RPE, aging and age-related macular degeneration: the role of oxidative stress

摘要 年导致老年人口不可逆转失明的最常见原因 工业化国家是老年性黄斑变性(AMD)。退化，退化 视网膜色素上皮(RPE)细胞与氧化应激和 炎症是AMD的一个重要标志。然而，详细的分子机制 AMD的基础在很大程度上仍不清楚，早期尚无有效的治疗方法 或疾病的晚期萎缩期。氧化应激对生理学的影响 RPE细胞的功能和导致焦点丢失被认为是一种 导致老年性黄斑变性地理萎缩和视力丧失的重要因素。因此， 这意味着限制RPE内的活性氧物种的形成可能 有效预防或减少AMD患者的RPE功能障碍。这项建议 试图了解DJ-1，一种具有抗氧化功能的多功能蛋白质， 调节RPE细胞的氧化应激反应。我们观察到增加了 AMD供体RPE中功能性失活磺酸氧化DJ-1的含量 在氧化应激引起的急性RPE变性的小鼠模型中也是如此。这些 这些发现为开展抗氧化剂的研究提供了坚实的临床基础 DJ-1对RPE细胞的调控机制。根据我们的初步数据，我们 RPE退变的可能机制途径的推论 整体水平较低的原生DJ-1或翻译后修饰(PTM)会损害其 功能。本项目中要调查的三个重叠区域是：1-测试 氧化应激引起的RPE变性受抗氧化剂调节的假说 DJ-1的功能；2-检验视网膜色素上皮线粒体功能障碍 氧化应激受DJ-1调节；3-测试DJ-1的治疗潜力。我们 将评估DJ-1在体内保护RPE的有效性，在两种氧化应激状态下 相关的临床前小鼠模型：急性RPE变性模型和慢性RPE变性模型 在视网膜外部产生AMD样病变的模型。在此过程中使用的方法 研究项目包括建立人和小鼠RPE细胞原代培养， 免疫沉淀、免疫染色、活细胞成像、共聚焦显微镜、动物 视网膜色素变性模型、扫描激光眼底镜、光谱域光学 相干断层扫描和视网膜电流图。

Abstract The most common cause of irreversible blindness in the elderly population in industrialized countries is age-related macular degeneration (AMD). Degeneration of retinal pigment epithelium (RPE) cells in association with oxidative stress and inflammation is a key hallmark of AMD. However, the detailed molecular mechanisms underlying AMD remain largely unknown and no effective treatment exists for the early or late atrophic stages of the disease. Oxidative stress affecting the physiological function and leading to focal loss of the RPE cells has been suggested to be an important factor contributing to geographic atrophy and vision loss in AMD. Thus implying that limiting the formation of reactive oxygen species within the RPE may effectively prevent or reduce RPE dysfunction observed in AMD patients. This proposal seeks to understand how DJ-1, a multifunctional protein with an antioxidant function, regulates oxidative stress responses in RPE cells. We have observed increased amounts of the functional inactive sulfonic oxidized DJ-1 in the RPE of AMD donors as well as in a mouse model of acute RPE degeneration due to oxidative stress. These findings provide a solid clinical foundation for pursuing studies on the antioxidant mechanisms regulated by DJ-1 in RPE cells. Based on our preliminary data we reasoned of a possible mechanistic pathway via which RPE degeneration results from overall low levels of native DJ-1 or posttranslational modifications (PTMs) that impair its function. The three overlapping areas to be investigated in this project are: 1- To test the hypothesis that RPE degeneration due to oxidative stress is regulated by antioxidant function of DJ-1; 2- To test the hypothesis that mitochondria dysfunction of the RPE due to oxidative stress is regulated by DJ-1; 3- To test the therapeutic potential of DJ-1. We will evaluate the effectiveness of DJ-1 to protect RPE in vivo, in two oxidative stress relevant pre-clinical mice models: an acute RPE degeneration model and a chronic model that generates AMD-like lesions in the outer retina. The methods utilized in this research project include establishment of human and mouse primary RPE cell cultures, immunoprecipitation, immunostaining, live cell imaging, confocal microscopy, animal models of RPE degeneration, scanning laser ophthalmoscopy, spectral-domain optical coherence tomography and electroretinograms.