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: 9164808
• 负责人: Manas R Biswal
• 金额: $ 12.88万
• 依托单位: UNIVERSITY OF 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 2018-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9164808
• 关键词: Affect; Affinity Chromatography; Age; Age related macular degeneration; Area; Atrophic; Binding; Biochemical; Bioinformatics; Biological Assay; Biology; Biotinylation; Blindness; Bruch' s basal membrane structure; CASP1 gene; CCL2 gene; Cathepsins B; Cell Culture Techniques; Cell Surface Proteins; Cell surface; Cells; Cessation of life; Choroidal Neovascularization; Chronic; Confocal Microscopy; Data; Data Analyses; 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; Immune; Immunoelectron Microscopy; Immunofluorescence Immunologic; Immunohistochemistry; In Vitro; Individual; Inflammation; Inflammatory; Injury; Interleukin-1 alpha; Interleukin-18; Interleukin-6; Label; Lasers; LysoTracker; Lysosomes; Magic; Maintenance; Measures; Membrane Proteins; Mitochondria; Modeling; Molecular; Mus; Ovalbumin; Oxidative Stress; Pathogenesis; Pathway interactions; Patients; Pattern; Phagocytosis; Phagolysosome; Phagosomes; Photoreceptors; Play; Postdoctoral Fellow; Prevention; Process; Protein Isoforms; Proteins; Proteomics; Public Health; Reactive Oxygen Species; Reporting; Research; Research Personnel; Research Training; Retinal; Retinal Degeneration; Retinal Photoreceptors; Retinoids; Reverse Transcriptase Polymerase Chain Reaction; Rhodopsin; Risk; Role; Running; SOD2 gene; Scanning; Signal Pathway; Site; Source; Staining method; Stains; Streptavidin; Structure; Structure of retinal pigment epithelium; System; Systems Biology; Techniques; Testing; Time; Training; Vacuole; Western Blotting; age related; base; career development; cytokine; differential expression; effective therapy; geographic atrophy; improved; in vivo; loss of function; macula; mouse model; new therapeutic target; novel; 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： 与年龄相关的黄斑变性（AMD）导致许多年龄较大的人的视力丧失， 视网膜色素上皮（RPE）被认为是损伤的关键部位。由于 感光细胞变性和/或脉络膜新生血管形成。地理萎缩（GA），高级形式 干燥AMD的特征是RPE，绒毛膜毛细血管和光感受器的分解，尤其是在 黄斑。对GA的分子机制缺乏明确的了解会阻碍治疗的发展。 对于终身维持感光器，RPE细胞在吞噬和降解中起着至关重要的作用 从光感受器外部段（POS）脱落的尖端。感光细胞和RPE细胞易受损伤 来自线粒体氧化应激。该项目的核心目标是了解光感受器如何 变性发生在GA中。我假设氧化应激会损害吞噬作用和溶酶体功能 并最终激活刺激地理萎缩的RPE中的炎症过程。我会测试我的 RPE细胞培养和新的依赖年龄依赖性RPE萎缩的小鼠模型中的假设 在我们的实验室中开发。在此模型中，我们使用CRE/LOX系统来生成SOD2的RPE特异性缺失， 锰超氧化物歧化酶（MNSOD）的线粒体基因。这些小鼠会形成正常的RPE， 但是加班的RPE具有升高的氧化应激，导致表型变化通常是 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 GA，我有以下目的：（1）表征氧化应激对吞噬作用，溶酶体的影响 RPE中的功能和炎性体激活； （2）在氧化应激下确定RPE的分子变化。 这些研究将阐明驱动感光器和RPE损失的信号通路，并提供 建立新的治疗靶标，以防止AMD疾病进展。 总体而言，该建议不仅将开始揭示光感受器的新分子机制 GA的退化，但也将在候选人的培训和职业发展中发挥作用。 Manas Biswal。拟议的培训计划将使他能够分支到新的研究领域 吞噬作用，溶酶体生物学和眼部炎症，它将通过新技术训练他：FACS， 免疫EM，LC-MS，激光扫描单和多光共聚焦显微镜，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。该建议也将 与他的研究和培训相关的支持课程，讲习班和会议，并将使他能够 从博士后研究员过渡到一个独立的研究人员，经营自己的实验室。