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Contribution of tauopathy to bioplar disorders: Clinical and pathological studies
Poster Presentations: P3
P3-392
INDUCTION OF NEURONAL TAU EXPRESSION BY CONDITIONED MEDIUM FROM ACTIVATED MICROGLIAL CELLS
Moonhee Lee1, Claudia Schwab1, Sheng Yu1, Edith McGeer1, Patrick McGeer2, 1Kinsmen Laboratory of Neurological Research, Vancouver, British Columbia, Canada; 2University of British Columbia, Vancouver, British Columbia, Canada. Background: The major hallmark of tauopathies, including Alzheimer’s disease (AD), is the accumulation of neurofibrillary tangles (NFTs). These are aggregates of tau which develop intracellularly, eventually causing the neurons to die. Since the tau is phosphorylated, it has generally been considered that the phosphorylation causes the aggregation. Another potential cause, which is relatively unexplored, is that neurons under stress, as occurs in AD from activated microglia, upregulate expression of tau and that excessive tau is the cause. We explored this possibility by measuring the levels of tau expression in human neurons following exposure to medium from stimulated human microglia. Methods: We exposed both differentiated NT-2 cells and SH-SY5Y cells to conditioned medium from human microglia that had been stimulated for 2 days with LPS/IFN g. We measured the expression levels of total tau and tau isoforms by Western blotting and fluorescence-labelled immunostaining with anti-human tau antibodies. We compared these levels with that of tubulin, the protein that tau normally binds to in vivo. Results: Increased expression of total tau, with the same of 3R/4R isoform distribution found in AD was observed in both NT-2 cells and SH-SY5Y cells following incubation in conditioned medium from human microglia. There was no upregulation of tubulin. Treatment of microglia with A b proteins (10 ng/ml) and proinflammatory cytokines such as IL-6 and IL-1 b (1 ng/ml each) also increased tau expression, but TNFa did not. However treatment with an anti-inflammatory cytokine IL-10 attenuated the increase in tau expression. Conclusions: Activated microglia can secrete inflammatory components which upregulate expression of 3R/ 4R tau but not tubulin. An excess of tau upregulated in this manner may be the cause of neurofibrillary tangle development in AD.
P3-393
CONTRIBUTION OF TAUOPATHY TO BIOPLAR DISORDERS: CLINICAL AND PATHOLOGICAL STUDIES
Yuko Saito1, Ayako Shioya1, Kunimasa Arima1, Masato Hasegawa2, Akira Tamaoka3, Shigeo Murayama4, 1NCNP, Kodaira, Japan; 2Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan; 3Tsukuba University, Tsukuba, Japan; 4Brain Bank for Aging Research TMGHIG, Tokyo, Japan. Background: Recent studies including lithium blocking GSK 3 beta and increased expression of SFPQ which interferes exon 10 splicing of tau gene in postmortem brain suffering from bipolar disorders may indicate that tauopathy may contribute to pathogenesis of bipolar disorders. Methods: Six consecutive autopsy cases, who received clinical diagnosis of bipolar disorders were examined. Serial six- um- thick sections were obtained from archival paraffin blocks of medulla oblongata, pons, midbrain, cerebellum, amygdala, hippocampus, basal ganglia, thalamus and frontal, temporal and parietal neocortex. The sections were stained with H.E., K.B. and Gallyas-Braak silver staining (GB), as well as immunocytochemistry of phosphorylated tau (AT8) with Ventana NX20 autoimmunostainer. Clinical information was retrospectively collected from medical charts. Results: Six cases were all males and average age of death was 67 years (52- 84) and average clinical course was 27 years (18- 33). One case received diagnosis of corticobasal degeneration (CBD) at age 73 and two cases presented with mild cognitive impairment. Neuropathologically, one case, who died of suicide with hanging, showed global hypoxic ischemic changes. CBD cases presented typical pathological picture of CBD, which accompanied Stage 1 argyrophilic grains (AGs). Other four cases also contained AGs with Stage 2 to 3 (average 2.5), emphasized in the brain stem, including inferior olivary nucleus, locus ceruleus, raphe nucleus, abducent and oculomotor nucleui and substantia nigra. One case did not contain AG. Braak NFT stage of these five
P593
cases was entorhinal. Conclusions: Our study newly proved high prevalence of 4R tauopathy in bipolar disorders (5/6 cases). Distribution of AGs were unique in predisposition in the brain stem. In one case of CBD, onset of bipolar disorders heralded 22 years before the onset of CBD and may suggest that bipolar disorders could be a risk factor for CBD rather than bipolar disorder is the onset of CBD. The presence of one case who escaped four repeat tauopathy may indicate that pathogenesis of bipolar disorders is not single.
P3-394
HYPERPHOSPHORYLATION AND CLEAVAGE OF TAU AT THE C-TERMINAL ENHANCE THE SECRETION OF TAU
Nguyen-Vi Mohamed, Vanessa Plouffe, Jessica Rivest-McGraw, Johanne Bertrand, Michel Lauzon, Nicole Leclerc, University of Montreal, Montreal, Quebec, Canada. Background: It is well established that tau pathology propagates in a predictable manner in Alzheimer’s disease (AD). Moreover, tau accumulates in the cerebrospinal fluid (CSF) of AD’s patients. The mechanisms underlying the propagation of tau pathology and its accumulation in the CSF remain to be elucidated. Recent studies have reported that human tau was secreted by neurons and non-neuronal cells when it was overexpressed indicating that tau secretion could contribute to the spreading of tau pathology in the brain and could lead to its accumulation in the CSF. In the present study, we showed that the overexpression of human tau resulted in its secretion by Hela cells. Methods: Human tau fused to the GFP tag (GFP-tau) was overexpressed in Hela cells. Forty-eight hrs after transfection, tau expression was analyzed in the cell lysate and culture medium with several anti-tau antibodies. To demonstrate that the presence of tau in the culture medium was not caused by membrane leakage from dying cells but rather by an active process of secretion, three approaches were used. First, the presence of a cytosolic protein such as tubulin in the culture medium from control and cells overexpressing tau was analyzed. Second, cell death was evaluated by the Trypan blue exclusion method and by the lactate dehydrogenase (LDH) activity measurement in the medium. Third, to demonstrate that tau was secreted by an active process by Hela cells, the secretion of tau was examined when the cells were incubated at low temperature, a condition known to decrease secretion by exocytosis. Results: The main form of tau secreted by these cells was cleaved at the C-terminal. Secreted tau was phosphorylated at several sites as reported for tau found in the CSF. Our data also revealed that the mimicking of phosphorylation at 12 sites known to be phosphorylated in AD enhanced tau secretion. Similarly, when tau was cleaved at the C-terminal, a significant increase of tau secretion was observed. Conclusions: Taken together, our results indicate that hyperphosphorylation and cleavage of tau by favoring its secretion could contribute to the propagation of tau pathology in the brain and its accumulation in the CSF.
P3-395
REANIMATING BASAL FOREBRAIN CHOLINERGIC NEURONS IN ELECTROPHYSIOLOGY BIOSIMULATIONS USING COPASI SOFTWARE
Clyde Phelix1, Greg Villareal2, Miriam Perez-Cordova3, Sofia Hernandez3, George Perry4, Luis Colom5, 1University of Texas at San Antonio, San Antonio, Texas, United States; 2CFPAL Biomedical Consultants, San Antonio, Texas, United States; 3UTB, Brownsville, Texas, United States; 4 UTSA, San Antonio, Texas, United States; 5The University of Texas at Brownsville, Brownsville, Texas, United States. Background: Alzheimer’s disease (AD) is a devastating progressive brain disorder that leads to dementia and ultimately death. Basal forebrain cholinergic neurons are mainly affected in AD. Understanding cellular and network changes is key to create rational and effective new therapeutics to correct dysfunction and prevent neuronal damage. Due to the difficulties of obtaining recordings from control and AD patient brains, we propose a novel and patent-pending approach to reconstruct neurons and network
P3-392
INDUCTION OF NEURONAL TAU EXPRESSION BY CONDITIONED MEDIUM FROM ACTIVATED MICROGLIAL CELLS
Moonhee Lee1, Claudia Schwab1, Sheng Yu1, Edith McGeer1, Patrick McGeer2, 1Kinsmen Laboratory of Neurological Research, Vancouver, British Columbia, Canada; 2University of British Columbia, Vancouver, British Columbia, Canada. Background: The major hallmark of tauopathies, including Alzheimer’s disease (AD), is the accumulation of neurofibrillary tangles (NFTs). These are aggregates of tau which develop intracellularly, eventually causing the neurons to die. Since the tau is phosphorylated, it has generally been considered that the phosphorylation causes the aggregation. Another potential cause, which is relatively unexplored, is that neurons under stress, as occurs in AD from activated microglia, upregulate expression of tau and that excessive tau is the cause. We explored this possibility by measuring the levels of tau expression in human neurons following exposure to medium from stimulated human microglia. Methods: We exposed both differentiated NT-2 cells and SH-SY5Y cells to conditioned medium from human microglia that had been stimulated for 2 days with LPS/IFN g. We measured the expression levels of total tau and tau isoforms by Western blotting and fluorescence-labelled immunostaining with anti-human tau antibodies. We compared these levels with that of tubulin, the protein that tau normally binds to in vivo. Results: Increased expression of total tau, with the same of 3R/4R isoform distribution found in AD was observed in both NT-2 cells and SH-SY5Y cells following incubation in conditioned medium from human microglia. There was no upregulation of tubulin. Treatment of microglia with A b proteins (10 ng/ml) and proinflammatory cytokines such as IL-6 and IL-1 b (1 ng/ml each) also increased tau expression, but TNFa did not. However treatment with an anti-inflammatory cytokine IL-10 attenuated the increase in tau expression. Conclusions: Activated microglia can secrete inflammatory components which upregulate expression of 3R/ 4R tau but not tubulin. An excess of tau upregulated in this manner may be the cause of neurofibrillary tangle development in AD.
P3-393
CONTRIBUTION OF TAUOPATHY TO BIOPLAR DISORDERS: CLINICAL AND PATHOLOGICAL STUDIES
Yuko Saito1, Ayako Shioya1, Kunimasa Arima1, Masato Hasegawa2, Akira Tamaoka3, Shigeo Murayama4, 1NCNP, Kodaira, Japan; 2Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan; 3Tsukuba University, Tsukuba, Japan; 4Brain Bank for Aging Research TMGHIG, Tokyo, Japan. Background: Recent studies including lithium blocking GSK 3 beta and increased expression of SFPQ which interferes exon 10 splicing of tau gene in postmortem brain suffering from bipolar disorders may indicate that tauopathy may contribute to pathogenesis of bipolar disorders. Methods: Six consecutive autopsy cases, who received clinical diagnosis of bipolar disorders were examined. Serial six- um- thick sections were obtained from archival paraffin blocks of medulla oblongata, pons, midbrain, cerebellum, amygdala, hippocampus, basal ganglia, thalamus and frontal, temporal and parietal neocortex. The sections were stained with H.E., K.B. and Gallyas-Braak silver staining (GB), as well as immunocytochemistry of phosphorylated tau (AT8) with Ventana NX20 autoimmunostainer. Clinical information was retrospectively collected from medical charts. Results: Six cases were all males and average age of death was 67 years (52- 84) and average clinical course was 27 years (18- 33). One case received diagnosis of corticobasal degeneration (CBD) at age 73 and two cases presented with mild cognitive impairment. Neuropathologically, one case, who died of suicide with hanging, showed global hypoxic ischemic changes. CBD cases presented typical pathological picture of CBD, which accompanied Stage 1 argyrophilic grains (AGs). Other four cases also contained AGs with Stage 2 to 3 (average 2.5), emphasized in the brain stem, including inferior olivary nucleus, locus ceruleus, raphe nucleus, abducent and oculomotor nucleui and substantia nigra. One case did not contain AG. Braak NFT stage of these five
P593
cases was entorhinal. Conclusions: Our study newly proved high prevalence of 4R tauopathy in bipolar disorders (5/6 cases). Distribution of AGs were unique in predisposition in the brain stem. In one case of CBD, onset of bipolar disorders heralded 22 years before the onset of CBD and may suggest that bipolar disorders could be a risk factor for CBD rather than bipolar disorder is the onset of CBD. The presence of one case who escaped four repeat tauopathy may indicate that pathogenesis of bipolar disorders is not single.
P3-394
HYPERPHOSPHORYLATION AND CLEAVAGE OF TAU AT THE C-TERMINAL ENHANCE THE SECRETION OF TAU
Nguyen-Vi Mohamed, Vanessa Plouffe, Jessica Rivest-McGraw, Johanne Bertrand, Michel Lauzon, Nicole Leclerc, University of Montreal, Montreal, Quebec, Canada. Background: It is well established that tau pathology propagates in a predictable manner in Alzheimer’s disease (AD). Moreover, tau accumulates in the cerebrospinal fluid (CSF) of AD’s patients. The mechanisms underlying the propagation of tau pathology and its accumulation in the CSF remain to be elucidated. Recent studies have reported that human tau was secreted by neurons and non-neuronal cells when it was overexpressed indicating that tau secretion could contribute to the spreading of tau pathology in the brain and could lead to its accumulation in the CSF. In the present study, we showed that the overexpression of human tau resulted in its secretion by Hela cells. Methods: Human tau fused to the GFP tag (GFP-tau) was overexpressed in Hela cells. Forty-eight hrs after transfection, tau expression was analyzed in the cell lysate and culture medium with several anti-tau antibodies. To demonstrate that the presence of tau in the culture medium was not caused by membrane leakage from dying cells but rather by an active process of secretion, three approaches were used. First, the presence of a cytosolic protein such as tubulin in the culture medium from control and cells overexpressing tau was analyzed. Second, cell death was evaluated by the Trypan blue exclusion method and by the lactate dehydrogenase (LDH) activity measurement in the medium. Third, to demonstrate that tau was secreted by an active process by Hela cells, the secretion of tau was examined when the cells were incubated at low temperature, a condition known to decrease secretion by exocytosis. Results: The main form of tau secreted by these cells was cleaved at the C-terminal. Secreted tau was phosphorylated at several sites as reported for tau found in the CSF. Our data also revealed that the mimicking of phosphorylation at 12 sites known to be phosphorylated in AD enhanced tau secretion. Similarly, when tau was cleaved at the C-terminal, a significant increase of tau secretion was observed. Conclusions: Taken together, our results indicate that hyperphosphorylation and cleavage of tau by favoring its secretion could contribute to the propagation of tau pathology in the brain and its accumulation in the CSF.
P3-395
REANIMATING BASAL FOREBRAIN CHOLINERGIC NEURONS IN ELECTROPHYSIOLOGY BIOSIMULATIONS USING COPASI SOFTWARE
Clyde Phelix1, Greg Villareal2, Miriam Perez-Cordova3, Sofia Hernandez3, George Perry4, Luis Colom5, 1University of Texas at San Antonio, San Antonio, Texas, United States; 2CFPAL Biomedical Consultants, San Antonio, Texas, United States; 3UTB, Brownsville, Texas, United States; 4 UTSA, San Antonio, Texas, United States; 5The University of Texas at Brownsville, Brownsville, Texas, United States. Background: Alzheimer’s disease (AD) is a devastating progressive brain disorder that leads to dementia and ultimately death. Basal forebrain cholinergic neurons are mainly affected in AD. Understanding cellular and network changes is key to create rational and effective new therapeutics to correct dysfunction and prevent neuronal damage. Due to the difficulties of obtaining recordings from control and AD patient brains, we propose a novel and patent-pending approach to reconstruct neurons and network