- Email: [email protected]
P3-290
Poster P3:: Tuesday Posters such as gene regulation, chromosome condensation, recombination, and replication. P3-290
ENHANCED TAU PATHOLOGY, CEREBRAL ATROPHY AND IRON DEPOSITION INDUCED BY REPETITIVE MILD BRAIN INJURY IN A TAU TRANSGENIC MOUSE
Yasumasa Yoshiyama1,2, Uryu Kunihiro2, Makoto Higuchi2, Tracy McIntosh2, Virginia M-Y Lee2, John Q. Trojanowski2, 1National Chiba East Hospital, Chiba, Japan; 2University of Pennsylvania, Philadelphia, PA, USA. Contact e-mail: [email protected] Background: Traumatic brain injury (TBI) is a risk factor for Alzheimer’s disease (AD), and repetitive TBI (rTBI) may culminate in dementia pugilistica (DP), a syndrome characterized by progressive dementia, parkinsonism, and the hallmark brain lesions of AD, including neurofibrillary tangles (NFTs), and senile plaques (SPs), suggesting similar pathological pathways between AD and TBI. Although previous study showed that mild rTBI (mrTBI) accelerated the deposition of A in a mouse model of AD-like amyloidosis Tg2576, it is still unclear that mrTBI enhance tau pathology. Objectives and Methods: We examined the effects of mrTBI on AD-like tau pathologies in Tg mice expressing the shortest human tau isoform subjected to mrTBI, causing brain concussion without structural brain damage to simulate injuries linked to DP. Twelve-month-old Tg T44 (n ⫽ 18) and wild-type (WT; n ⫽ 24) mice were subjected to mrTBI (four times a day, 1 day per week, for 4 weeks; n ⫽ 24) or sham treatment (n ⫽ 18). Results: Histopathological analysis of mice at 9 months after mrTBI revealed that one of the Tg T44 mice showed extensive telencephalic NFTs and cerebral atrophy. Although the critical difference between that mouse and other T44 remained unclear, the positive iron staining was found only in the brain surface of that mouse. Since iron is a major free radical source and there is circumstantial evidence linking abnormal iron metabolism to mechanisms underlying AD, we speculated that iron deposition might contribute to the enhanced tau pathology. To address this possibility, we micro-injected ferrin chloride (0.5 l 100mM FeCl3) into the hippocampus of T44 mice (n ⫽ 8), and then diachronically examined histological changes for 6 months. However, we could not affirm the enhancement of NFT formation in any FeCl3-injected T44 mice. Conclusions: We cannot conclude that iron directly relates to the enhanced tau pathology, though possibilities that some blood components other than iron, or combination of several blood components including iron, might enhance tau pathology cannot be excluded because the iron-positive staining is convincing evidence of BBB breakdown. P3-291
REAL-TIME IMAGE ACQUISITION TO MEASURE THE EFFECTS OF MODULATING PHOSPHORYLATION ON THE OVERALL RATE OF TAU AXONAL TRANSPORT IN INDIVIDUAL NEURONS
Catherine J. Evans1, Nicholas A. Evans2, Davina E. Mitchell2, Karen L. Philpott2, Diane P. Hanger1, 1MRC Centre for Neurodegeneration Research, Kings College London, Institute of Psychiatry, London, United Kingdom; 2Neurology and GI CEDD, GlaxoSmithKline, Harlow, United Kingdom. Contact e-mail: [email protected] Background: Tau is a neuronal cytoskeletal protein, the most well characterised function of which is its binding to and stabilisation of microtubules, which serve as tracks for axonal transport in neurons. Abnormalities in tau have been linked to a number of neurodegenerative disorders including Alzheimer’s disease (AD). In AD brain tau is the main constituent of neurofibrillary tangles, which are composed of paired helical filaments of insoluble tau. Tau in these inclusions is abnormally phosphorylated, although the mechanisms linking phosphorylation to the formation of aggregates remain unclear. Phosphorylation may induce aggregation by changing the physical properties of tau, or may mediate tangle formation
S461
through a change in other properties of tau such as its microtubule binding or axonal transport. Objectives: To develop a method for the study of the axonal transport of tau in individual cultured neurons. To examine the effect of modulating tau phosphorylation on its axonal transport. Methods: cDNA constructs of enhanced green fluorescent protein (EGFP) tagged tau were transfected into primary cultures of rat cortical neurons and images of individual neurons were collected at different timepoints after transfection. The rate of transport of tau-EGFP was calculated by measuring the distance from the axon hilum to the front of EGFP fluorescence in individual neurons. Results: We were able to track net anterograde axonal transport in individual live neurons over several hours in culture. The rate of tau transport was comparable both to that previously observed in vivo and in vitro. Metabolic inhibition using 2-deoxy-D-glucose and sodium azide inhibited transport of tau thus validating our experimental system. Treatment of neurons with agents affecting tau phosphorylation was found to modulate tau transport. Conclusions: We have developed and validated a novel method to determine the rate of axonal transport of tau in individual neurons in culture. This experimental system can be used to investigate the effects of modulators of tau phosphorylation on its transport. P3-292
HIGH THROUGHPUT SCREEN ASSAY TO DEVELOP DRUGS FOR INHIBITION OF ABNORMAL HYPERPHOSPHORYLATION OF TAU
Sabiha Khatoon, Inge Grundke-Iqbal, Khalid Iqbal, New York State Institute for Basic Research in Developmental Disabilities, Staten Island, NY, USA. Contact e-mail: [email protected] Background: In Alzheimer disease (AD) and related tauopathies, the microtubule associated protein tau is abnormally hyperphosphorylated and in this state it is polymerized into filaments, i.e. paired helical filaments/ straight filaments. The activity of protein phosphatase (PP)-2A, which regulates the phosphorylation of tau and some of the tau kinases, is compromised in AD brain. The activity of PP-2A, in turn, is regulated by two small endogenous inhibitor proteins, namely I1PP2A and I2PP2A Objective(s): To develop a high throughput screening assay for the development of drugs that can restore PP-2A activity by inhibiting the activities of I1PP2A and/or I2PP2A. Methods: We have combined, in a microtiter plate-based assay, biochemical dephosphorylation of abnormally hyperphosphorylated tau/tau peptides by PP-2A and its inhibition by I2PP2A, with ELISA that measures the level of dephosphorylated peptide by monoclonal antibody Tau-1; Tau-1 recognizes tau unphosphorylated at Ser-195/198/199/202. Results: Dephosphorylation of tau/tau peptide at Tau-1 site by PP-2A, and its inhibition by I2PP2A, could be quantitated by the high throughput screening assay. Memantine, in the nanomolar concentration, was found to antagonize the effect of I2PP2A on PP-2A activity. This drug had no significant direct effect on PP-2A activity. Conclusions: A high throughput screen assay has been developed by which compounds can be identified that restore the PP-2A activity by inhibiting the activity of I2PP2A and, consequently, the abnormal hyperphosphorylation of tau. (Supported in part by the New York State Office of Mental Retardation and Developmental Disabilities, NIH Grant AG019158 and a grant from the Institute for the Study of Aging, New York, NY.) P3-293
TAU CLEARANCE THROUGH PROTEIN DEGRADATION PATHWAYS
Jason Eriksen, Cynthia Zehr, Zdenek Berger, Todd Robinson, Jada Lewis, Michael Hutton, Mayo Clinic Jacksonville, Jacksonville, FL, USA. Contact e-mail: [email protected] Background: During the aging process, rates of protein degradation decrease, and this effect can contribute to the accumulation of potentially toxic protein species. This observation suggests that, in Alzheimer’s disease and other tauopathies, the accumulation of hyperphosphorylated tau may be due to the impaired clearance of abnormal tau by cellular protein
ENHANCED TAU PATHOLOGY, CEREBRAL ATROPHY AND IRON DEPOSITION INDUCED BY REPETITIVE MILD BRAIN INJURY IN A TAU TRANSGENIC MOUSE
Yasumasa Yoshiyama1,2, Uryu Kunihiro2, Makoto Higuchi2, Tracy McIntosh2, Virginia M-Y Lee2, John Q. Trojanowski2, 1National Chiba East Hospital, Chiba, Japan; 2University of Pennsylvania, Philadelphia, PA, USA. Contact e-mail: [email protected] Background: Traumatic brain injury (TBI) is a risk factor for Alzheimer’s disease (AD), and repetitive TBI (rTBI) may culminate in dementia pugilistica (DP), a syndrome characterized by progressive dementia, parkinsonism, and the hallmark brain lesions of AD, including neurofibrillary tangles (NFTs), and senile plaques (SPs), suggesting similar pathological pathways between AD and TBI. Although previous study showed that mild rTBI (mrTBI) accelerated the deposition of A in a mouse model of AD-like amyloidosis Tg2576, it is still unclear that mrTBI enhance tau pathology. Objectives and Methods: We examined the effects of mrTBI on AD-like tau pathologies in Tg mice expressing the shortest human tau isoform subjected to mrTBI, causing brain concussion without structural brain damage to simulate injuries linked to DP. Twelve-month-old Tg T44 (n ⫽ 18) and wild-type (WT; n ⫽ 24) mice were subjected to mrTBI (four times a day, 1 day per week, for 4 weeks; n ⫽ 24) or sham treatment (n ⫽ 18). Results: Histopathological analysis of mice at 9 months after mrTBI revealed that one of the Tg T44 mice showed extensive telencephalic NFTs and cerebral atrophy. Although the critical difference between that mouse and other T44 remained unclear, the positive iron staining was found only in the brain surface of that mouse. Since iron is a major free radical source and there is circumstantial evidence linking abnormal iron metabolism to mechanisms underlying AD, we speculated that iron deposition might contribute to the enhanced tau pathology. To address this possibility, we micro-injected ferrin chloride (0.5 l 100mM FeCl3) into the hippocampus of T44 mice (n ⫽ 8), and then diachronically examined histological changes for 6 months. However, we could not affirm the enhancement of NFT formation in any FeCl3-injected T44 mice. Conclusions: We cannot conclude that iron directly relates to the enhanced tau pathology, though possibilities that some blood components other than iron, or combination of several blood components including iron, might enhance tau pathology cannot be excluded because the iron-positive staining is convincing evidence of BBB breakdown. P3-291
REAL-TIME IMAGE ACQUISITION TO MEASURE THE EFFECTS OF MODULATING PHOSPHORYLATION ON THE OVERALL RATE OF TAU AXONAL TRANSPORT IN INDIVIDUAL NEURONS
Catherine J. Evans1, Nicholas A. Evans2, Davina E. Mitchell2, Karen L. Philpott2, Diane P. Hanger1, 1MRC Centre for Neurodegeneration Research, Kings College London, Institute of Psychiatry, London, United Kingdom; 2Neurology and GI CEDD, GlaxoSmithKline, Harlow, United Kingdom. Contact e-mail: [email protected] Background: Tau is a neuronal cytoskeletal protein, the most well characterised function of which is its binding to and stabilisation of microtubules, which serve as tracks for axonal transport in neurons. Abnormalities in tau have been linked to a number of neurodegenerative disorders including Alzheimer’s disease (AD). In AD brain tau is the main constituent of neurofibrillary tangles, which are composed of paired helical filaments of insoluble tau. Tau in these inclusions is abnormally phosphorylated, although the mechanisms linking phosphorylation to the formation of aggregates remain unclear. Phosphorylation may induce aggregation by changing the physical properties of tau, or may mediate tangle formation
S461
through a change in other properties of tau such as its microtubule binding or axonal transport. Objectives: To develop a method for the study of the axonal transport of tau in individual cultured neurons. To examine the effect of modulating tau phosphorylation on its axonal transport. Methods: cDNA constructs of enhanced green fluorescent protein (EGFP) tagged tau were transfected into primary cultures of rat cortical neurons and images of individual neurons were collected at different timepoints after transfection. The rate of transport of tau-EGFP was calculated by measuring the distance from the axon hilum to the front of EGFP fluorescence in individual neurons. Results: We were able to track net anterograde axonal transport in individual live neurons over several hours in culture. The rate of tau transport was comparable both to that previously observed in vivo and in vitro. Metabolic inhibition using 2-deoxy-D-glucose and sodium azide inhibited transport of tau thus validating our experimental system. Treatment of neurons with agents affecting tau phosphorylation was found to modulate tau transport. Conclusions: We have developed and validated a novel method to determine the rate of axonal transport of tau in individual neurons in culture. This experimental system can be used to investigate the effects of modulators of tau phosphorylation on its transport. P3-292
HIGH THROUGHPUT SCREEN ASSAY TO DEVELOP DRUGS FOR INHIBITION OF ABNORMAL HYPERPHOSPHORYLATION OF TAU
Sabiha Khatoon, Inge Grundke-Iqbal, Khalid Iqbal, New York State Institute for Basic Research in Developmental Disabilities, Staten Island, NY, USA. Contact e-mail: [email protected] Background: In Alzheimer disease (AD) and related tauopathies, the microtubule associated protein tau is abnormally hyperphosphorylated and in this state it is polymerized into filaments, i.e. paired helical filaments/ straight filaments. The activity of protein phosphatase (PP)-2A, which regulates the phosphorylation of tau and some of the tau kinases, is compromised in AD brain. The activity of PP-2A, in turn, is regulated by two small endogenous inhibitor proteins, namely I1PP2A and I2PP2A Objective(s): To develop a high throughput screening assay for the development of drugs that can restore PP-2A activity by inhibiting the activities of I1PP2A and/or I2PP2A. Methods: We have combined, in a microtiter plate-based assay, biochemical dephosphorylation of abnormally hyperphosphorylated tau/tau peptides by PP-2A and its inhibition by I2PP2A, with ELISA that measures the level of dephosphorylated peptide by monoclonal antibody Tau-1; Tau-1 recognizes tau unphosphorylated at Ser-195/198/199/202. Results: Dephosphorylation of tau/tau peptide at Tau-1 site by PP-2A, and its inhibition by I2PP2A, could be quantitated by the high throughput screening assay. Memantine, in the nanomolar concentration, was found to antagonize the effect of I2PP2A on PP-2A activity. This drug had no significant direct effect on PP-2A activity. Conclusions: A high throughput screen assay has been developed by which compounds can be identified that restore the PP-2A activity by inhibiting the activity of I2PP2A and, consequently, the abnormal hyperphosphorylation of tau. (Supported in part by the New York State Office of Mental Retardation and Developmental Disabilities, NIH Grant AG019158 and a grant from the Institute for the Study of Aging, New York, NY.) P3-293
TAU CLEARANCE THROUGH PROTEIN DEGRADATION PATHWAYS
Jason Eriksen, Cynthia Zehr, Zdenek Berger, Todd Robinson, Jada Lewis, Michael Hutton, Mayo Clinic Jacksonville, Jacksonville, FL, USA. Contact e-mail: [email protected] Background: During the aging process, rates of protein degradation decrease, and this effect can contribute to the accumulation of potentially toxic protein species. This observation suggests that, in Alzheimer’s disease and other tauopathies, the accumulation of hyperphosphorylated tau may be due to the impaired clearance of abnormal tau by cellular protein