Genetically testing interactions of ER and oxidative stresses in retinal disease

描述(由申请人提供)：IV型胶原α1基因(Col4a1)基因突变导致小鼠视网膜表型类似于年龄相关性黄斑变性(AMD)。了解从突变到视网膜病变的细胞机制(S)将确定AMD和/或其他视网膜疾病的基础路径。突变的COL4A1错误折叠，分泌受损，导致细胞内蓄积。COL4A1是最丰富的基底膜蛋白，由视网膜色素上皮细胞(RPE)和脉络膜血管内皮细胞分泌，对Bruchs膜有贡献。RPE/Bruch膜/脉络膜复合体是多种视网膜疾病的重要发病部位。我们的初步数据结合了内质网(ER)的动态平衡和活性氧对视网膜疾病的解毒的基本细胞过程。生理上的侮辱，包括错误折叠的蛋白质，可能会导致内质网中的压力。内质网应激激活信号级联，寻求恢复蛋白质生产需求和内质网蛋白质折叠能力之间的动态平衡。慢性内质网应激会产生活性氧物种，导致氧化损伤。活性氧的解毒能力随着年龄的增长而降低，氧化损伤是几种衰老疾病的关键机制。氧化损伤导致慢性炎症和先天免疫替代补体途径的失调。在这里，我们检验了我们的假设，即错误折叠的突变体COL4A1通过RPE和/或脉络膜血管内皮细胞中ER和氧化应激的机制导致视网膜病变。在目标1中，我们将通过临床、功能、组织学、超微结构和分子分析对Col4a1诱导的视网膜病变进行彻底和详细的检查。在目标2中，我们将通过对致病途径的分子询问来测试ER和氧化应激的参与。RPE和脉络膜内皮细胞将被比较潜在的分子差异，以确定原发疾病过程的位置。在目标3中，我们将使用缓解内质网应激能力受损的小鼠和解毒活性氧物种能力受损的小鼠来从遗传学角度测试这些途径的关系和相对重要性。该提案中概述的实验将揭示在视网膜病理生理学中重要的有形细胞过程，并具有更广泛的应用潜力。对ER和氧化应激相关作用的知识可以被用来开发靶向疗法，从而防止或减少全球数百万人的视力丧失。公共卫生相关性：这项应用将研究导致小鼠视网膜疾病的两个关键细胞通路，这种疾病类似于人类老年性黄斑变性。了解这些途径何时、何地以及如何相互作用，可能使它们成为全球数百万人预防或治疗失明的新药物靶点。

DESCRIPTION (provided by applicant): Mutation of the type IV collagen alpha 1 gene (Col4a1) gene cause retinal phenotypes in mice that resemble age-related macular degeneration (AMD). Understanding the cellular mechanism(s) that lead from mutation to retinopathy will identify pathways that underlie AMD and/or other retinal diseases. Mutant COL4A1 is misfolded, and secretion is impaired, leading to intracellular accumulation. COL4A1 is the most abundant basement membrane protein and is secreted both from retinal pigment epithelial cells (RPE) and choroidal vascular endothelial cells to contribute to Bruch's membrane. The RPE/Bruch's membrane/choroid complex is an important site of pathogenesis in several retinal diseases. Our preliminary data couple the basic cellular processes of endoplasmic reticulum (ER) homeostasis and detoxification of reactive oxygen species to retinal disease. Physiological insults, including misfolded proteins, can lead to stress in the ER. ER stress activates signaling cascades that seek to restore a homeostasis between the demands of protein production and the protein folding capacity of the ER. Chronic ER stress generates reactive oxygen species that lead to oxidative damage. The ability to detoxify reactive oxygen species decreases with age and oxidative damage is a key mechanism in several diseases of aging. Oxidative damage contributes to chronic inflammation and de-regulation of the alternate complement pathway of innate immunity. Here, we test our hypothesis that misfolded mutant COL4A1 leads to retinopathy via a mechanism of ER and oxidative stress in the RPE and/or choroidal vascular endothelial cells. In Aim 1 we will conduct a thorough and detailed examination of Col4a1-induced retinopathy via clinical, functional, histological, ultrastructural and molecular analyses. In Aim 2 we will test the involvement of ER and oxidative stress by molecular interrogation of the pathogenic pathways. RPE and choroidal endothelial cells will be compared for potential molecular differences that could identify the site of primary disease processes. In Aim 3 we will use mice with impaired ability to alleviate ER stress and mice with impaired ability to detoxify reactive oxygen species to genetically test the relationship and relative importance of each of these pathways. The experiments outlined in this proposal will reveal tangible cellular processes important in pathophysiology of the retina and with potential for broader application. Knowledge of the relative roles of ER and oxidative stress can be exploited in development of targeted therapeutics that could prevent or reduce vision loss in millions of people worldwide. PUBLIC HEALTH RELEVANCE: This application will study two key cellular pathways that lead to a retinal disease in mice that resembles human age-related macular degeneration. Understanding when, where and how these pathways interact could expose them as novel pharmaceutical targets for prevention or treatment of blindness in millions of people worldwide.