Calpain as a Therapeutic Target Following Spinal Cord Injury

This revised proposal is based on findings from our lab and other groups that calcium-activated proteases (calpains) are rapidly activated after spinal cord injury (SCI), with activity peaking at 1-2h postinjury. Transient calpain activation may participate in cell signaling and cellular plasticity, whereas prolonged calpain activation becomes pathological and contributes to cell death. Calpain substrates include cytoskeletal proteins, signal transduction proteins, and other proteins important for cell survival. The previous studies provide substantial evidence that calpain inhibition represents a rationale and feasible therapeutic target for the treatment of spinal cord injury. The goals of the experiments outlined in this proposal are to examine the ability of synthetic calpain inhibitors to decrease cellular damage and locomotor impairment when administered postinjury; to develop novel inhibitors based on the potent and specific endogenous calpain inhibitor, calpastatin, fused to the protein transduction domain of the HIV TAT protein; and to begin to examine the relative roles of!!- vs. m- calpain in the cellular degeneration that occurs following spinal cord injury. To examine the hypothesis that calpain inhibition represents a rationale target for intervention following spinal cord injury, the first aim of this proposal will use two synthetic calpain inhibitors, MDL28170 and calpeptin. We will determine the optimal drug, dose, route of administration, and postinjury therapeutic window for inhibition of calpain activity. These conditions will then be used to test the hypothesis that postinjury drug administration attenuates the cellular damage and loss of locomotor function resulting from SCI. Currently available calpain inhibitors also inhibit other cysteine proteases. The endogenous calpain inhibitor, calpastatin, is much more potent and very specific for calpain but is not cell permeable. In Aim 2, we will construct fusion proteins consisting of calpastatin or its inhibitory domain, combined with the 11 amino acid protein transduction domain (PTD) from the HIV TAT protein. The ability of the resultant fusion proteins to cross cell membranes and inhibit calpain will be evaluated both in vivo and in vitro. The ability of the TAT-calpastatin fusion proteins to inhibit calpain activation following spinal cord injury will also be examined. Although the m-calpain isoform is predominant in many tissues including the CNS, !!-calpain is most strongly implicated in the neurodegeneration associated excitotoxic insult, ischemia, and spinal cord injury. Dr. Athar Chishti and colleagues at Tufts University recently developed a mouse without functional !!-calpain. In Aim 3, we will compare the extent of cellular damage and calpain substrate proteolysis following SCI in !!-calpainCI-)mice and with wild-type littermate controls. The goal of these studies is to determine if selective inhibition of one calpain isoform could block the pathologic consequences of excessive calpain activation without being detrimental to normal cellular function. Together, the proposed studies will examine the suitability of current synthetic calpain inhibitors as a possible postinjury therapeutic intervention, develop novel calpain inhibitors that are more selective and potent than current drugs, and begin to examine the role of individual calpain isoforms in the secondary damage that occurs following SCI.