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P2-167: Alzheimer disease tangles and threads display multiple tau phosphorylation sites
Poster Presentations P2: Background: Among the pathological hallmarks of Alzheimer’s disease (AD) there are the neurofibrillary tangles (NFTs) composed of intracellular filamentous aggregates of hyperphosphorylated protein tau. Generally, the level of tau phosphorylation is regulated by the equilibrium between the activities of its protein kinases and phosphatases. An imbalanced regulation in protein kinase and protein phosphatase is proposed to be a reasonable causative factor to the disease process. One protein kinase that play an important role in regulating the phosphorilation state of tau is glycogen synthase kinase (Gsk) 3. Recent studies show that overexpression of active Gsk3 results in AD. The major phosphatase that dephosphorylates tau is protein phosphatase 2A (PP2A). PP2A is a trimeric serine/threonine protein phosphatase composed of three subunits. Different groups showed that the highly conserved carboxyl-terminal sequence of PP2A C subunit is a focal point for phosphatase regulation. This is the site of a reversible methyl esterification reaction that controls the formation of AB␣C heterotrimers. Objective(s): The aim of this study is to demonstrate that hypomethylation regulates the equilibrium between protein kinase and phosphatase activities, leading to abnormal hyperphosphorylated tau. Methods: Both SK-N-BE neuroblastoma cell line and TgCRND8 mice were used. In vitro, we used culture media without folate, B12 and B6, whereas in vivo, mice were fed with a diet deficient of the same vitamins. We analyzed expression levels of kinase and phosphatase with quantitative Real-Time PCR, western blot and activity assay. Results: We demonstrated that Gsk3 and PP2A genes were upregulated by inhibiting methylation reactions. Western blot analysis illustrate that methylated PP2A was decreased leading to reduced PP2A activity. By contrast, the levels of Gsk3 protein increased in the same condition. Conclusions: The analysis of Tau and the toxic tau fragments demonstrate that lowered SAM levels are the cause of deregulation of the equilibrium between Gsk3 and PP2A activities. P2-166
ATOMIC STRUCTURE OF THE PHF CORE C-TERMINUS
Martina Handzusova1, Natalia Ivanovova1, Jozef Sevcik1,2, Rostislav Skrabana1, Khalid Iqbal3, Michal Novak1,4, 1Institute of Neuroimmunology Slovak Academy of Sciences, Bratislava, Slovakia; 2 Institute of Molecular Biology, Bratislava, Slovakia; 3New York State Institute for Basic Research in Developmental Disabilities, New York, NY, USA; 4Axon Neuroscience GmbH, Vienna, Slovakia. Contact e-mail: [email protected] Background: The major constituent of Alzheimer’s disease paired helical filaments (PHF) core is intrinsically disordered protein (IDP) tau. In spite of a considerable effort, insoluble character of PHF together with inherent physical properties of IDP tau have precluded so far reconstruction of PHF 3-D structure by X-ray crystallography or NMR spectroscopy. Methods: Using monoclonal antibody MN423 specific to the tertiary structure of the PHF core, the in vivo PHF structure was imprinted into recombinant core PHF tau. Crystallization of the complex led to determination of the structure of the core PHF tau protein fragment 386TDHGAE391 at 1.65 Å resolution. Results: Here we present first crystallographic study of PHF core C-terminus. PHF core C-terminal hexapeptide 386TDHGAE391 has a form of a loop, which is not stabilized by any direct intrachain hydrogen bonds (Fig. 1). As the MN423 was raised against a compact, protease resistant core PHF tau, the hexapeptide directly interacting with MN423 is an integral part of the compact PHF core. Its close association with the compact PHF core suggests a potential role of the C-terminal core peptide in PHF assembly.
T419
Conclusions: The approach presented here suggests a new possibility in crystallographic analysis of intrinsically disordered proteins involved in pathogenesis of neurodegenerative diseases. It is reasonable to expect that this approach will help to reveal the structural principles underlying the tau protein assembly into PHF and possibly will facilitate rationale drug design for inhibition of Alzheimer neurofibrillary changes. P2-167
ALZHEIMER DISEASE TANGLES AND THREADS DISPLAY MULTIPLE TAU PHOSPHORYLATION SITES
J. J. Guo1, Claudia Schwab1, D. H. Li2, J. Liu2, C. Po2, P. L. McGeer1, 1University of BC, Vancouver, BC, Canada; 2 Anaspec Inc, San Jose, CA, USA. Contact e-mail: [email protected] Background: Insoluble tau accumulations are characteristic of AD and many other tauopathies. They occur primarily in the form of neurofibrillary tangles (NFTs) and threads. Tau is known to be phosphorylated in these abnormal accumulations but it is still uncertain as to which phosphokinases are primarily involved. To explore this question, we epitope mapped a series of anti-tau antibodies directed at specific sites of phosphorylation and compared their ability to detect NFTs and threads in paraformaldehyde fixed sections of AD brain. Methods: We synthesized on cellulose membranes by robotic methods, arrays of 11-mer peptides covering specific sequences in tau where each peptide was shifted by one amino acid. Matching arrays were prepared where one array was phosphorylated at key threonine and serine sites and the other non phosphorylated. Results: We found that specific antibodies raised against peptides phosphorylated at threonine 181, 205, 212 and 217 and at serine 198, 214, and 396 each recognized the specific phosphorylated peptides but not the corresponding non phosphorylated peptides. By immunohistochemistry, each antibody strongly recognized both NFTs and threads in AD tissue with no apparent qualitative differences. Conclusions: We conclude that each of these threonine and serine sites of tau is abundantly phosphorylated in the threads and NFTs that develop in AD brain. The phosphokinase GSK3b has been reported to phosphorylate recombinant tau at each of these sites and the phosphokinase cdk5 has been reported to phosphorylated the same sites except for serine 198. These phosphokinases could be playing a key role in the development of tau pathology in AD. Supported by the Pacific Alzheimer Research Foundation.
ATOMIC STRUCTURE OF THE PHF CORE C-TERMINUS
Martina Handzusova1, Natalia Ivanovova1, Jozef Sevcik1,2, Rostislav Skrabana1, Khalid Iqbal3, Michal Novak1,4, 1Institute of Neuroimmunology Slovak Academy of Sciences, Bratislava, Slovakia; 2 Institute of Molecular Biology, Bratislava, Slovakia; 3New York State Institute for Basic Research in Developmental Disabilities, New York, NY, USA; 4Axon Neuroscience GmbH, Vienna, Slovakia. Contact e-mail: [email protected] Background: The major constituent of Alzheimer’s disease paired helical filaments (PHF) core is intrinsically disordered protein (IDP) tau. In spite of a considerable effort, insoluble character of PHF together with inherent physical properties of IDP tau have precluded so far reconstruction of PHF 3-D structure by X-ray crystallography or NMR spectroscopy. Methods: Using monoclonal antibody MN423 specific to the tertiary structure of the PHF core, the in vivo PHF structure was imprinted into recombinant core PHF tau. Crystallization of the complex led to determination of the structure of the core PHF tau protein fragment 386TDHGAE391 at 1.65 Å resolution. Results: Here we present first crystallographic study of PHF core C-terminus. PHF core C-terminal hexapeptide 386TDHGAE391 has a form of a loop, which is not stabilized by any direct intrachain hydrogen bonds (Fig. 1). As the MN423 was raised against a compact, protease resistant core PHF tau, the hexapeptide directly interacting with MN423 is an integral part of the compact PHF core. Its close association with the compact PHF core suggests a potential role of the C-terminal core peptide in PHF assembly.
T419
Conclusions: The approach presented here suggests a new possibility in crystallographic analysis of intrinsically disordered proteins involved in pathogenesis of neurodegenerative diseases. It is reasonable to expect that this approach will help to reveal the structural principles underlying the tau protein assembly into PHF and possibly will facilitate rationale drug design for inhibition of Alzheimer neurofibrillary changes. P2-167
ALZHEIMER DISEASE TANGLES AND THREADS DISPLAY MULTIPLE TAU PHOSPHORYLATION SITES
J. J. Guo1, Claudia Schwab1, D. H. Li2, J. Liu2, C. Po2, P. L. McGeer1, 1University of BC, Vancouver, BC, Canada; 2 Anaspec Inc, San Jose, CA, USA. Contact e-mail: [email protected] Background: Insoluble tau accumulations are characteristic of AD and many other tauopathies. They occur primarily in the form of neurofibrillary tangles (NFTs) and threads. Tau is known to be phosphorylated in these abnormal accumulations but it is still uncertain as to which phosphokinases are primarily involved. To explore this question, we epitope mapped a series of anti-tau antibodies directed at specific sites of phosphorylation and compared their ability to detect NFTs and threads in paraformaldehyde fixed sections of AD brain. Methods: We synthesized on cellulose membranes by robotic methods, arrays of 11-mer peptides covering specific sequences in tau where each peptide was shifted by one amino acid. Matching arrays were prepared where one array was phosphorylated at key threonine and serine sites and the other non phosphorylated. Results: We found that specific antibodies raised against peptides phosphorylated at threonine 181, 205, 212 and 217 and at serine 198, 214, and 396 each recognized the specific phosphorylated peptides but not the corresponding non phosphorylated peptides. By immunohistochemistry, each antibody strongly recognized both NFTs and threads in AD tissue with no apparent qualitative differences. Conclusions: We conclude that each of these threonine and serine sites of tau is abundantly phosphorylated in the threads and NFTs that develop in AD brain. The phosphokinase GSK3b has been reported to phosphorylate recombinant tau at each of these sites and the phosphokinase cdk5 has been reported to phosphorylated the same sites except for serine 198. These phosphokinases could be playing a key role in the development of tau pathology in AD. Supported by the Pacific Alzheimer Research Foundation.