Genetic analysis of axonal regeneration

描述（申请人提供）：在人类中，视力是最重要的感觉，视网膜或视神经的损伤会导致不可逆的视力丧失。这是因为视网膜和视神经是中枢神经系统（CNS）的一部分，而中枢神经系统在成年哺乳动物中已经失去了再生能力。相比之下，两栖动物和鱼类，包括斑马鱼，保留了产生新的中枢神经系统神经元的非凡能力，并重新生长切断或受损的轴突和神经，包括视神经。将视觉信息从视网膜传递到大脑的视神经只包含一种视网膜细胞的轴突，即视网膜神经节细胞（RGC）。在视神经横断后，斑马鱼RGC神经元存活，并且独立于神经发生，轴突再生到它们最初的突触目标，在那里它们形成功能性突触。令人惊讶的是，分子遗传途径的这种显著能力再生轴突在体内，不是很清楚。在这里，我建议利用这种再生能力，并进行遗传和小分子筛选，以确定体内视神经再生所需的基因和途径。我们之所以选择这个系统，是因为它非常容易实现简单、快速和可复制的神经横切，而且对屏幕友好，因为我们对视觉系统的发展和功能有长期的兴趣和专业知识，因为所有关于视觉再生的发现也可以转化为一般的中枢神经系统再生。这项研究计划的长期目标是确定轴突再生的遗传、分子和细胞途径。本实验将：(1)筛选约970个化学诱变基因组，以寻找视神经轴突再生中的缺陷；(2)鉴定至少30个突变体的分子性质（通过全基因组测序方法）；(3)对已知靶点的1760个小分子进行小分子先导筛选，以确定延迟轴突断裂、减少或增加视神经轴突再生的因素，以确定轴突再生通路的进入点。这些研究与研究引起视神经损伤的人类疾病有关，包括遗传性视神经病变、癌症或多发性硬化症，以及可导致不可逆视神经变性和视力丧失的眼压升高的情况。鉴于目前缺乏针对视神经损伤或脊髓损伤的治疗干预措施，我们建议采用一种更公正的遗传方法来确定轴突再生的分子机制，并利用这些机制来开发哺乳动物的治疗策略。

DESCRIPTION (provided by applicant): In humans, vision is the most important sense and damage to the retina or the optic nerve can cause irreversible vision loss. This is because the retina and the optic nerve are part of the central nervous system (CNS), which in adult mammals has lost its regenerative capacity. In contrast, amphibians and fish, including zebrafish, have retained a remarkable capacity to generate new CNS neurons, and to re-grow severed or damage axons and nerves, including the optic nerve. The optic nerve, which conveys visual information from the retina into the brain contains axons from only one retinal cell type, the retinal ganglion cells (RGC). After optic nerve transection, zebrafish RGC neurons survive, and -independently of neurogenesis- regrow axons to their original synaptic targets where they form functional synapses. Surprisingly, the molecular genetic pathways for this remarkable capacity to regenerate axons in vivo, are not well understood. Here, I propose to take advantage of this regenerative capacity and to perform a genetic and a small molecule screens to identify genes and pathways required for optic nerve regeneration in vivo. We have chosen this system because it is very accessible to simple, rapid and reproducible nerve transection amiable to screens, because of our longstanding interest and expertise in visual system development and function, and because all findings regarding regeneration in the visual are also translatable to CNS regeneration in general. The long term-goal of this research proposal is to define the genetic, molecular and cellular pathways underlying axonal regeneration. The experiments in this proposal will: (1) screen an equivalent of ~970 chemically mutagenized genomes for defects specifically in optic nerve axonal regeneration; (2) identify the molecular nature of at least 30 mutants (through a whole genome sequencing approach); and (3) perform a small molecule pilot screen of 1760 small molecules with known targets to identify factors that delay axon fragmentation, and de- or increase optic nerve axonal regeneration to define entry points into pathways underlying axonal regeneration. These studies are relevant to the study of human diseases that cause damage to the optic nerve, including hereditary optic neuropathies, cancer or multiple sclerosis as well conditions of increased intraocular pressure which can cause irreversible optic nerve degeneration and vision loss. Given the current lack of therapeutic interventions for optic nerve damage or for spinal cord injury in general, we propose to apply a more unbiased genetic approach to determine the molecular mechanisms underlying axonal regeneration, and to exploit these mechanisms towards the development of therapeutic strategies in mammals.