The long term objective of this proposal is to understand the mechanism of neurofibrillary degeneration in Alzheimer's disease (AD).
The specific aims of this project are to reveal the nature and extent of glycosylation of microtubule associated protein tau during various stages of Alzheimer neurofibrillary degeneration, and to understand the role of tau glycosylation in the conversion of tau into tangles of paired helical filaments (PHF) in AD brain. Towards these aims it is proposed: (1) to identify the saccharides and the glycosylation sites of various species of tau representing different stages of neurofibrillary degeneration. Normal tau from normal human brains, and AD non- hyperphosphorylated tau, soluble AD abnormally phosphorylated tau, readily detergent soluble PHF-tau and sparingly detergent soluble PHF-tau from AD brains will be bulk isolated. The presence and the quantity of saccharides conjugated to these proteins will be determined by lectin binding techniques and gas chromatography-Mass spectrometry, respectively. The types of glycosidic linkages will be identified by their sensitivity to selective glycosidases. The glycoylation sites will be mapped by generation and isolation of glycopeptides, and their amino acid sequencing and matrix-assisted laser desorption/ionization-Mass spectrometry. (2) To study the interactions between abnormal glycosylation and abnormal hyperphosphorylation of tau and the role of tau glycosylation in the AD pathogenesis. The effects of one of the two tau posttranslational modifications (glycosylation and phosphorylation) on each other will be investigated by comparing the kinetics of the modification with and without the prior presence of the other modification. The sensitivity of glycosylated tau to proteolysis and its tendency to polymerize into filaments will be studied before and after removal of glycans from the proteins. Finally, the structure of PHF tangles will be examined by electron microscopy after the tangles are deglycosylated. These studies will elucidate the abnormal tau glycosylation and its impact on the formation of PHF tangles and, therefore, should provide a new inside view of the mechanism of Alzheimer neurofibrillary degeneration.
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