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Role of astroglial glycolysis in apolipoprotein E lipidation and secretion
S662 P3-456
Poster Presentations P3 EXAMINING THE ROLE OF C-JUN N-TERMINAL KINASE ON HYPOTHERMIA-INDUCED TAU PHOSPHORYLATION IN CORTICAL NEURONS AND MOUSE BRAIN
Jonathan Papworth*1, Eimear Howley1, Laura Barden1, Ryota Taguchi1, Anthony Groom1, Peter Atkinson1, Vishal Sahni1, Gregory Keen2, Gurpreet Bhatia1, Darren Medland1, Paschalis Dimopoulos1, Chris Farthing1, Piotr Graczyk1, Andrew Payne1, Louise Morgan1, Avril Treacy1, Adam Kline2, Toshal Patel1, Lee Dawson1, 1Eisai Limited, Hatfield, United Kingdom; 2Eisai Limited, London, United Kingdom. Background: One of the principal features of Alzheimer’s disease (AD) pathology is the presence of neurofibrillary tangles composed of abnormally hyper-phosphorylated tau. Numerous kinases have been implicated in the modulation of tau phosphorylation and therefore represent potential targets for pharmacological intervention. c-Jun N-terminal kinase (JNK), a key stress-response protein, has been shown to directly phosphor late tau in vitro. Furthermore, the JNK pathway can mediate apoptosis and has been shown to be activated in animal models of AD. Methods: In the current study we investigated the role of JNK on tau phosphorylation in primary cortical neurons and in vivo using a previously described mouse model of hypothermia. In these experiments, incubation of primary cortical neurons or C57/Bl6 mice for 1 h at 28 oC induced a robust (w2-fold) increase in phospho-tau [pS202/pT205] immunoreactivity compared to control. Results: A broad spectrum kinase inhibitor, ER-358063 (JNK3 IC50 63 nM) significantly attenuated tau phosphorylation in vitro (60% inhibition at 3 uM; 1 h pre-treatment). Treatment with a JNK -selective inhibitor, ER-417258 (JNK3 IC50 161 nM), had a somewhat reduced effect on tau phosphorylation (40% inhibition at 3 uM; 1 h pre-treatment) on the hypothermic challenge. Concurrently, both compounds significantly inhibited c-Jun phosphorylation (>60% at 3 uM) in the cortical neurons. In vivo ER-358063 (40 mg/kg, p.o.; pre-treatment 15 min) significantly attenuated (w20% reduction) the hypothermia-induced tau phosphorylation in the hippocampus and cortex. However, treatment with the JNK-selective inhibitor, ER-417258 (40 mg/kg, p.o.; pre-treatment 15 min), had no effect on the hypothermia-induced tau phosphorylation in either brain region. Conclusions: ER-358063 exhibits activity at a number of other tau phosphorylating kinases including glycogen synthase kinase (GSK3b). Detailed examination of the phosphorylated epitopes of tau indicated that while ER-358063 significantly inhibited phosphorylation of pS235 in addition to pS202, ER-417258 treatment did not affect either of these epitopes in vivo. Both epitopes have been reported to be phosphorylated by JNK and GSK3b. These data suggest that JNK inhibition alone, although having a weak effect in vitro, may not be sufficient to attenuate hypothermia-induced tau phosphorylation in vivo. P3-457
DECAFFEINATED COFFEE PROMOTES SUCCESSFUL AGING THROUGH EPIGENETIC MODIFICATIONS ASSOCIATED WITH PROMOTION OF MITOCHONDRIAL ENERGY METABOLISM IN THE BRAIN
Giulio Pasinetti*1, 1The Mount Sinai School Of Medicine, New York, New York, United States. Background: Coffee is one of the world’s most popular beverages. However, consumption of caffeinated is known to be associated with increased coronary heart disease risk, including elevation of serum cholesterol, blood pressure and stroke. Bioactive coffee components responsible for these negative effects on cardiovascular functions include caffeine, diterpenes, etc. On the contrary numerous health benefits resulted from decaffeinated coffee consumption have received significant scientific attention in several chronic diseases including metabolic and neurodegenerative disorders. Alzheimer’s disease (AD) is a progressive neurodegenerative disorder. The sporadic nature of 90% of AD cases, varying susceptibility, as well as the late age of onset suggest that the epigenetic and environmental factors play an important role in the etiology of late-onset AD. We hypothesize that the beneficial role of decaffeinated coffee may result from modulation of specific DNA promoters. Methods: To investigate the beneficial effects of decaffeinated -non roasted (green coffee)
on both metabolic and epigenetic changes in AD, mice were exposed to Svetol, a gift from NATUREX Inc, for 6 months. Body weight and food and drink consumption were monitored weekly, and glucose tolerance assays were carried bimonthly. The frontal cortex was isolated after 6 months of treatment; mRNA of the frontal cortex was extracted for microarray assay and further qPCR confirmation, followed by ChIP-Chip. Mitochondria were also isolated to evaluate the role of Svetol on oxygen consumption rate (OCR) using Seahorse technology. Results: We found that glucose tolerance was significantly improved in green-coffee treated mice compared to control, as was mitochondrial function (as measured by OCR). Further analysis using microarray and qPCR suggested functional expression level changes in certain epigenetic genes, including Histone deactylase 1 (HDAC1) and DNA methyltrasferase 3a (Dnmt3a) in the brain. ChIP-Chip of these two genes is currently under investigation, and will elucidate the mechanism(s) of green coffee’s effect in this mouse model of normal aging and in AD mice. Conclusions: Our data suggest that the green coffee may benefits AD by improving mitochondrial functions and through epigenetic alterations in certain genes and might further developed as secondary preventative measure in order to maintain healthy aging. P3-458
ROLE OF ASTROGLIAL GLYCOLYSIS IN APOLIPOPROTEIN E LIPIDATION AND SECRETION
Sachin Patil*1, 1Widener University, Chester, Pennsylvania, United States. Background: Alzheimer’s disease (AD) is a progressive, neurodegenerative disease mostly affecting basal forebrain, cortex and hippocampus. One of the central pathological markers of AD brain is the extracellular deposits of amyloid beta (Aß) protein in the affected brain regions. Based on a recent, real-time Aß turnover study in AD patients, the increased Aß deposition in AD brains can be specifically attributed to decreased Aß clearance. Here, it is noteworthy that Apolipoprotein E (ApoE), one of the significant genetic risk factors for AD, has been shown to play a major role in brain Aß clearance. The ability of ApoE to facilitate Aß degradation is dependent upon its lipidation status. Thus, our research plan is to discover novel drug molecules that will increase ApoE lipidation. These molecules will form the basis of novel “disease-modifying” therapeutics against AD. Here, we hypothesize possible role of glycolysis in affecting the ApoE lipidation status as the published data imply decreased glucose metabolism and failure to sustain critical glucose-dependent functions such as biosynthesis of lipids (& in turn, ApoE lipidation) in AD-affected brain regions may be responsible for decreased clearance and thus increased deposition of Aß in those brain regions. Thus, our specific research plan is to discover and develop drugs that will enhance astroglial glycolysis, which may in turn help in increasing ApoE lipidation status. Methods: A) Isolation and Culture of Primary RatCortical Astroglia- The cortical astroglia are isolated from 1-day-old Sprague- Dawley rat pups and cultured as described earlier [1]. B) Measurement of glucose uptake and lactate production- The uptake of glucose and the production of lactate are measured as markers of glycosis, as explained in our previously published paper [1]. C) Non-denaturing gradient gel electrophoresis for ApoE lipidation status: ApoE lipidation status is determined as described previously by Jiang et al. [2]. 1. Patil, S.P. et al., Eur. J. Neurosci., 26(8):2131-2141 (2007). 2. Jiang, Q. et al., Neuron, 58(5):681-693 (2008). Results: Complete results are expected to be obtained by May 2011. Conclusions: Final conclusions will be derived after receiving the complete results. P3-459
GLYCOGEN SYNTHASE KINASE 3 INHIBITORS PROMOTE ADULT HIPPOCAMPAL NEUROGENESIS
Jose A. Morales-Garcia*1, Valle Palomo2, Carmen Gil2, Ana Martinez2, Angel Santos3, Ana Perez-Castillo4, 1Consejo Superior de Investigaciones Cientıficas-CIBERNED, Madrid, Spain; 2Instituto de Quımica Medica, Consejo Superior de Investigaciones Cientıficas, Madrid, Spain; 3Facultad de Medicina, Universidad Complutense, Madrid, Spain; 4Instituto de Investigaciones Biomedicas, Consejo Superior de Investigaciones Cientıficas and CIBERNED, Madrid, Spain. Background: In the central nervous system, developing neurons are derived from quiescent multipotent or neural stem cells. Throughout life, neural
Poster Presentations P3 EXAMINING THE ROLE OF C-JUN N-TERMINAL KINASE ON HYPOTHERMIA-INDUCED TAU PHOSPHORYLATION IN CORTICAL NEURONS AND MOUSE BRAIN
Jonathan Papworth*1, Eimear Howley1, Laura Barden1, Ryota Taguchi1, Anthony Groom1, Peter Atkinson1, Vishal Sahni1, Gregory Keen2, Gurpreet Bhatia1, Darren Medland1, Paschalis Dimopoulos1, Chris Farthing1, Piotr Graczyk1, Andrew Payne1, Louise Morgan1, Avril Treacy1, Adam Kline2, Toshal Patel1, Lee Dawson1, 1Eisai Limited, Hatfield, United Kingdom; 2Eisai Limited, London, United Kingdom. Background: One of the principal features of Alzheimer’s disease (AD) pathology is the presence of neurofibrillary tangles composed of abnormally hyper-phosphorylated tau. Numerous kinases have been implicated in the modulation of tau phosphorylation and therefore represent potential targets for pharmacological intervention. c-Jun N-terminal kinase (JNK), a key stress-response protein, has been shown to directly phosphor late tau in vitro. Furthermore, the JNK pathway can mediate apoptosis and has been shown to be activated in animal models of AD. Methods: In the current study we investigated the role of JNK on tau phosphorylation in primary cortical neurons and in vivo using a previously described mouse model of hypothermia. In these experiments, incubation of primary cortical neurons or C57/Bl6 mice for 1 h at 28 oC induced a robust (w2-fold) increase in phospho-tau [pS202/pT205] immunoreactivity compared to control. Results: A broad spectrum kinase inhibitor, ER-358063 (JNK3 IC50 63 nM) significantly attenuated tau phosphorylation in vitro (60% inhibition at 3 uM; 1 h pre-treatment). Treatment with a JNK -selective inhibitor, ER-417258 (JNK3 IC50 161 nM), had a somewhat reduced effect on tau phosphorylation (40% inhibition at 3 uM; 1 h pre-treatment) on the hypothermic challenge. Concurrently, both compounds significantly inhibited c-Jun phosphorylation (>60% at 3 uM) in the cortical neurons. In vivo ER-358063 (40 mg/kg, p.o.; pre-treatment 15 min) significantly attenuated (w20% reduction) the hypothermia-induced tau phosphorylation in the hippocampus and cortex. However, treatment with the JNK-selective inhibitor, ER-417258 (40 mg/kg, p.o.; pre-treatment 15 min), had no effect on the hypothermia-induced tau phosphorylation in either brain region. Conclusions: ER-358063 exhibits activity at a number of other tau phosphorylating kinases including glycogen synthase kinase (GSK3b). Detailed examination of the phosphorylated epitopes of tau indicated that while ER-358063 significantly inhibited phosphorylation of pS235 in addition to pS202, ER-417258 treatment did not affect either of these epitopes in vivo. Both epitopes have been reported to be phosphorylated by JNK and GSK3b. These data suggest that JNK inhibition alone, although having a weak effect in vitro, may not be sufficient to attenuate hypothermia-induced tau phosphorylation in vivo. P3-457
DECAFFEINATED COFFEE PROMOTES SUCCESSFUL AGING THROUGH EPIGENETIC MODIFICATIONS ASSOCIATED WITH PROMOTION OF MITOCHONDRIAL ENERGY METABOLISM IN THE BRAIN
Giulio Pasinetti*1, 1The Mount Sinai School Of Medicine, New York, New York, United States. Background: Coffee is one of the world’s most popular beverages. However, consumption of caffeinated is known to be associated with increased coronary heart disease risk, including elevation of serum cholesterol, blood pressure and stroke. Bioactive coffee components responsible for these negative effects on cardiovascular functions include caffeine, diterpenes, etc. On the contrary numerous health benefits resulted from decaffeinated coffee consumption have received significant scientific attention in several chronic diseases including metabolic and neurodegenerative disorders. Alzheimer’s disease (AD) is a progressive neurodegenerative disorder. The sporadic nature of 90% of AD cases, varying susceptibility, as well as the late age of onset suggest that the epigenetic and environmental factors play an important role in the etiology of late-onset AD. We hypothesize that the beneficial role of decaffeinated coffee may result from modulation of specific DNA promoters. Methods: To investigate the beneficial effects of decaffeinated -non roasted (green coffee)
on both metabolic and epigenetic changes in AD, mice were exposed to Svetol, a gift from NATUREX Inc, for 6 months. Body weight and food and drink consumption were monitored weekly, and glucose tolerance assays were carried bimonthly. The frontal cortex was isolated after 6 months of treatment; mRNA of the frontal cortex was extracted for microarray assay and further qPCR confirmation, followed by ChIP-Chip. Mitochondria were also isolated to evaluate the role of Svetol on oxygen consumption rate (OCR) using Seahorse technology. Results: We found that glucose tolerance was significantly improved in green-coffee treated mice compared to control, as was mitochondrial function (as measured by OCR). Further analysis using microarray and qPCR suggested functional expression level changes in certain epigenetic genes, including Histone deactylase 1 (HDAC1) and DNA methyltrasferase 3a (Dnmt3a) in the brain. ChIP-Chip of these two genes is currently under investigation, and will elucidate the mechanism(s) of green coffee’s effect in this mouse model of normal aging and in AD mice. Conclusions: Our data suggest that the green coffee may benefits AD by improving mitochondrial functions and through epigenetic alterations in certain genes and might further developed as secondary preventative measure in order to maintain healthy aging. P3-458
ROLE OF ASTROGLIAL GLYCOLYSIS IN APOLIPOPROTEIN E LIPIDATION AND SECRETION
Sachin Patil*1, 1Widener University, Chester, Pennsylvania, United States. Background: Alzheimer’s disease (AD) is a progressive, neurodegenerative disease mostly affecting basal forebrain, cortex and hippocampus. One of the central pathological markers of AD brain is the extracellular deposits of amyloid beta (Aß) protein in the affected brain regions. Based on a recent, real-time Aß turnover study in AD patients, the increased Aß deposition in AD brains can be specifically attributed to decreased Aß clearance. Here, it is noteworthy that Apolipoprotein E (ApoE), one of the significant genetic risk factors for AD, has been shown to play a major role in brain Aß clearance. The ability of ApoE to facilitate Aß degradation is dependent upon its lipidation status. Thus, our research plan is to discover novel drug molecules that will increase ApoE lipidation. These molecules will form the basis of novel “disease-modifying” therapeutics against AD. Here, we hypothesize possible role of glycolysis in affecting the ApoE lipidation status as the published data imply decreased glucose metabolism and failure to sustain critical glucose-dependent functions such as biosynthesis of lipids (& in turn, ApoE lipidation) in AD-affected brain regions may be responsible for decreased clearance and thus increased deposition of Aß in those brain regions. Thus, our specific research plan is to discover and develop drugs that will enhance astroglial glycolysis, which may in turn help in increasing ApoE lipidation status. Methods: A) Isolation and Culture of Primary RatCortical Astroglia- The cortical astroglia are isolated from 1-day-old Sprague- Dawley rat pups and cultured as described earlier [1]. B) Measurement of glucose uptake and lactate production- The uptake of glucose and the production of lactate are measured as markers of glycosis, as explained in our previously published paper [1]. C) Non-denaturing gradient gel electrophoresis for ApoE lipidation status: ApoE lipidation status is determined as described previously by Jiang et al. [2]. 1. Patil, S.P. et al., Eur. J. Neurosci., 26(8):2131-2141 (2007). 2. Jiang, Q. et al., Neuron, 58(5):681-693 (2008). Results: Complete results are expected to be obtained by May 2011. Conclusions: Final conclusions will be derived after receiving the complete results. P3-459
GLYCOGEN SYNTHASE KINASE 3 INHIBITORS PROMOTE ADULT HIPPOCAMPAL NEUROGENESIS
Jose A. Morales-Garcia*1, Valle Palomo2, Carmen Gil2, Ana Martinez2, Angel Santos3, Ana Perez-Castillo4, 1Consejo Superior de Investigaciones Cientıficas-CIBERNED, Madrid, Spain; 2Instituto de Quımica Medica, Consejo Superior de Investigaciones Cientıficas, Madrid, Spain; 3Facultad de Medicina, Universidad Complutense, Madrid, Spain; 4Instituto de Investigaciones Biomedicas, Consejo Superior de Investigaciones Cientıficas and CIBERNED, Madrid, Spain. Background: In the central nervous system, developing neurons are derived from quiescent multipotent or neural stem cells. Throughout life, neural