Novel Gene Targets for CNS Axonal Regeneration

描述（由申请人提供）：中枢神经系统（CNS）损伤后恢复的主要障碍是轴突不能有效再生。各种外在和内在因素导致了这一问题。外在因素包括在损伤部位内和周围发现的抑制性蛋白，例如来自神经胶质瘢痕的那些以及与完整或受损的髓鞘相关的那些。关于内在因素，激励这项工作的一个关键发现是，背根神经节（DRG）神经元可以响应外周损伤，即使在损伤部位周围的抑制环境中，也可以通过基因表达的变化促进CNS再生。相比之下，CNS神经元通常不能通过这种抑制区域再生轴突。这意味着CNS神经元具有限制CNS再生能力的固有分子差异。最近发现的PTEN，SOCS 3，KLF 4和KLF 7作为重要的中枢神经系统轴突再生的内在调节剂验证了这一假设。然而，即使在这些基因已经被操纵的动物中，也只有一小部分CNS轴突能够在损伤后再生，这表明还需要发现其他的调节因子。目前的建议是使用高通量技术，以确定基因调控中枢神经系统的再生，通过检查两个相关的假说的内在因素。第一种是直接延续 驱动先前授权的假设;即，DRG神经元表达的RNA在CNS神经元中以显著较低的水平表达，并允许DRG轴突再生。第二个是经历了条件性外周损伤的DRG神经元表达允许再生的RNA，并且在受损的CNS神经元（例如皮质脊髓神经元）中缺失（或非常低表达）。目的1将使用RNA-Seq结合生物信息学方法鉴定这些分子差异。这些方法在鉴定罕见的和潜在的新亚型方面是有效的，允许鉴定重要的但可能不丰富或先前鉴定的靶标;实例包括miRNA和特异性转录因子亚型。将使用体外表型分析对候选物进行检测。目标2将使用病毒 载体来转导皮质脊髓束神经元，并使用轴突生长的锥体切开术模型单独和组合测试目标1中的候选物。这些实验将提供有关DRG神经元中表达的基因的新信息，这些基因使DRG神经元在受损的CNS中再生。这些靶点的鉴定和多种候选物的体外和体内测试不仅会导致SCI的潜在治疗，而且还会导致其他CNS疾病，如创伤性脑损伤和中风。

DESCRIPTION (provided by applicant): A major impediment to recovery after central nervous system (CNS) injury is the failure of axons to regrow effectively. A variety of extrinsic and intrinsic factors contribute to this problem. Extrinsic factors include inhibitory proteins found i and around the injury site such as those from the glial scar as well as those associated with intact or damaged myelin. Regarding intrinsic factors, a key finding motivating this work is that Dorsal Root Ganglion (DRG) neurons can respond to peripheral injury with changes in gene expression that promote CNS regeneration, even in the inhibitory environment around the injury site. In contrast, CNS neurons typically fail to regenerate axons through such inhibitory regions. This implies that CNS neurons have inherent molecular differences that limit CNS regenerative capacity. The recent discovery of PTEN, SOCS3, KLF4 and KLF7 as important intrinsic regulators of CNS axon regeneration validates this hypothesis. However, the small fraction of CNS axons able to regenerate after injury, even in animals in which these genes have been manipulated, indicates that additional regulators remain to be discovered. The present proposal is to use high throughput technologies to identify genes regulating CNS regeneration by examining two related hypothesis about intrinsic factors. The first is a direct continuation of the hypothesis driving the preceding grant; i.e., that DRG neurons express RNAs that are expressed at significantly lower levels in CNS neurons and allow DRG axon regeneration. The second is that DRG neurons that have experienced a conditioning peripheral lesion express RNAs that allow regeneration and are missing (or very lowly expressed) in lesioned CNS neurons, such as the corticospinal neurons. Aim 1 will identify these molecular differences using RNA-Seq combined with bioinformatics approaches. These methods are effective at identifying rare and potentially novel isoforms, allowing identification of targets that are important but may not be abundant or previously identified; examples include miRNAs and specific transcription factor isoforms. Candidates will be tested using phenotypic analysis in vitro. Aim 2 will use viral vectors to transduce corticospinal tract neurons and test candidates from Aim 1, alone and in combination, using a pyramidotomy model of axonal growth. These experiments will provide novel information about the genes expressed in DRG neurons that allow them regenerate in the injured CNS. The identification of these targets and the testing of multiple candidates both in vitro and in vivo should lead to potential treatments not only for SCI, but also for other CNS disorders such as traumatic brain injury and stroke.