Targeting Multiple Kinases to Treat Experimental Spinal Cord Injury

靶向多种激酶治疗实验性脊髓损伤 项目总结： 脊髓损伤(SCI)患者功能恢复有限，部分原因是轴突缺乏。 受损的中枢神经系统神经元的再生。缺乏有效的治疗方法，可能是由于多种因素，内在的和 抑制轴突生长的外在因素。因此，我们需要针对不止一个再生故障来源的代理。 蛋白激酶是一种普遍存在的信号转导分子，它调节包括轴突生长在内的大多数细胞过程。要开始 为了确定正向调节轴突生长的化合物，我们筛选了1600个小分子激酶抑制剂(Kis)。 体外中枢神经系统神经突起生长试验，并鉴定出可重复性强促进突起生长的“Hit”Kis。到期 由于催化结构域的同源性，Kis典型地抑制多个激酶。这使得鉴定该激酶变得困难(S) 它介导了KI对细胞的影响。我们使用信息论和机器学习来分析抑制谱 Kis与它们对轴突生长的影响有关。这使我们能够识别并在以后通过siRNA敲除进行验证 在初级神经元中，多个激酶靶点(即应该被抑制以促进轴突生长的激酶)。这些 包括先前已知的调节内在和外在抑制因子的靶点，此外还有几个新的 候选人。相反，我们确定了其活性对轴突生长至关重要，并且其抑制作用必须 被避开(反目标)。我们发现了几种抑制多靶点的KI，而不是反靶点。这些KI强烈地 促进体外培养的轴突生长。 我们在两个体内模型中测试了体外作用最大的KI，RO48。我们的初步实验 说明RO48在体内具有显著的药效。促进大鼠皮质脊髓束(CST)轴突生长 三种不同的CST损伤模型(锥体切断术、脊髓索切断术、背侧半横断)和背侧半横断 模型，改进了前肢功能。我们建议以这些显著的结果为基础来检验工作假说 同时抑制RO48‘S五种靶向激酶(ROCK、PKC、PRKG1、PRKX和RPS6K)促进萌发 和CST轴突的再生。这将通过使用病毒载体来下调不同基因的表达来实现 单独和组合使用目标激活器。我们将对大脑皮质中的CST神经元进行击倒。我们将评估CST 光镜观察损伤部位轴突生长情况。我们还将进行实验，以确定RO48诱导的 CST轴突生长促进轴突萌发、再生或两者兼而有之，以及RO48是否改善了行为结果 就像挫伤后的抓握和行走一样。 这些实验将1)验证新的激酶作为未来脊髓损伤治疗发展的体内靶点2) 确定这些激酶是否调节CST轴突的萌发、再生或两者兼而有之，以及3)确认 RO48治疗对轴突生长的实质性刺激改善了临床相关的运动结果 挫伤模型。

Title: Targeting Multiple Kinases to Treat Experimental Spinal Cord Injury Project Summary: Spinal cord injury (SCI) patients experience limited functional recovery, owing in part to the paucity of axon regrowth from injured CNS neurons. Effective treatments are lacking, likely because of multiple factors, intrinsic and extrinsic, that inhibit axon growth. Thus we require agents that target more than one source of regeneration failure. Kinases are ubiquitous signal transducers that regulate most cellular processes, including axon growth. To begin to identify compounds that positively regulate axon growth, we screened 1600 small-molecule kinase inhibitors (KIs) in an in vitro CNS neurite outgrowth assay and identified “hit” KIs that reproducibly and strongly promote outgrowth. Due to homology of catalytic domains, KIs typically inhibit multiple kinases. This makes it difficult to identify the kinase(s) that mediate a KI's effects on cells. We used information theory and machine learning to analyze the inhibition profiles of KIs in relation to their effects on neurite outgrowth. This enabled us to identify, and later validate via siRNA knockdown in primary neurons, multiple kinase targets (i.e. kinases that should be inhibited to promote neurite outgrowth). These included previously known targets that regulate intrinsic and extrinsic inhibitor factors, in addition to several novel candidates. Conversely, we identified kinases whose activity is critical for neurite outgrowth, and whose inhibition must be avoided (anti-targets). We discovered several KIs that inhibit multiple targets and no anti-targets. These KIs strongly promoted neurite outgrowth in vitro. We tested the KI, RO48, that had the largest effect in vitro in two in vivo models. Our preliminary experiments indicate that RO48 is remarkably effective in vivo. It promoted robust axonal growth of the corticospinal tract (CST) in three separate models of CST injury (pyramidotomy, funiculotomy, dorsal hemisection), and in the dorsal hemisection model, improved forelimb function. We propose to build on these remarkable results to test the working hypothesis that the simultaneous inhibition of RO48's five target kinases (ROCK, PKC, PRKG1, PRKX, and RPS6K) promotes sprouting and regeneration of CST axons. This will be accomplished using viral vectors to knock down expression of the different target kinases individually and in combination. We will do knockdown in CST neurons in the cortex. We will assess CST axon growth at the injury site using light microscopy. We will also perform experiments to determine if RO48-induced CST axon growth promotes axon sprouting, regeneration, or both, and whether RO48 improves behavioral outcomes such as grasping and walking after a contusion injury. These experiments will 1) validate novel kinases as in vivo targets for future development of SCI therapeutics 2) determine whether these kinases regulate CST axon sprouting, regeneration, or both, and 3) confirm whether the substantial stimulation of axon growth induced by treatment with RO48 improves motor outcomes in a clinically relevant contusion model.