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

项目总结/摘要 引起视网膜神经节细胞（RGC）损伤和不可逆损失的青光眼是青光眼的主要原因。 全世界失明在美国，超过300万人患有这种疾病。的一种形式 青光眼，正常眼压性青光眼（NTG），其中存在RGC损失，但没有眼睛增加的证据 压力，通常与视神经磷酸酶（OPTN）E50 K突变有关。对小鼠的研究表明， E50 K突变可导致RGC死亡和视神经凹陷。迄今为止，所有青光眼治疗都是针对 降低眼压，不直接促进RGC健康和存活（神经保护）。这里我们有 建立了一种从人胚胎干细胞中大量分离纯化视网膜节细胞的方法 细胞研究视神经氨酸相关的RGC损伤的分子机制。视神经磷酸酶是一个关键的球员， 自噬途径中的线粒体降解，这被称为线粒体自噬。我们的核心假设是 具有OPTN E50 K突变的干细胞衍生的RGC将重演RGC变性， 通过破坏线粒体质量控制（MQC）途径治疗青光眼。拟议的研究分为 三个具体目标：1）确定OPTN E50 K突变对线粒体功能和降解的影响 在RGC中。2)研究E50 K介导的线粒体缺陷的分子机制，并进行小规模的 分子筛选以寻找RGC保护化合物。3)用E50 K在3D视网膜中建立RGC变性模型 突变来研究神经保护剂的作用。这一建议具有多方面的创新性：第一， 与啮齿动物模型不同，该干细胞衍生的RGC模型更可能反映人RGC生物学。第二、 我们将研究青光眼相关OPTN突变体中MQC缺陷的分子机制 RGC是近年来发现的对RGC生物学和病理学有重要意义的蛋白质。第三，小分子 用突变报告细胞系筛选将为青光眼以及其他视网膜病变提供药物筛选平台。 疾病这个项目是在轨道上的成功发展的研究资助局分化和纯化 方法、基于流式细胞术的线粒体自噬测定和3D视网膜杯形成。我们获得了OPTN突变体， 基于Aim 1和CRISPR产生OPTN突变体的合作者正在进行目标2和3的研究。PI的合作- 导师Debasish Sinha博士是自噬领域的领先科学家，他的办公室在同一栋楼里， 将在辅导阶段提供所需的一切支助。建议项目是根据PI设计的 细胞生物学、分子生物学、显微镜和干细胞方面的专业知识，沿着视网膜病变方面的持续培训， 唐·扎克实验室的生物学（导师）。PI是在理想的环境中提出的研究作为唐扎克有 一个建立的干细胞为基础的视网膜研究计划沿着与国家的最先进的HCS设施，干细胞 核心，几个共聚焦和电子显微镜与著名的视觉科学家可在威尔默 眼睛/霍普金斯。这将有助于PI建立合作和学习新技术，以建立独立的研究 使用干细胞衍生疾病模型对人类视网膜疾病进行机制研究的计划。

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.