Defining oxidative stress induced changes in RPE that control RPE and photoreceptor degeneration

摘要： 老年性黄斑变性(AMD)会导致许多老年人的视力丧失， 视网膜色素上皮(RPE)被认为是损伤的关键部位。AMD患者视力丧失的原因是 光感受器变性和/或脉络膜新生血管。进展型地理性萎缩(GA) 干性AMD的特点是RPE、脉络膜毛细血管和光感受器的破坏，尤其是在 黑斑。由于对GA的分子机制缺乏清晰的认识，阻碍了治疗的发展。 对于光感受器的终生维持，RPE细胞在吞噬和降解过程中起着至关重要的作用 从光感受器外节(POS)脱落的尖端。光感受器和RPE细胞对损伤很敏感 线粒体氧化应激所致。该项目的中心目标是了解光感受器如何 退行性变发生在GA。我假设氧化应激损害吞噬和溶酶体功能 并最终激活RPE中的炎症过程，刺激地理萎缩。我要测试我的 RPE细胞培养和一种新的年龄依赖性RPE萎缩小鼠模型的假说 这是我们实验室研发的。在该模型中，我们使用CRE/LOX系统来生成Sod2的RPE特定删除， 锰超氧化物歧化酶(MnSOD)线粒体基因。这些小鼠会产生正常的RPE， 但随着时间的推移，RPE氧化应激增加，导致通常情况下的表型变化 在AMD中观察到，包括RPE损伤、功能丧失和随后的视网膜变性。在…的背景下 GA，我有以下目的：(1)表征氧化应激对吞噬功能、溶酶体的影响 RPE的功能和炎性小体的激活；(2)确定RPE在氧化应激下的分子变化。 这些研究将阐明驱动光感受器和RPE丢失的信号通路，并将提供 基金会开发新的治疗目标，以防止AMD的疾病进展。 总体而言，这一提议不仅将开始解开光感受器的新分子机制 GA的退化，但也将有助于候选人的培训和职业发展，Dr。 玛纳斯·比斯瓦尔。拟议的培训计划将使他能够进入新的研究领域，包括 吞噬、溶酶体生物学和眼部炎症，这将训练他新的技术：FACS， 免疫-EM、LC-MS、激光扫描单光子和多光子共聚焦显微镜、RNA-SEQ数据分析 使用生物信息学工具、生物素化和基于2D-DGE的定量蛋白质组学。这项提议还将 支持与他的研究和培训相关的课程、讲习班和会议，并将使他能够 从博士后过渡到独立研究人员，管理着自己的实验室。

ABSTRACT: Age-related macular degeneration (AMD) causes vision loss among many older individuals, and the retinal pigment epithelium (RPE) is thought to be a critical site of injury. Vision loss in AMD occurs due to photoreceptor degeneration and/or choroidal neovascularization. Geographic atrophy (GA), the advanced form of dry AMD, is characterized by the breakdown of RPE, choriocapillaris, and photoreceptors, especially in the macula. Lack of clear understanding of the molecular mechanisms of GA hinders the development of therapy. For lifelong maintenance of photoreceptors, RPE cells play an essential role in phagocytosis and degradation of tips shed from photoreceptor outer segments (POS). Photoreceptors and RPE cells are susceptible to injury from mitochondrial oxidative stress. The central goal of the project is to understand how photoreceptor degeneration occurs in GA. I hypothesize that oxidative stress impairs phagocytosis and lysosome function and ultimately activates inflammatory processes in RPE that stimulate geographic atrophy. I will test my hypothesis in RPE cell culture and in a new mouse model of age-dependent RPE atrophy that was recently developed in our lab. In this model we used the cre/lox system to generate an RPE-specific deletion of Sod2, the mitochondrial gene for manganese superoxide dismutase (MnSOD). These mice develop a normal RPE, but overtime the RPE has elevated oxidative stress resulting in phenotypic changes that are commonly observed in AMD, including RPE injury, loss of function and subsequent retinal degeneration. In the context of GA, I have following aims: (1) To characterize the impact of oxidative stress on phagocytosis, lysosomal function and inflammasome activation in RPE; (2) Identify molecular changes in RPE under oxidative stress. These studies will illuminate signaling pathways that drive photoreceptor and RPE loss and will provide a foundation to develop new therapeutic targets to prevent disease progression in AMD. Overall, this proposal will not only begin to unravel the novel molecular mechanisms of photoreceptor degeneration in GA but will also be instrumental in the training and career development of the candidate, Dr. Manas Biswal. The proposed training plan will allow him to branch into new areas of research including phagocytosis, lysosome biology, and ocular inflammation, and it will train him in new techniques: FACS, Immuno-EM, LC-MS, laser scanning single and multiphoton confocal microscopy, RNA-seq data analysis using bioinformatics tools, biotinylation and 2D-DIGE based quantitative proteomics. This proposal will also support courses, workshops and conferences relevant to his research and training, and will allow him to transition from a postdoctoral fellow to an independent researcher running his own lab.