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

摘要： 视网膜相关性黄斑变性（AMD）在许多老年人中引起视力丧失， 视网膜色素上皮（RPE）被认为是损伤的关键部位。AMD的视力丧失是由于 光感受器变性和/或脉络膜新生血管形成。地图样萎缩（GA），晚期形式 干性AMD的特征在于RPE、脉络膜毛细血管和光感受器的破坏，特别是在 黄斑。对GA的分子机制缺乏明确的理解阻碍了治疗的发展。 对于光感受器的终身维持，RPE细胞在吞噬和降解中发挥重要作用 光感受器外节（POS）脱落。光感受器和RPE细胞易受损伤 来自线粒体氧化应激。该项目的中心目标是了解光感受器如何 GA中发生退化。我推测氧化应激损害吞噬作用和溶酶体功能 并最终激活RPE中刺激地图状萎缩的炎症过程。我将测试我的 在RPE细胞培养和最近发现的年龄依赖性RPE萎缩的新小鼠模型中， 在我们的实验室里开发的。在该模型中，我们使用cre/lox系统生成RPE特异性的Sod 2缺失， 锰超氧化物歧化酶（MnSOD）的线粒体基因。这些小鼠产生正常的RPE， 但随着时间的推移，RPE具有升高的氧化应激，导致表型变化， 在AMD中观察到的视网膜变性包括RPE损伤、功能丧失和随后的视网膜变性。背景下 本论文的主要目的是：（1）研究氧化应激对大鼠巨噬细胞吞噬功能、溶酶体吞噬功能和细胞凋亡的影响。 （2）研究氧化应激对RPE细胞的影响。 这些研究将阐明驱动光感受器和RPE损失的信号通路，并将提供一种新的方法。 基金会开发新的治疗靶点，以防止AMD的疾病进展。 总的来说，这一提议不仅将开始揭开感光细胞的新分子机制， 退化，但也将有助于在培训和职业发展的候选人，博士。 玛纳斯·比斯瓦尔拟议的培训计划将使他能够分支到新的研究领域，包括 吞噬作用，溶酶体生物学和眼部炎症，这将训练他在新的技术：流式细胞仪， 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-DIGE的定量蛋白质组学。该提案还将 支持与他的研究和培训有关的课程、讲习班和会议，并使他能够 从博士后研究员到独立研究员的转变，经营自己的实验室。

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.