- Email: [email protected]
P3-303: PP2A activates the antiapoptotic function of BCL-2, while the dephosphorylation of tau by PP2A facilitates apoptosis
Poster Presentations P3: P3-302
TAU OVEREXPRESSION DEPRESSED APOPTOSIS BY INHIBITING P53 AND CYTOCHROME C
Honglian Li, Department of Histology and Embryology, Tongji Medical College of Huazhong University of Science and Technology, Wuhan, Hubei Province, China. Contact e-mail: [email protected] Background: Formation of tau inclusions in the brain neurons is one of the hallmarks of Alzheimer’s disease (AD) and related tauopathies. In our previous study, we showed that cells overexpressing tau exhibited marked resistance to apoptosis induced by various apoptotic stimuli (PNAS 2007, 104: 3591-3596). But it is little known how the tau inclusions-bearing neurons not dying of apoptosis but rather degeneration regulate the apoptotic factors, such as P53 and cytochrome C. Methods: we utilized mouse neuroblastoma N2a cells (N2a/wt) and stably expressing wild-type tau (N2a/tau) to study the level of phosphorylated tau, apoptotic rate and the alterations of the related proteins. Results: We found that cells overexpressing tau protein are more resistant to apoptosis induced by apoptotic inducers. The anti-apoptotic effect of tau overexpression involves depression of P53, arrest of cytochrome C release from mitochondrial and inhibition of caspases-9/-3 activity. Inverse staining pattern of hyperphosphorylated tau and activated caspase-3 or fragmented nuclei was observed in cells overexpressing tau. Further studies demonstrated that tau overexpression decreases phosphorylated -catenin, increases -catenin and nuclear translocation of -catenin. Knock down of -catenin antagonized the antiapoptotic effect of tau. Conclusions: These findings reveal an antiapoptotic function of tau which involves P53-mediated pathway and stabilization of -catenin. P3-303
PP2A ACTIVATES THE ANTIAPOPTOTIC FUNCTION OF BCL-2, WHILE THE DEPHOSPHORYLATION OF TAU BY PP2A FACILITATES APOPTOSIS
Xin-an Liu, Kai Liao, Jian-zhi Wang, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China. Contact e-mail: [email protected] Background: Hyperphosphorylated tau is the major protein subunit of neurofibrillary tangles in Alzheimer’s disease (AD) and related tauopathies. Our previous research has proved that phosphorylation of tau antagonizes apoptosis by stabilizing beta-catenin. Tau dephosphorylation is a general feature of the end stage of apoptosis and tau dephosphorylation during apoptosis is inhibited by okadaic acid. Dephosphorylation of Bcl-2 by protein phosphatase 2A results in apoptosis resistance. Objective: To explore the effect of PP2A and dephosphorylation of tau on apoptosis and the possible underlying mechanism. Methods: Annexin V-PI staining and flow cytometry were used to detect cell apoptosis. Western blot was used for the phosphorylation level of tau and the activity of PP2A. Results: Transfection of PP2A in HEK293 cells resulted in an obvious reduction of cell apoptosis induced by various apoptotic stimuli which also causes correlated reduction of the level of S87P Bcl-2, while PP2A transfection in HEK293 cells stably expressing tau441(HEK293/tau441) increased apoptosis and the level of S87P Bcl-2. We also found that the level of S87P Bcl-2 is positively correlated to Tau-1 in HEK293/tau441 cells. Conclusions: Overexpression of PP2A has an antiapoptotic effect in HEK293 cells, while the dephosphorylation of tau by PP2A facilitates apoptosis in HEK293 cells stably expressing tau441, which involves the apoptosis resistant effects of phosphorylation of tau and Bcl-2 dephosphorylation by protein phosphatase 2A. P3-304
DEATH ASSOCIATED PROTEIN KINASE (DAPK): A CALCIUM/CALMODULIN (CAM) REGULATOR OF NEURONAL APOPTOSIS THAT IS A DRUG DISCOVERY TARGET FOR NEURODEGENERATIVE DISORDERS
Laurie K. McNamara, James P. Schavocky, Joseph S. Brunzelle, D. Martin Watterson, Northwestern University, Chicago, IL, USA. Contact e-mail: [email protected]
T611
Background: DAPK is a pro-apoptotic CaM-regulated serine/threonine protein kinase that is a drug discovery target for neurodegenerative disorders. Most recently, DAPK gene polymorphisms have been linked to late onset AD susceptibility. Treatments with our novel DAPK inhibitor after chronic exposure to toxic forms of human amyloid beta or after brain ischemia attenuate brain injury and synaptic dysfunction in animal models. Therefore, there is a need to further define the relationship between DAPK’s structure, activity and in vivo functions relevant to neuronal apoptosis. Methods: We used an interdisciplinary approach that included the convergence of informatics, genetics, structural biology, in vivo chemical biology and enzymology in order to identify the first protein substrates related to DAPK’s role as an integrator of neuronal survival responses and potential therapeutic target. Results: These studies identified CaM-regulated protein kinase kinase (CaMKK) and ribosomal S6 protein as DAPK substrates relevant to these in vivo roles. On-going studies combine molecular and structural biology investigations with enzymology and cell biology to test prevailing hypotheses about the linkage among DAPK’s structure, catalytic activity and potential cellular functions. Conclusions: These investigations allow a refinement of current models of DAPK as an enzymic regulator of neuronal function and provide insight into the molecular basis of DAPK-mediated phosphorylation events. P3-305
INCREASED EXPRESSION OF THE PKR MODULATORS, PACT AND TRBP, IN THE BRAINS OF PATIENTS WITH ALZHEIMER’S DISEASE (AD)
Claire Paquet1,2, Anindita Bose3, Anne Gatignol4, Eliane Meurs5, Franc¸oise Gray6, Jacques Hugon6,2, 1Alzheimer Center Hospital Lariboisie`re-Fernand Widal / Department of Pathology, Paris, France; 2 INSERM 839, Paris, France; 3INSERM 839, Paris, France; 4 Departments of Medicine and Microbiology & Immunology McGill University, Montreal, QC, Canada; 5Hepacivirus Unit, Pasteur Institute, Paris, France; 6Alzheimer Center Hoˆpital Lariboisie`re-Fernand Widal / Department of Pathology, Paris, France. Contact e-mail: [email protected] Background: AD is characterized by extensive neuronal loss in different brain regions including the hippocampus. The levels of the pro-apoptotic kinase PKR (double stranded RNA dependent protein kinase) are increased in the brains of AD patients. PKR is expressed in degenerating neurons as well as in senile plaques. The protein PACT is a PKR activator and the protein TRBP reduces PKR activation. The aim of the current study is to assess with immunohistochemical techniques, the staining of PKR PACT and TRBP in the brains of patients suffering from AD and in the brains of control individuals. Methods: Five AD brains and five age-matched control brains were included in this research. The immunohistochemical analysis was carried out in hippocampal areas using a polyclonal antibody against phosphorylated PKR (thr 451), a monoclonal TRBP antibody developed by one of the co-authors (AG) and a polyclonal antibody raised against PACT. Results: The staining of the activated and phosphorylated kinase PKR was enhanced in neurons of the entorhinal cortex and hippocampus in AD brains compared with controls. Many neurons displayed features of neurodegeneration including granulovacuolar degeneration In addition,in AD, increased cytoplasmic PACT immunolabellings were detected in neurons of the enthorinal cortex and the hippocampus and neuronal nuclear membranes were intensely stained with the TRBP antibody. Marked TRBP labeling were also noted in the cytoplasm of degenerating neurons in AD brains and not in control brains. Granular or granulovacuolar accumulations were frequently detected in these TRBP positive neurons. Conclusions: Our results show an accumulation of the proteins phosphorylated PKR, PACT and TRBP in the brains of AD patients suggesting that PACT could contribute to PKR activation and neuronal degeneration. TRBP immunostainings could be associated with an ineffective cellular reaction for neurons to attenuate the deleterious consequences of the triggering of the PKR pro-apoptotic pathway.
TAU OVEREXPRESSION DEPRESSED APOPTOSIS BY INHIBITING P53 AND CYTOCHROME C
Honglian Li, Department of Histology and Embryology, Tongji Medical College of Huazhong University of Science and Technology, Wuhan, Hubei Province, China. Contact e-mail: [email protected] Background: Formation of tau inclusions in the brain neurons is one of the hallmarks of Alzheimer’s disease (AD) and related tauopathies. In our previous study, we showed that cells overexpressing tau exhibited marked resistance to apoptosis induced by various apoptotic stimuli (PNAS 2007, 104: 3591-3596). But it is little known how the tau inclusions-bearing neurons not dying of apoptosis but rather degeneration regulate the apoptotic factors, such as P53 and cytochrome C. Methods: we utilized mouse neuroblastoma N2a cells (N2a/wt) and stably expressing wild-type tau (N2a/tau) to study the level of phosphorylated tau, apoptotic rate and the alterations of the related proteins. Results: We found that cells overexpressing tau protein are more resistant to apoptosis induced by apoptotic inducers. The anti-apoptotic effect of tau overexpression involves depression of P53, arrest of cytochrome C release from mitochondrial and inhibition of caspases-9/-3 activity. Inverse staining pattern of hyperphosphorylated tau and activated caspase-3 or fragmented nuclei was observed in cells overexpressing tau. Further studies demonstrated that tau overexpression decreases phosphorylated -catenin, increases -catenin and nuclear translocation of -catenin. Knock down of -catenin antagonized the antiapoptotic effect of tau. Conclusions: These findings reveal an antiapoptotic function of tau which involves P53-mediated pathway and stabilization of -catenin. P3-303
PP2A ACTIVATES THE ANTIAPOPTOTIC FUNCTION OF BCL-2, WHILE THE DEPHOSPHORYLATION OF TAU BY PP2A FACILITATES APOPTOSIS
Xin-an Liu, Kai Liao, Jian-zhi Wang, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China. Contact e-mail: [email protected] Background: Hyperphosphorylated tau is the major protein subunit of neurofibrillary tangles in Alzheimer’s disease (AD) and related tauopathies. Our previous research has proved that phosphorylation of tau antagonizes apoptosis by stabilizing beta-catenin. Tau dephosphorylation is a general feature of the end stage of apoptosis and tau dephosphorylation during apoptosis is inhibited by okadaic acid. Dephosphorylation of Bcl-2 by protein phosphatase 2A results in apoptosis resistance. Objective: To explore the effect of PP2A and dephosphorylation of tau on apoptosis and the possible underlying mechanism. Methods: Annexin V-PI staining and flow cytometry were used to detect cell apoptosis. Western blot was used for the phosphorylation level of tau and the activity of PP2A. Results: Transfection of PP2A in HEK293 cells resulted in an obvious reduction of cell apoptosis induced by various apoptotic stimuli which also causes correlated reduction of the level of S87P Bcl-2, while PP2A transfection in HEK293 cells stably expressing tau441(HEK293/tau441) increased apoptosis and the level of S87P Bcl-2. We also found that the level of S87P Bcl-2 is positively correlated to Tau-1 in HEK293/tau441 cells. Conclusions: Overexpression of PP2A has an antiapoptotic effect in HEK293 cells, while the dephosphorylation of tau by PP2A facilitates apoptosis in HEK293 cells stably expressing tau441, which involves the apoptosis resistant effects of phosphorylation of tau and Bcl-2 dephosphorylation by protein phosphatase 2A. P3-304
DEATH ASSOCIATED PROTEIN KINASE (DAPK): A CALCIUM/CALMODULIN (CAM) REGULATOR OF NEURONAL APOPTOSIS THAT IS A DRUG DISCOVERY TARGET FOR NEURODEGENERATIVE DISORDERS
Laurie K. McNamara, James P. Schavocky, Joseph S. Brunzelle, D. Martin Watterson, Northwestern University, Chicago, IL, USA. Contact e-mail: [email protected]
T611
Background: DAPK is a pro-apoptotic CaM-regulated serine/threonine protein kinase that is a drug discovery target for neurodegenerative disorders. Most recently, DAPK gene polymorphisms have been linked to late onset AD susceptibility. Treatments with our novel DAPK inhibitor after chronic exposure to toxic forms of human amyloid beta or after brain ischemia attenuate brain injury and synaptic dysfunction in animal models. Therefore, there is a need to further define the relationship between DAPK’s structure, activity and in vivo functions relevant to neuronal apoptosis. Methods: We used an interdisciplinary approach that included the convergence of informatics, genetics, structural biology, in vivo chemical biology and enzymology in order to identify the first protein substrates related to DAPK’s role as an integrator of neuronal survival responses and potential therapeutic target. Results: These studies identified CaM-regulated protein kinase kinase (CaMKK) and ribosomal S6 protein as DAPK substrates relevant to these in vivo roles. On-going studies combine molecular and structural biology investigations with enzymology and cell biology to test prevailing hypotheses about the linkage among DAPK’s structure, catalytic activity and potential cellular functions. Conclusions: These investigations allow a refinement of current models of DAPK as an enzymic regulator of neuronal function and provide insight into the molecular basis of DAPK-mediated phosphorylation events. P3-305
INCREASED EXPRESSION OF THE PKR MODULATORS, PACT AND TRBP, IN THE BRAINS OF PATIENTS WITH ALZHEIMER’S DISEASE (AD)
Claire Paquet1,2, Anindita Bose3, Anne Gatignol4, Eliane Meurs5, Franc¸oise Gray6, Jacques Hugon6,2, 1Alzheimer Center Hospital Lariboisie`re-Fernand Widal / Department of Pathology, Paris, France; 2 INSERM 839, Paris, France; 3INSERM 839, Paris, France; 4 Departments of Medicine and Microbiology & Immunology McGill University, Montreal, QC, Canada; 5Hepacivirus Unit, Pasteur Institute, Paris, France; 6Alzheimer Center Hoˆpital Lariboisie`re-Fernand Widal / Department of Pathology, Paris, France. Contact e-mail: [email protected] Background: AD is characterized by extensive neuronal loss in different brain regions including the hippocampus. The levels of the pro-apoptotic kinase PKR (double stranded RNA dependent protein kinase) are increased in the brains of AD patients. PKR is expressed in degenerating neurons as well as in senile plaques. The protein PACT is a PKR activator and the protein TRBP reduces PKR activation. The aim of the current study is to assess with immunohistochemical techniques, the staining of PKR PACT and TRBP in the brains of patients suffering from AD and in the brains of control individuals. Methods: Five AD brains and five age-matched control brains were included in this research. The immunohistochemical analysis was carried out in hippocampal areas using a polyclonal antibody against phosphorylated PKR (thr 451), a monoclonal TRBP antibody developed by one of the co-authors (AG) and a polyclonal antibody raised against PACT. Results: The staining of the activated and phosphorylated kinase PKR was enhanced in neurons of the entorhinal cortex and hippocampus in AD brains compared with controls. Many neurons displayed features of neurodegeneration including granulovacuolar degeneration In addition,in AD, increased cytoplasmic PACT immunolabellings were detected in neurons of the enthorinal cortex and the hippocampus and neuronal nuclear membranes were intensely stained with the TRBP antibody. Marked TRBP labeling were also noted in the cytoplasm of degenerating neurons in AD brains and not in control brains. Granular or granulovacuolar accumulations were frequently detected in these TRBP positive neurons. Conclusions: Our results show an accumulation of the proteins phosphorylated PKR, PACT and TRBP in the brains of AD patients suggesting that PACT could contribute to PKR activation and neuronal degeneration. TRBP immunostainings could be associated with an ineffective cellular reaction for neurons to attenuate the deleterious consequences of the triggering of the PKR pro-apoptotic pathway.