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）或中风后恢复的主要障碍是中枢神经系统（CMS）轴突通过白质长距离有效再生的失败。许多因素被认为是造成这个问题的原因。这些包括神经胶质瘢痕的抑制剂，与髓磷脂或受损髓磷脂相关的抑制物质以及发育过程中神经元的分子变化，这些变化降低了它们轴突生长的潜力。在过去的几年里，研究表明，如果使用最小化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，它们可以在白质中发送很远的距离的轴突。相反，以同样的方式移植中枢神经系统神经元不会产生相同的结果；也就是说，它们不能通过白质轨迹发出长轴突。这表明DRG神经元和CNS神经元具有内在的分子差异，限制了CNS的再生效率。我们提出验证一个特定的假设，即DRG神经元表达不同于CNS神经元的基因，这使得DRG神经元能够在CNS中再生。在具体目标1中，我们将使用cDNA文库的连续减法来识别这些分子差异。我们将在DRG神经元中寻找可能促进再生的独特基因，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。这种方法在鉴定罕见的和可能新的cdna方面非常有效，确保鉴定重要靶标，如转录因子。我们还将搜索公共微阵列数据库以搜索其他候选对象。在特定目标中，2个候选基因将使用一种成熟的体外实验进行测试，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来评估靶基因在抑制性底物上轴突生长中的作用。特异性目标3将使用神经元转染和体内微移植。这将使我们能够在最相关的实验中直接测试每个候选基因的作用，即哺乳动物中枢神经系统的再生。这些实验将为DRG神经元中表达的蛋白质提供全新的信息，这些蛋白质允许它们在中枢神经系统中延伸长轴突。确定这些靶点并使用体外方法测试多个候选靶点，随后使用体内方法进行优化，将直接导致脊髓损伤、TBI和中风的潜在治疗。这些实验的目的是确定与控制成人大脑白质再生有关的基因。这些基因将在不能正常生长轴突的神经元中进行测试，通过细胞培养和动物移植来确定这些基因是否能促进再生。

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.