The long term objective of this project is to learn the etiology and the pathogenesis of Alzheimer's disease/senile dementia of the Alzheimer's type (AD) which constitutes one of the major public health problems in our country since over two million people are affected. Increasing evidence suggests that microtubule-associated protein tau is abnormally phosphorylated in AD brain and is a major component of the Alzheimer paired helical filaments (PHF). Our working hypothesis is (1) that the protein phosphorylation-dephosphorylation system is defective in AD brain leading to abnormally phosphorylated tau, and (2) that this abnormal phosphorylation is at least partly due to a deficit in the phosphoprotein phosphatase system. Towards this hypothesis we propose to: (1) determine in AD and control brains the levels of activities of phosphoprotein phosphatases using in vitro phosphorylated phosphorylase kinase and light chain of myosin as exogenous substrates, and in vitro phosphorylated normal tau and AD brain abnormally phosphorylated tau as endogenous substrates; (2) isolate phosphoprotein phosphatases from AD and control brains and determine their enzyme kinetics towards standard exogenous substrates, phosphorylase kinase and light chain of myosin and towards PHF, unpolymerized ,abnormally phosphorylated tau, normal tau and in vitro phosphorylated tau; (3) generate rabbit antibodies to isolated phosphatases, and determine immunocytochemical distribution, and immunoassay the levels of each phosphatase in various areas of AD and control brains; (4) study stimulation of microtubule assembly from tubulin with PHF-tau, unpolymerized abnormal tau, and normal tau, before and after dephosphorylation with different phosphatases from Specific Aim #2. The enzyme activities in Specific Aim #1 will be assayed radiometrically towards in vitro phosphorylated [32P] substrates. The phosphatases indicated from Specific Aim #1 will be isolated by tissue fractionation followed by salting or ethanol precipitation, liquid and affinity chromatographies. Immunocytochemical distribution of phosphatases (Aim #3) will be studied by light microscopy. Microtubule assembly in Specific Aim #4 will be determined both by turbidimetric measurements and by negative stain electron microscopy. Studies proposed in this application are on the identification of the protein phosphatase/s responsible for the abnormal phosphorylation in Alzheimer disease brain which information is critical to devise a rational approach in correcting this defect.
