Traumatic brain injury (TBI) results in the prolonged activation of calpains, which contributes to cytoskeletaldamage, neuronal death and behavioral dysfunction. However, surprisingly few in vivo cellular substrates ofcalpains have been identified in the traumatically injured brain and, consequently, little is understood aboutthe pathways through which calpains mediate posttraumatic morbidity. Calpastatin, the endogenous inhibitorof calpains, is the only known protein that exclusively inhibits calpains. As such, calpastatin represents anideal molecular tool with which to isolate the actions of calpains within the injured brain. Efforts to translateexciting preclinical data demonstrating functional improvement in brain-injured rodents treated withexogenous calpain inhibitors have been slowed by challenges with solubility, specificity and bioavailability ofsmall molecule inhibitors. Enhancing endogenous calpastatin activity may represent a novel and potenttherapeutic approach. The overall goals of Project 1, then, are to evaluate the role of calpastatin inregulating posttraumatic calpain-mediated proteolysis and to assess the neuroprotective and behavioralefficacy of increasing calpastatin activity in the setting of TBI. Using genetically altered mice that eitheroverexpress human calpastatin or are calpastatin deficient, Project 1 will: 1) evaluate the role of calpastatinin modulating behavioral outcome following focal or diffuse brain injury, 2) quantify the effects of alteredcalpastatin expression on neuronal survival and axonal injury after focal or diffuse brain injury, 3) determinethe in vivo role of calpastatin in limiting trauma-induced proteolysis of neuronal cytoskeletal proteins, and 4)evaluate the role of posttraumatic calpain activation in modifying membrane proteins involved in calciuminflux and in modulating mitochondria-related cell death events. Our central hypothesis is that calpastatinoverexpression will prevent calpain-mediated cleavage of neuronal substrates critical for cell survival,thereby attenuating posttraumatic neuronal death and dysfunction. The proposed experiments will providethe first evidence for a functional role for calpastatin in posttraumatic pathology and elucidate differentialroles for the calpain/calpastatin system in focal and diffuse TBI. In addition, this Project will provide thegroundwork for novel therapeutic approaches, based on manipulation of the calpastatin system, aimed atattenuating brain damage and dysfunction due to TBI as well as other CMS injury and disease states.
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