- Email: [email protected]
Subregion-specific vulnerability to endoplasmic reticulum stress-induced neurotoxicity in rat hippocampal neurons
P310
Poster Presentations P2
with serum free medium for another 24 h (starvation). Results: The N2a cells overexpressing tau were more resistent to serum withdrawal-induced apoptosis. The protective effect was positively correlated with the relatively increased phosphrylation level of tau. We also found that the Akt was activated in cells overexpressing tau. Simultaneously, the phosphorylation level of FOXO3 increased (representing decreased activity) and the expression of p53 and Bax decreased. The antiapoptotic effects of tau almost disappeared by inhibition of Akt. Upon serum starvation, the expression level of catalytic subunit of PI3K (p110) increased significantly in cells overexpressing tau, whereas the relative level of the phosphorylated p85 was not changed. Conclusions: Our findings suggest that tau leads the cells to escape apoptosis by activating PI3K/Akt pathway and the activation of PI3K. P2-166
VARIOUS LENGTHS OF C-TERMINAL FRAGMENTS OF AMYLOID PRECURSOR PROTEIN INDUCE NEUROTOXICITY, NOT ONLY C31
Yoori Choi1, Minji Kim1, Hye-Sun Kim2, Yoo-Hun Suh1, 1Creative Research Initiative Center for Alzheimer dementia, Seoul, Republic of Korea; 2 Department of Pharmacology, Bundang Hospital, College of Medicine, Seoul National University, Seoul, Republic of Korea. Contact e-mail: [email protected] Background: Abnormal proteolytic cleavages of amyloid precursor protein (APP) generate amyloid beta peptide (Ab) and carboxy-terminal fragments of APP (APP-CTFs). As ‘amyloid cascade hypotheses’ have been lead the cause of Alzheimer’s disease (AD), senile plaque composed with Ab is one of the pathological hallmarks of AD. A recent study has shown that C31, derived from cleavage of APP at Asp664 site by caspase, is inducer of apoptosis and linked Ab-mediated toxicity. The APP transgenic mice with D664A mutation still produce Ab and senile plaque, but prevention of C31 generation protects neuronal proliferation, synaptic transmission, synaptic plasticity deficits and behavioral abnormality. In this study, we observed that neurotoxicity of CTFs is involved whether C31 downstream or NPTY domain which interact with other adaptor proteins. Methods: Primary cortical neuron cuture, transfection, LDH assay. Results: We show that LDH release was increased by various APP-CTFs versus it was reduced by various APP-CTFs with mutated cleavage site at amino acid 664 or NPTY deletion. There is no significant difference in LDH release between various lengths of APP-CTFs. Conclusions: These results indicate that various APP-CTFs including NPTY domain play a critical role in neuronal cell death, not only APP-C31 generation. P2-167
SUBREGION-SPECIFIC VULNERABILITY TO ENDOPLASMIC RETICULUM STRESS-INDUCED NEUROTOXICITY IN RAT HIPPOCAMPAL NEURONS
Kumiko Ishige, Yasuhiro Kosuge, Yoshihisa Ito, Nihon University, Funabashi-shi, Japan. Contact e-mail: [email protected] Background: It is well known that in certain disease states, including ischemia and Alzheimer’s disease, neurodegeneration occurs in the hippocampus, and that vulnerability to neuronal death is area-dependent. The present study investigated the mechanism of area-dependent vulnerability to neuronal death under endoplasmic reticulum stress conditions induced by tunicamycin (TM), using rat organotypic hippocampal cultures (OHC) and hippocampal slices. Methods: The OHC was prepared from 6-7-day-old Wistar rats, in accordance with the Ethical Guidelines for Animal Experiments at Nihon University. Animals were deeply anesthetized, and the hippocampi were removed by dissection from brains. Transverse slices (400 mm thick) were prepared and were placed on a membrane insert. Cultures were fed with 1 ml of culture medium consisting of 50% minimal essential medium, 25% horse serum, and 25% Hanks’ balanced salt solution. Experiments were performed using the OHC cultured for 3 weeks in vitro. Results: Analysis of propidium iodide uptake showed that TM induced neuronal death in a concentration-dependent manner (20w80 mg/mL), and that the rank order of vulnerability among hippocampal subregions was dentate gyrus (DG) > CA1 > CA3. Results of immu-
nohistochemistry using hippocampal slices also showed that procaspase12-positive cells in area CA3 were significantly fewer than those in area CA1 and the DG. Moreover, procurement of neurons in areas CA1, CA3 and the DG by laser microdissection, followed by Western blot analysis, also revealed that the level of procaspase-12 in area CA3 was significantly lower than those in area CA1 and the DG. Pretreatment with zATAD-fmk, a cell-permeable caspase-12-selective inhibitor significantly attenuated the TM-induced increase of PI fluorescence in the CA1 and DG subregion but not in area CA3. Conclusions: In conclusion, the present study has revealed that TM-induced neuronal death is subregion-dependent in the hippocampus, and that the subregion-dependent vulnerability to TM is attributable, at least in part, to the expression level of endogenous procaspase-12 in each area of the hippocampus. The present results also demonstrate that a combination of LMD and Western blotting allows direct measurement of the levels of specific proteins in each subregion in hippocampal slices. P2-168
REGULATOR OF CALCINEURIN 1 (RCAN1) IS DEGRADED BY UBIQUITIN-PROTEASOME AND CHAPERONE-MEDIATED AUTOPHAGY PATHWAY
Xiulian Sun1, Heng Liu1, Pin Wang1, Weihong Song2, 1Shandong University, Jinan, China; 2University of British Columbia, Vancouver, BC, Canada. Contact e-mail: [email protected] Background: Regulator of calcineurin 1 (RCAN1) is a gene identified from the critical region of Down syndrome (DS) and has been implied in pathogenesis of Alzheimer’s disease (AD). RCAN1 expression is increased in AD brains; however, the mechanism of which is unknown. Methods: Co-ip and immunostaning were used to study the RCAN1 ubiquitination. Drug treatment as well as gene knockout by siRNA were used to interrupt macroautophagy pathway and CMA pathway. Results: Here our study investigated the molecular mechanism of RCAN1 protein degradation. Our results showed that RCAN1 is ubiquitinated and degraded by ubiquitin-proteasome pathway. Furthermore, we showed that RCAN1 protein expression is remarkably increased by inhibition of lysosome in a time- and dosage-dependent manner. Inhibition of macroautophagy reduced RCAN1 expression, indicating that RCAN1 degradation is not through macroautophagy pathway. Further evidence showed that disruption of chaperone-mediated autophagy (CMA) increased RCAN1 expression. Our results showed that RCAN1 contains two CMA recognition motifs, by which RCAN1 can be degraded through CMA-lysosome pathway. Conclusions: Dysfunctions of ubiquitin-proteasome and autophagy-lysosome pathways have been implicated in neurodegenerative diseases. Elucidation of RCAN1 degradation by ubiquitin-proteasome and CMA-lysosome pathways greatly advances our understanding of AD pathogenesis. P2-169
PROTEIN PHOSPHATASE-2A INHIBITOR-2 UPREGULATES P53 AND AKT-IMPLICATION FOR A NEW MECHANISM FOR ALZHEIMER’S NEURODEGENERATION
Liu Gongping, Wei Wei, Xin Zhou, Yao Zhang, Hairong Shi, Jun Yin, Xiuqing Yao, Qun Wang, Jianzhi Wang, Pathophysiology Department, Key Laboratory of Neurological Disease of Hubei Province, wuhan, China. Contact e-mail: [email protected] Background: The protein phosphatase-2A inhibitor-2 (I2PP-2A) is a nuclearoriginated protein participating in a wide variety of cellular functions and elevated in Alzheimer disease (AD) brain. However, the pathophysiological role for the elevation is illusive. Methods: We transfected pc-DNA I2PP-2A or pSUP-si I2PP-2A plasmids into N2a and HEK293 cells and detected cell viability by MTT. The levels of I2PP-2A, p53 and Akt were detected by western blots and immunohistochemistry. The transcription activity of p53 or Akt was detected by Chromatin immunoprecipitation. Results: We found that I2PP-2A could regulate the expression of multiple AD-related proteins. Among them, only p53 and Akt expression in mRNA and protein levels was regulated synchronously by I2PP-2A in N2a and HEK293 cells; the
Poster Presentations P2
with serum free medium for another 24 h (starvation). Results: The N2a cells overexpressing tau were more resistent to serum withdrawal-induced apoptosis. The protective effect was positively correlated with the relatively increased phosphrylation level of tau. We also found that the Akt was activated in cells overexpressing tau. Simultaneously, the phosphorylation level of FOXO3 increased (representing decreased activity) and the expression of p53 and Bax decreased. The antiapoptotic effects of tau almost disappeared by inhibition of Akt. Upon serum starvation, the expression level of catalytic subunit of PI3K (p110) increased significantly in cells overexpressing tau, whereas the relative level of the phosphorylated p85 was not changed. Conclusions: Our findings suggest that tau leads the cells to escape apoptosis by activating PI3K/Akt pathway and the activation of PI3K. P2-166
VARIOUS LENGTHS OF C-TERMINAL FRAGMENTS OF AMYLOID PRECURSOR PROTEIN INDUCE NEUROTOXICITY, NOT ONLY C31
Yoori Choi1, Minji Kim1, Hye-Sun Kim2, Yoo-Hun Suh1, 1Creative Research Initiative Center for Alzheimer dementia, Seoul, Republic of Korea; 2 Department of Pharmacology, Bundang Hospital, College of Medicine, Seoul National University, Seoul, Republic of Korea. Contact e-mail: [email protected] Background: Abnormal proteolytic cleavages of amyloid precursor protein (APP) generate amyloid beta peptide (Ab) and carboxy-terminal fragments of APP (APP-CTFs). As ‘amyloid cascade hypotheses’ have been lead the cause of Alzheimer’s disease (AD), senile plaque composed with Ab is one of the pathological hallmarks of AD. A recent study has shown that C31, derived from cleavage of APP at Asp664 site by caspase, is inducer of apoptosis and linked Ab-mediated toxicity. The APP transgenic mice with D664A mutation still produce Ab and senile plaque, but prevention of C31 generation protects neuronal proliferation, synaptic transmission, synaptic plasticity deficits and behavioral abnormality. In this study, we observed that neurotoxicity of CTFs is involved whether C31 downstream or NPTY domain which interact with other adaptor proteins. Methods: Primary cortical neuron cuture, transfection, LDH assay. Results: We show that LDH release was increased by various APP-CTFs versus it was reduced by various APP-CTFs with mutated cleavage site at amino acid 664 or NPTY deletion. There is no significant difference in LDH release between various lengths of APP-CTFs. Conclusions: These results indicate that various APP-CTFs including NPTY domain play a critical role in neuronal cell death, not only APP-C31 generation. P2-167
SUBREGION-SPECIFIC VULNERABILITY TO ENDOPLASMIC RETICULUM STRESS-INDUCED NEUROTOXICITY IN RAT HIPPOCAMPAL NEURONS
Kumiko Ishige, Yasuhiro Kosuge, Yoshihisa Ito, Nihon University, Funabashi-shi, Japan. Contact e-mail: [email protected] Background: It is well known that in certain disease states, including ischemia and Alzheimer’s disease, neurodegeneration occurs in the hippocampus, and that vulnerability to neuronal death is area-dependent. The present study investigated the mechanism of area-dependent vulnerability to neuronal death under endoplasmic reticulum stress conditions induced by tunicamycin (TM), using rat organotypic hippocampal cultures (OHC) and hippocampal slices. Methods: The OHC was prepared from 6-7-day-old Wistar rats, in accordance with the Ethical Guidelines for Animal Experiments at Nihon University. Animals were deeply anesthetized, and the hippocampi were removed by dissection from brains. Transverse slices (400 mm thick) were prepared and were placed on a membrane insert. Cultures were fed with 1 ml of culture medium consisting of 50% minimal essential medium, 25% horse serum, and 25% Hanks’ balanced salt solution. Experiments were performed using the OHC cultured for 3 weeks in vitro. Results: Analysis of propidium iodide uptake showed that TM induced neuronal death in a concentration-dependent manner (20w80 mg/mL), and that the rank order of vulnerability among hippocampal subregions was dentate gyrus (DG) > CA1 > CA3. Results of immu-
nohistochemistry using hippocampal slices also showed that procaspase12-positive cells in area CA3 were significantly fewer than those in area CA1 and the DG. Moreover, procurement of neurons in areas CA1, CA3 and the DG by laser microdissection, followed by Western blot analysis, also revealed that the level of procaspase-12 in area CA3 was significantly lower than those in area CA1 and the DG. Pretreatment with zATAD-fmk, a cell-permeable caspase-12-selective inhibitor significantly attenuated the TM-induced increase of PI fluorescence in the CA1 and DG subregion but not in area CA3. Conclusions: In conclusion, the present study has revealed that TM-induced neuronal death is subregion-dependent in the hippocampus, and that the subregion-dependent vulnerability to TM is attributable, at least in part, to the expression level of endogenous procaspase-12 in each area of the hippocampus. The present results also demonstrate that a combination of LMD and Western blotting allows direct measurement of the levels of specific proteins in each subregion in hippocampal slices. P2-168
REGULATOR OF CALCINEURIN 1 (RCAN1) IS DEGRADED BY UBIQUITIN-PROTEASOME AND CHAPERONE-MEDIATED AUTOPHAGY PATHWAY
Xiulian Sun1, Heng Liu1, Pin Wang1, Weihong Song2, 1Shandong University, Jinan, China; 2University of British Columbia, Vancouver, BC, Canada. Contact e-mail: [email protected] Background: Regulator of calcineurin 1 (RCAN1) is a gene identified from the critical region of Down syndrome (DS) and has been implied in pathogenesis of Alzheimer’s disease (AD). RCAN1 expression is increased in AD brains; however, the mechanism of which is unknown. Methods: Co-ip and immunostaning were used to study the RCAN1 ubiquitination. Drug treatment as well as gene knockout by siRNA were used to interrupt macroautophagy pathway and CMA pathway. Results: Here our study investigated the molecular mechanism of RCAN1 protein degradation. Our results showed that RCAN1 is ubiquitinated and degraded by ubiquitin-proteasome pathway. Furthermore, we showed that RCAN1 protein expression is remarkably increased by inhibition of lysosome in a time- and dosage-dependent manner. Inhibition of macroautophagy reduced RCAN1 expression, indicating that RCAN1 degradation is not through macroautophagy pathway. Further evidence showed that disruption of chaperone-mediated autophagy (CMA) increased RCAN1 expression. Our results showed that RCAN1 contains two CMA recognition motifs, by which RCAN1 can be degraded through CMA-lysosome pathway. Conclusions: Dysfunctions of ubiquitin-proteasome and autophagy-lysosome pathways have been implicated in neurodegenerative diseases. Elucidation of RCAN1 degradation by ubiquitin-proteasome and CMA-lysosome pathways greatly advances our understanding of AD pathogenesis. P2-169
PROTEIN PHOSPHATASE-2A INHIBITOR-2 UPREGULATES P53 AND AKT-IMPLICATION FOR A NEW MECHANISM FOR ALZHEIMER’S NEURODEGENERATION
Liu Gongping, Wei Wei, Xin Zhou, Yao Zhang, Hairong Shi, Jun Yin, Xiuqing Yao, Qun Wang, Jianzhi Wang, Pathophysiology Department, Key Laboratory of Neurological Disease of Hubei Province, wuhan, China. Contact e-mail: [email protected] Background: The protein phosphatase-2A inhibitor-2 (I2PP-2A) is a nuclearoriginated protein participating in a wide variety of cellular functions and elevated in Alzheimer disease (AD) brain. However, the pathophysiological role for the elevation is illusive. Methods: We transfected pc-DNA I2PP-2A or pSUP-si I2PP-2A plasmids into N2a and HEK293 cells and detected cell viability by MTT. The levels of I2PP-2A, p53 and Akt were detected by western blots and immunohistochemistry. The transcription activity of p53 or Akt was detected by Chromatin immunoprecipitation. Results: We found that I2PP-2A could regulate the expression of multiple AD-related proteins. Among them, only p53 and Akt expression in mRNA and protein levels was regulated synchronously by I2PP-2A in N2a and HEK293 cells; the