Calcium-activated neutral proteases (calpains) are excessively activated soon after spinal cord injury (SCI), with elevated activity persisting for at least 24h postinjury. Calpain substrates include cytoskeletal proteins, signaling proteins, and cell life/death proteins. While calpains play normal roles in cell signaling and plasticity, their over activation following SCI is strongly implicated in secondary neuron death, axonal degeneration, and oligodendrocyte death and demyelination. Calpain inhibition represents a rational and feasible therapeutic target for postinjury intervention following SCI. In previous studies, postinjury administration of calpain inhibitors attenuated axonal damage and loss of neurofilament proteins, but functional outcome was not examined and the magnitude of calpain inhibition was not measured. In some studies the inhibitors used were not cell permeable, and in others the low inhibitor concentrations may not have inhibited calpain activity. Therefore, the hypothesis that calpain inhibition is neuroprotective following SCI has not been tested. Effective calpain inhibition following SCI presents a number of challenges. Synthetic calpain inhibitors are not specific for calpain, have relatively weak potency, and have a plasma half-life of only 2h. While short-term calpain inhibition may be beneficial, long-term inhibition can be detrimental to normal cell function. The first Specific Aim of this proposal will examine the hypothesis that postinjury administration of synthetic calpain inhibitors can significantly attenuate calpain activity and the proteolysis of calpain substrates following spinal cord injury. We will use both active site-directed peptide calpain inhibitors and a non-peptide inhibitor acting at the calcium-binding domain, and will examine both intravenous, intrathecal, and intraspinal delivery of the calpain inhibitors. Having identified conditions that provide effective calpain inhibition, the second Aim will examine the hypothesis that postinjury calpain inhibition can significantly attenuate lesion volume and improve functional outcome. The third Specific Aim will focus on the endogenous calpain inhibitor, calpastatin, and will examine the hypothesis that fusion proteins consisting of calpastatin and the Tat protein transduction domain will result in a potent and specific, cell-permeable, calpain inhibitor. The fourth Specific Aim examines the hypothesis that expression and secretion of the Tat-catpastatin will provide effective calpain inhibition in neighboring cells. Together, the goal of the proposed studies is to develop effective strategies for calpain inhibition following SCI.
