Deficits of cholinergic neurons and altered membrane composition and function are prominent characteristics of Alzheimer's Disease. This project is focussed on the regulation of choline availability, which is both an important precursor and product of both acetylcholine and phosphatidylcholine, the dominant membrane phospholipid in brain. Phosphatidylcholine in brain membrane is hydrolyzed by phospholipase D to produce free choline and phosphatidic acid, which may have regulatory properties itself and which can be rapidly cleaved to form diacylglycerol, and activator of protein kinase C. Thus this reaction is a central point linking acetylcholine, membrane lipids, and second messenger production, each of which appears to be abnormal in Alzheimer's Disease. Virtually nothing is known about the regulation of phospholipase D in brain tissue. Because of this lack of knowledge and its potential importance in Alzheimer's Disease, the central goal of this project is to fully characterize the regulation of choline release and directly address several specific hypotheses relating to this system. Preliminary data suggest that there may be some similarities between phosphatidylcholine hydrolysis and that of phosphoinositides, a well- characterized second messenger source. Thus many of the approaches used in this project are based on knowledge of phosphoinositide hydrolysis, a system with which this laboratory has much experience. An unique advantage that will be utilized is the ability to use gas chromatography mass spectrometry to measure the mass of choline released, thus obviating the more common requirement of only measuring radiolabeled products. The initial goal will be to characterize in rat hippocampal and cortical membranes the release of choline and phosphocholine, ionic requirements, and to test the hypothesis that a G-protein modulates this reaction. The second goal will be to use modulate choline release. The third goal is to test the hypothesis that choline release is modulated by other intracellular messengers. The final three goals are to study the link of choline production to protein kinase C activity, to identify age-related changes, and to test the feasibility of making these measurements in postmortem tissue.
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