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Toxic effect of the carboxyl-terminal peptide of beta-amyloid precursor protein (APPC31) on Neuro2a cells
Poster Presentations: P2 addition, the peptides protected ischemia induced brain damage via i.v. injection. Conclusions: These results suggest that TDP-43 cleavage may play a pivotal role in caspase-dependent apoptosis in degenerative neurological diseases such as ALS and FTLD.
P2-142
TOXIC EFFECT OF THE CARBOXYL-TERMINAL PEPTIDE OF BETA-AMYLOID PRECURSOR PROTEIN (APPC31) ON NEURO2A CELLS
Shengdi Chen, Department of Neurology, Ruijin Hospital Shanghai Jiao Tong University, Shanghai, China. Background: Alzheimer’s disease is the most common neurodegenerative disease in the elderly. The pathogenesis of the disease is still unkown. b-amyloid (Ab) has been demonstrated to involve the molecular mechanism of the disease. The aim of the present study is to investigate the toxic effect of the carboxyl-terminal peptide of b-amyloid precursor protein (APPC31) on Neuro2a cells as well as its role in the toxic process in Neuro2a cells induced by Ab 42 in vitro. Methods: The plasmid vector and the APPC31 construct were transiently transfected into Neuro2a cells respectively by lipofectamine 2000. The viability of the cells was measured by the methyl thiazolyl tetrazolium (MTT) assay at 48 h after transfection, and their morphocytology was observed by 4,6-diamino-2-phenyl indole (DAPI) nucleus staining. Afterword different constructs including vector, WTAPP695, APP(D664A), the amino-terminal peptide of b-amyloid precursor protein (APPDC31) and APPC31 were transiently transfected into Neuro2a cells respectively via the same method. At 24 h after transfection Ab42 was added into the culture medium of Neuro2a cells with the desired concentration for another 24 h for cell studies. The viability and morphocytology of the cells were measured by using the MTT assay and DAPI nucleus staining, respectively. Results: When incubated in the absence of Ab42, the viability of cells transfected with vector and APPC31 construct were respective 0.81 6 0.10 and 0.88 6 0.12, and accordingly there was no significant difference between the these two groups (t ¼ -0.78, P ¼ 0.48); meanwhile no obvious cell nuclear morphological changes of apoptosis or death occurred. However when incubated in the presence of Ab42, the viability of cells transfected with vector, WTAPP695, APP(D664A), APP–3C31 and APPC31 constructs were respective 0.82 6 0.01, 0.78 6 0.03, 0.55 6 0.04, 0.81 6 0.04, 0.78 6 0.02 and 0.54 6 0.02. The viability of cells transfected with WTAPP construct and APPC31 construct decreased significantly (F ¼ 47.53, P<0.05) compared with the control group, meanwhile cells displayed condensed nuclear and even nuclear fragmentation. Conclusions: The overexpression of a certain level of APPC31 in Neuro2a cells failed to cause cell death, but this short peptide enhanced cytotoxicity induced by Ab42 in Neuro2a cells. Thus, these results provided the experimental basis for the further explication of the pathogenesis of Alzheimer’s disease.
P2-143
INTRACELLULAR SEEDED AGGREGATION AND CYTOTOXIC MODEL OF TDP-43
Takashi Nonaka, Masami Suzukake, Makiko Yamashita, Masato Hosokawa, Haruhiko Akiyama, Masato Hasegawa, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan. Background: TAR-DNA binding protein of 43 kDa (TDP-43) is the major component protein of ubiquitin-positive inclusions observed in brains of patients with frontotemporal lobar degeneration (FTLD-U) and amyotrophic lateral sclerosis (ALS). Here we report the establishment of seeded TDP43 aggregation model using cultured SH-SY5Y cells by introducing detergent-insoluble TDP-43 prepared from ALS brains as seeds for aggregation into cells. Methods: HA-tagged TDP-43 was transiently expressed in SHSY5Y cells. After incubation overnight, cells were treated with Sarkosylinsoluble fractions prepared from FTLD-U or ALS brains, using the MultiFectam reagent (Promega). Cells were harvested after 3 days-incubation, and analyzed by immunoblotting and/or using a confocal microscopy. Results: When we transduced insoluble TDP-43 of diseased brains
P309
into cells transiently expressing HA-tagged TDP-43, round inclusions composed of phosphorylated and ubiquitinated exogenous HA-tagged TDP-43 were observed. Biochemical fractionation revealed the presence of sarkosyl-insoluble HA-tagged TDP-43 positive for anti-phospho TDP-43 antibody. C-terminal fragments of TDP-43 were also observed in sarkosyl-insoluble fractions, which were very similar patterns to those of TDP-43 seeds prepared from brains using for transduction. Time course observation of cells expressing HA-tagged TDP-43 treated with brain extracts showed that full-length TDP-43 but not C-terminal fragment was first accumulated, followed by the production of C-terminal fragments, suggesting that cleavage of full-length TDP-43 to produce C-terminal fragments is not essential for intracellular TDP-43 inclusions formation. In these cells with TDP-43 inclusions, significant cell death and proteasome dysfunction were shown. Conclusions: This unique seeding model has characteristic features of affected neurons in TDP-43 proteinopathy brains including aggregation of phosphorylated and ubiquitinated TDP-43, the production of C-terminal fragments, and cell death caused by TDP-43 aggregates, and is useful for drug screening of chemicals to treat these diseases.
P2-144
ACCELERATED NEURODEGENERATION AND PHENOTYPIC SWITCH OF NEUROINFLAMMATION IN TRANSGENIC MICE EXPRESSING P301L TAU-MUTANT AND TAUTUBULIN KINASE 1
Hirohide Asai1, Maya Woodbury2, Seiko Ikezu1, Megan Varnum1, Tsuneya Ikezu1, 1Boston University School of Medicine, Boston, Massachusetts, United States; 2Boston University, Boston, Massachusetts, United States. Background: Tau-tubulin kinase-1 (TTBK1) is a serine/threonine/tyrosine protein kinase specifically expressed in the central nervous system and testis. TTBK1 directly phosphorylates the tau protein at the sites known in paired helical filament (PHF)-tau. Genetic variations of the TTBK1 gene are associated with late-onset AD in two large cohorts of Chinese and Spanish populations, further validating the importance of the TTBK1 gene in AD. We created a transgenic mouse model harboring a human genomic fragment of the TTBK1 gene. The major phenotypes of TTBK1 mice are enhanced neuroinflammation in cortical and hippocampal regions and loss of spatial learning. A double transgenic mice expressing FTDP-17-linked tau mutant and TTBK1 (Tau/TTBK1 mice) show increased accumulation of oligomeric tau protein in the forebrain and spinal cord, and enhanced reduction in motoneurons in the anterior horn of the lumber spinal cord. However, the exact role of neuroinflammation in this pathogenesis of motor neuron degeneration is poorly understood. Methods: We have generated systematically characterized the neuropathology of Tau/TTBK1, Tau, TTBK1, and non-transgenic littermates in the spinal cord. Behavioral tests include rotarod, balance beam, and grip tests. Immunohistochemistry and immunofluorescence were performed using L3-L4 spinal cord for motorneuron loss, microgliosis, astrogliosis, and their alternative/classical activation status. Primary cultured mouse motoneurons and microglia were also prepared for analyzing the effect of TTBK1 expression and silencing on axonal growth and neurotoxicity of classically activated microglia in vitro. Results: Immunohistochemical examination reveals that there is a striking switch in mononuclear phagocytes (resident microglia and infiltrating macrophages) and their activation phenotype in the spinal cord: it is mainly alternatively activated microglia in Tau mice, whereas it is predominantly classically activated peripheral macrophages in TTBK1 or TTBK1/Tau mice. Further, primary cultured mouse mortoneurons show axonal degeneration after the treatment with conditioned media derived from lipopolysaccharide-stimulated microglia, which is partially blocked by silencing of endogenous TTBK1 gene in neurons. Conclusions: These data suggest that TTBK1 plays a key role in accelerating neurodegeneration by switching the type and activation phenotypes of mononuclear phagocytes in the spinal cord, and enhancing the sensitivity to the glial neurotoxicity in inflammatory conditions. Thus, TTBK1 is a potential therapeutic target of disease.
P2-142
TOXIC EFFECT OF THE CARBOXYL-TERMINAL PEPTIDE OF BETA-AMYLOID PRECURSOR PROTEIN (APPC31) ON NEURO2A CELLS
Shengdi Chen, Department of Neurology, Ruijin Hospital Shanghai Jiao Tong University, Shanghai, China. Background: Alzheimer’s disease is the most common neurodegenerative disease in the elderly. The pathogenesis of the disease is still unkown. b-amyloid (Ab) has been demonstrated to involve the molecular mechanism of the disease. The aim of the present study is to investigate the toxic effect of the carboxyl-terminal peptide of b-amyloid precursor protein (APPC31) on Neuro2a cells as well as its role in the toxic process in Neuro2a cells induced by Ab 42 in vitro. Methods: The plasmid vector and the APPC31 construct were transiently transfected into Neuro2a cells respectively by lipofectamine 2000. The viability of the cells was measured by the methyl thiazolyl tetrazolium (MTT) assay at 48 h after transfection, and their morphocytology was observed by 4,6-diamino-2-phenyl indole (DAPI) nucleus staining. Afterword different constructs including vector, WTAPP695, APP(D664A), the amino-terminal peptide of b-amyloid precursor protein (APPDC31) and APPC31 were transiently transfected into Neuro2a cells respectively via the same method. At 24 h after transfection Ab42 was added into the culture medium of Neuro2a cells with the desired concentration for another 24 h for cell studies. The viability and morphocytology of the cells were measured by using the MTT assay and DAPI nucleus staining, respectively. Results: When incubated in the absence of Ab42, the viability of cells transfected with vector and APPC31 construct were respective 0.81 6 0.10 and 0.88 6 0.12, and accordingly there was no significant difference between the these two groups (t ¼ -0.78, P ¼ 0.48); meanwhile no obvious cell nuclear morphological changes of apoptosis or death occurred. However when incubated in the presence of Ab42, the viability of cells transfected with vector, WTAPP695, APP(D664A), APP–3C31 and APPC31 constructs were respective 0.82 6 0.01, 0.78 6 0.03, 0.55 6 0.04, 0.81 6 0.04, 0.78 6 0.02 and 0.54 6 0.02. The viability of cells transfected with WTAPP construct and APPC31 construct decreased significantly (F ¼ 47.53, P<0.05) compared with the control group, meanwhile cells displayed condensed nuclear and even nuclear fragmentation. Conclusions: The overexpression of a certain level of APPC31 in Neuro2a cells failed to cause cell death, but this short peptide enhanced cytotoxicity induced by Ab42 in Neuro2a cells. Thus, these results provided the experimental basis for the further explication of the pathogenesis of Alzheimer’s disease.
P2-143
INTRACELLULAR SEEDED AGGREGATION AND CYTOTOXIC MODEL OF TDP-43
Takashi Nonaka, Masami Suzukake, Makiko Yamashita, Masato Hosokawa, Haruhiko Akiyama, Masato Hasegawa, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan. Background: TAR-DNA binding protein of 43 kDa (TDP-43) is the major component protein of ubiquitin-positive inclusions observed in brains of patients with frontotemporal lobar degeneration (FTLD-U) and amyotrophic lateral sclerosis (ALS). Here we report the establishment of seeded TDP43 aggregation model using cultured SH-SY5Y cells by introducing detergent-insoluble TDP-43 prepared from ALS brains as seeds for aggregation into cells. Methods: HA-tagged TDP-43 was transiently expressed in SHSY5Y cells. After incubation overnight, cells were treated with Sarkosylinsoluble fractions prepared from FTLD-U or ALS brains, using the MultiFectam reagent (Promega). Cells were harvested after 3 days-incubation, and analyzed by immunoblotting and/or using a confocal microscopy. Results: When we transduced insoluble TDP-43 of diseased brains
P309
into cells transiently expressing HA-tagged TDP-43, round inclusions composed of phosphorylated and ubiquitinated exogenous HA-tagged TDP-43 were observed. Biochemical fractionation revealed the presence of sarkosyl-insoluble HA-tagged TDP-43 positive for anti-phospho TDP-43 antibody. C-terminal fragments of TDP-43 were also observed in sarkosyl-insoluble fractions, which were very similar patterns to those of TDP-43 seeds prepared from brains using for transduction. Time course observation of cells expressing HA-tagged TDP-43 treated with brain extracts showed that full-length TDP-43 but not C-terminal fragment was first accumulated, followed by the production of C-terminal fragments, suggesting that cleavage of full-length TDP-43 to produce C-terminal fragments is not essential for intracellular TDP-43 inclusions formation. In these cells with TDP-43 inclusions, significant cell death and proteasome dysfunction were shown. Conclusions: This unique seeding model has characteristic features of affected neurons in TDP-43 proteinopathy brains including aggregation of phosphorylated and ubiquitinated TDP-43, the production of C-terminal fragments, and cell death caused by TDP-43 aggregates, and is useful for drug screening of chemicals to treat these diseases.
P2-144
ACCELERATED NEURODEGENERATION AND PHENOTYPIC SWITCH OF NEUROINFLAMMATION IN TRANSGENIC MICE EXPRESSING P301L TAU-MUTANT AND TAUTUBULIN KINASE 1
Hirohide Asai1, Maya Woodbury2, Seiko Ikezu1, Megan Varnum1, Tsuneya Ikezu1, 1Boston University School of Medicine, Boston, Massachusetts, United States; 2Boston University, Boston, Massachusetts, United States. Background: Tau-tubulin kinase-1 (TTBK1) is a serine/threonine/tyrosine protein kinase specifically expressed in the central nervous system and testis. TTBK1 directly phosphorylates the tau protein at the sites known in paired helical filament (PHF)-tau. Genetic variations of the TTBK1 gene are associated with late-onset AD in two large cohorts of Chinese and Spanish populations, further validating the importance of the TTBK1 gene in AD. We created a transgenic mouse model harboring a human genomic fragment of the TTBK1 gene. The major phenotypes of TTBK1 mice are enhanced neuroinflammation in cortical and hippocampal regions and loss of spatial learning. A double transgenic mice expressing FTDP-17-linked tau mutant and TTBK1 (Tau/TTBK1 mice) show increased accumulation of oligomeric tau protein in the forebrain and spinal cord, and enhanced reduction in motoneurons in the anterior horn of the lumber spinal cord. However, the exact role of neuroinflammation in this pathogenesis of motor neuron degeneration is poorly understood. Methods: We have generated systematically characterized the neuropathology of Tau/TTBK1, Tau, TTBK1, and non-transgenic littermates in the spinal cord. Behavioral tests include rotarod, balance beam, and grip tests. Immunohistochemistry and immunofluorescence were performed using L3-L4 spinal cord for motorneuron loss, microgliosis, astrogliosis, and their alternative/classical activation status. Primary cultured mouse motoneurons and microglia were also prepared for analyzing the effect of TTBK1 expression and silencing on axonal growth and neurotoxicity of classically activated microglia in vitro. Results: Immunohistochemical examination reveals that there is a striking switch in mononuclear phagocytes (resident microglia and infiltrating macrophages) and their activation phenotype in the spinal cord: it is mainly alternatively activated microglia in Tau mice, whereas it is predominantly classically activated peripheral macrophages in TTBK1 or TTBK1/Tau mice. Further, primary cultured mouse mortoneurons show axonal degeneration after the treatment with conditioned media derived from lipopolysaccharide-stimulated microglia, which is partially blocked by silencing of endogenous TTBK1 gene in neurons. Conclusions: These data suggest that TTBK1 plays a key role in accelerating neurodegeneration by switching the type and activation phenotypes of mononuclear phagocytes in the spinal cord, and enhancing the sensitivity to the glial neurotoxicity in inflammatory conditions. Thus, TTBK1 is a potential therapeutic target of disease.