Genetic analysis of axonal regeneration

描述(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会导致不可逆转的视力丧失。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，保持了生成新的中枢神经系统神经元的非凡能力，并重新生长被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只包含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神经元存活下来，并独立于神经发生，将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，这种在体内再生轴突的非凡能力的分子遗传途径还没有被很好地了解。在这里，我建议利用这种再生能力，进行遗传和小分子筛查，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，是因为我们对视觉系统开发和功能的长期兴趣和专业知识，以及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，因此它非常容易进行简单、快速和可重复性的神经横断。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。这项计划中的实验将：(1)筛选相当于970个化学突变的基因组，以寻找特定于视神经轴突再生的缺陷；(2)确定至少30个突变体的分子性质(通过全基因组测序方法)；以及(3)对1760个具有已知靶点的小分子进行小分子初步筛选，以确定推迟轴突碎裂的因素，并取消或增加视神经轴突再生，以确定进入轴突再生途径的入口点。这些研究与人类导致视神经损害的疾病相关，包括遗传性视神经疾病、癌症或多发性硬化症，以及可导致不可逆转的视神经退化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，我们建议应用一种更公正的遗传学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.