Novel Gene Targets for CNS Axonal Regeneration

描述（由申请人提供）：脊髓损伤（SCI）、创伤性脑损伤（TBI）或中风后恢复的一个主要障碍是中枢神经系统（CMS）轴突无法通过长距离的白质有效再生。据信多种因素导致了这个问题。这些包括神经胶质疤痕中的抑制剂、与髓磷脂或受损髓磷脂相关的抑制物质以及神经元在发育过程中的分子变化，这些变化降低了轴突生长的潜力。过去几年的研究表明，如果使用最大限度减少损伤的技术将背根神经节 (DRG) 神经元移植到中枢神经系统中，它们可以在白质中将轴突发送很长的距离。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道发出长轴突。这意味着 DRG 神经元和 CNS 神经元具有固有的分子差异，限制了 CNS 再生效率。我们建议测试一个特定的假设，即 DRG 神经元表达与 CNS 神经元不同的基因，这允许 DRG 神经元在 CNS 中再生。在具体目标 1 中，我们将使用 cDNA 文库的连续扣除来识别这些分子差异。我们将在 DRG 神经元中寻找可能增强再生的独特基因，以及在海马神经元和皮质脊髓神经元中可能抑制再生的独特基因。这种方法对于识别稀有且可能新颖的 cDNA 非常有效，确保识别重要靶标，例如转录因子。我们还将搜索公共微阵列数据库以寻找其他候选者。在具体目标中，将使用成熟的体外测定来测试 2 个候选基因，其中神经元在髓磷脂或蛋白聚糖上生长，并测量其神经突的长度。 CNS 神经元将被 DRG 特异性基因转染或使用 CNS 特异性基因的 RNAi，以评估靶基因在抑制基质上轴突生长中的作用。具体目标3将使用神经元转染和体内微移植。这将使我们能够直接测试每个候选基因在最相关的测定中的作用，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中枢神经系统中延伸长轴突。这些靶点的识别和使用体外方法测试多个候选者，以及随后使用体内方法的细化子集，应直接导致 SCI、TBI 和中风的潜在治疗。简单总结：这些实验旨在识别参与控制成人大脑白质再生的基因。这些基因将在不能正常生长轴突的神经元中进行测试，通过细胞培养和移植到动物体内，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.