THROMBIN REGULATION OF CNS CELLS AFTER INJURY

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Thrombin is a multifunctional protease that regulates gene expression and process outgrowt in cultured neurons and astrocytes as well as proliferation of cultured astrocytes. Our recent studies have shown that thrombin prevents cell death in neurons cultured under hypoglycemic conditions or in the absence of growth factors. Thrombin also prevents cell death in astrocytes cultured under hypoglycemic conditions or in the presence of H202. In contrast, high concentrations of thrombin can kill both neurons and astrocytes cultured under Nnormal conditions. The effects of thrombin on these cells are receptor-mediated and can be blocked by the brain thrombin inhibitor, protease nexin-1 (PN-1). The first goal of these studies is to explore the mechanisms by which thrombin protects or kills cultured rat neurons and astrocytes. Experiments will focus on protective effects, since they occur over a broad concentration range of thrombin (10pM to 500nM) and because thrombin is one of the first molecules present at sites of trauma. Since the diverse cellular responses produced by thrombin are due to differential activation of a variety of second messenger systems, experiments will examine the role of phospholipase C, adenylyl cyclase, protein kinase G, intracellular calcium, and protein tyrosine kinase in protection and killing of neurons and astrocytes by thrombin. We will also examine the ability of thrombin to regulate certain genes, including bcl-2 and bax, which have been shown to mediate cell protection or death. Studies will also be conducted to determine if cell killing by thrombin occurs by programmed cell death or by necrosis and if it involves oxidative mechanisms. The second major goal of these studies is to test the hypothesis that thrombin might help orchestrate certain activities of astrocytes and microglia that are involved in repair/inflammatory processes, based on findings that injection of thrombin into rat brain causes an increase in reactive astrocytes and infiltration of inflammatory cells. Experiments will be conducted on cultured rat astrocy!es to determine if thrombin increases GFAP and vimentin expression as well as secretion of growth factors and cytokines such as bFGF, IL-1, IL-6 and TGF-beta. Studies will also be carried out to determine if thrombin is chemotactic for microglia and if it stimulates them to proliferate, to phagocytose particles or to secrete cytokines. The final goal of this project is to conduct in vivo experiments to evalute results obtained from studies on cultured cells. In collaboration with Dr. Cotman and his colleagues, we will administer the thrombin inhibitor PN-1 to excitotoxic lesions in rat brain that range from threshold to severe. This will enable us to examine effects of thrombin inhibition on cell death and repair processes. In collaboration with Dr. Gall, prothrombin, thrombin receptor and PN-1 mRNA will be examined by in situ hybridization in specific regions of rat brain as a function of aging. These studies will test the hypothesis that age-related chanaes in these molecules miaht compromise the abilitv to respond to certain kinds of stress or trauma.