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

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得主要资金，因此可以在其他CRISP条目中表示。所列机构为中心，不一定是研究者所在机构。脊髓损伤后神经胶质瘢痕的形成是轴突再生失败的主要原因。硫酸软骨素蛋白聚糖（CSPG）是SCI后上调的一类主要抑制分子。我们假设CSPG触发PKC激活，从而诱导SCI后感觉轴突中小GTdR Rho和Rho相关激酶（ROK）的下游激活。我们发现：1）软骨素酶ABC，一种去除CSPG的糖胺聚糖（GAG）侧链的酶，对DRG轴突再生具有有限的有益作用，2）应用于脊髓的Rho抑制剂细菌外毒素C3转移酶在高剂量下是神经毒性的，3）PKC磷酸化是RhoA激活所需的，（4）常规PKC亚型的激活导致体外神经突起生长和体内DRG轴突再生。了解PKC和Rho/ROK激活之间的相互作用以及它们如何影响体外神经突生长和体内轴突再生是与CNS损伤和再生相关的机制问题。我们将使用挫伤和撕裂SCI模型以及KO转基因小鼠来研究PKC及其下游Rho/ROK抑制对DRG轴突再生的影响。目的1将使用体外细胞培养系统表征常规PKC和Rho/ROK抑制对CSPG底物上DRG神经突生长的影响。目的2将研究在小鼠挫伤性SCI后形成的富含CSPG的胶质瘢痕环境中移植的DRG神经元的轴突生长。目的3将探索成年小鼠体内背侧脊髓撕裂伤后，移植的背根神经节向富含CSPG的梯度的轴突再生。目的4将研究在各种药物治疗下，包括PKC和RhoA/ROK抑制剂，通过和超越脊髓背侧撕裂损伤的宿主DRG感觉轴突再生。我们希望通过对PKC-Rho-ROK信号通路调控的研究，为神经胶质瘢痕轴突再生机制的研究提供新的思路，并为脊髓损伤的修复提供新的方向。

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.