- Email: [email protected]
P3-301
Poster P3:: Tuesday Posters tion degree of PHF-. We purified bovine to homogeneity with affinity chromatography using monoclonal antibody (mAb) BT-2, which binds all -versions independent of their posttranslational modifications, and analyzed its N-glycosylation state after two-dimensional gel electrophoresis (2-DE) using sensitive immuno- and lectin-blot analyses. Blots from crude resulted mostly in N- and O-glycosylated neurofilaments that incidentally comigrated with in SDS-PAGE, but were separated by the pH-gradient in 2-DE. Thus N-glycosylated versions present in the affinity purified were enriched by a lectin-based affinity chromatography. The eluate was separated by 2-DE and probed with an anti- mAb and lectins. Taken together, appears to be O- and N-glycosylated at a low level. This raises the question, if the glycosylation levels detected in PHF- are really diseasespecific or if it is only related to different aggregation characteristics unand N-glycosylated species. P3-298
IN VIVO EFFICACY OF GSK3 INHIBITORS IN THE POSTNATAL RAT MODEL OF TAU HYPERPHOSPHORYLATION
Maj-Linda Selenica, H. Lundbeck A/S, Copenhagen, Denmark. Contact e-mail: [email protected] Glycogen synthase kinase-3 (GSK3) is involved in several neuropathological events associated with Alzheimer’s disease (AD), including tau hyperphosphorylation. Hyperphosphorylated tau (p-tau) is the major constituent of paired-helical filaments (PHF) and neurofibrillary tangels found in AD brains. The present study applied a postnatal rat model of tau hyperphosphorylation to study the efficacy of different classes of GSK3 inhibitors in decreasing tau phosphorylation in vivo. Western blot analysis of cortex, hippocampus and whole rat brain showed a robust increase in p-tau levels during early postnatal development (P0-P17) at both Ser396 and Thr181 epitopes followed by a rapid decrease in p-tau with age. Likewise, immunohistochemical examination of phosphorylated Ser202/ Thr205 epitopes on tau demonstrated a significant increase in p-tau immunoreactivity both in the dentate gyrus and CA3 region when compared to adult animals. Per-oral administration of LiCl (100 and 200 mg/kg) significantly decreased 3R0N and 4R0N Ser396 p-tau levels when compared with NaCl-treated animals as detected by Western blotting. In this study we also tested whether the aminothiazole GSK3 inhibitor AR-A014418 could decrease levels of p-tau. P12 rats were treated with two doses of AR-A014418 by two routes of administration and using different vehicles (30 mg/kg per-oral in PEG-400 and 30 mg/kg or 100 mg/kg sub-cutaneous in 25% Cremaphor EL). No effect of AR-A014418 using either paradigm was observed. However, different vehicles are currently being tested. In addition, the effect of two other GSK3 inhibitors, SB216763 (arylindolemaleimide class) and CHIR98014 (aminopyrimidine class) on phosphorylated tau levels in both hippocampus and cortex is currently being tested. P3-299
PROTEIN DEGRADATION RELATED WITH POST-MORTEM DELAY IN THE STUDY OF HUMAN NEURODEGENERATIVE DISEASES. A PROTEOMICS APPROACH
Gabriel Santpere, Berta Puig, Isidre Ferrer, Institut de Neuropatologia de Bellvitge, Barcelona, Spain. Contact e-mail: [email protected] Background: Research on the human brain is basic to increase understanding about human neurological disorders such as Alzheimer disease (AD). Yet post-mortem delay is an unavoidable factor in the studies based on human post-mortem brain tissue. Brain proteins, when exposed to long post-mortem interval, may be altered by endogenous proteases and phosphatases, thus resulting in pitfalls and confusing results. Objective(s): The present study stresses the use of proteomics in the identification of vulnerable proteins to post-mortem delay, and illustrates the necessity for careful control of protein degradation when using post-mortem brain tissues. Methods: In the present work, brains of AD and age-matched control cases
S463
were analysed by two-dimensional electrophoresis at different artificial post-mortem intervals (0, 3, 6, 12, 18, 24 and 48 hours) subjected to variable temperature conditions. Silver-stained gels of the same case with increased artificial post-mortem delay disclosed differential spot expression which was the result of target protein degradation as revealed with mass spectrometry. We also studied by Western blot the post-mortemdependent degradation of tau protein in paired helical filament (PHF)enriched fractions from AD brains. Conclusions: Several proteins were identified and categorized as differentially vulnerable to post-mortem delay. Following standard paradigms (post-mortem delay at 4°C, beginning two h after death) phospho-tau degradation, manifested as a reduction in the number and intensity of the bands, occurred between 6 and 24 h of post-mortem and was universal in samples with post-mortem delays of 48 h. Again, these studies indicate the importance in considering postmortem delay artefacts related with post-mortem delay in the study of phospho-tau patterns in AD and other tauopathies. P3-300
INCREASED TYROSINE 307 PHOSPHORYLATION OF PROTEIN PHOSPHATASE-2A CORRELATES WITH TAU ASSOCIATED PATHOLOGIES IN ALZHEIMER’S DISEASE
Rong Liu1,2, Cecilia Bjo¨rkdahl1, Xin-Wen Zhou1,2, Irina Alafuzoff3, Hilkka Soininen3, Nenad Bogdanovic1, Khalid Iqbal4, Inge Grundke-Iqbal4, Bengt Winblad Winblad1, Jian-Zhi Wang2, Jin-Jing Pei1, 1Karolinska Institutet, Stockholm, Sweden; 2Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China; 3Kuopio University, Kuopio, Finland; 4New York State Institute for Basic Research in Developmental Disabilities, New York, USA. Contact e-mail: [email protected] Background: The activity of protein phosphatase (PP)-2A is compromised towards dephosphorylation of abnormally hyperphosphorylated tau in Alzheimer’s disease (AD) brain but the mechanism for decreased PP-2A activity remained unclear. Previous in vitro studies have shown that phosphorylation of PP-2A catalytic subunit at the tyrosine 307 (Tyr307) site potently inactivates PP-2A. Objective(s): To understand the mechanism of reduced PP-2A activity in AD brain. Methods: Level of PP-2A phosphorylation at Tyr307 and its relationship with tau phosphorylation were investigated in AD and control brains by Western blotting, immunohistochemistry and confocal microscopy using a specific antibody against inactive / phosphorylated (p) -PP-2A at Tyr307. Cultured mouse N2a neuroblastoma cells were treated with 100 nM okadaic acid to confirm the role of Tyr307 phosphorylation of PP-2A in tau hyperphosphorylation. Conclusions: The results showed a significant increase of inactive form of PP-2A in 100, 000 xg pellets of AD brains as compared with controls. By confocal microscopy, the inactive form of PP-2A was observed to aberrantly accumulate in neurons bearing neurofibrillary tangles and pretangles. In pellets, the level of inactive form of PP-2A positively correlated with tau phosphorylation level at AT8 (Ser202/Thr205) and PHF-1 (Ser396/Ser404) sites, and negatively correlated with tau dephosphorylation at Tau-1 (Ser199/Ser202) sites, but not correlated with total tau. The coincidence of increase of both inactivated PP-2A and tau phosphorylation could be repeated in mouse N2a neuroblastoma cells treated with 100 nM okadaic acid, a specific PP-2A inhibitor. We hypothesized that inactivation of PP-2A via phosphorylation by unknown upstream tyrosine kinases might be involved in formation of tau abnormal hyperphosphorylation in AD. P3-301
A MEDIATES TAU HYPERPHOSPHORYLATION VIA DOWN-REGULATING PP2A IN ALZHEIMER’S DISEASE
Xin-Wen Zhou1,2, Heikki Tanila3, Rong Liu1,2, Bengt Winblad1, Jin-Jing Pei1, 1Karolinska Institutet, Huddinge, Stockholm, Sweden; 2 Dept. of Pathophysiology of Tongji Medical College, Huazhong
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Poster P3:: Tuesday Posters
University of Science and Technology, Wuhan, China; 3Dept. of Neurobiology, A. I. Virtanen Institute, Kuopio, Finland. Contact e-mail: [email protected] Background: In Alzheimer’s disease brains (AD), amyloid A is abnormally deposited in senile plaques (SPs) and hyperphosphorylated tau in neurons bearing neurofibrillary tangles (NFTs). In vivo evidence has demonstrated that A can induce tau hyperphosphorylation and tangle formation, but the underlying mechanism remains largely elusive. Objective(s): Whether or not in AD brains A plays a causal role in the reduced PP2A activity towards to tau, and PP2A bridges A and PHF-tau pathologies are of imperative to clarify. Methods: Our results showed that in the homogenates of the medial temporal cortex, levels of phosphorylated (p) PP2A catalytic subunit (PP2AC) (Y307) / total PP2AC (⬃2-fold) and demethylated (-m) PP2AC (L309) /total PP2AC (⬃1.5-fold), PHF-1, A42, and A43 were significantly increased in AD relative to control. p-PP2AC (Y307) co-existed with NFTs (AT8) that encircled SPs labeled by 4G8. In both mouse APPswe N2a and transgenic APPswe /PS1 (A246E) mice that have overexpress A, levels of p-PP2AC (Y307), p-GSK-3 (S9 and/or Y216), p-P70S6K (T421/S424 and T389) and p-tau (S205, PHF-1, AT180) were dramatically increased, but levels of methylated (⫹m) PP2AC (L309) and de-p-tau at Tau-1 sites were significantly decreased relative to controls. A similar pattern of responses of p-PP2AC (Y307) and -m-PP2AC (L309), p-GSK-3 (S9 and/or Y216), p-P70S6K (T421/S424 and T389), tau (S205, Tau-1, PHF-1, and T180) was seen in wild-type N2a cells and APPswe N2a cells treated with okadaic acid. Conclusions: Together, it suggested that up-regulated p-PP2AC (Y307) and down-regulated ⫹m-PP2AC contribute to the reduced PP2A activity in AD brains, resulting in activation of GSK-3 and p70S6K and the compromised dephosphorylation of abnormally hyperphosphorylated tau. The deregulation of PP2AC might play a critical role in A-mediated tau hyperphosphorylation in AD. P3-302
AGGREGATIONAL PROPERTIES OF WILD-TYPE AND MUTATED TAU PROTEIN
Cecilia Fa¨gerblad, Johan Fredriksson, Lena Skoglund, Joakim Bergstro¨m, Lars Lannfelt, Martin Ingelsson, Uppsala University, Uppsala, Sweden. Contact e-mail: [email protected] Background: Mutations in the tau gene cause frontotemporal dementia with parkinsonism (FTDP-17). Some of the tau mutations are intronic and influence tau exon 10 splicing, leading to a shift in the ratio of tau with four (4R tau) and three (3R tau) microtubule binding repeats. However, a majority of tau mutations are located in exons and have instead been proposed to increase tau aggregation. The aggregational properties of the different tau isoforms have only been partly elucidated and it also remains unclear whether the presence of mutated tau may induce aggregation of wild-type tau isoforms. Objective(s): To investigate different aspects of wild-type and mutated tau protein aggregation in vitro. Methods: A tau aggregation assay, based on the fluorescent dye thioflavine T and with heparin as inducer, was designed. Single and mixed preparations of wild type tau isoforms as well as 4R tau versions of the three common tau mutations P301L, V337M and R406W were studied. Results: In accordance with previous studies, the tau mutants aggregated faster and to a greater extent than wild-type tau. Tau R406W was the most rapidly aggregating mutant, followed by P301L and V337M, respectively. Moreover, mixed samples with 75% 4R tau or 75% 3R tau had a slightly higher propensity to aggregate compared to samples with equal representation of 4R tau and 3R tau. Finally, cryo transmission electron microscopy demonstrated that fibrils formed by the various tau mutants were much more numerous, but had a roughly similar morphological appearance as fibrils formed by wild-type tau. Conclusions: Our results confirm previous findings of tau mutants aggregating faster and to a greater extent than wild-type tau. Moreover, preliminary evidence suggest that preparations with a inbalance of 4R tau and 3R tau isoforms are particularly prone to aggregate,
which may reflect a pathogenic mechanism for tauopathies with a shift in tau exon 10 splicing. P3-303
INHIBITION OF TAU AGGREGATION AND DISAGGREGATION OF PHFS BY LOW MW COMPOUNDS IN VITRO AND IN CELLS
Marcus Pickhardt, Zuzana Gazova, Martin von Bergen, Jacek Biernat, Inna Khlistunova, Yi-Peng Wang, Antje Hascher, Eva-Maria Mandelkow, Eckhard Mandelkow, Max-Planck Society, Hamburg, Germany. Contact e-mail: [email protected] The pathological aggregation of tau protein is one of the hallmarks of Alzheimer’s disease and other neurodegenerative diseases (‘tauopathies’) and correlates closely with the clinical progression of the disease. The aim of this project is to identify compounds capable of preventing or reversing the aggregation of tau. Such compounds could form the basis for the development of drugs for treating the disease. We have performed a large screen of 200,000 compounds and identified hits from different chemical groups like anthraquinones, isothiazol-carbonitriles, benzoxazolyl-phenyls, indols, rhodanins and a series of singletons. We established a battery of in vitro techniques to monitor PHF assembly which has been used to analyse the effect of compounds on tau aggregation like ThS fluorescence (Friedhoff et al., 1998), tryptophan fluorescence (Li et al., 2002), pelleting assay, filter assay and electron microscopy (Pickhardt et al., 2005). Based on the active compounds coming out of the initial screen an in silico screen was performed and the group of N-phenlyamines besides others was identified as potential inhibitors of tau aggregation. For optimization the structures will be further modified and the resulting derivatives will be tested by the assays. The results will be used to build up a structure-activity relationship. We also tested the activity of these compounds in a cell model consisting of a neuroblastoma cell line which expresses tau in an inducible fashion, with subsequent aggregation of tau. The results show that PHF aggregation from recombinant tau protein is reversible and can be inhibited by low MW compounds. P3-304
MNB/DYRK1A PRIMES PHOSPHORYLATION OF TAU AT SEVERAL PHOSPHORYLATION SITES BY GSK-3
Zhihou Liang, Fei Liu, Yu-Wen Hwang, Jerzy Wegiel, Khalid Iqbal, Inge Grundke-Iqbal, Cheng-Xin Gong, New York State Institute for Basic Research in Developmental Disabilities, Staten Island, NY, USA. Contact e-mail: [email protected] All patients with Down syndrome develop histopathological changes characteristic of Alzheimer disease, i.e. senile plaques of -amyloid and neurofibrillary tangles of abnormally hyperphosphorylated tau, during the fourth decade of life. The early onset of brain amyloidosis with fibriliar -amyloid deposition in plaques and vessels is due to the extra copy of chromosome 21 that bears the gene of -amyloid precursor protein, from which -amyloid peptide is produced. However, why neurofibrillary tangles develop in the brains of Down syndrome patients is not understood. The Down syndrome critical region of chromosome 21 includes a gene encoding minibrain kinase/dual-specificity tyrosine-phosphorylated and regulated kinase 1A (Mnb/Dyrk1A), which has been reported to phosphorylate tau at Thr212. Here, we report that Mnb/Dyrk1A can phosphorylate tau at Ser199 and Ser396 in addition to Thr212 in vitro. More importantly, phosphorylation by Mnb/Dyrk1A primes tau for subsequent phosphorylation at Ser199, Ser202, Thr205 and Thr217 by glycogen synthase kinase-3 (GSK-3). Phosphorylation at most of these sites of tau was found to be elevated in the brain of adult patients with Down syndrome as compared to the age-matched controls. These studies suggest that overexpression of Mnb/Dyrk1A due to an extra copy of the gene might contribute to the abnormal hyperphosphorylation of tau and neurofibrillary degeneration in Down syndrome.
IN VIVO EFFICACY OF GSK3 INHIBITORS IN THE POSTNATAL RAT MODEL OF TAU HYPERPHOSPHORYLATION
Maj-Linda Selenica, H. Lundbeck A/S, Copenhagen, Denmark. Contact e-mail: [email protected] Glycogen synthase kinase-3 (GSK3) is involved in several neuropathological events associated with Alzheimer’s disease (AD), including tau hyperphosphorylation. Hyperphosphorylated tau (p-tau) is the major constituent of paired-helical filaments (PHF) and neurofibrillary tangels found in AD brains. The present study applied a postnatal rat model of tau hyperphosphorylation to study the efficacy of different classes of GSK3 inhibitors in decreasing tau phosphorylation in vivo. Western blot analysis of cortex, hippocampus and whole rat brain showed a robust increase in p-tau levels during early postnatal development (P0-P17) at both Ser396 and Thr181 epitopes followed by a rapid decrease in p-tau with age. Likewise, immunohistochemical examination of phosphorylated Ser202/ Thr205 epitopes on tau demonstrated a significant increase in p-tau immunoreactivity both in the dentate gyrus and CA3 region when compared to adult animals. Per-oral administration of LiCl (100 and 200 mg/kg) significantly decreased 3R0N and 4R0N Ser396 p-tau levels when compared with NaCl-treated animals as detected by Western blotting. In this study we also tested whether the aminothiazole GSK3 inhibitor AR-A014418 could decrease levels of p-tau. P12 rats were treated with two doses of AR-A014418 by two routes of administration and using different vehicles (30 mg/kg per-oral in PEG-400 and 30 mg/kg or 100 mg/kg sub-cutaneous in 25% Cremaphor EL). No effect of AR-A014418 using either paradigm was observed. However, different vehicles are currently being tested. In addition, the effect of two other GSK3 inhibitors, SB216763 (arylindolemaleimide class) and CHIR98014 (aminopyrimidine class) on phosphorylated tau levels in both hippocampus and cortex is currently being tested. P3-299
PROTEIN DEGRADATION RELATED WITH POST-MORTEM DELAY IN THE STUDY OF HUMAN NEURODEGENERATIVE DISEASES. A PROTEOMICS APPROACH
Gabriel Santpere, Berta Puig, Isidre Ferrer, Institut de Neuropatologia de Bellvitge, Barcelona, Spain. Contact e-mail: [email protected] Background: Research on the human brain is basic to increase understanding about human neurological disorders such as Alzheimer disease (AD). Yet post-mortem delay is an unavoidable factor in the studies based on human post-mortem brain tissue. Brain proteins, when exposed to long post-mortem interval, may be altered by endogenous proteases and phosphatases, thus resulting in pitfalls and confusing results. Objective(s): The present study stresses the use of proteomics in the identification of vulnerable proteins to post-mortem delay, and illustrates the necessity for careful control of protein degradation when using post-mortem brain tissues. Methods: In the present work, brains of AD and age-matched control cases
S463
were analysed by two-dimensional electrophoresis at different artificial post-mortem intervals (0, 3, 6, 12, 18, 24 and 48 hours) subjected to variable temperature conditions. Silver-stained gels of the same case with increased artificial post-mortem delay disclosed differential spot expression which was the result of target protein degradation as revealed with mass spectrometry. We also studied by Western blot the post-mortemdependent degradation of tau protein in paired helical filament (PHF)enriched fractions from AD brains. Conclusions: Several proteins were identified and categorized as differentially vulnerable to post-mortem delay. Following standard paradigms (post-mortem delay at 4°C, beginning two h after death) phospho-tau degradation, manifested as a reduction in the number and intensity of the bands, occurred between 6 and 24 h of post-mortem and was universal in samples with post-mortem delays of 48 h. Again, these studies indicate the importance in considering postmortem delay artefacts related with post-mortem delay in the study of phospho-tau patterns in AD and other tauopathies. P3-300
INCREASED TYROSINE 307 PHOSPHORYLATION OF PROTEIN PHOSPHATASE-2A CORRELATES WITH TAU ASSOCIATED PATHOLOGIES IN ALZHEIMER’S DISEASE
Rong Liu1,2, Cecilia Bjo¨rkdahl1, Xin-Wen Zhou1,2, Irina Alafuzoff3, Hilkka Soininen3, Nenad Bogdanovic1, Khalid Iqbal4, Inge Grundke-Iqbal4, Bengt Winblad Winblad1, Jian-Zhi Wang2, Jin-Jing Pei1, 1Karolinska Institutet, Stockholm, Sweden; 2Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China; 3Kuopio University, Kuopio, Finland; 4New York State Institute for Basic Research in Developmental Disabilities, New York, USA. Contact e-mail: [email protected] Background: The activity of protein phosphatase (PP)-2A is compromised towards dephosphorylation of abnormally hyperphosphorylated tau in Alzheimer’s disease (AD) brain but the mechanism for decreased PP-2A activity remained unclear. Previous in vitro studies have shown that phosphorylation of PP-2A catalytic subunit at the tyrosine 307 (Tyr307) site potently inactivates PP-2A. Objective(s): To understand the mechanism of reduced PP-2A activity in AD brain. Methods: Level of PP-2A phosphorylation at Tyr307 and its relationship with tau phosphorylation were investigated in AD and control brains by Western blotting, immunohistochemistry and confocal microscopy using a specific antibody against inactive / phosphorylated (p) -PP-2A at Tyr307. Cultured mouse N2a neuroblastoma cells were treated with 100 nM okadaic acid to confirm the role of Tyr307 phosphorylation of PP-2A in tau hyperphosphorylation. Conclusions: The results showed a significant increase of inactive form of PP-2A in 100, 000 xg pellets of AD brains as compared with controls. By confocal microscopy, the inactive form of PP-2A was observed to aberrantly accumulate in neurons bearing neurofibrillary tangles and pretangles. In pellets, the level of inactive form of PP-2A positively correlated with tau phosphorylation level at AT8 (Ser202/Thr205) and PHF-1 (Ser396/Ser404) sites, and negatively correlated with tau dephosphorylation at Tau-1 (Ser199/Ser202) sites, but not correlated with total tau. The coincidence of increase of both inactivated PP-2A and tau phosphorylation could be repeated in mouse N2a neuroblastoma cells treated with 100 nM okadaic acid, a specific PP-2A inhibitor. We hypothesized that inactivation of PP-2A via phosphorylation by unknown upstream tyrosine kinases might be involved in formation of tau abnormal hyperphosphorylation in AD. P3-301
A MEDIATES TAU HYPERPHOSPHORYLATION VIA DOWN-REGULATING PP2A IN ALZHEIMER’S DISEASE
Xin-Wen Zhou1,2, Heikki Tanila3, Rong Liu1,2, Bengt Winblad1, Jin-Jing Pei1, 1Karolinska Institutet, Huddinge, Stockholm, Sweden; 2 Dept. of Pathophysiology of Tongji Medical College, Huazhong
S464
Poster P3:: Tuesday Posters
University of Science and Technology, Wuhan, China; 3Dept. of Neurobiology, A. I. Virtanen Institute, Kuopio, Finland. Contact e-mail: [email protected] Background: In Alzheimer’s disease brains (AD), amyloid A is abnormally deposited in senile plaques (SPs) and hyperphosphorylated tau in neurons bearing neurofibrillary tangles (NFTs). In vivo evidence has demonstrated that A can induce tau hyperphosphorylation and tangle formation, but the underlying mechanism remains largely elusive. Objective(s): Whether or not in AD brains A plays a causal role in the reduced PP2A activity towards to tau, and PP2A bridges A and PHF-tau pathologies are of imperative to clarify. Methods: Our results showed that in the homogenates of the medial temporal cortex, levels of phosphorylated (p) PP2A catalytic subunit (PP2AC) (Y307) / total PP2AC (⬃2-fold) and demethylated (-m) PP2AC (L309) /total PP2AC (⬃1.5-fold), PHF-1, A42, and A43 were significantly increased in AD relative to control. p-PP2AC (Y307) co-existed with NFTs (AT8) that encircled SPs labeled by 4G8. In both mouse APPswe N2a and transgenic APPswe /PS1 (A246E) mice that have overexpress A, levels of p-PP2AC (Y307), p-GSK-3 (S9 and/or Y216), p-P70S6K (T421/S424 and T389) and p-tau (S205, PHF-1, AT180) were dramatically increased, but levels of methylated (⫹m) PP2AC (L309) and de-p-tau at Tau-1 sites were significantly decreased relative to controls. A similar pattern of responses of p-PP2AC (Y307) and -m-PP2AC (L309), p-GSK-3 (S9 and/or Y216), p-P70S6K (T421/S424 and T389), tau (S205, Tau-1, PHF-1, and T180) was seen in wild-type N2a cells and APPswe N2a cells treated with okadaic acid. Conclusions: Together, it suggested that up-regulated p-PP2AC (Y307) and down-regulated ⫹m-PP2AC contribute to the reduced PP2A activity in AD brains, resulting in activation of GSK-3 and p70S6K and the compromised dephosphorylation of abnormally hyperphosphorylated tau. The deregulation of PP2AC might play a critical role in A-mediated tau hyperphosphorylation in AD. P3-302
AGGREGATIONAL PROPERTIES OF WILD-TYPE AND MUTATED TAU PROTEIN
Cecilia Fa¨gerblad, Johan Fredriksson, Lena Skoglund, Joakim Bergstro¨m, Lars Lannfelt, Martin Ingelsson, Uppsala University, Uppsala, Sweden. Contact e-mail: [email protected] Background: Mutations in the tau gene cause frontotemporal dementia with parkinsonism (FTDP-17). Some of the tau mutations are intronic and influence tau exon 10 splicing, leading to a shift in the ratio of tau with four (4R tau) and three (3R tau) microtubule binding repeats. However, a majority of tau mutations are located in exons and have instead been proposed to increase tau aggregation. The aggregational properties of the different tau isoforms have only been partly elucidated and it also remains unclear whether the presence of mutated tau may induce aggregation of wild-type tau isoforms. Objective(s): To investigate different aspects of wild-type and mutated tau protein aggregation in vitro. Methods: A tau aggregation assay, based on the fluorescent dye thioflavine T and with heparin as inducer, was designed. Single and mixed preparations of wild type tau isoforms as well as 4R tau versions of the three common tau mutations P301L, V337M and R406W were studied. Results: In accordance with previous studies, the tau mutants aggregated faster and to a greater extent than wild-type tau. Tau R406W was the most rapidly aggregating mutant, followed by P301L and V337M, respectively. Moreover, mixed samples with 75% 4R tau or 75% 3R tau had a slightly higher propensity to aggregate compared to samples with equal representation of 4R tau and 3R tau. Finally, cryo transmission electron microscopy demonstrated that fibrils formed by the various tau mutants were much more numerous, but had a roughly similar morphological appearance as fibrils formed by wild-type tau. Conclusions: Our results confirm previous findings of tau mutants aggregating faster and to a greater extent than wild-type tau. Moreover, preliminary evidence suggest that preparations with a inbalance of 4R tau and 3R tau isoforms are particularly prone to aggregate,
which may reflect a pathogenic mechanism for tauopathies with a shift in tau exon 10 splicing. P3-303
INHIBITION OF TAU AGGREGATION AND DISAGGREGATION OF PHFS BY LOW MW COMPOUNDS IN VITRO AND IN CELLS
Marcus Pickhardt, Zuzana Gazova, Martin von Bergen, Jacek Biernat, Inna Khlistunova, Yi-Peng Wang, Antje Hascher, Eva-Maria Mandelkow, Eckhard Mandelkow, Max-Planck Society, Hamburg, Germany. Contact e-mail: [email protected] The pathological aggregation of tau protein is one of the hallmarks of Alzheimer’s disease and other neurodegenerative diseases (‘tauopathies’) and correlates closely with the clinical progression of the disease. The aim of this project is to identify compounds capable of preventing or reversing the aggregation of tau. Such compounds could form the basis for the development of drugs for treating the disease. We have performed a large screen of 200,000 compounds and identified hits from different chemical groups like anthraquinones, isothiazol-carbonitriles, benzoxazolyl-phenyls, indols, rhodanins and a series of singletons. We established a battery of in vitro techniques to monitor PHF assembly which has been used to analyse the effect of compounds on tau aggregation like ThS fluorescence (Friedhoff et al., 1998), tryptophan fluorescence (Li et al., 2002), pelleting assay, filter assay and electron microscopy (Pickhardt et al., 2005). Based on the active compounds coming out of the initial screen an in silico screen was performed and the group of N-phenlyamines besides others was identified as potential inhibitors of tau aggregation. For optimization the structures will be further modified and the resulting derivatives will be tested by the assays. The results will be used to build up a structure-activity relationship. We also tested the activity of these compounds in a cell model consisting of a neuroblastoma cell line which expresses tau in an inducible fashion, with subsequent aggregation of tau. The results show that PHF aggregation from recombinant tau protein is reversible and can be inhibited by low MW compounds. P3-304
MNB/DYRK1A PRIMES PHOSPHORYLATION OF TAU AT SEVERAL PHOSPHORYLATION SITES BY GSK-3
Zhihou Liang, Fei Liu, Yu-Wen Hwang, Jerzy Wegiel, Khalid Iqbal, Inge Grundke-Iqbal, Cheng-Xin Gong, New York State Institute for Basic Research in Developmental Disabilities, Staten Island, NY, USA. Contact e-mail: [email protected] All patients with Down syndrome develop histopathological changes characteristic of Alzheimer disease, i.e. senile plaques of -amyloid and neurofibrillary tangles of abnormally hyperphosphorylated tau, during the fourth decade of life. The early onset of brain amyloidosis with fibriliar -amyloid deposition in plaques and vessels is due to the extra copy of chromosome 21 that bears the gene of -amyloid precursor protein, from which -amyloid peptide is produced. However, why neurofibrillary tangles develop in the brains of Down syndrome patients is not understood. The Down syndrome critical region of chromosome 21 includes a gene encoding minibrain kinase/dual-specificity tyrosine-phosphorylated and regulated kinase 1A (Mnb/Dyrk1A), which has been reported to phosphorylate tau at Thr212. Here, we report that Mnb/Dyrk1A can phosphorylate tau at Ser199 and Ser396 in addition to Thr212 in vitro. More importantly, phosphorylation by Mnb/Dyrk1A primes tau for subsequent phosphorylation at Ser199, Ser202, Thr205 and Thr217 by glycogen synthase kinase-3 (GSK-3). Phosphorylation at most of these sites of tau was found to be elevated in the brain of adult patients with Down syndrome as compared to the age-matched controls. These studies suggest that overexpression of Mnb/Dyrk1A due to an extra copy of the gene might contribute to the abnormal hyperphosphorylation of tau and neurofibrillary degeneration in Down syndrome.