Cellular and molecular analysis of spontaneous optic nerve regeneration

自发视神经再生的细胞和分子分析

• 批准号: 10450086
• 负责人: Michael Granato
• 金额: $ 52.51万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-08-01 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10450086
• 关键词: Adult; Amphibia; Animals; Area; Axon; Behavior; Biological Assay; Blindness; Brain; Cell Death; Cell Proliferation; Cells; Cellular Morphology; Collagen; Critical Pathways; Cues; Data; Defect; Deposition; Development; Disease; Environment; Excision; Extracellular Matrix; Eye; Fishes; Foundations; Functional Regeneration; Genes; Genetic Screening; Glaucoma; Goals; Growth; Hour; Human; Immune; Individual; Inherited; Injury; Knowledge; Mammals; Mediating; Modeling; Molecular; Molecular Analysis; Molecular Mechanisms of Action; Monitor; Morphology; Mutation; Natural regeneration; Nerve Degeneration; Neuraxis; Neuroglia; Neurons; Optic Chiasm; Optic Nerve; Pathway interactions; Phenotype; Process; Protein Isoforms; Regenerative capacity; Reporter; Research; Retina; Retinal Ganglion Cells; Rodent; Signal Pathway; Site; System; Time; Transgenic Organisms; Vertebrates; Vision; Visual; Work; Zebrafish; axon growth; axon guidance; axon regeneration; base; cell behavior; cell type; experimental study; glycosyltransferase; human disease; imaging genetics; in vivo; injured; live cell imaging; mutant; neurogenesis; optic nerve disorder; optic nerve regeneration; peripheral nerve regeneration; prevent; regeneration function; regenerative; relating to nervous system; retinal axon; retinal damage; superior colliculus Corpora quadrigemina; tool

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.

对于人类来说，视觉是最重要的感觉，视网膜或视神经的损伤可能会导致 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 （中枢神经系统），在成年哺乳动物中已经失去了再生能力。在啮齿类动物中，一些神经元内在信号传导 现已确定主要促进受损视网膜神经节细胞 (RGC) 轴突生长的途径 轴突，然而这是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， 它们指导 RGC 轴突再生的机制，以及神经胶质细胞和其他细胞类型的身份 提供指导的视神经路径尚不十分清楚。令人惊讶的是，即使是细胞行为 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 轴突尚不清楚，主要是由于哺乳动物活细胞成像的挑战。相比之下 哺乳动物、两栖动物和鱼类，包括斑马鱼，保留了非凡的视神经能力 再生。我们已经建立了一种强大的检测方法来横切斑马鱼幼虫的视神经，并监测 轴突和功能再生。 RGC 轴突在几天内再生，独立于神经发生， 提供了一个独特的机会来研究独立于自发再生的关键基因 神经存活和神经发生的混淆。通过基因筛选，我们发现了两个基因的突变体， 糖基转移酶 lh3 及其底物之一 col18a1 对于引导受损的 RGC 轴突至关重要。我们的 初步数据支持以下假设：lh3 和 col18a1 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。目标是 该提案旨在定义在其原生状态下再生轴突、神经胶质细胞和免疫细胞的基本行为 环境，并确定 lh3 和 Col18a1 指导的细胞和分子机制 再生视神经轴突。本提案中的实验将：（1）首次揭示和定义 任何脊椎动物系统视神经轴突、神经胶质细胞和免疫细胞再生的基本行为 他们的原生环境； (2)通过lh3和col18a1直接光学确定细胞和分子机制 神经再生； (3) 确定 col18a1 功能如何与 RGC 轴突的轴突引导相连接。 这些研究与导致视神经损伤的人类疾病的研究相关，包括 遗传性视神经病和青光眼。尽管自发性视神经再生基本上不存在 在哺乳动物中，通过神经元内在操纵促进轴突再生经常会导致误导， 强调定义受损 RGC 轴突与周围神经胶质细胞相互作用的重要性，并 破译再生引导背后的分子机制。最终，预期结果将形成 为全面制定视神经再生的具体假设奠定了坚实的基础。