Novel Gene Targets for CNS Axonal Regeneration

描述(申请人提供)：中枢神经系统(CNS)损伤后恢复的一个主要障碍是轴突不能有效地再生。造成这一问题的原因有多种，既有外部因素，也有内在因素。外在因素包括在损伤部位和周围发现的抑制蛋白，例如来自胶质瘢痕的抑制蛋白，以及与完整或受损髓鞘有关的抑制蛋白。关于内在因素，这项工作的一个关键发现是背根神经节(DRG)神经元可以通过促进中枢神经系统再生的基因表达的变化来响应外周损伤，即使在损伤部位周围的抑制环境中也是如此。相比之下，中枢神经系统神经元通常不能通过这样的抑制区域再生轴突。这意味着中枢神经系统神经元具有固有的分子差异，限制了中枢神经系统的再生能力。最近发现的PTEN、SOCS3、KLF4和KLF7作为中枢神经系统轴突再生的重要内在调节因子证实了这一假说。然而，中枢神经系统轴突的一小部分能够在损伤后再生，即使在这些基因被操纵的动物中也是如此，这表明还有更多的调控因素有待发现。目前的建议是利用高通量技术，通过检验关于内在因素的两个相关假说来寻找调控中枢神经系统再生的基因。第一个是直接延续 驱动前一项授权的假设；即，DRG神经元表达在中枢神经系统神经元中表达水平显著降低的RNA，并允许DRG轴突再生。第二，经历了外周条件性损伤的DRG神经元表达允许再生的RNA，并且在受损的中枢神经系统神经元中缺失(或表达非常低)，如皮质脊髓神经元。目标1将使用RNA-Seq和生物信息学相结合的方法识别这些分子差异。这些方法在识别稀有和潜在的新的异构体方面是有效的，允许识别重要但可能不丰富或以前识别的靶标；例子包括miRNAs和特定的转录因子异构体。候选人将在体外使用表型分析进行测试。AIM 2将使用病毒 载体转导皮质脊髓束神经元和测试来自目标1的候选，单独和组合，使用锥体切割法轴突生长模型。这些实验将提供有关DRG神经元表达的基因的新信息，这些基因允许它们在受损的CNS中再生。这些靶点的确定以及在体外和体内对多个候选者的测试应该不仅会导致对脊髓损伤的潜在治疗，而且还会导致其他中枢神经系统疾病的潜在治疗，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.