This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Neuroinflammation is an important factor in the pathogenesis of a number of human neurodegenerative disorders resulting in neuronal cell loss and dementia. The objectives of this proposal are to determine specifically the contribution that phospholipases A2 (PLA2)- and phospholipases C (PLC)-mediated signal transduction have in the progression of cell loss in a rat model of neuroinflammation. It is known that the activation of these enzymes participate in the neuroinflammatory cascade by releasing second messenger molecules that influence ionic channels, glutamate release, and that can be converted to biologically active eicosanoids. Induction of these cascades also disrupts brain metabolism and increases the expression of enzymes found in the inflammatory cascades. However, little is known concerning the contribution that these signaling pathways have in the sequel of events that result in cell death during neuroinflammation. The goals of this project are to test our central hypothesis that phospholipase activation is an initial convergence point of the neuroinflammatory reaction and is essential for the progression of injury that results in neural cell death. This will be accomplished by pursuing the following Specific Aims: 1) Identify brain inflammatory signaling pathways in vivo, 2) Determine the contribution of PLC/PI3 kinase activation in TNF-a release and glial activation, and 3) Evaluate strategies for intervention by disrupting PLA2 expression in vivo. To test our hypothesis, we will use steady-state radiotracer kinetic analysis, biochemical assays, gene expression analysis, immuno histochemistry, behavioral studies, and enzyme inhibiting strategies. During these studies we will determine the contribution that both PLA2 and PLC and the subsequent turnover of membrane phospholipids have in initiating cascades that result in injury associated with neuroinflammation, determine the contribution that receptor/G-protein coupled activation of the PLC/PI3 kinase pathway has in altering lipid-mediated cascades that result in the induction of inflammatory cascades, and determine specifically the contribution that type IVA cytosolic PLA2 has in the progression of inflammatory cell death by inhibiting its activity and expression using inhibitor cocktails and siRNA technology. These studies will provide the information needed to evaluate the contribution that PLA2- and PLC-mediated signaling has in the temporal progression of cell loss in neuroinflammation.
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