- Email: [email protected]
reperfusion induces hippocampal tau hyperphosphorylation in rats via ER stress
Poster Presentations P1
P1-351
SPLICING FACTOR SC35 REGULATES THE ALTERNATIVE SPLICING OF TAU EXON 10
Wei Qian1, Hongwei Liang2, Jianhua Shi2, Jianlan Gu1, Xiaolin Wu2, Shibao Wang2, Cheng-Xin Gong3, Fei Liu2,3, 1Department of Biochemistry, Medical School, Nantong University, Nantong, China; 2Jiangsu Key Laboratory of Neuroregeneration, Medical School, Nantong University, Nantong, China; 3Department of Neurochemistry, New York State Institute for Basic Research in Developmental Disabilities, Staten Island, NY, USA. Contact e-mail: [email protected] Background: Abnormal alternative splicing of tau exon 10 results in imbalance of 3R-tau and 4R-tau expression, which is sufficient to cause neurofibrillary degeneration. Splicing factor SC35, a member of the superfamily of the serine/arginine-rich (SR) proteins, promotes tau exon 10 inclusion. The function of SC35 is tightly regulated by phosphorylation. Recently, we have found that 3R-tau and 4R-tau is imbalanced and Dyrk1A (dual-specificity tyrosine-phosphorylated and regulated kinase 1A) phosphorylates splicing factor SF2/ASF and suppresses its role of promotion of tau exon 10 inclusion. Overexpression of Dyrk1A due to trisome in Down syndrome brain may contribute to the dysregulation of tau exon 10 splicing. Methods: By mutation of SC35 and mini-tau gene, pCI/SI9-SI10 which consists of exons 9, 10, 11, a part of intron 9 and intron 10 and RNA-chromatin immunoprecipitation (RNA-CHIP), we elucidated the molecular mechanism by which SC35 works on tau exon 10 splicing and whether and how Dyrk1A regulates SC35-promoted tau exon 10 inclusion in vitro and in cultured cells. Results: We observed that tau pre-mRNA could be precipitated by antiSC35 antibody. Mutation of the SC35-like exonic splicing enhancer located at exon 10 of tau affected both the binding of SC35 to tau pre-mRNA and promotion of tau exon 10 inclusion, suggesting that SC35 acts on the SC35-like exonic splicing enhancer to promote tau exon 10 inclusion. By deletion mutation, we found that RS domain of SC35 is required for the promotion of tau exon 10 inclusion. Dyrk1A phosphorylated SC35 in vitro and interacted with it in cultured cells. Overexpression of Dyrk1A affected the interaction between SC35 and tau pre-mRNA and suppressed SC35-promoted tau exon 10 inclusion. Conclusions: Upregulation of Dyrk1A in Down syndrome brain may cause dysregulation of tau exon 10 splicing through SF2/ASF, SC35, and other splicing factors, leading to the imbalance in 3R-tau and 4R-tau expression, which may initiate or accelerate tau pathology and cause neurofibrillary degeneration in DS brain. P1-352
ACCUMULATION OF PHOSPHORYLATED TAU IN NEURITES IS THE EARLIEST CYTOSKELETAL ABNORMALITY IN AGING AND ALZHEIMER’S DISEASE
Ramez Hoveydai, David Riascos, Changiz Geula, Northwestern University, Chicago, IL, USA. Contact e-mail: [email protected] Background: Cytoskeletal abnormalities in the form of pre-tangles and tangles composed of abnormally phosphorylated tau (PH-Tau) are a constant feature of the brain in Alzheimer’s disease (AD) and normal aging. In AD, PH-Tau is also present in dystrophic neurites and neuropil threads thought to represent primarily dendritic abnormalities. Given that dendrites are the primary sites of synaptic contact, we sought to determine the time course and epitope of PH-Tau accumulation in neurites in the aging-AD continuum as a potential mechanism of synaptic dysfunction and loss. Methods: Sections from brains of young (20-45), middle aged (46-60), old (65 and above) and AD cases were stained immunohistochemically using antibodies to the earliest appearing PH-Tau epitopes (Alz-50 and AT8), an intermediate appearing epitope (PHF-1) and a late appearing epitope (PH-Tau Ser-262). The temporal appearance of PH-Tau in neurites was compared to that in pre-tangles and tangles. The total length of neurites in the entorhinal cortex of each case / epitope was estimated using the Space Balls unbiased stereological probe. Results: All brains examined contained PH-Tau-positive neurites in the entorhinal cortex. The total length of neurites was lowest in the young group and displayed a progressive increase in the middle-aged, old and AD cases. In the youngest cases, PH-Tau-positive neurites were present in the absence of pre-tangles and tangles. As the density of neurites increased,
S275
a few tangles or pre-tangles were observed. PH-Tau-positive neurites then spread to temporal neocortex in the absence of local tangles or pre-tangles. Only in the aged and AD cases were thioflavin S-positive neurites observed. The AT8 epitope was present in the largest total length of neurites. AT8, Alz50 and PHF-1 were observed in early neurites and increased during aging and AD. The PH-Ser262 epitope was sparse in normal cases and visualized a sizeable length of neurites only in AD. Conclusions: PH-tau in neurites represents the first cytoskeletal abnormality in aging and AD and displays the same epitope map and progression as that of tangles and pre-tangles. Accumulation of PH-tau in neurites, which most likely represent dendrites that are sites of synaptic contact, is likely to contribute to synaptic abnormalities in AD. P1-353
ISCHEMIA/REPERFUSION INDUCES HIPPOCAMPAL TAU HYPERPHOSPHORYLATION IN RATS VIA ER STRESS
Zan-chao Liu, Qing Tian, Key Laboratory of Neurological Disease of National Education Ministry,Tongji Medical College, Wu Han, China. Contact e-mail: [email protected] Background: The main clinical symptoms of Alzhermer’s Disease (AD) patients are cognitive impairment and the degraded learning and memory ability. The amount of intracellular neurofibrillary (NFTs) formed by hyperphosphorylated tau is closely positivecorrelated with the clinical dementia degree of AD patients. Phosphorylation of tau is mainly regulated by protein kinase glycogen synthase kinase-3 (GSK-3b) and phosphatase protein phosphatase 2A (PP2A). Brain Ischemia/reperfusion is a frequent important pathophysiological process of brain injury. Some scholars have proposed ‘‘ischemia / reperfusion’’ hypothesis of AD and regard the reischemia/reperfusion as an important pathophysiological pathogenesis in the sporadic AD. Ischemia/reperfusion can cause cell stress response, such as endoplasmic reticulum stress (ER Stress). GRP78/Bip as an inherent molecular chaperone in the endoplasmic reticulum is the marker of ER stress. The increased ER stress and expression of Bip were found in the AD brain. Objective: To investigate that whether ischemia or ischemia/reperfusion can affect tau phosphorylation via the ER stress. Methods: The SD rats were used to make the animal models of cerebral ischemia 20min and ischemia/reperfusion (1d, 3d, 5d) by bilateral carotid artery occlusion and the inhibitor of ER stress, 4-phenyl butyric acid (PBA) was used. The phosphorylation levels of tau protein, the expressions and activities of GSK-3b, PP2A catalytic subunit (PP2Ac) were observed. Results: The results showed that reperfusion after ischemia 20min induced significantly increased tau phosphorylation in hippocampus at the Ser396, Ser404, Ser198/199/202 sites and increased Bip expression after ischemia 20min and reperfusion (1d, 3d, 5d). The expressions of GSK-3b and PP2Ac had no significant change after ischemia and ischemia/reperfusion. The increased phosphorylation levels of GSK-3b at Tyr216 after ischemia and reperfusion and decreased phosphorylation levels at Ser9 after reperfused 3d and 5d, and increased phosphorylation levels at Tyr307 of PP2Ac after reperfusion were observed. We used PBA to treat the ischemia/reperfusion rat models and found that PBA could significantly reduce the expression of Bip and the phosphorylation of tau at Ser396, Ser198/199/202 phosphorylation sites. Conclusions: These results confirmed that ischemia/reperfusion could cause ER stress, which induce hyperphosphorylation of tau via activating GSK-3b and inhibiting PP2A. P1-354
THE MISLOCALIZATION OF TAU TO DENDRITIC SPINES MEDIATES SYNAPTIC DYSFUNCTION INDEPENDENT OF NEURODEGENERATION
Brian R. Hoover, Jianjun Su, Miranda N. Reed, Rachel D. Penrod, Lorene M. Lanier, Li-Lian Yuan, Karen H. Ashe, Dezhi Liao, University of Minnesota, Minneapolis, MN, USA. Contact e-mail: [email protected] Background: The microtubule-associated protein tau accumulates in the somatodendritic compartment of neurons in patients with tauopathies, but its effects, if any, on the function of dendritic spines has not yet been reported. Although several mechanisms have been proposed to explain how the accumulation of tau leads to neurodegeneration, few studies have attempted to understand how tau affects neuronal function, independent of
P1-351
SPLICING FACTOR SC35 REGULATES THE ALTERNATIVE SPLICING OF TAU EXON 10
Wei Qian1, Hongwei Liang2, Jianhua Shi2, Jianlan Gu1, Xiaolin Wu2, Shibao Wang2, Cheng-Xin Gong3, Fei Liu2,3, 1Department of Biochemistry, Medical School, Nantong University, Nantong, China; 2Jiangsu Key Laboratory of Neuroregeneration, Medical School, Nantong University, Nantong, China; 3Department of Neurochemistry, New York State Institute for Basic Research in Developmental Disabilities, Staten Island, NY, USA. Contact e-mail: [email protected] Background: Abnormal alternative splicing of tau exon 10 results in imbalance of 3R-tau and 4R-tau expression, which is sufficient to cause neurofibrillary degeneration. Splicing factor SC35, a member of the superfamily of the serine/arginine-rich (SR) proteins, promotes tau exon 10 inclusion. The function of SC35 is tightly regulated by phosphorylation. Recently, we have found that 3R-tau and 4R-tau is imbalanced and Dyrk1A (dual-specificity tyrosine-phosphorylated and regulated kinase 1A) phosphorylates splicing factor SF2/ASF and suppresses its role of promotion of tau exon 10 inclusion. Overexpression of Dyrk1A due to trisome in Down syndrome brain may contribute to the dysregulation of tau exon 10 splicing. Methods: By mutation of SC35 and mini-tau gene, pCI/SI9-SI10 which consists of exons 9, 10, 11, a part of intron 9 and intron 10 and RNA-chromatin immunoprecipitation (RNA-CHIP), we elucidated the molecular mechanism by which SC35 works on tau exon 10 splicing and whether and how Dyrk1A regulates SC35-promoted tau exon 10 inclusion in vitro and in cultured cells. Results: We observed that tau pre-mRNA could be precipitated by antiSC35 antibody. Mutation of the SC35-like exonic splicing enhancer located at exon 10 of tau affected both the binding of SC35 to tau pre-mRNA and promotion of tau exon 10 inclusion, suggesting that SC35 acts on the SC35-like exonic splicing enhancer to promote tau exon 10 inclusion. By deletion mutation, we found that RS domain of SC35 is required for the promotion of tau exon 10 inclusion. Dyrk1A phosphorylated SC35 in vitro and interacted with it in cultured cells. Overexpression of Dyrk1A affected the interaction between SC35 and tau pre-mRNA and suppressed SC35-promoted tau exon 10 inclusion. Conclusions: Upregulation of Dyrk1A in Down syndrome brain may cause dysregulation of tau exon 10 splicing through SF2/ASF, SC35, and other splicing factors, leading to the imbalance in 3R-tau and 4R-tau expression, which may initiate or accelerate tau pathology and cause neurofibrillary degeneration in DS brain. P1-352
ACCUMULATION OF PHOSPHORYLATED TAU IN NEURITES IS THE EARLIEST CYTOSKELETAL ABNORMALITY IN AGING AND ALZHEIMER’S DISEASE
Ramez Hoveydai, David Riascos, Changiz Geula, Northwestern University, Chicago, IL, USA. Contact e-mail: [email protected] Background: Cytoskeletal abnormalities in the form of pre-tangles and tangles composed of abnormally phosphorylated tau (PH-Tau) are a constant feature of the brain in Alzheimer’s disease (AD) and normal aging. In AD, PH-Tau is also present in dystrophic neurites and neuropil threads thought to represent primarily dendritic abnormalities. Given that dendrites are the primary sites of synaptic contact, we sought to determine the time course and epitope of PH-Tau accumulation in neurites in the aging-AD continuum as a potential mechanism of synaptic dysfunction and loss. Methods: Sections from brains of young (20-45), middle aged (46-60), old (65 and above) and AD cases were stained immunohistochemically using antibodies to the earliest appearing PH-Tau epitopes (Alz-50 and AT8), an intermediate appearing epitope (PHF-1) and a late appearing epitope (PH-Tau Ser-262). The temporal appearance of PH-Tau in neurites was compared to that in pre-tangles and tangles. The total length of neurites in the entorhinal cortex of each case / epitope was estimated using the Space Balls unbiased stereological probe. Results: All brains examined contained PH-Tau-positive neurites in the entorhinal cortex. The total length of neurites was lowest in the young group and displayed a progressive increase in the middle-aged, old and AD cases. In the youngest cases, PH-Tau-positive neurites were present in the absence of pre-tangles and tangles. As the density of neurites increased,
S275
a few tangles or pre-tangles were observed. PH-Tau-positive neurites then spread to temporal neocortex in the absence of local tangles or pre-tangles. Only in the aged and AD cases were thioflavin S-positive neurites observed. The AT8 epitope was present in the largest total length of neurites. AT8, Alz50 and PHF-1 were observed in early neurites and increased during aging and AD. The PH-Ser262 epitope was sparse in normal cases and visualized a sizeable length of neurites only in AD. Conclusions: PH-tau in neurites represents the first cytoskeletal abnormality in aging and AD and displays the same epitope map and progression as that of tangles and pre-tangles. Accumulation of PH-tau in neurites, which most likely represent dendrites that are sites of synaptic contact, is likely to contribute to synaptic abnormalities in AD. P1-353
ISCHEMIA/REPERFUSION INDUCES HIPPOCAMPAL TAU HYPERPHOSPHORYLATION IN RATS VIA ER STRESS
Zan-chao Liu, Qing Tian, Key Laboratory of Neurological Disease of National Education Ministry,Tongji Medical College, Wu Han, China. Contact e-mail: [email protected] Background: The main clinical symptoms of Alzhermer’s Disease (AD) patients are cognitive impairment and the degraded learning and memory ability. The amount of intracellular neurofibrillary (NFTs) formed by hyperphosphorylated tau is closely positivecorrelated with the clinical dementia degree of AD patients. Phosphorylation of tau is mainly regulated by protein kinase glycogen synthase kinase-3 (GSK-3b) and phosphatase protein phosphatase 2A (PP2A). Brain Ischemia/reperfusion is a frequent important pathophysiological process of brain injury. Some scholars have proposed ‘‘ischemia / reperfusion’’ hypothesis of AD and regard the reischemia/reperfusion as an important pathophysiological pathogenesis in the sporadic AD. Ischemia/reperfusion can cause cell stress response, such as endoplasmic reticulum stress (ER Stress). GRP78/Bip as an inherent molecular chaperone in the endoplasmic reticulum is the marker of ER stress. The increased ER stress and expression of Bip were found in the AD brain. Objective: To investigate that whether ischemia or ischemia/reperfusion can affect tau phosphorylation via the ER stress. Methods: The SD rats were used to make the animal models of cerebral ischemia 20min and ischemia/reperfusion (1d, 3d, 5d) by bilateral carotid artery occlusion and the inhibitor of ER stress, 4-phenyl butyric acid (PBA) was used. The phosphorylation levels of tau protein, the expressions and activities of GSK-3b, PP2A catalytic subunit (PP2Ac) were observed. Results: The results showed that reperfusion after ischemia 20min induced significantly increased tau phosphorylation in hippocampus at the Ser396, Ser404, Ser198/199/202 sites and increased Bip expression after ischemia 20min and reperfusion (1d, 3d, 5d). The expressions of GSK-3b and PP2Ac had no significant change after ischemia and ischemia/reperfusion. The increased phosphorylation levels of GSK-3b at Tyr216 after ischemia and reperfusion and decreased phosphorylation levels at Ser9 after reperfused 3d and 5d, and increased phosphorylation levels at Tyr307 of PP2Ac after reperfusion were observed. We used PBA to treat the ischemia/reperfusion rat models and found that PBA could significantly reduce the expression of Bip and the phosphorylation of tau at Ser396, Ser198/199/202 phosphorylation sites. Conclusions: These results confirmed that ischemia/reperfusion could cause ER stress, which induce hyperphosphorylation of tau via activating GSK-3b and inhibiting PP2A. P1-354
THE MISLOCALIZATION OF TAU TO DENDRITIC SPINES MEDIATES SYNAPTIC DYSFUNCTION INDEPENDENT OF NEURODEGENERATION
Brian R. Hoover, Jianjun Su, Miranda N. Reed, Rachel D. Penrod, Lorene M. Lanier, Li-Lian Yuan, Karen H. Ashe, Dezhi Liao, University of Minnesota, Minneapolis, MN, USA. Contact e-mail: [email protected] Background: The microtubule-associated protein tau accumulates in the somatodendritic compartment of neurons in patients with tauopathies, but its effects, if any, on the function of dendritic spines has not yet been reported. Although several mechanisms have been proposed to explain how the accumulation of tau leads to neurodegeneration, few studies have attempted to understand how tau affects neuronal function, independent of