- Email: [email protected]
Role of the amyloid precursor protein intracellular domain for the cellular proteome
S402 P2-301
Poster Presentations P2 MOLECULAR MECHANISMS UNDERLYING SONIC HEDGEHOG INDUCTION BY AMYLOID BETA-PEPTIDE
Yu-Hsing Hong, Ding-I Yang, Institute of Brain Science, National Yang-Ming University, Taipei, Taiwan. Contact e-mail: smallcat501@ hotmail.com Background: Alzheimer’s disease (AD) is a neurodegenerative disorder that causes cognitive impairment. Neuronal cell death in AD is caused by accumulation of senile plaques mainly composed of amyloid beta-peptide (Ab). Sonic hedgehog (Shh) is a morphogen that plays critical roles in regulation of neural development. Recent studies have revealed several nuclear factor-kappaB (NF-kB) binding sites in the promoter region of Shh gene. Ab is known to activate NF-kB. However, whether Ab may directly increase Shh expression has never been explored. The goal of the present study is therefore to test whether Ab may induce Shh expression and the underlying mechanisms that involve NF-kB and cGMP-dependent protein kinase (PKG) in rat cortical neurons. Methods: Primary neuronal cultures were prepared from fetal rat cortices at embryonic day 18. Cortical cultures were exposed to Ab25-35 (10 microM) and Ab1-42 (5 microM) at various time points. Quantitative real-time RT-PCR and Western blotting were used to evaluate Ab induction of Shh at mRNA and protein levels, respectively. Confocal immunocytochemistry was applied to identify the cell type that expresses Shh in our mixed cortical culture. Pharmacological inhibitors including SN50 and KT5823, the respective inhibitor of NF-kB and PKG, were used to establish the causative relationship between these mediators and Shh induction mediated by Ab. Results: We observed a time-dependent increase of Shh expression, at both mRNA and protein levels, following exposure to Ab25-35 and Ab1-42. Expression of Shh was localized in cortical neurons as revealed by confocal microscopy. This Ab effect could be abolished by co-treatment with the NF-kB inhibitor SN50 (18 microM) and the PKG inhibitor KT5823 (2 microM). Exogenous application of the 8-Br-cGMP, a cGMP analogue capable of activating PKG, directly induced Shh expression in cortical neurons. Conclusions: These results suggest that NF-kB and PKG mediate Ab-induced Shh expression in cultured rat cortical neurons.
P2-302
PROGRANULIN ACTIVATES MAP KINASE AND PROTECTS NEURONS AGAINST GLUTAMATE TOXICITY AND OXIDATIVE STRESS
Junichi Shioi, Jindong Xu, Patty Lan, Anastasios Georgakopoulos, Nikolaos K. Robakis, Mount Sinai School of Medicine, New York, NY, USA. Contact e-mail: [email protected] Background: Frontotemporal lobar degeneration (FTLD) accounts for about 5-10% of all dementia. Clinically, FTLD is characterized by personality disturbances, progressive language impairment, cognitive deterioration and dementia. Pathologically, it is marked by severe neurodegeneration in the frontal and/or temporal regions of the cerebral cortex. About 20-30% of all FTLD cases display autosomal dominant patterns of inheritance caused by mutations in the genes encoding microtubule-associated protein tau, progranulin (PGRN) and to a lesser extent in the genes of VCP and CHMP2B. PGRN is a secreted protein composed of 593 amino acids. Although the functions of PGRN have been studied extensively in peripheral tissues, its function in the central nervous system is mostly unknown. Interestingly, a PGRN role is also suggested in other neurodegenerative diseases including Alzheimer’s disease. Methods: His-tagged recombinant PGRN was prepared from the culture media of stably-transfected HEK293T cells and purified by Ni-beads column chromatography. Cortical neurons were prepared from 15 ˇ1/2 day rat embryos. Neuroprotection is evaluated by MTT assay, based on mitochondrial metabolic activity, and Hoechst staining, a commonly used protocol that evaluates cell death by nuclear fragmentation. To identify neuronal signaling pathways modulated by PGRN, cell extracts from treated neurons were subjected to Western blotting using relevant anti-phosphoprotein antibodies. Results: MTT assay showed that treatment of neuronal cultures with 50mM glutamate decreased neuronal cell
viability to w60% while pretreatment with 35nM PGRN for 24 hours significantly decreased the glutamate-induced neuronal damage. PGRN pretreatment alone resulted in no significant change of cell viability. Nuclear staining also showed that PGRN rescued neurons from glutamate-induced neurotoxicity while bovine serum albumin had no protective effect on this toxicity. In MTT assay, treatment with 25mM of H2O2 reduced neuronal viability to w45% while pretreatment with 35nM PGRN significantly protected neurons from oxidative damage and increased cell survival to w55%. As a negative control, BSA had no effect on cell viability. PGRN treatment at 35nM on primary neurons promoted a significant increase in phosphorylation of p42/44 MAPK at Thr202 and Tyr204 residues while Src kinase phosphorylation was not affected. Conclusions: PGRN may promote neuronal survival by stimulating MAPK signaling pathways.
P2-303
ROLE OF THE AMYLOID PRECURSOR PROTEIN INTRACELLULAR DOMAIN FOR THE CELLULAR PROTEOME.
Thorsten Mu¨ller1, Andreas Schro¨tter1, Christina Looße1, Anke Schnabel1, Carsten Theiß2, Katrin Marcus1, 1Functional Proteomics, Ruhr-University Bochum, Bochum, Germany; 2Department of Cytology, Ruhr-University Bochum, Bochum, Germany. Contact e-mail: [email protected] Background: Within the course of Alzheimer’s disease, APP undergoes proteolytic processing. Successive cleavage by ß- and g-secretase activity results in the generation of the amyloid precursor protein intracellular domain (AICD). This domain is believed to translocate to the nucleus and to establish a nuclear complex in combination with the adapter protein Fe65 and the histone acetyl transferase TIP60. Methods: Using transfection, microinjection, and fluorescence microscopy we studied the cellular localization of AICD and its associated proteins in HEK293T and Purkinje cells. A 1D gel - mass spectrometry based approach was used to analyze the proteome of AICD, FE65, and TIP60 transfected HEK293T cells vs. controls. For data analysis we applied spectral counting which is a well established method in our lab. Results: Fluorescence imaging demonstrates the characteristic nuclear spot-like phenotype of Fe65/TIP60 transfected HEK293T and Purkinje cells. Nuclear microinjected AICD is able to change the phenotype to more speckle-like aggregates within the nucleus as demonstrated by video imaging. Proteomic analysis of AICD, FE65, TIP60 transfected cells vs. controls revealed significant changes on the cellular proteome. Of note, we found significant changes for proteins involved in vesicle transport. Conclusions: Studying the proteome of cells transfected with the AICD-dependent transcriptionally active complex identified proteins with differences in abundance. Some of the candidates are already well known in the context of Morbus Alzheimer pathophysiology. In addition, we identified AICD-dependent proteins for the first time, which have a crucial impact in cellular transport mechanisms. Latter were suggested to play a role in early stages of Alzheimer’s disease pointing to the importance of studying AICD-dependent mechanisms.
P2-304
CHOLESTEROL CAUSES ALZHEIMER PATHOLOGY THROUGH AKT/MTOR INHIBITION
Othman Ghribi, Gurdeep Marwarha, UND School of Medicine, Grand Forks, ND, USA. Contact e-mail: [email protected] Background: Alzheimer’s disease (AD) is a complex disorder for which there is currently no medicine that can prevent its progression. The causes of AD are not yet completely understood but genetic, environmental, and dietary factors may contribute to the pathogenesis of this devastating disorder. Epidemiological, genome-wide association, postmortem, animal, and cellular studies strongly suggest that high plasma cholesterol is a risk factor for AD. However, the mechanisms by which cholesterol may cause AD are ill-defined. We tested the hypothesis that cholesterol-enriched diets cause AD hallmarks by mechanisms involving the down-regulation of the Akt/ mTOR pathway. Methods: New Zealand white male rabbits (4 6 0.4 kg
Poster Presentations P2 MOLECULAR MECHANISMS UNDERLYING SONIC HEDGEHOG INDUCTION BY AMYLOID BETA-PEPTIDE
Yu-Hsing Hong, Ding-I Yang, Institute of Brain Science, National Yang-Ming University, Taipei, Taiwan. Contact e-mail: smallcat501@ hotmail.com Background: Alzheimer’s disease (AD) is a neurodegenerative disorder that causes cognitive impairment. Neuronal cell death in AD is caused by accumulation of senile plaques mainly composed of amyloid beta-peptide (Ab). Sonic hedgehog (Shh) is a morphogen that plays critical roles in regulation of neural development. Recent studies have revealed several nuclear factor-kappaB (NF-kB) binding sites in the promoter region of Shh gene. Ab is known to activate NF-kB. However, whether Ab may directly increase Shh expression has never been explored. The goal of the present study is therefore to test whether Ab may induce Shh expression and the underlying mechanisms that involve NF-kB and cGMP-dependent protein kinase (PKG) in rat cortical neurons. Methods: Primary neuronal cultures were prepared from fetal rat cortices at embryonic day 18. Cortical cultures were exposed to Ab25-35 (10 microM) and Ab1-42 (5 microM) at various time points. Quantitative real-time RT-PCR and Western blotting were used to evaluate Ab induction of Shh at mRNA and protein levels, respectively. Confocal immunocytochemistry was applied to identify the cell type that expresses Shh in our mixed cortical culture. Pharmacological inhibitors including SN50 and KT5823, the respective inhibitor of NF-kB and PKG, were used to establish the causative relationship between these mediators and Shh induction mediated by Ab. Results: We observed a time-dependent increase of Shh expression, at both mRNA and protein levels, following exposure to Ab25-35 and Ab1-42. Expression of Shh was localized in cortical neurons as revealed by confocal microscopy. This Ab effect could be abolished by co-treatment with the NF-kB inhibitor SN50 (18 microM) and the PKG inhibitor KT5823 (2 microM). Exogenous application of the 8-Br-cGMP, a cGMP analogue capable of activating PKG, directly induced Shh expression in cortical neurons. Conclusions: These results suggest that NF-kB and PKG mediate Ab-induced Shh expression in cultured rat cortical neurons.
P2-302
PROGRANULIN ACTIVATES MAP KINASE AND PROTECTS NEURONS AGAINST GLUTAMATE TOXICITY AND OXIDATIVE STRESS
Junichi Shioi, Jindong Xu, Patty Lan, Anastasios Georgakopoulos, Nikolaos K. Robakis, Mount Sinai School of Medicine, New York, NY, USA. Contact e-mail: [email protected] Background: Frontotemporal lobar degeneration (FTLD) accounts for about 5-10% of all dementia. Clinically, FTLD is characterized by personality disturbances, progressive language impairment, cognitive deterioration and dementia. Pathologically, it is marked by severe neurodegeneration in the frontal and/or temporal regions of the cerebral cortex. About 20-30% of all FTLD cases display autosomal dominant patterns of inheritance caused by mutations in the genes encoding microtubule-associated protein tau, progranulin (PGRN) and to a lesser extent in the genes of VCP and CHMP2B. PGRN is a secreted protein composed of 593 amino acids. Although the functions of PGRN have been studied extensively in peripheral tissues, its function in the central nervous system is mostly unknown. Interestingly, a PGRN role is also suggested in other neurodegenerative diseases including Alzheimer’s disease. Methods: His-tagged recombinant PGRN was prepared from the culture media of stably-transfected HEK293T cells and purified by Ni-beads column chromatography. Cortical neurons were prepared from 15 ˇ1/2 day rat embryos. Neuroprotection is evaluated by MTT assay, based on mitochondrial metabolic activity, and Hoechst staining, a commonly used protocol that evaluates cell death by nuclear fragmentation. To identify neuronal signaling pathways modulated by PGRN, cell extracts from treated neurons were subjected to Western blotting using relevant anti-phosphoprotein antibodies. Results: MTT assay showed that treatment of neuronal cultures with 50mM glutamate decreased neuronal cell
viability to w60% while pretreatment with 35nM PGRN for 24 hours significantly decreased the glutamate-induced neuronal damage. PGRN pretreatment alone resulted in no significant change of cell viability. Nuclear staining also showed that PGRN rescued neurons from glutamate-induced neurotoxicity while bovine serum albumin had no protective effect on this toxicity. In MTT assay, treatment with 25mM of H2O2 reduced neuronal viability to w45% while pretreatment with 35nM PGRN significantly protected neurons from oxidative damage and increased cell survival to w55%. As a negative control, BSA had no effect on cell viability. PGRN treatment at 35nM on primary neurons promoted a significant increase in phosphorylation of p42/44 MAPK at Thr202 and Tyr204 residues while Src kinase phosphorylation was not affected. Conclusions: PGRN may promote neuronal survival by stimulating MAPK signaling pathways.
P2-303
ROLE OF THE AMYLOID PRECURSOR PROTEIN INTRACELLULAR DOMAIN FOR THE CELLULAR PROTEOME.
Thorsten Mu¨ller1, Andreas Schro¨tter1, Christina Looße1, Anke Schnabel1, Carsten Theiß2, Katrin Marcus1, 1Functional Proteomics, Ruhr-University Bochum, Bochum, Germany; 2Department of Cytology, Ruhr-University Bochum, Bochum, Germany. Contact e-mail: [email protected] Background: Within the course of Alzheimer’s disease, APP undergoes proteolytic processing. Successive cleavage by ß- and g-secretase activity results in the generation of the amyloid precursor protein intracellular domain (AICD). This domain is believed to translocate to the nucleus and to establish a nuclear complex in combination with the adapter protein Fe65 and the histone acetyl transferase TIP60. Methods: Using transfection, microinjection, and fluorescence microscopy we studied the cellular localization of AICD and its associated proteins in HEK293T and Purkinje cells. A 1D gel - mass spectrometry based approach was used to analyze the proteome of AICD, FE65, and TIP60 transfected HEK293T cells vs. controls. For data analysis we applied spectral counting which is a well established method in our lab. Results: Fluorescence imaging demonstrates the characteristic nuclear spot-like phenotype of Fe65/TIP60 transfected HEK293T and Purkinje cells. Nuclear microinjected AICD is able to change the phenotype to more speckle-like aggregates within the nucleus as demonstrated by video imaging. Proteomic analysis of AICD, FE65, TIP60 transfected cells vs. controls revealed significant changes on the cellular proteome. Of note, we found significant changes for proteins involved in vesicle transport. Conclusions: Studying the proteome of cells transfected with the AICD-dependent transcriptionally active complex identified proteins with differences in abundance. Some of the candidates are already well known in the context of Morbus Alzheimer pathophysiology. In addition, we identified AICD-dependent proteins for the first time, which have a crucial impact in cellular transport mechanisms. Latter were suggested to play a role in early stages of Alzheimer’s disease pointing to the importance of studying AICD-dependent mechanisms.
P2-304
CHOLESTEROL CAUSES ALZHEIMER PATHOLOGY THROUGH AKT/MTOR INHIBITION
Othman Ghribi, Gurdeep Marwarha, UND School of Medicine, Grand Forks, ND, USA. Contact e-mail: [email protected] Background: Alzheimer’s disease (AD) is a complex disorder for which there is currently no medicine that can prevent its progression. The causes of AD are not yet completely understood but genetic, environmental, and dietary factors may contribute to the pathogenesis of this devastating disorder. Epidemiological, genome-wide association, postmortem, animal, and cellular studies strongly suggest that high plasma cholesterol is a risk factor for AD. However, the mechanisms by which cholesterol may cause AD are ill-defined. We tested the hypothesis that cholesterol-enriched diets cause AD hallmarks by mechanisms involving the down-regulation of the Akt/ mTOR pathway. Methods: New Zealand white male rabbits (4 6 0.4 kg