Cellular and molecular analysis of spontaneous optic nerve regeneration

在人类中，视觉是最重要的感觉，对视网膜或视神经的损害可能会导致 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的一部分。 (CNS)，它在成年哺乳动物中已经失去了再生能力。在啮齿动物中，几个神经元的内在信号 现已发现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，这伴随着认识到，增强的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，即 它们引导再生RGC轴突的机制，以及胶质细胞和其他细胞类型的识别 提供指导的视神经路径还没有被很好地理解。令人惊讶的是，即使是细胞行为 常驻胶质细胞和免疫细胞被召集到损伤部位，以及它们如何与再生的RGC相互作用 轴突还不是很清楚，主要是由于哺乳动物活体细胞成像的挑战。与之形成鲜明对比的是 哺乳动物、两栖动物和鱼类，包括斑马鱼，一直保持着非凡的视神经能力。 再生。我们已经建立了一种有效的方法来横切斑马鱼幼体的视神经，并监测 轴突和功能再生。RGC轴突在几天内再生，不依赖于神经发生， 为研究自然再生的关键基因提供了一个独特的机会，而不依赖于 神经存活和神经发生的混淆。从基因筛查中，我们发现了两个基因的突变 糖基转移酶1h3及其底物之一col18a1对损伤的RGC轴突的引导至关重要。我们的 初步数据支持一种假设，即Lh3和col18a1参与一条途径，以提供外源性 引导--可能是通过周围的胶质细胞--引导再生的RGC轴突走向中枢神经系统中线。的目标是 这项提议是为了定义再生轴突、神经胶质细胞和免疫细胞的基本行为 并确定LH3和Col18a1引导的细胞和分子机制。 再生视神经轴突。本方案中的实验将：(1)首次揭示和定义 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 它们的自然环境；(2)通过lh3和col18a1的直接光学作用确定细胞和分子机制。 神经再生；以及(3)确定col18a1功能如何与RGC轴突的轴突指导联系。 这些研究与对人类造成视神经损害的疾病的研究有关，包括 遗传性视神经疾病和青光眼。尽管在很大程度上没有自发的视神经再生 在哺乳动物中，通过神经元内部操作促进轴突再生往往会导致误导， 强调确定受损的RGC轴突与周围神经胶质细胞的细胞相互作用的重要性 破译再生引导背后的分子机制。最后，预期的结果将形成一个 为全面阐述视神经再生的具体假设奠定了强大的基础。

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.