The identification and characterization of cellular mechanisms responsible for expressing ischemic pathology are essential for understanding the fate of vulnerable neurons and for designing rational therapeutic strategies. The studies in this proposal will characterize the role of calcium- activated proteolysis in ischemia-induced brain damage and will evaluate the therapeutic efficacy of targeting this biochemical mechanism. The calcium-activated protease, calpain, cleaves several major cytoskeletal proteins and alters the regulation of key enzyme systems in the brain. Activation of calpain in situ results in the cleavage of brain spectrin and MAP2, both prominent cytoskeletal proteins. The uncontrolled activation of calpain would thus be expected to have detrimental effects upon cellular morphology and function. Recent studies support this idea. Spectrin breakdown products (BDPs) are markedly elevated in response to manipulations which lead to neurodegenerative responses (i.e., electrolytic lesions, colchicine injections, application of excitatory amino acids). Calpain is therefore in a position to provide a link between transient ischemia and cell death because: 1) it is activated by an appropriate signal (elevated intracellular calcium), 2) it produces appropriate effects (breakdown of cytoskeleton) and, 3) it is known to be activated in conjunction with several types of neurodegenerative responses. We have recently shown that transient ischemia induces a rapid breakdown of cytoskeletal proteins in vulnerable regions of the brain and that this proteolytic response precedes overt signs of neuronal degeneration. Data are presented here indicating that treatments which inhibit calcium- activated proteolysis attenuate morphological and functional pathologies occurring in vivo and in vitro model systems of ischemia. These results provide the first evidence of the benefits (i.e. neuroprotection) that can be derived from targeting ischemia-induced proteolysis. Furthermore, these findings strongly support the hypothesis that calcium-activated proteolysis represents a critical mechanism in the process of ischemia-induced neuronal pathology. The studies proposed in this application will evaluate and extend this hypothesis by: 1) testing the correlation between ischemia-induced proteolysis and neuronal vulnerability in multiple models of CNS ischemia; 2) characterizing the calpain response to ischemia with regard to its time course, auto-activation and relationship to its endogenous inhibitor; 3) refining techniques for suppressing calpain activity and applying these techniques to investigate the timing and critical phases of calcium- activated proteolysis, and; 4) analyzing the mechanisms involved in the activation and regulation of calpain in an in vitro model system.