Distribution and Gene Expression of Extravascular Fibrinolytic Enzyme Inhibitors (Serpins) in the Nervous System

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Dr. Festoff proposes to study the distribution and gene expression for protease nexin I (PNI), a potent serine protease inhibitor of thrombin, plasmin and plasminogen activators (PAs) and PAI-1, a specific PA inhibitor, in the developing rodent nervous system. The discovery, isolation, sequencing and gene- cloning for PNI have been accomplished. PNI is identical to glial-derived neurite promoting factor. Dr. Festoff has isolated PNI and PAI-1-like molecules from muscle and glial cells of mice. Protein (antigen) and functional (protease-inhibitory) assays will be performed by Dr. Festoff during embryonic and post-natal development in mouse brain, spinal cord and skeletal muscle. Determination of active versus inactive PNI and PAI-1 will be made in these tissues and will be correlated with immunocytochemical studies to localize the PNI and PAI-1 antigens to specific cell types at different stages of development. Since tissue culture studies show that glial cells contain both PNI and PAI-1 and secrete these inhibitors into the media, Dr. Festoff proposes that glial cells, probably astrocytes and Schwann cells, will contain the antigen in vivo as well. Using a cDNA probe for human PNI, Dr. Festoff will obtain complementary RNA and then screen mouse cDNA libraries for cross-hybridizing clones. He will perform slot-blot, Northern analysis and in situ hybridization to identify PNI mRNA in those regions of the nervous system found to contain the highest concentration of antigen and functional inhibitor. For PAI-1, Dr. Festoff will screen a GT11 library using monospecific antisera and isolate cDNA for it to perform the same studies. A number of recent studies suggests that extravascular serine proteases, notably thrombin and plasminogen activators, may actively participate in neural development. Since these enzymes are rapidly and dramatically inhibited by PNI and PAI-1 the proposed studies may reveal more precise information concerning the role of these intriguing proteins in nervous system development.