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）轴突无法通过长距离白色脑膜有效再生。据信，造成这一问题的因素很多。这些包括神经胶质瘢痕中的抑制剂、与髓鞘或受损髓鞘相关的抑制物质以及发育期间神经元中降低其轴突生长潜力的分子变化。在过去的几年中，已经表明，背根神经节（DRG）神经元可以在白色物质中发送轴突非常长的距离，如果它们被移植到CNS中，使用最小化损伤的技术。相比之下，以同样的方式移植CNS神经元不会产生相同的结果;即它们不能通过白色物质轨迹发出长轴突。这意味着DRG神经元和CNS神经元具有限制CNS再生效率的固有分子差异。我们建议测试一个特定的假设，DRG神经元表达不同的基因比中枢神经系统神经元，这使得DRG神经元在中枢神经系统再生。在具体目标1中，我们将使用cDNA文库的连续消减来鉴定这些分子差异。我们将寻找DRG神经元中可能增强再生的独特基因，以及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。这种方法在鉴定罕见的，也许是新的cDNA方面非常有效，确保鉴定重要的靶点，如转录因子。我们还将搜索公共微阵列数据库，以寻找其他候选人。具体而言，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。将用DRG特异性基因转染CNS神经元或使用CNS特异性基因的RNAi以评估靶基因在抑制性底物上轴突生长中的作用。具体目标3将在体内使用神经元转染和微移植。这将使我们能够直接测试每个候选基因在最相关的检测中的作用，即哺乳动物中枢神经系统的再生。这些实验将提供有关DRG神经元中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。这些目标的识别和使用体外方法测试多个候选物，随后使用体内方法进行细化子集，应该直接导致SCI，TBI和中风的潜在治疗。这些实验的目的是鉴定成年人大脑中参与控制白色物质再生的基因。这些基因将在不能正常生长轴突的神经元中进行测试，使用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.