Cellular and molecular analysis of spontaneous optic nerve regeneration

在人类中，视觉是最重要的感觉，视网膜或视神经的损伤可能导致 不可逆转的视力丧失这是因为视网膜和视神经都是中枢神经系统的一部分 (CNS)在成年哺乳动物中，它已经失去了再生能力。在啮齿类动物中，几种神经元内在信号 现在已经确定了主要促进受损视网膜神经节细胞（RGC）轴突生长的途径 轴突，然而，这已经认识到，增强轴突再生经常导致广泛的 误导，不利于功能再生。目前，外在指导线索的身份， 它们指导再生RGC轴突的机制，以及胶质细胞和其他细胞类型的特性，沿着 提供引导的视神经路径还没有被很好地理解。令人惊讶的是，即使是 常驻神经胶质细胞和免疫细胞被召唤到损伤部位，以及它们如何与再生的RGC相互作用 轴突还没有得到很好的理解，主要是由于哺乳动物活细胞成像的挑战。相比 哺乳动物，两栖动物和鱼类，包括斑马鱼，都保留了视觉神经的非凡能力， 再生我们已经建立了一个强大的分析，以横切斑马鱼幼鱼的视神经，并监测 轴突和功能再生。RGC轴突在几天内独立于神经发生再生， 提供了一个独特的机会，研究基因的关键自发再生独立的 神经存活和神经发生的混淆。从基因筛选中，我们发现了两个基因的突变体， 糖基转移酶lh 3和其底物之一col 18 a1对于引导受损的RGC轴突至关重要。我们 初步数据支持一种假设，即lh 3和col 18 a1参与提供外源性的途径 引导-可能是通过周围的神经胶质细胞-引导再生的RGC轴突朝向CNS中线。的目标 这项提议是为了确定轴突、神经胶质和免疫细胞在其自然状态下再生的基本行为， 环境，并确定lh 3和Col 18 a1引导的细胞和分子机制 再生视神经轴突该方案的实验将：（1）首次揭示和定义了 任何脊椎动物系统再生视神经轴突，神经胶质细胞和免疫细胞的基本行为， （2）通过LH 3和Col 18 A1直接光学显微镜观察， （3）确定col 18 a1功能如何连接到RGC轴突的轴突引导。 这些研究与导致视神经损伤的人类疾病的研究有关，包括 遗传性视神经病和青光眼。虽然自发性视神经再生在很大程度上是不存在的 在哺乳动物中，通过神经元内在操纵来促进轴突再生经常导致误导， 强调了确定受损RGC轴突与周围神经胶质细胞的细胞相互作用以及 破译再生指导的分子机制。最后，预期的结果将形成一个 强大的基础，制定具体的假设，视神经再生的全面。

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 rodents, several neuron intrinsic signaling pathways have now been identified that majorly boost axonal growth of injured retinal ganglion cells (RGC) axons, yet this has come with the realization that enhanced axonal regrowth frequently results in extensive misguidance, detrimental to function regeneration. Currently, the identity of extrinsic guidance cues, the mechanisms by which they direct regenerating RGC axons, and the identity of glia and other cell types along the optic nerve path that provide guidance are not well understood. Surprisingly, even the cellular behaviors of resident glial cells and immune cells summoned to the injury site, and how they interact with regenerating RGC axons is not well understood, mainly due to challenges of live cell imaging in mammals. In contrast to mammals, amphibians and fish, including zebrafish, have retained a remarkable capacity for optic nerve regeneration. We have established a powerful assay to transect the optic nerve in larval zebrafish, and monitor axonal and functional regeneration. RGC axons regenerate within a few days independently of neurogenesis, providing a unique opportunity to study the genes critical for spontaneous regeneration independently of the confound of neural survival and neurogenesis. From a genetic screen we identified mutants in two genes, the glycosyltransferase lh3 and one of its substrate col18a1 critical for the guidance of injured RGC axons. Our preliminary data support a hypothesis by which lh3 and col18a1 participate in a pathway to provide extrinsic guidance –likely by surrounding glia- to guide regenerating RGC axons towards the CNS midline. The goal of this proposal are to define fundamental behaviors of regenerating axons, glia and immune cells in their native environment, and to determine the cellular and molecular mechanism by which lh3 and Col18a1 guide regenerating optic nerve axons. The experiments in this proposal will: (1) reveal and define for the first time in any vertebrate system the fundamental behaviors of regenerating optic nerve axons, glia and immune cells in their native environment; (2) determine the cellular and molecular mechanisms by lh3 and col18a1 direct optic nerve regeneration; and (3) determine how col18a1 function connects to axonal guidance of RGC axons. These studies are relevant to the study of human diseases that cause damage to the optic nerve, including hereditary optic neuropathies and glaucoma. Although spontaneous optic nerve regeneration is largely absent in mammals, boosting axonal regeneration via neuron intrinsic manipulation frequently results in misguidance, underscoring the importance to define the cellular interplay of injured RGC axons with surrounding glia and to decipher the molecular mechanism underlying regenerative guidance. Finally, the expected results will form a powerful foundation to formulate specific hypotheses of optic nerve regeneration across the board.