The objective of this proposal is to investigate the activity and potential mechanism of action of kallikreins in traumatic spinal cord injury with a focus on their roles in mediating axon injury, astrogliosis, and alteration of the environment available for nerve regeneration. Preliminary studies of all 15 members of this newly identified gene family show select kallikreins are differentially expressed across the CNS-immune axis and therein are differentially regulated by injury. We are particularly interested in the activity of kallikrein 6 (K6) since we have shown that: 1) K6 is up regulated in resident spinal cord cells and infiltrating macrophages at acute and chronic time points post-injury in both animal models and cases of human traumatic spinal cord injury; 2) K6 is a trypsin-like enzyme that rapidly hydrolyzes myelin and extracellular matrix proteins; 3) K6 hydrolyzes growth facilitatory substrates such as laminin to inhibit neurite outgrowth, while hydrolysis of inhibitory substrates, such as aggrecan, promote neurite extension; 4) K6-function blocking antibodies attenuate clinical and pathological disease in murine models of inflammatory spinal cord injury. Further, preliminary data demonstrate that kallikreins may exert their effects not only at the level of the substrate, but in addition may cleave select protease activate receptors (PARs) to trigger intracellular signaling cascades including calcium flux and Erk- phosphorylation, which may directly contribute to altered neurite outgrowth and astrogliosis. Based on these results, we hypothesize that kallikrein-mediated proteolysis plays fundamental roles in the response of the spinal cord to injury, including secondary tissue destructive events and modification of the capacity for nerve regeneration. To test this hypothesis, we intend to fulfill the following specific aims: 1) determine the dynamics and cellular specificity of expression of all 15 kallikreins in human and murine traumatic spinal cord injury; 2) using cell culture systems compare the consequences of elevations in the level of 2 kallikreins, K1 or K6, on axon integrity and neurite outgrowth, astrogliosis, and the potential mechanism of action; 3) determine the range of action of K1 and K6 in spinal cord pathogenesis and whether their effects are mediated by PAR following direct injection into the spinal cord of wild-type or PAR-deficient mice; and 4) determine whether altering the activity of K1, K6, or PAR at the time of SCI, alone or in combination, alters secondary tissue destruction and promotes locomotor recovery in a murine clip compression model. Our long-term goal is to understand the role and possible mechanisms by which kallikreins participate in events secondary to spinal cord trauma, including failure of axon regeneration. This study should generate new and important information providing the impetus for development of novel therapeutic regimes to promote recovery in cases of SCI.
The goal of this project is to delineate novel pathophysiologic mechanisms mediated by a family of proteases referred to as kallikreins, in human traumatic spinal cord injury, to model these in mice, and to determine whether altering their activity promotes neurobehavioral recovery. These studies have the potential to identify new therapeutic interventions for SCI patients with both acute and more chronic injuries. ? ? ?
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