Truman C. Gamblin - US grants

oxidative stress; tau proteins; neurofilament proteins; free fatty acids; polymerization; molecular assembly /self assembly; gas chromatography mass spectrometry; genetically modified animals; laboratory mouse;

DESCRIPTION (provided by applicant): The long term goals of the Principal Investigator are to investigate the changes in the tau molecule that lead to a decrease in its normal functions and increase its pathological ability to self-assemble into filaments found in many neurodegenerative disorders. The purpose of this proposal is to determine the effects of changes in phosphorylation that are observed in Alzheimer's disease (AD) on the functions of the tau protein. These experiments will have an immediate impact in AD research since there is a current controversy over the role of phosphorylation and "hyperphosphorylation" in the polymerization of tau into filaments found in AD pathology. We hypothesize that site-specific phosphorylation events can change the conformation of tau and create variants of the molecule that have an increased ability to polymerize, a decreased ability to bind to and stabilize microtubules, or both. We also hypothesize that the phosphorylation of the tau molecule will increase the stability of tau filaments, which could account for their longevity in affected neurons in AD. We will test these hypotheses by accomplishing the following specific aims: 1) The effects of site-specific phosphorylation events found in AD on the polymerization of the longest isoform of tau and the stability of those filaments will be assessed using an in vitro polymerization paradigm monitored by laser light scattering and quantitative electron microscopy. 2) The effects of AD-like phosphorylation on the ability of tau to bind to and stabilize microtubules will be assessed using tubulin binding assays and microtubule polymerization assays in the presence of phospho-variants of tau. 3) The effects of site-specific phosphorylation events will be modeled in the background of the six major isoforms of tau since these isoforms can be differentially involved in neurodegenerative diseases. 4) The effects of "hyperphosphorylation" will be modeled to determine the effects of multiple phosphorylation events on the polymerization and stability of tau filaments. 5) The effects of phosphorylation that occur after tau polymerization on the stability of tau filaments will be investigated using quantitative electron microscopy.

[unreadable] DESCRIPTION (provided by applicant): My long term goal is to become a leader in the field of Alzheimer's disease research. I plan to reach this objective by studying the changes in the microtubule-associated protein tau that lead to a decrease in its normal functions and increase its pathological ability to self-assemble into filaments found in Alzheimer's disease and many other neurodegenerative disorders. We hypothesize that changes in the phosphorylation state of tau alters its biochemical characteristics of microtubule binding and self- assembly. We propose to test this hypothesis by accomplishing the following specific aims: 1) We will determine the effects of phosphorylation events known to occur in Alzheimer's disease on the ability of' tau to form fibrils; 2) We will measure the effects of phosphorylation sites generated in Aim 1 on their interactions with microtubules and their ability to polymerize in the presence of microtubules; 3) We will determine whether phosphorylation events have differential effects on the fibril formation or microtubule binding of the six isoforms of the tau protein; 4) We will determine whether combining phosphorylation events in a single protein to generate a "hyperphosphorylated" form of tau will lead to larger effects on tau's functional properties; and 5) We will use kinases that target tau protein in order to determine the effects of phosphate addition on its fibril formation and fibril stability both pre- and post-polymerization. The first step in reaching this objective was the funding of an R01 (AG022428) to study these Aims using biochemical techniques such as dynamic light scattering, right angle laser light scattering, fluorescence assays, and electron microscopy. In order to move this research to a nationally prominent level, I am seeking the Independent Scientist Award in order to obtain time off from teaching to provide the necessary training to the relatively inexperienced workers in the laboratory. Specifically, I hope to provide training in the proper execution of the experiments, responsible conduct in the recording of the data, and assistance in the interpretation of the data. I feel that this time will greatly increase the productivity in the laboratory and enhance the development of my career. [unreadable] [unreadable] [unreadable]

Antibodies; Antigenic Determinants; Arachidonic Acids; Binding Determinants; CRISP; Coloring Agents; Computer Retrieval of Information on Scientific Projects Database; Degenerative Diseases, Nervous System; Degenerative Neurologic Disorders; Disease; Disorder; Dyes; Epitopes; Filament; Funding; Grant; Heparin; Heparinic Acid; In Vitro; Institution; Investigators; Isoforms; Knowledge; Lead; Length; MT-bound tau; Molecular; NIH; National Institutes of Health; National Institutes of Health (U.S.); Nerve Degeneration; Neurodegenerative Diseases; Neurodegenerative Disorders; Neurofibrillary Tangles; Neurologic Degenerative Conditions; Neurologic Diseases, Degenerative; Neuron Degeneration; Numbers; Patients; Pb element; Play; Post-Translational Modifications; Post-Translational Protein Processing; Posttranslational Modifications; Process; Protein Isoforms; Protein Modification; Protein Modification, Post-Translational; Protein Processing, Post-Translational; Protein Processing, Posttranslational; Protein/Amino Acid Biochemistry, Post-Translational Modification; Proteins; Research; Research Personnel; Research Resources; Researchers; Resources; Role; Shapes; Solid; Source; Structure; Tauopathies; United States National Institutes of Health; abnormally aggregated tau protein; aggregation of microtubule associated protein tau; aggregation of microtubule-associated protein tau; disease/disorder; filamentous tau inclusion; gene product; heavy metal Pb; heavy metal lead; microtubule associated protein tau; microtubule associated protein tau aggregation; microtubule associated protein tau deposit; microtubule bound tau; microtubule-associated protein tau; microtubule-associated protein tau aggregation; microtubule-associated protein tau deposit; microtubule-bound tau; neural degeneration; neurodegeneration; neurodegenerative illness; neurofibrillary degeneration; neurofibrillary lesion; neurofibrillary pathology; neuronal degeneration; paired helical filament of tau; polymerization; self-aggregate tau; size; social role; tangle; tau; tau PHF; tau PHF formation; tau Proteins; tau accumulation; tau aggregate; tau aggregation; tau associated neurodegeneration; tau associated neurodegenerative process; tau factor; tau fibrillization; tau filament; tau filament assembly; tau induced neurodegeneration; tau mediated neurodegeneration; tau neurodegenerative disease; tau neurofibrillary tangle; tau neuropathology; tau oligomer; tau paired helical filament; tau paired helical filament formation; tau polymerization; tau self-aggregate; tau-tau interaction; tauopathic neurodegenerative disorder; tauopathy

DESCRIPTION (provided by applicant): Alzheimer's disease is the most common neurodegenerative disorder and is characterized pathologically by the intraneuronal deposition of abnormally phosphorylated and aggregated tau protein and by the formation of extracellular amyloid plaques. Abnormal deposition of tau into neurofibrillary tangles is also the primary pathologic feature of a group of less common disorders, collectively termed the tauopathies. To define the molecular mechanisms controlling tau-induced neurodegeneration we (Dr. Gamblin) have performed extensive biochemical characterization of tau variants, including splicing isoforms and mutants linked to the familial tauopathy frontotemporal dementia and parkinsonism linked to chromosome 17. Importantly, these biochemical studies have defined tau variants with altered aggregation and microtubule binding properties. In parallel we (Dr. Feany) have created Drosophila models of tauopathy. Our models recapitulate key features of the human diseases, including age-dependent neurodegeneration, abnormal tau phosphorylation, aggregation of tau into fibrillary tangle-like inclusions, and early death. We will now combine our strengths in biochemistry and in vivo tauopathy modeling to define the species of tau that cause cellular and organismal toxicity in tauopathies.