined LRP1 mRNA levels. Surprisingly, LRP1 mRNA was not altered in the Tg2576 mice compared to wildtype controls. An examination of LRP1 protein levels and/or LRP1 processing and their effects of LRP1 function is currently underway. Conclusions: Taken together these data provide evidence in support of the growing association between AD and cholesterol metabolism and further establish the biological link between APP and ApoE, the two major determinants of AD. P4-051
THE BIOLOGICAL EFFECTS OF R278IPRESENILIN-1 FAMILIAL MUTATION ON g-SECRETASE ACTIVITY AND AMYLOID PATHOLOGY
Takashi Saito1, Takahiro Suemoto1, Naomi Mihira1, Yukio Matsuba1, Jiro Takano1, Masaki Nishimura2, Nobuhisa Iwata1, Takaomi C. Saido1, 1 RIKEN Brain Science Institute, Wako-shi, Saitama, Japan; 2Shiga University of Medical Science, Shiga, Japan. Contact e-mail: takasai@ brain.riken.jp Background: Presenilin-1 (PS1) associated with familial Alzheimer’s disease (FAD)-causing mutations increase production or ratio of Ab42, the
potent pathogenic agent for Alzheimer’s disease (AD). Recently, R278I misssense mutation in PS1 gene was found to generate a high level of Ab43 in vitro. This mutation modulates a g-secretase activity and accelerates a prominent production of Ab42 and Ab43, particularly Ab43, rather than other same site FAD-linked mutations (R278K-, R278S- and R278T-PS1). To study the biological role of Ab43 is necessary to understand a mechanism for amlyoid pathology formation. Methods: We generated a R278I-PS1 mutant mice using gene knock-in strategy. The analysis such mice were performed by biochemical and immunohistchemical method. Results: Beyond our expectation, R278I-PS1-KI homozygous mice occurred embryonic lethal distinguish other PS1 mutant mice previously reported. The obvious phenotypes (e.g. shortened tail and hemorrhage in the brain) were identical to PS1 knock-out mice or Notch phenotype. Although the expression of R278I-PS1 mRNA and holoprotein were normal in the embryonic brain at E15, abnormal maturation of R278I-PS1 protein which is caused by failure of PS1-endoproteolysis were occurred in the brain and mouse embryonic fibroblast (MEF) derived from R278I-PS1-KI homozygous mice. Its failure of PS1-endoproteolysis also induced a modulation of g-secretase activity, and leaded to abnormal processing of APP, Notch and other substrates of g-secretase. On the other hand, amyloid pathology formations were accelerated by R278I-PS1 mutation. At 9 months age of R278I-PS1-KI heterozygous mice crossbred with APP transgenic mouse, accelerated Ab depositions appeared in the cortical and hippocampal area, while no depositions were found in APP transgenic mice. In such mice brain, the amounts of Ab42, Ab43, and the ratio of Ab42/Ab40, Ab43/Ab40 were significantly increased. Generated Ab43 accelerated amyloid plaque formation especially cored plaque of Ab as well as AD brain. Conclusions: R278I-PS1 mutation has newly biological effects which modulates a g-secretase activity in vivo dissimilar to other PS1 mutations, and accelerates a formation of amyloid pathology due to increase the ratio of Ab42 and Ab43, which shows very similar amyloid pathology to AD brain. Thus, mice generated here will be a useful mouse model for AD research.
P4-052
DISSOCIATION OF SYNAPTIC DYSFUNCTION AND BEHAVIOR IN A TAUOPATHY MOUSE MODEL (PRP-MAPT) OF ALZHEIMER’S DISEASE
Margaret Zaleska, Robert A. Crozier, Nathalie Breysse, Reka Hosszu, Mark R. Bowlby, Jonathan Bard, Wyeth Research, Monmouth Junction, NJ, USA. Contact e-mail: [email protected] Background: One major pathophysiological features of Alzheimer’s disease (AD) is the accumulation neurofibrillary tangles originating from aggregation of hyperphosphorylated tau. Tau binds to and stabilizes microtubules (MTs) which, in turn, provide structural support and a scaffold for shuttling of essential proteins between the cell body and axon. MT function is governed by a dynamic equilibrium of phosphorylation and dephosphorylation mechanisms; however, aberrant hyperphosphorylation of tau results in its dissociation from MTs, resulting in MT instability and tau aggregation, both of which may independently lead to neurotoxicity. Methods: Here, we used PrP-MAPT mice that over-express normal human fetal tau (3R/0N isoform) to better understand the temporal relationship between tau accumulation with synaptic and behavioral deficits. We initiated the electrophysiology studies in the hippocampus_a region critical for memory formation. Field potential recordings were obtained from the lateral perforant path of the dentate gyrus of either wild-type (WT) or heterozygous (Het) mice aged 2, 4 or 10 months, and assessments included basal transmission and long-term potentiation (LTP). Results: At 2 months of age, no difference in the magnitude of LTP between genotypes was observed but a subtle impairment in basal transmission was detected in the Het’s even at this early age. By 4 months of age, differences in LTP (WT: 178.266.4%, n¼6; Het: 148.1611.9%, n¼9) and basal synaptic transmission were observed. Notably, 4 month Het LTP data revealed a large variation in the magnitude of potentiation in that some slices had WT LTP magnitudes while others showed deficits. This duality suggests that at 4 months, Het mice may reside on a threshold between normal
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months (p<0.002), respectively. Likewise, IHC analysis revealed an 83% increase in PHF1 IR tau in the pyriform cortex at 8-9 months, (p¼0.02), and a comparable increase at 5-6 months and in AT8 IR (IHC and Western) that are being quantitated. Importantly, the htau/PS1/mtau-/- mice were more cognitively impaired than controls in the Radial Arm Maze (p<0.03). Analyses of other cortical and hippocampal regions with advanced tau pathology, with other tau antibodies as well as of insoluble tau is underway. Furthermore, older animals, tau-related pathology and the potential involvement of various signaling pathways are being assessed. Conclusions: The M146L mutation promotes age-related tau phosphorylation and aggregation, and impairs cognition compared to controls, suggesting that PS1/tau interaction may be important in the etiology and/or pathogenesis of AD. This novel model can be very useful for studying the onset and progression of AD as well as for therapeutic studies. Supported by AG032611. P4-050
DYSREGULATION OF BRAIN APOE AND CHOLESTEROL METABOLISM IN APP TRANSGENIC MICE
Helen K. Warwick, Julius Jefferson, Kevin Atchison, Hua Zhou, Yolanda Kirksey, Cathleen Gonzales, Suzan Aschmies, J. S. Jacobsen, Menelas N. Pangalos, Peter H. Reinhart, David R. Riddell, Wyeth Research, Monmouth Junction, NJ, USA. Contact e-mail: [email protected] Background: Recent evidence has suggested a potential role for APP in the regulation of brain Apolipoprotein E (ApoE) and cholesterol metabolism. In support of this function we have previously presented data from a transcriptional profiling study which demonstrates dysregulation of cholesterol-related genes in the Tg2576 APP transgenic mouse model of AD. We have now expanded these findings to investigate the effects of APP transgene expression on ApoE metabolism. Methods: ApoE protein levels in the cortex and hippocampus of wildtype and APP transgenic mice have been examined using western blotting and an ApoE-specific immunoassay. Results: In both brain regions ApoE protein levels were significantly increased in the transgenic animals compared to wildtype controls. This was not a result of increased ApoE transcription, as mRNA levels in the Tg2576 mice were unchanged. ApoE is one of a number of ligands for the low density lipoprotein-related receptor, LRP1. To investigate whether changes in ApoE protein levels arise as a result of decreased functionality of LRP1, we first investigated levels of other LRP1 ligands in APP transgenic mice. Immunohistochemical analysis demonstrated increased protein levels of two additional LRP1 ligands, tissue plasminogen activator (tPA) and plasminogen activator inhibitor-1 (PAI-1). Together these findings suggest that LRP1 function may be altered in the APP transgenic mice leading to a reduction in the catabolism of LRP1 ligands. Previously, LRP1 expression has been shown to be regulated at the transcriptional level by the APP intracellular domain (AICD). To investigate whether this mechanism underlies the differences in ApoE, tPA and PAI-1 protein levels observed in the Tg2576 mice we examined LRP1 mRNA levels. Surprisingly, LRP1 mRNA was not altered in the Tg2576 mice compared to wildtype controls. An examination of LRP1 protein levels and/or LRP1 processing and their effects of LRP1 function is currently underway. Conclusions: Taken together these data provide evidence in support of the growing association between AD and cholesterol metabolism and further establish the biological link between APP and ApoE, the two major determinants of AD. P4-051
THE BIOLOGICAL EFFECTS OF R278IPRESENILIN-1 FAMILIAL MUTATION ON g-SECRETASE ACTIVITY AND AMYLOID PATHOLOGY
Takashi Saito1, Takahiro Suemoto1, Naomi Mihira1, Yukio Matsuba1, Jiro Takano1, Masaki Nishimura2, Nobuhisa Iwata1, Takaomi C. Saido1, 1 RIKEN Brain Science Institute, Wako-shi, Saitama, Japan; 2Shiga University of Medical Science, Shiga, Japan. Contact e-mail: takasai@ brain.riken.jp Background: Presenilin-1 (PS1) associated with familial Alzheimer’s disease (FAD)-causing mutations increase production or ratio of Ab42, the
potent pathogenic agent for Alzheimer’s disease (AD). Recently, R278I misssense mutation in PS1 gene was found to generate a high level of Ab43 in vitro. This mutation modulates a g-secretase activity and accelerates a prominent production of Ab42 and Ab43, particularly Ab43, rather than other same site FAD-linked mutations (R278K-, R278S- and R278T-PS1). To study the biological role of Ab43 is necessary to understand a mechanism for amlyoid pathology formation. Methods: We generated a R278I-PS1 mutant mice using gene knock-in strategy. The analysis such mice were performed by biochemical and immunohistchemical method. Results: Beyond our expectation, R278I-PS1-KI homozygous mice occurred embryonic lethal distinguish other PS1 mutant mice previously reported. The obvious phenotypes (e.g. shortened tail and hemorrhage in the brain) were identical to PS1 knock-out mice or Notch phenotype. Although the expression of R278I-PS1 mRNA and holoprotein were normal in the embryonic brain at E15, abnormal maturation of R278I-PS1 protein which is caused by failure of PS1-endoproteolysis were occurred in the brain and mouse embryonic fibroblast (MEF) derived from R278I-PS1-KI homozygous mice. Its failure of PS1-endoproteolysis also induced a modulation of g-secretase activity, and leaded to abnormal processing of APP, Notch and other substrates of g-secretase. On the other hand, amyloid pathology formations were accelerated by R278I-PS1 mutation. At 9 months age of R278I-PS1-KI heterozygous mice crossbred with APP transgenic mouse, accelerated Ab depositions appeared in the cortical and hippocampal area, while no depositions were found in APP transgenic mice. In such mice brain, the amounts of Ab42, Ab43, and the ratio of Ab42/Ab40, Ab43/Ab40 were significantly increased. Generated Ab43 accelerated amyloid plaque formation especially cored plaque of Ab as well as AD brain. Conclusions: R278I-PS1 mutation has newly biological effects which modulates a g-secretase activity in vivo dissimilar to other PS1 mutations, and accelerates a formation of amyloid pathology due to increase the ratio of Ab42 and Ab43, which shows very similar amyloid pathology to AD brain. Thus, mice generated here will be a useful mouse model for AD research.
P4-052
DISSOCIATION OF SYNAPTIC DYSFUNCTION AND BEHAVIOR IN A TAUOPATHY MOUSE MODEL (PRP-MAPT) OF ALZHEIMER’S DISEASE
Margaret Zaleska, Robert A. Crozier, Nathalie Breysse, Reka Hosszu, Mark R. Bowlby, Jonathan Bard, Wyeth Research, Monmouth Junction, NJ, USA. Contact e-mail: [email protected] Background: One major pathophysiological features of Alzheimer’s disease (AD) is the accumulation neurofibrillary tangles originating from aggregation of hyperphosphorylated tau. Tau binds to and stabilizes microtubules (MTs) which, in turn, provide structural support and a scaffold for shuttling of essential proteins between the cell body and axon. MT function is governed by a dynamic equilibrium of phosphorylation and dephosphorylation mechanisms; however, aberrant hyperphosphorylation of tau results in its dissociation from MTs, resulting in MT instability and tau aggregation, both of which may independently lead to neurotoxicity. Methods: Here, we used PrP-MAPT mice that over-express normal human fetal tau (3R/0N isoform) to better understand the temporal relationship between tau accumulation with synaptic and behavioral deficits. We initiated the electrophysiology studies in the hippocampus_a region critical for memory formation. Field potential recordings were obtained from the lateral perforant path of the dentate gyrus of either wild-type (WT) or heterozygous (Het) mice aged 2, 4 or 10 months, and assessments included basal transmission and long-term potentiation (LTP). Results: At 2 months of age, no difference in the magnitude of LTP between genotypes was observed but a subtle impairment in basal transmission was detected in the Het’s even at this early age. By 4 months of age, differences in LTP (WT: 178.266.4%, n¼6; Het: 148.1611.9%, n¼9) and basal synaptic transmission were observed. Notably, 4 month Het LTP data revealed a large variation in the magnitude of potentiation in that some slices had WT LTP magnitudes while others showed deficits. This duality suggests that at 4 months, Het mice may reside on a threshold between normal