Use of Human Stem Cell-derived RGCs to Study the Mechanism of Optineurin-associated Glaucoma

项目摘要/摘要 青光眼是导致视网膜神经节细胞(RGC)损伤和不可逆转的丢失的主要原因 世界范围内的失明。在美国，有300多万人患有这种疾病。一种形式的 青光眼，正常眼压性青光眼(NTG)，视网膜神经节细胞丢失，但没有证据表明眼睛增加 压力，通常与视神经磷酸酶(OPTN)E50K突变有关。对小鼠的研究表明， E50K突变可导致RGC死亡和视神经挖出。到目前为止，所有的青光眼治疗都是针对 降低眼压，而不是直接促进RGC的健康和生存(神经保护)。这就是我们的 建立一种从人胚胎干细胞中分离和纯化大量视网膜神经节细胞的方法 目的探讨视神经磷酸核苷相关RGC损伤的分子机制。Optineurin是一个关键的参与者 线粒体在自噬途径中的降解，这就是众所周知的有丝分裂。我们的中心假设是 具有OPTN E50K突变的干细胞来源的视网膜神经节细胞将概括为视网膜神经节细胞变性。 通过破坏线粒体质量控制(MQC)途径导致青光眼。这项拟议的研究被分解为 三个特定目标：1)确定OPTN E50K突变对线粒体功能和降解的影响 在RGC里。2)研究E50K介导的线粒体缺陷的分子机制，并进行小鼠 分子筛选寻找RGC保护性化合物。3)E50K 3D视网膜RGC变性模型 突变研究神经保护剂的作用。这一建议在多个方面具有创新性：第一， 与啮齿动物模型不同，这种干细胞来源的RGC模型更有可能反映人类RGC生物学。第二, 我们将研究人类青光眼相关OPTN突变的MQC缺陷的分子机制 RGC，最近被认为对RGC的生物学和病理学具有重要作用。第三，一种小分子 带有突变报告线的筛查将为青光眼和其他视网膜提供药物筛选平台 疾病。随着RGC分化和纯化的成功开发，该项目正在进行中 方法：基于Flow的有丝分裂实验和3D视网膜视杯形成。我们从我们的OPTN突变体 Aim1和CRISPR为基础的OPTN突变体的生成的合作者正在为目标2和3而进行。 导师德巴西什·辛哈博士是自噬领域的领先科学家，他的办公室在同一栋楼里， 将提供指导阶段所需的所有支持。建议的项目是基于PI的设计 在细胞生物学、分子生物学、显微镜和干细胞方面的专业知识，以及正在进行的视网膜培训 唐·扎克实验室里的生物学(导师)。与唐·扎克一样，PI处于拟议研究的理想环境中 已建立的基于干细胞的视网膜研究计划以及最先进的HCS设施，干细胞 核心，几个共焦和电子显微镜，著名的视觉科学家在威尔默 眼科/霍普金斯。这将帮助PI建立协作并学习新技术来建立独立研究 使用干细胞衍生疾病模型进行人类视网膜疾病机制研究的计划。

Project Summary/Abstract Glaucoma, which causes damage to and irreversible loss of retinal ganglion cells (RGCs), is a leading cause of blindness worldwide. In America, more than 3 million people are living with this disease. One form of glaucoma, normal tension glaucoma (NTG), in which there is loss of RGCs without evidence of increased eye pressure, is commonly associated with the optineurin (OPTN) E50K mutation. Studies in mice show that the E50K mutation can cause RGC death and optic nerve excavation. To date, all glaucoma therapy is directed at lowering eye pressure, not directly promoting RGC health and survival (neuroprotection). Here we have developed a method to differentiate and purify large amounts of RGCs from human embryonic stem cells to investigate molecular mechanism of optineurin-associated RGC injury. Optineurin is a critical player for mitochondrial degradation in the autophagy pathway, which is known as mitophagy. Our central hypothesis is that the stem cell derived RGCs with the OPTN E50K mutation will recapitulate RGC degeneration in glaucoma by disrupting the mitochondrial quality control (MQC) pathway. The proposed study is broken into three specific aims: 1) Determine the effect of OPTN E50K mutation on mitochondrial function and degradation in RGCs. 2) Investigate molecular mechanism of E50K mediated mitochondrial defect and perform a small molecule screen to find RGC protective compounds. 3) Model RGC degeneration in 3D retina with E50K mutation to study the effect of neuroprotective reagents. This proposal is innovative in multiple ways: first, unlike rodent models this stem cell derived RGC model is more likely to reflect human RGC biology. Second, we will investigate the molecular mechanism of MQC defect in glaucoma associated OPTN mutant in human RGC, which is recently indicated to be important for RGC biology and pathology. Third, a small molecule screen with a mutant reporter line will provide drug-screening platform for glaucoma as well as for other retinal diseases. This project is on track with the successful development of the RGC differentiation and purification method, flow based mitophagy assay and 3D retinal cup formation. We obtained OPTN mutants from our collaborator for the Aim1 and CRISPR based generation of OPTN mutant is underway for aim 2 and 3. PI's co- mentor Dr. Debasish Sinha is a leading scientist in autophagy field, whose office is in the same building and will provide all the support required during the mentored phase. Proposed project is deigned based on PI's expertise in cell biology, molecular biology, microscopy and stem cell along with ongoing training in retinal biology in Don Zack's lab (mentor). PI is in the ideal environment for the proposed research as Don Zack has an established stem cell based retinal research program along with state-of-the-art HCS facilities, stem cell core, several confocal and electron microscopes with renowned vision scientists available at Wilmer Eye/Hopkins. This will help PI to set-up collaboration and learn new techniques to built independent research program on mechanistic investigation of human retinal disease using stem cell derived disease models.