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

项目摘要 视觉信息通过视神经从每只眼睛的视网膜传递到大脑， 由视网膜神经节细胞（RGC）和相关神经胶质的轴突组成。增加眼内的疾病 压力和损害RGC体及其轴突，如青光眼，最终可导致不可逆的 失明在RGC轴突受损后，哺乳动物视神经的再生由于以下原因而在很大程度上缺乏： 有限的RGC轴突再生长与大面积损伤复合诱导RGC死亡。几种RGC内在 已知信号传导途径增加损伤后RGC存活和增加长距离轴突生长。 然而，增强RGC轴突生长常常导致轴突错误引导， 再生，因为轴突在视交叉之前和视交叉处从视束不适当地伸出。目前 对于引导再生RGC轴突的关键的外部线索和机制的身份还没有很好地理解。 此外，定位于视神经的神经胶质细胞、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。在采取充分 斑马鱼系统的优势，这里提出的研究的总体目标是使用视神经 由Michael Granato博士的实验室在光学透明的斑马鱼幼虫中开发的横切测定， 识别和表征促进视神经再生的机制。A的初步研究 使用该测定法进行的候选遗传筛选鉴定了三个基因中的突变，这三个基因对于指导 再生RGC轴突：lh 3，一种对翻译后胶原修饰至关重要的糖基转移酶， 胶原蛋白18 a1，Lh 3的假定底物，和Wnt配体分泌所需的Wntless 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 相反，沿着沿着异常的轨迹投射，揭示了RGC轴突生长朝向和跨越 视交叉在再生期间需要关键的外部引导线索。 为了定义引导RGC轴突向神经元方向航行的外在线索和细胞-细胞相互作用， 再生过程中的交叉，我建议使用活细胞成像来表征RGC轴突和胶质细胞动力学 在视神经再生的初始阶段。我也会确定细胞和分子 Lh 3、胶原蛋白18 a1和Wntless促进视神经过程中正确轴突引导的机制 再生和我将使用一个公正的转录组学方法，以确定额外的基因需要这一点 过程我将在格拉纳达大学的格拉纳托实验室完成本提案的辅导K99阶段。 宾夕法尼亚州，一所顶尖的研究型大学，拥有尖端技术和优秀的指导资源。的 该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相关见解。

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.