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

定义氧化应激诱导的 RPE 变化，控制 RPE 和光感受器变性

• 批准号: 10222694
• 负责人: Manas R Biswal
• 金额: $ 24.05万
• 依托单位: UNIVERSITY OF SOUTH FLORIDA
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-08-01 至 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10222694
• 关键词: Affect; Affinity Chromatography; Age; Age related macular degeneration; Area; Atrophic; Binding; Biochemical; Biological Assay; Biology; Biotinylation; Blindness; Bruch' s basal membrane structure; CASP1 gene; CCL2 gene; Cathepsins B; Cell Culture Techniques; Cell Surface Proteins; Cell surface; Cessation of life; Choroidal Neovascularization; Chronic; Confocal Microscopy; Data Analyses; Data Set; Defect; Deposition; Development; Disease; Disease Progression; ERCC2 gene; Educational workshop; Elderly; Electron Microscopy; Enzyme-Linked Immunosorbent Assay; Event; Exudative age-related macular degeneration; Eye; Fluorescein-5-isothiocyanate; Foundations; Functional disorder; Gene Expression; Genes; Goals; Health; Healthcare Systems; Immunoelectron Microscopy; Immunofluorescence Immunologic; Immunohistochemistry; Impairment; In Vitro; Individual; Inflammasome; Inflammation; Inflammatory; Injury; Interleukin-1 alpha; Interleukin-18; Interleukin-6; Label; Lasers; LysoTracker; Lysosomes; Magic; Maintenance; Measures; Membrane Proteins; Mitochondria; Modeling; Molecular; Mus; Nonexudative age-related macular degeneration; Ovalbumin; Oxidative Stress; Pathogenesis; Pathway interactions; Patients; Pattern; Phagocytes; Phagocytosis; Phagolysosome; Phagosomes; Phenotype; Photoreceptors; Play; Postdoctoral Fellow; Prevention; Process; Protein Isoforms; Proteins; Proteomics; Public Health; Reactive Oxygen Species; Reporting; Research; Research Personnel; Research Training; Retina; Retinal Degeneration; Retinal Photoreceptors; Retinoids; Reverse Transcriptase Polymerase Chain Reaction; Rhodopsin; Risk; Role; Running; SOD2 gene; Scanning; Signal Pathway; Site; Source; Stains; Streptavidin; Structure; Structure of retinal pigment epithelium; System; Systems Biology; Techniques; Testing; Time; Training; Vacuole; Western Blotting; age related; base; bioinformatics tool; career development; cytokine; differential expression; effective therapy; epithelial injury; geographic atrophy; improved; in vivo; loss of function; macula; mouse model; new therapeutic target; novel; novel therapeutic intervention; novel therapeutics; photoreceptor cell outer segment; photoreceptor degeneration; prevent; recombinase-mediated cassette exchange; socioeconomics; symposium; therapy development; tool; transcriptome sequencing

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.

抽象的： 年龄相关性黄斑变性（AMD）导致许多老年人视力丧失，并且 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。地理萎缩（GA），高级形式 干性 AMD 的特点是 RPE、脉络膜毛细血管和光感受器的损坏，尤其是在 黄斑。对 GA 分子机制缺乏清晰的了解阻碍了治疗的发展。 对于光感受器的终生维持，RPE 细胞在吞噬和降解中发挥着重要作用 尖端从光感受器外节（POS）脱落。感光细胞和视网膜色素上皮细胞容易受到损伤 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 中发生变性。我假设氧化应激会损害吞噬作用和溶酶体功能 最终激活 RPE 中的炎症过程，刺激地理萎缩。我会测试我的 RPE 细胞培养和最近提出的年龄依赖性 RPE 萎缩的新小鼠模型中的假设 我们实验室开发的。在此模型中，我们使用 cre/lox 系统生成 Sod2 的 RPE 特异性删除， 锰超氧化物歧化酶（MnSOD）的线粒体基因。这些小鼠发育出正常的 RPE， 但随着时间的推移，RPE 会升高氧化应激，导致常见的表型变化 AMD 中观察到的症状包括 RPE 损伤、功能丧失和随后的视网膜变性。在这样的背景下 GA，我有以下目标：（1）表征氧化应激对吞噬作用、溶酶体的影响 RPE 中的功能和炎症小体激活； (2) 识别氧化应激下RPE的分子变化。 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 基金会开发新的治疗靶点以预防 AMD 疾病进展。 总的来说，这一提议不仅将开始揭示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涉足新的研究领域，包括 吞噬作用、溶酶体生物学和眼部炎症，这将训练他新技术：FACS、 免疫电镜、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 支持与其研究和培训相关的课程、研讨会和会议，并使他能够 从博士后研究员转变为经营自己实验室的独立研究员。