Cellular and molecular mechanisms promoting retinal ganglion cell axonal guidance during optic nerve regeneration

视神经再生过程中促进视网膜神经节细胞轴突引导的细胞和分子机制

• 批准号: 10191752
• 负责人: Beth Mee Ra Harvey
• 金额: $ 11.79万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-01 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10191752
• 关键词: Animals; Axon; Behavior; Biological Assay; Blindness; Brain; Candidate Disease Gene; Cell Communication; Cell Death; Cell Survival; Cells; Cellular Morphology; Collagen; Complement; Cues; Development; Disease; Exhibits; Extracellular Space; Eye; Foundations; Gene Expression Profiling; Genes; Genetic; Genetic Screening; Glaucoma; Goals; Hour; Image; Immune; Individual; Injury; Laboratories; Larva; Lead; Ligands; Mammals; Mentors; Modification; Molecular; Monitor; Morphology; Mutation; Natural regeneration; Neuroglia; Neurons; Optic Chiasm; Optic Nerve; Optic Nerve Injuries; Optic Nerve Transections; Optic tract structure; Optics; Pathway interactions; Pattern; Pennsylvania; Phase; Physiologic Intraocular Pressure; Process; Regenerative capacity; Research; Resources; Retina; Retinal Ganglion Cells; Role; Signal Pathway; Site; Solid; Supporting Cell; System; Techniques; Technology; Testing; Time; Tissues; Training; Transgenic Organisms; Trauma; Universities; WNT Signaling Pathway; Work; Zebrafish; axon growth; axon guidance; axon regeneration; cell type; experimental study; follow-up; glycosyltransferase; in vivo; insight; live cell imaging; loss of function; mutant; optic nerve regeneration; programs; regenerative; response; retinal axon; retinal damage; sight restoration; superior colliculus Corpora quadrigemina; transcriptomics; vertebrate genome; visual information

PROJECT SUMMARY Visual information is transmitted from the retina in each eye to the brain through the optic nerve, which is composed of the axons of retinal ganglion cells (RGCs) and associated glia. Diseases that increase intraocular pressure and damage the RGC bodies and their axons, such as glaucoma, can ultimately result in irreversible blindness. After damage to RGC axons, regeneration of the mammalian optic nerve is largely deficient due to limited RGC axonal regrowth compounded by massive injury induced RGC death. Several RGC intrinsic signaling pathways are known to increase RGC survival and increase long range axonal growth after injury. However, enhancing RGC axonal growth often results in axonal misguidance during the initial stages of regeneration, as axons project inappropriately from the optic tract before and at the optic chiasm. Currently, the identity of extrinsic cues and mechanisms critical for guiding regenerating RGC axons are not well understood. In addition, the cellular responses and behaviors of glia, immune and other support cells that localize to the optic tract, which potentially provide guidance cues to regenerating RGC axons, have not yet been described. In contrast to most mammals, zebrafish exhibit a remarkable capacity for regeneration. In taking full advantage of the zebrafish system, the overall goal of the proposed research here is to use an optic nerve transection assay developed by the laboratory of Dr. Michael Granato in the optically transparent larval zebrafish, to identify and characterize mechanisms that promote optic nerve regeneration. Preliminary studies from a candidate genetic screen conducted using this assay identified mutations in three genes critical for guiding regenerating RGC axons: lh3, a glycosyltransferase critical for posttranslational collagen modifications, collagen18a1, a presumptive substrate of Lh3, and wntless, which is required for the secretion of Wnt ligands into the extracellular space. In all three mutants, regenerating RGC axons extend but fail to cross the optic chiasm, and instead project along aberrant trajectories, revealing that RGC axonal growth toward and across the optic chiasm requires critical extrinsic guidance cues during regeneration. To define the extrinsic cues and cell-cell interactions that guide RGC axons as they navigate towards the chiasm during regeneration, I propose to use live cell imaging to characterize RGC axonal and glial cell dynamics in vivo during the initial stages of optic nerve regeneration. I will also determine the cellular and molecular mechanisms by which Lh3, Collagen18a1, and Wntless promote correct axonal guidance during optic nerve regeneration and I will use an unbiased transcriptomics approach to identify additional genes required for this process. I will complete the mentored K99 phase of this proposal in the Granato lab at the University of Pennsylvania, a top research university with cutting-edge technologies and excellent mentoring resources. The results from this proposal will create a strong foundation to establish a focused independent research program and provide relevant insight to fundamental questions in the field of optic nerve regeneration.

项目总结 视觉信息从每只眼睛的视网膜通过视神经传递到大脑，视神经是 由视网膜神经节细胞(RGC)的轴突和相关的胶质细胞组成。眼内增多的疾病 压迫和损害RGC小体及其轴突，如青光眼，最终可能导致不可逆转的 失明。视神经RGC轴突损伤后，哺乳动物视神经再生严重不足 有限的RGC轴突再生合并大量损伤导致RGC死亡。几个RGC内部特性 众所周知，信号通路可以增加RGC的存活率，并增加损伤后长时间的轴突生长。 然而，促进RGC轴突生长往往导致轴突在早期阶段被误导。 再生，因为轴突不适当地从视交叉前和视交叉处的视束投射出来。目前， 对于引导再生RGC轴突的外在线索和机制的识别还不是很清楚。 此外，定位于视神经的胶质细胞、免疫细胞和其他支持细胞的细胞反应和行为 可能为再生RGC轴突提供指导线索的束尚未被描述。 与大多数哺乳动物不同，斑马鱼表现出非凡的再生能力。在充分利用 斑马鱼系统的优势，这里拟议的研究的总体目标是使用视神经 迈克尔·格拉纳托博士的实验室在光学透明的斑马鱼幼体上开发了横切法， 识别和描述促进视神经再生的机制。一项初步研究 使用这种方法进行的候选基因筛查发现了三个对引导基因至关重要的基因突变 再生RGC轴突：LH3，一种对翻译后胶原修饰至关重要的糖基转移酶， 胶原蛋白18a1，可能是Lh3的底物，以及Wnnless，是分泌Wnt配体所必需的 进入细胞外空间。在所有三个突变体中，再生的RGC轴突延伸但不能穿过视神经 交叉，而不是沿着反常的轨迹投射，揭示了RGC轴突生长朝向和横跨 在再生过程中，视交叉需要关键的外在引导信号。 为了定义引导RGC轴突导航到 在再生过程中的交叉，我建议使用活细胞成像来表征RGC轴突和神经胶质细胞的动力学 在体内视神经再生的初始阶段。我还将确定细胞和分子 Lh3、胶原蛋白18a1和Wnless促进视神经正确轴突引导的机制 再生和我将使用一种无偏见的转录组学方法来确定这一过程所需的其他基因 进程。我将在加州大学格拉纳托实验室完成该建议书的K99指导阶段 宾夕法尼亚州，一所拥有尖端技术和优秀导师资源的顶尖研究型大学。这个 这项提案的结果将为建立一个有重点的独立研究计划奠定坚实的基础 并为视神经再生领域的基本问题提供了相关的见解。