Targeting Multiple Kinases to Treat Experimental Spinal Cord Injury.

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 以及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来源的代理 失败了。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。 为了开始鉴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。 抑制物(KIS)在体外中枢神经系统神经突起生长实验中的作用和鉴定重复性强的“击中”KIS 促进企业成长。由于催化结构域的同源性，Kis通常抑制多种激酶。这就是它 很难确定介导KI对细胞影响的激酶(S)。我们使用了信息论和机器 学会分析KIS的抑制谱与其对轴突生长的影响。这使我们能够 为了识别并随后通过siRNA敲除初级神经元中的多个激酶靶点(即 应该被抑制以促进轴突生长)。这些包括先前已知的调节内源性的靶点。 和外在抑制因素，以及几个新的候选因素。相反，我们确定了其 活性对神经突起的生长至关重要，必须避免其抑制(抗靶)。我们发现 抑制多个靶点和无反靶点的几个KI。这些KI强烈促进了神经突起的生长 体外培养。我们在两个体内模型中测试了体外作用最大的KI，RO48。我们的预赛 实验表明，RO48在体内具有显著的效果。促进了大脑皮层轴突的强健生长。 三种不同的皮质脊髓束损伤模型(锥体切开术、脊髓索切断术、背侧切断术)中的CST 在背侧半横断模型中，前肢功能得到了改善。我们建议在这些基础上 显著结果检验工作假说RO48‘S同时抑制五种靶向激酶 (ROCK、PKC、PRKG1、PRKX和RPS6K)促进CST轴突的萌发和再生。这将是 使用病毒载体分别敲除不同靶蛋白激酶的表达 组合。我们将对大脑皮质中的CST神经元进行击倒。我们将评估损伤处CST轴突的生长情况 网站使用光学显微镜。我们还将进行实验，以确定RO48是否诱导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，如 挫伤后的抓握和行走。这些实验将1)验证新的激酶作为体内靶点 对于脊髓损伤治疗的未来发展，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)确认治疗对轴突生长的实质性刺激是否 在临床相关的挫伤模型中，RO48可改善运动结果。

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.