Genetic analysis of axonal regeneration

描述（由申请人提供）：在人类中，视觉是最重要的感觉，视网膜或视神经的损伤可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，成年哺乳动物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显着能力，并重新生长切断或损坏的轴突和神经，包括视神经。将视觉信息从视网膜传递到大脑的视神经仅包含来自一种视网膜细胞类型的轴突，即视网膜神经节细胞（RGC）。视神经切断后，斑马鱼RGC神经元存活，并且-独立于神经发生-轴突再生到它们原始的突触靶，在那里它们形成功能性突触。令人惊讶的是，这种在体内再生轴突的非凡能力的分子遗传途径还没有得到很好的理解。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。我们选择了这个系统，因为它是非常容易获得的简单，快速和可重复的神经横切友好的屏幕，因为我们长期的兴趣和专业知识在视觉系统的发展和功能，因为所有的发现有关再生的视觉也可以翻译为中枢神经系统的再生一般。这项研究的长期目标是确定轴突再生的遗传，分子和细胞途径。本计划的实验将：（1）筛选相当于约970个化学诱变基因组的视神经轴突再生缺陷;（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）;和（3）用已知靶点对1760个小分子进行小分子中试筛选以鉴定延迟轴突断裂的因子，和减少或增加视神经轴突再生以限定进入轴突再生下的通路的入口点。这些研究与导致视神经损伤的人类疾病的研究有关，包括遗传性视神经病、癌症或多发性硬化症以及可导致不可逆的视神经变性和视力丧失的眼内压升高状况。鉴于目前缺乏视神经损伤或脊髓损伤的治疗干预一般，我们建议应用一个更公正的遗传方法来确定轴突再生的分子机制，并利用这些机制对哺乳动物的治疗策略的发展。

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.