These studies are based on findings that: (a) protease nexin-1 (PN-1) is a thrombin inhibitor present in human brain that produces neurite extension in cultured neuroblastoma cells, (b) thrombin produces neurite retraction in cultured neuroblastoma cells, (c) PN-1 and thrombin, reversibly regulate the stellation of cultured astrocytes, (d) PN-1 activity is reduced about 6-fold in AD brain, and (e) PN-1-protease complexes (probably PN-1 thrombin complexes appear to be increased in AD brain. Together, these results suggest the hypothesis that decreases in PN-1 (and increases in thrombin or a thrombin-like protease) could contribute to the pathology of AD by altering neurons and astrocytes. A major goal of our proposed studies is to learn more about the decrease in PN-1 and increase in PN-1-protease complexes in AD brain and to learn whether similar changes occur in other neurodegenerative diseases. WE will determine if the changes are global, or if they localized to certain brain structures. Accordingly, we will measure PN-1 activity, PN-1 protein, PN-1 protease complexes and PN-1 mRNA in various brain structures in a large number control brains and brains from individuals who died from various neurological diseases. In these studies we will also examine the microscopic distribution of PN-1 protein and PN-1 mRNA using immunohistochemistry and in situ hybridization. Another major goal is to identify the protease present in PN-1 protease complexes found in AD brain. The studies in the paragraph above will determine what brain sections contain the complexes, and if they occur in other neurodegenerative diseases. We will purify the complexes, disrupt them to release the intact protease, and identify the protease by amino acid sequence analysis. The other major goal is based on our observation that thrombin brings about shortening of neurites on cultured rat hippocampal neurons. We will determine if thrombin affects neurite branching and the number of neurite segments and if it modulates neuronal regulation by EGF, FGF and the amyloid b-protein. We will also determine if the effects of thrombin on neuroblastoma cells and neurons are mediated by cell surface thrombin binding sites. We will also purify rat PN-1 and isolate a panel of monoclonal antibodies to it for Dr. Cotman's studies on the response of PN-1 to entorhinal and hippocampal lesions in the rat brain.
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