Novel Gene Targets for CNS Axonal Regeneration

描述(申请人提供)：脊髓损伤(SCI)、创伤性脑损伤(TBI)或中风后康复的一个主要障碍是中枢神经系统(CMS)轴突在长时间内无法通过白质有效再生。有多种因素被认为是造成这一问题的原因。这些包括胶质瘢痕中的抑制物，与髓鞘或受损髓鞘相关的抑制物质，以及神经元发育过程中降低其轴突生长潜力的分子变化。在过去的几年中，研究表明，背根神经节(DRG)神经元如果使用最小化损伤的技术移植到中枢神经系统中，可以在白质中发出非常远距离的轴突。相比之下，以同样的方式移植中枢神经系统神经元并不会产生同样的结果；即它们无法通过白质轨迹发出长轴突。这表明DRG神经元和CNS神经元存在固有的分子差异，限制了CNS的再生效率。我们建议检验一个特定的假设，即DRG神经元表达不同于CNS神经元的基因，这允许DRG神经元在CNS再生。在特定的目标1中，我们将通过连续消减cDNA文库来鉴定这些分子差异。我们将在DRG神经元中寻找可能促进再生的独特基因，以及在海马神经元和皮质脊髓神经元中寻找可能抑制再生的独特基因。这种方法在识别稀有甚至可能是新的cDNA方面非常有效，确保识别重要的靶标，如转录因子。我们还将搜索公共微阵列数据库以寻找更多的候选基因。在特定的目标中，将使用成熟的体外实验来测试2个候选基因，在体外实验中，神经元生长在髓鞘或蛋白多糖上，并测量其轴突的长度。将DRG特异性基因导入中枢神经系统神经元，或利用CNS特异性基因的RNAi技术，在抑制底物上评价靶基因在轴突生长中的作用。特效靶点3将在体内采用神经元转染法和显微移植法。这将使我们能够直接测试每个候选基因在最相关的测试中的作用，即哺乳动物中枢神经系统的再生。这些实验将提供有关DRG神经元表达的蛋白质的全新信息，这些蛋白质使它们能够在CNS中延伸长轴突。识别这些靶点并使用体外方法测试多个候选对象，然后使用体内方法改进子集，应该会直接导致对脊髓损伤、脑损伤和中风的潜在治疗。Lay摘要：这些实验旨在识别参与控制成人大脑中白质再生的基因。这些基因将通过细胞培养和移植到动物体内，在不能正常生长轴突的神经元中进行测试，以确定这些基因是否促进再生。

DESCRIPTION (provided by applicant): A major impediment to recovery after spinal cord injury (SCI), traumatic brain injury (TBI), or stroke is the failure of central nervous system (CMS) axons to regenerate effectively through white mater over long distances. A variety of factors are believed to contribute to this problem. These include inhibitors in glial scars, inhibitory material associated with myelin or damaged myelin and molecular changes in neurons during development that reduce their potential for axon growth. Over the past several years it has been shown that Dorsal Root Ganglion (DRG) neurons can send axons very long distances in white mater if they are transplanted into the CNS using techniques that minimize damage. In contrast, transplanting CNS neurons the same way does not produce the same result; i.e. they fail to send out long axons through white matter tracks. This implies that DRG neurons and CNS neurons have inherent molecular differences that limit CNS regenerative efficiency. We propose to test a specific hypothesis that DRG neurons express different genes than CNS neurons, which permit DRG neurons to regeneration in the CNS. In specific aim 1 we will identify these molecular differences using serial subtraction of cDNA libraries. We will look for unique genes in DRG neurons that might enhance regeneration and unique genes in hippocampal neurons and corticospinal neurons that might inhibit regeneration This method is extremely effective at identifying rare and perhaps novel cDNAs, ensuring identification of important targets, such as transcription factors. We will also search public microarray databases to search for additional candidates. In specific aim 2 candidate genes will be tested using a well-established in vitro assay where neurons are grown on myelin or proteoglycans and the lengths of their neurites measured. CNS neurons will be transfected with DRG specific genes or use RNAi of CNS specific genes in order to evaluate the target genes roles in axon growth on inhibitory substrates. Specific aim 3 will use neuronal transfection and microtransplantation in vivo. This will permit us to directly test the role of each candidate gene in the most relevant assay, regeneration in the mammalian central nervous system. These experiments will provide entirely new information about the proteins expressed in DRG neurons that allows them to extend long axons in the CNS. The identification of these targets and testing multiple candidates using in vitro methods and subsequently a refined subset using in vivo approaches should lead directly to potential treatments for SCI, TBI and stroke. Lay Summary: These experiments are designed to identify genes involved in controlling regeneration in white matter in the adult brain. The genes will be tested in neurons that cannot normally grow axons using cell culture and transplantation into animals to determine if the genes promote regeneration.