The long term objective of this proposal is to understand the etiology and the pathogenesis of Alzheimer's disease (AD). It is a progressive degenerative disorder of the brain which destroys the cognitive functioning of the patient, leading to a total dependence on caregivers. With rapid advances in life saving procedures, the increase in elderly population will lead to increase in expenditure for long term care. Such costs can be reduced by addressing issues related to the understanding and prevention of AD. In AD brain, there appears to be a derangement in the phosphorylation/dephosphorylation mechanism of neuronal proteins, including the microtubule associated protein, Tau. The paired helical filaments (PHF) in AD brain may contain abnormally phosphorylated tau.
The Specific Aims of this project are: 1) to determine the total phosphate and the levels of each phosphoaminoacid in PHF, AD tau and control tau, as well as in the individual polypeptides of tau and PHF obtained from Western blots and identified by immunoblots from sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The total phosphate content will be determined, by measuring calorimetrically, the amount of inorganic phosphate released from the ashed phosphoproteins, PHF and tau, and their polypeptides. The phosphoaminoacids will be qualitatively and quantitavely analysed by high performance liquid chromatography (HPLC) of the hydrolysed phosphoproteins. 2) to study the accessibility of tau and PHF polypeptides to exogenous phophoprotein phosphatases. The cleavage of the phosphate groups by the phosphatases will be assessed by measuring the release of free inorganic phosphate calorimetrically and by determining the phosphoaminoacids remaining unaffected by this treatment. This proposal might provide a lead to the type of protein kinase/s (serine or tyrosine protein kinases), that are responsible for abnormal phosphorylation of tau and PHF in AD, and will provide information as to whether, the phosphate groups in the abnormally phosphorylated tau and PHF, are accessible to cleavage by specific phosphatases. Identification of the nature of the phosphorylation in PHF and in altered tau in AD will help understand the role of this biochemical lesion in neurofibrillary pathology and eventually help develop a rational approach in rectifying this defect in AD.
