The activities of diacylglycerol and monoacylglycerol lipases are markedly elevated in the nucleus basalis and hippocampus of Alzheimer disease brains as compared to controls. Neuron-enriched primary cultures show a marked stimulation of diacylglycerol and monoacylglycerol lipase and phospholipase A2 activities after treatment with glutamate or NMDA. The proposed work seeks to determine a mechanism for the stimulation of lipases and phospholipases A2. The stimulation may be due to (a) induction, (b) translocation, and/or (c) covalent modification (phosphorylation) of lipases and phospholipases. The amount of diacylglycerol lipase and phospholipase A2 will be quantified by Western blotting. Homogeneous preparations of diacylglycerol lipase and Ca2+- independent phospholipase A2 will be phosphorylated using various kinases. Activities and kinetic properties of phosphorylated diacylglycerol lipase and Ca2+-independent phospholipase A2 will be compared to native enzymes. The effects of inhibitors of protein kinases on the stimulation of lipases and phospholipases by glutamate and its analogs will be studied. Activity of nitric oxide synthase and effects of its inhibitors on the stimulation of lipases and phospholipases will be evaluated. Finally, levels of lipid peroxides and the phospholipid composition of neuron-enriched cultures will be determined in cultures treated with glutamate and its analogs. Results of these experiments will test our specific hypothesis that stimulation of lipases and phospholipases may produce changes in membrane permeability and fluidity, and this may be responsible for neurodegeneration.
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