Timing and Duration of Calpain Inhibition Following SCI

The pathogenesis of traumatic spinal cord injury (SCI) involves two components: the initial primary mechanical injury and subsequent secondary mechanisms; including elevations in intracellular Ca2 +, vasoconstriction, edema, oxidative stress, and inflammation; which contribute to extensive neuronal degeneration over the next few hours to days. Calcium-activated neutral proteases (calpains) are excessively activated soon after spinal cord injury (SCI), with elevated activity persisting for up to two weeks postinjury. Calpains are ideally positioned in signaling and proteolytic cascades to help coordinate the postinjury secondary mechanisms. Calpain overactivation is strongly implicated in neuron death, axonal degeneration, necrotic and apoptotic oligodendrocyte death and demyelination, and in cell migration, cytokine activation, and signal transduction associated with the postinjury inflammatory response. Unfortunately, current calpain inhibitors suffer from weak potency, poor specificity, and short in vivo half lives. In vivo administration of the inhibitors has been limited to a single postinjury administration and results in very modest calpain inhibition. As a result, it has not been possible to test the hypothesis that effective, sustained calpain inhibition will protect against the secondary damage and improve functional outcome following spinal cord injury. Since calpain is involved in normal cell signaling and plasticity, the challenge is to sufficiently inhibit calpain overactivation but not be detrimental to normal cell function or viability. The endogenous calpain inhibitor, calpastatin, is a potent and specific calpain inhibitor, but does not cross cell membranes. In the studies described in this application, intraspinal microinjection of viral vectors encoding tetracycline-regulated calpastatin will enable us to evaluate the timing and duration of calpain inhibition on functional outcomes and tissue sparing. Specific Aim 1 will evaluate the hypothesis that tetracycline-regulated lentiviral vectors can provide regulatable, robust, calpastatin expression and calpain inhibition in cultured cells and and in both gray and white matter in the spinal cord. Specific Aim 2 will evaluate the most effective timing of calpastatin expressionlcalpain inhibition following experimental spinal cord contusion injury for protection against the secondary damage and improvement of functional outcome.