University of Kentucky Research Foundation (Kentucky)
James W. Geddes, Ph.D.
Associate Director, SCoBIRC

Calpain Knockdown to Minimize Damage and Deficits Following SCI

Considerable evidence implicates overactivation of calpains (calcium-activated neutral proteases) as key components of the secondary injury cascade following spinal cord injury. However, calpains also play critical physiologic roles, with knockouts of several calpain isoforms being embryonically lethal. Thus, the challenge is to target the pathologic calpain overactivation but preserve physiologic calpain function following spinal cord injury. The major calpains in the spinal cord are the ubiquitous mu- and m-isoforms, composed of the catalytic 80 kDa subunit, calpain 1 and calpain 2 respectively, along with a common regulatory 28 kDa small subunit. We recently found that mu-calpain (calpain 1 plus calpain small subunit 1) is localized to the mitochondrial intermembrane space. One putative substrate of mu-calpain is apoptosis inducing factor, which when cleaved in and released from mitochondria induces caspase-independent cell death. Calpain 1 knockout mice are viable and have a minimal phenotype. By knocking down the calpain 1 levels, we hypothesize that injury induced calpain overactivation will be attenuated, but that the essential physiologic functions of calpain 2/m-calpain will be maintained. We therefore hypothesize that neurons deficient in calpain 1 will exhibit resistance to excitotoxic death (Aim 1) and that neurodegeneration and functional impairment following spinal cord injury will be markedly attenuated in mice and rats deficient in mu-calpain (Aim 2). These aims will also examine the alternate hypothesis, that knockdown of calpain 2 will protect neurons from exicitotoxin and injury induced degeneration. Calpain 1 knockdown will be achieved using lentiviral shRNA and calpain 1 null mice. Calpain 2 knockdown will utilize lentiviral shRNA vectors, as the knockout mice are embryonically lethal. Together the experiments outlined in this proposal will enhance our understanding of the role of mu- and m- calpain isoforms in neuron death and dysfunction following spinal cord injury, and determine their suitability as therapeutic targets.