COBRE: UL: PKC REGULATION OF REGENERATION ACROSS CSPG AFTER SCI

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Glial scar formation after spinal cord injury (SCI) is central in the failure of axonal regeneration. Chondroitin sulfate proteoglycan (CSPG) is a class of major inhibitory molecules that is upregulated after SCI. We hypothesize that CSPG triggers PKC activation which induces a downstream activation of a small GTPase Rho and Rho-associated kinase (ROK) in sensory axons following SCI. We found that: 1) chondroitinase ABC, an enzyme that removes CSPG's glycosaminoglycan (GAG) side chains, had limited beneficial effect on DRG axon regeneration, 2) the Rho inhibitor bacterial exotoxin C3 transferase applied to the spinal cord was neurotoxic at high doses, 3) PKC phosphorylation is required for RhoA activation, and 4) inhibitation of conventional PKC isoforms resulted in neurite outgrowth in vitro and regeneration of DRG axons in vivo. Understanding interactions between PKC and Rho/ROK activation and how they affect neurite outgrowth in vitro and axonal regeneration in vivo are mechanistic issues related to CNS injury and regeneration. We will use contusion and laceration SCI models as well as KO transgenic mice to investigate effects of PKC and its downstream Rho/ROK inhibition on DRG axon regeneration. Aim 1 will characterize the effect of conventional PKC and Rho/ROK inhibition on DRG neurite outgrowth on a CSPG substrate using an in vitro cell culture system. Aim 2 will investigate neurite outgrowth of engrafted DRG neurons within a CSPG-enriched glial scar environment developed after a contusive SCI in mice. Aim 3 will explore axonal regeneration of engrafted DRGs towards a CSPG-enriched gradient developed after a dorsal spinal cord laceration injury in adult mice in vivo. Aim 4 will study sensory axon regeneration of host DRGs through and beyond a spinal cord dorsal laceration injury under various pharmacological treatments including the PKC and RhoA/ROK inhibitors. We hope that understanding regulation of PKC-Rho-ROK signaling pathway may provide new insights into axon regeneraton through a glial scar and provide new directions for SCI repair.