Genetically testing interactions of ER and oxidative stresses in retinal disease

描述（由申请人提供）：IV型胶原蛋白α 1基因（Col 4a 1）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将确定AMD和/或其他视网膜疾病的基础途径。突变型COL 4A 1错误折叠，分泌受损，导致细胞内蓄积。COL 4A 1是最丰富的基底膜蛋白，并且从视网膜色素上皮细胞（RPE）和脉络膜血管内皮细胞分泌以贡献于布鲁赫膜。RPE/Bruch膜/脉络膜复合体是几种视网膜疾病的重要发病部位。我们的初步数据耦合内质网（ER）稳态和活性氧解毒视网膜疾病的基本细胞过程。生理损伤，包括错误折叠的蛋白质，可导致ER中的压力。ER应激激活信号级联，其寻求恢复蛋白质产生的需求与ER的蛋白质折叠能力之间的稳态。慢性内质网应激产生活性氧，导致氧化损伤。解毒活性氧的能力随着年龄的增长而下降，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的替代补体途径的失调。在这里，我们测试我们的假设，错误折叠的突变COL 4A 1导致视网膜病变的机制，ER和氧化应激的RPE和/或脉络膜血管内皮细胞。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Col 4a 1诱导的视网膜病变进行彻底和详细的检查。在目标2中，我们将通过致病途径的分子询问来测试ER和氧化应激的参与。将比较RPE和脉络膜内皮细胞的潜在分子差异，以确定原发性疾病过程的部位。在目标3中，我们将使用缓解ER应激能力受损的小鼠和解毒活性氧自由基能力受损的小鼠来从遗传上测试这些途径中每一种的关系和相对重要性。本提案中概述的实验将揭示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。ER和氧化应激的相对作用的知识可以用于开发靶向治疗，可以预防或减少全球数百万人的视力丧失。

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.