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NMDA increases phosphorylated tau immunoreactivity in primary neuronal cultures: a confocal laser microscopic study
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FOURTHINTERNATIONALCONFERENCEONALZHEIMER'S DISEASE
106 GLUTAMATE ENHANCES TAU mRNA EXPRESSION IN PRIMARY NEURONAL CULTURES. F. Esclaire, P. Sindou, M. Lesort, P. Couratier, J. Hugon Unite de Neurobiologie Cellulaire - Lab. d’tiistologie - Faculta de Medecine, 2 rue du Dr Marcland - 87025 LIMOGES cadex FRANCE In Alzheimer’s disease, degenerating neurons are characterized by the accumulation of paired helical filaments (PHF) mainly formed by abnormally phosphorylated TAU protein. Primary neuronal cultures exposed to high concentrations of glutamate display a neuronal degeneration with an increase in abnormally phosphorylated TAU immunoreactivity, observed immediatly as well as 12 hours after glutamate exposure. In order to determine the influence of TAU gene expression in this model, a quantitative in situ hybridization study of TAU mRNA using an oligonucleotidic probe was performed on mature rat neuronal cultures previously exposed for 5 min to glutamate (200pM). Cultures fixed immediatly, 30 set, 1 min or 2 min after glutamate exposure show no modification of hybridization signal, but cultures fixed 12 hours after glutamate exposure show an augmentation of TAU mRNA levels (150 % of controls). The increase in TAU immunoreactivity during the initial phase of excitotoxic cascade could be related to antigenic modifications (eg : abnormal phosphorylation) but the augmentation of TAU immunolabelling observed during the delayed phase seems also to result from an enhancement of TAU gene expression in addition to antigenic modifications. The observed increase in TAU transcription could be mediated by activation of immediate early genes which are known to be stimulated by a brief excitatory amino acid exposure.
107 NMDA INCREASES PHOSPHORYLATED TAU IMMUNOREACTIVITY IN PRIMARY NEURONAL CULTURES : A CONFOCAL LASER MICROSCOPIC STUDY. P. Couratier, M. Lesort, P. Sindou, F. Esclaire, J. Hugon Unite de Neurobiologie Cellulaire - Lab. d’Histologie - Faculte de Medecine - 2 rue du Dr Marcland 87025 LIMOGES Cadex FRANCE Abnormally phosphorylated Tau is a major component of paired helical neurofilaments (PHF). A previous study revealed that calcium-mediated glutamate toxicity in primary neuronal cultures induced a dose-dependent increase of Tau immunoreactivity. The goal of the present series of experiments was to analyse the effects of an acute or a chronic NMDA toxicity on the magnitude of neuronal Tau protein immunolabelling using AT8 monoclonal antibody which recognizes the abnormal PHF Tau and the phosphorylated fetal Tau. Cultures were assessed for NMDA toxicity 8 days from initial plating. Exposure to NMDA (500 pM) was carried out in a Mg++ free glycine supplemented medium in some dishes during 5, 10, 15, 20, 25, 30 min. Other cultures were treated with a low concentration of NMDA (20 FM) for 18 hours. Neurons were fixed and processed for confocal laser microscopic study using AT8 monoclonal antibody. An acute NMDA toxicity induced a significant increase in Tau immunoreactivity related to the duration of exposure marked by a maximum effect after 15 min (150 %).The chronic NMDA exposure produced a dramatic increase of Tau immunolabelling (130 %). Acute modifications appeared mainly mediated by a calcium influx through NMDA receptors since the suppression of calcium from the medium or the blockade by MK801 decreased phosphorylated Tau immunoreactivity. (AT8 provided by Innogenetics).
108 NMDA ACTIVATION INCREASES TAU PHOSPHORYLATION IN PRIMARY NEURONAL CULTURES : AN IMMUNOBLOT STUDY. M. Lesort, P. Couratier, F. Esclaire, P. Sindou, J. Hugon Unite de Neurobiologie Cellulaire, Lab. d’Histologie. - Faculte de Medecine, 2 rue du Dr Marcland 87025 LIMOGES cedex FRANCE.
In Alzheimer’s disease, paired helical filaments (PHF,) are composed of the abnormally phosphorylated microtubule associated protein Tau and form the principal component of the neurofibrillary tangles (NFT). AT8 is a monoclonal antibody which recognizes the abnormal PHF Tau and Tau from fetal brain in a phosphorylated-dependent manner. The objective of this study was to analyse in primary neuronal cultures Tau immunochemical modifications related to NMDA toxicity, using AT8 antibody. Rat cortical cell cultures were exposed to 500 f.tM of NMDA for 5, 10, 15, 20, 25, 30 mn and to 20 pM of NMDA during 16 hours. Then, cells were separated by SDSpolyactylamide gel (10 %) and electrophoretically transfered for Tau immunodetection. Using AT8 monoclonal antibody, Tau immunodetection (single band 55Kd) in acute treatment revealed a marked increase of Tau immunoreactivity after NMDA exposure (10 mn) as compared to control cultures not exposed to NMDA. Then immunolabelling returns to basic level after 30 mn. In chronic exposures, Tau immunoreactivity is strongly increased. Pretreatment with the non competitive NMDA antagonist MK801 (20pM) or exposure in Gas+ free medium reversed the augmentation of Tau immunostaining, suggesting that intracellular calcium influx could play a major role. This study shows that, in primary neuronal cultures, acute and chronic NMDA exposures induce a dramatic increase in phosphotylated Tau protein. NMDA receptors are able to trigger the activation of phosphorylases acting on a Tau protein site, abnormally phosphorylated in Alzheimer’s Disease. (AT8 was provided by Innogenetics).
109 MODULATION OF THE TAU PHF-1 EPITOPE IN VITRO C.K. Combs, P.D. Coleman and M.K. O’Banion Department of Neurobiology and Anatomy, University of Rochester, Rochester, New York 14642 Due to the lack of a satisfactory model for studying the progression of tau phosphorylation and/or paired helical filament formation in Alzheimer’s disease, much interest has been focused on systems that model the expression of certain Alzheimer specific tau epitopes. We have utilized primary embryonic rat hippocampal cultures to investigate the temporal expression of phosphorylated tau epitopes during neuronal differentiation. In our system, hippocampal neurons express a phosphorylated 5erine 3% tau epitope on Western blots and immunocytochemically. A robust pattern of expression appears during days 1-3 in vitro. Our preliminary data using a calcium channel blocker suggests that this phosphorylation site is selectively sensitive to intracellular calcium concentration compared to other selected potential phosphorylation sites. Further investigation of the modulation of this phosphorylation site in vitro may offer clues to both the normal and pathological role of tau phosphorylation. This work was supported by LEAD award A609016 and ROl A601121 to P.D.C., a Markey Foundation grant to M.K.O. and an NIA training grant to C.K.C.
110 NGF-STIMULATION OF ERKs IN UNDIFFERENTIATED PC1 2 CELLS DOES NOT LEAD TO PHF-TYPE TAUHYPERPHOSPHORYLATION. R.P. Fracasso, F.J. Hoffman and H.M. coder. Institute for Dementia Research, Miles Inc., West Haven, CT 06516, USA. MAP-kinases, particularly of the ERK2 type, have been implicated in the tau hyperphosphorylation of Alzheimer’s disease (AD). The ability of established stimulation pathways to induce PHF-type phosphorylation of tau in PC12 cells was investigated by the criteria of phosphoepitope and gel shift analysis. PC12 cells plated on fibronectin (for maximal ERK phosphorylation) were treated for up to 4 hrs with NGF and 2 mM vanadate (to inhibit MAP-kinase phosphatase). Sustained ERK phosphorylation was monitored by phosphotyrosine immunoblotting and ERKP gel shift assay. Incubations for initial rate assays of MAP2 and tau kinase activity of cell extracts were kept as brief as possible; controls incubations were performed to verify that the ERK phosphorylation state is not artificially altered during the kinase assay. Using these precautions, crude MAP2 kinase activity was found to be stimulated by NGF two-fold, however, tau kinase activity stimulations were
FOURTHINTERNATIONALCONFERENCEONALZHEIMER'S DISEASE
106 GLUTAMATE ENHANCES TAU mRNA EXPRESSION IN PRIMARY NEURONAL CULTURES. F. Esclaire, P. Sindou, M. Lesort, P. Couratier, J. Hugon Unite de Neurobiologie Cellulaire - Lab. d’tiistologie - Faculta de Medecine, 2 rue du Dr Marcland - 87025 LIMOGES cadex FRANCE In Alzheimer’s disease, degenerating neurons are characterized by the accumulation of paired helical filaments (PHF) mainly formed by abnormally phosphorylated TAU protein. Primary neuronal cultures exposed to high concentrations of glutamate display a neuronal degeneration with an increase in abnormally phosphorylated TAU immunoreactivity, observed immediatly as well as 12 hours after glutamate exposure. In order to determine the influence of TAU gene expression in this model, a quantitative in situ hybridization study of TAU mRNA using an oligonucleotidic probe was performed on mature rat neuronal cultures previously exposed for 5 min to glutamate (200pM). Cultures fixed immediatly, 30 set, 1 min or 2 min after glutamate exposure show no modification of hybridization signal, but cultures fixed 12 hours after glutamate exposure show an augmentation of TAU mRNA levels (150 % of controls). The increase in TAU immunoreactivity during the initial phase of excitotoxic cascade could be related to antigenic modifications (eg : abnormal phosphorylation) but the augmentation of TAU immunolabelling observed during the delayed phase seems also to result from an enhancement of TAU gene expression in addition to antigenic modifications. The observed increase in TAU transcription could be mediated by activation of immediate early genes which are known to be stimulated by a brief excitatory amino acid exposure.
107 NMDA INCREASES PHOSPHORYLATED TAU IMMUNOREACTIVITY IN PRIMARY NEURONAL CULTURES : A CONFOCAL LASER MICROSCOPIC STUDY. P. Couratier, M. Lesort, P. Sindou, F. Esclaire, J. Hugon Unite de Neurobiologie Cellulaire - Lab. d’Histologie - Faculte de Medecine - 2 rue du Dr Marcland 87025 LIMOGES Cadex FRANCE Abnormally phosphorylated Tau is a major component of paired helical neurofilaments (PHF). A previous study revealed that calcium-mediated glutamate toxicity in primary neuronal cultures induced a dose-dependent increase of Tau immunoreactivity. The goal of the present series of experiments was to analyse the effects of an acute or a chronic NMDA toxicity on the magnitude of neuronal Tau protein immunolabelling using AT8 monoclonal antibody which recognizes the abnormal PHF Tau and the phosphorylated fetal Tau. Cultures were assessed for NMDA toxicity 8 days from initial plating. Exposure to NMDA (500 pM) was carried out in a Mg++ free glycine supplemented medium in some dishes during 5, 10, 15, 20, 25, 30 min. Other cultures were treated with a low concentration of NMDA (20 FM) for 18 hours. Neurons were fixed and processed for confocal laser microscopic study using AT8 monoclonal antibody. An acute NMDA toxicity induced a significant increase in Tau immunoreactivity related to the duration of exposure marked by a maximum effect after 15 min (150 %).The chronic NMDA exposure produced a dramatic increase of Tau immunolabelling (130 %). Acute modifications appeared mainly mediated by a calcium influx through NMDA receptors since the suppression of calcium from the medium or the blockade by MK801 decreased phosphorylated Tau immunoreactivity. (AT8 provided by Innogenetics).
108 NMDA ACTIVATION INCREASES TAU PHOSPHORYLATION IN PRIMARY NEURONAL CULTURES : AN IMMUNOBLOT STUDY. M. Lesort, P. Couratier, F. Esclaire, P. Sindou, J. Hugon Unite de Neurobiologie Cellulaire, Lab. d’Histologie. - Faculte de Medecine, 2 rue du Dr Marcland 87025 LIMOGES cedex FRANCE.
In Alzheimer’s disease, paired helical filaments (PHF,) are composed of the abnormally phosphorylated microtubule associated protein Tau and form the principal component of the neurofibrillary tangles (NFT). AT8 is a monoclonal antibody which recognizes the abnormal PHF Tau and Tau from fetal brain in a phosphorylated-dependent manner. The objective of this study was to analyse in primary neuronal cultures Tau immunochemical modifications related to NMDA toxicity, using AT8 antibody. Rat cortical cell cultures were exposed to 500 f.tM of NMDA for 5, 10, 15, 20, 25, 30 mn and to 20 pM of NMDA during 16 hours. Then, cells were separated by SDSpolyactylamide gel (10 %) and electrophoretically transfered for Tau immunodetection. Using AT8 monoclonal antibody, Tau immunodetection (single band 55Kd) in acute treatment revealed a marked increase of Tau immunoreactivity after NMDA exposure (10 mn) as compared to control cultures not exposed to NMDA. Then immunolabelling returns to basic level after 30 mn. In chronic exposures, Tau immunoreactivity is strongly increased. Pretreatment with the non competitive NMDA antagonist MK801 (20pM) or exposure in Gas+ free medium reversed the augmentation of Tau immunostaining, suggesting that intracellular calcium influx could play a major role. This study shows that, in primary neuronal cultures, acute and chronic NMDA exposures induce a dramatic increase in phosphotylated Tau protein. NMDA receptors are able to trigger the activation of phosphorylases acting on a Tau protein site, abnormally phosphorylated in Alzheimer’s Disease. (AT8 was provided by Innogenetics).
109 MODULATION OF THE TAU PHF-1 EPITOPE IN VITRO C.K. Combs, P.D. Coleman and M.K. O’Banion Department of Neurobiology and Anatomy, University of Rochester, Rochester, New York 14642 Due to the lack of a satisfactory model for studying the progression of tau phosphorylation and/or paired helical filament formation in Alzheimer’s disease, much interest has been focused on systems that model the expression of certain Alzheimer specific tau epitopes. We have utilized primary embryonic rat hippocampal cultures to investigate the temporal expression of phosphorylated tau epitopes during neuronal differentiation. In our system, hippocampal neurons express a phosphorylated 5erine 3% tau epitope on Western blots and immunocytochemically. A robust pattern of expression appears during days 1-3 in vitro. Our preliminary data using a calcium channel blocker suggests that this phosphorylation site is selectively sensitive to intracellular calcium concentration compared to other selected potential phosphorylation sites. Further investigation of the modulation of this phosphorylation site in vitro may offer clues to both the normal and pathological role of tau phosphorylation. This work was supported by LEAD award A609016 and ROl A601121 to P.D.C., a Markey Foundation grant to M.K.O. and an NIA training grant to C.K.C.
110 NGF-STIMULATION OF ERKs IN UNDIFFERENTIATED PC1 2 CELLS DOES NOT LEAD TO PHF-TYPE TAUHYPERPHOSPHORYLATION. R.P. Fracasso, F.J. Hoffman and H.M. coder. Institute for Dementia Research, Miles Inc., West Haven, CT 06516, USA. MAP-kinases, particularly of the ERK2 type, have been implicated in the tau hyperphosphorylation of Alzheimer’s disease (AD). The ability of established stimulation pathways to induce PHF-type phosphorylation of tau in PC12 cells was investigated by the criteria of phosphoepitope and gel shift analysis. PC12 cells plated on fibronectin (for maximal ERK phosphorylation) were treated for up to 4 hrs with NGF and 2 mM vanadate (to inhibit MAP-kinase phosphatase). Sustained ERK phosphorylation was monitored by phosphotyrosine immunoblotting and ERKP gel shift assay. Incubations for initial rate assays of MAP2 and tau kinase activity of cell extracts were kept as brief as possible; controls incubations were performed to verify that the ERK phosphorylation state is not artificially altered during the kinase assay. Using these precautions, crude MAP2 kinase activity was found to be stimulated by NGF two-fold, however, tau kinase activity stimulations were