Role of unfolded protein response and COL8A2 in Fuchs corneal dystrophy

描述(申请人提供)：Fuchs内皮角膜营养不良(FECD)是美国人群中最常见和临床相关的遗传性角膜疾病，也是2007年角膜移植的第三大原因。FECD的原因是角膜内皮细胞的慢性丢失，这些内皮细胞排列在角膜内表面，维持角膜透明所需的生理水合。在某些情况下，FECD是由α2胶原蛋白VIII(COL8A2)基因的错义突变引起的。由于只有在角膜移植时获得的终末期角膜可用于研究，因此对FECD的发病机制知之甚少。有限的研究表明，晚期FECD角膜的内皮细胞正在发生凋亡，尽管导致凋亡的机制尚不清楚。因此，动物和细胞培养模型将是阐明FECD中导致内皮细胞凋亡的早期事件的宝贵工具。我们的初步工作包括：1)寻找FECD患者角膜内皮未折叠蛋白反应(UPR)的标志物。正确的蛋白质折叠对细胞的正常功能至关重要。UPR是一种高度保守的、广泛的细胞反应，由过量的错误折叠蛋白诱导。最初，UPR的功能是通过选择性地改变转录和翻译来缓解未折叠蛋白质的压力。如果蛋白质折叠需求和折叠能力保持不平衡，UPR会诱导细胞发生凋亡。此外，2)我们开发了两个基因靶向敲入小鼠品系，它们具有已知的导致人类FECD的不同COL8A2突变。初步结果显示，角膜内皮基底膜的病理变化与人类FECD角膜的病理变化非常相似。这项应用的总体假设是“角膜内皮细胞表达COL8A2突变激活了未折叠的蛋白反应，导致内皮细胞死亡。”获得具有相关病理表型的COL8A2 FECD敲入小鼠为检验这一假说提供了一个宝贵的机会，具体如下：AIM 1将评估COL8A2突变小鼠的角膜内皮细胞凋亡，并将更详细地表征与人类FECD角膜相似的角膜内皮和内皮基底膜的病理变化。目的研究COL8A2突变小鼠角膜和培养的角膜内皮细胞系中UPR的活性。目的3将评估紫外线(已知的UPR诱导物和角膜内皮的环境应激源)对角膜和培养的COL8A2突变小鼠角膜内皮细胞株UPR激活的协同作用。这项建议试图利用COL8A2 FECD突变小鼠和角膜内皮细胞系，在主要的细胞应激途径UPR和由COL8A2的FECD突变导致的角膜内皮细胞凋亡之间建立新的机制联系。这种联系的建立将代表着国家眼科研究所在改善遗传角膜疾病的知识和治疗方面的目标方面取得的重大进展。 公共卫生意义：Fuchs内皮性角膜营养不良(FECD)是一种常见疾病，由于角膜内层内的角膜内皮细胞死亡而导致角膜透明度降低，从而导致视力丧失(Borboli 2002)。FECD影响了大约0.6%的美国人口(Krachmer，1978)，是美国角膜移植手术的第三大原因(Van Meter，2007)。本研究的目的是探讨未折叠蛋白反应在FECD中导致内皮细胞死亡的作用，这是一个主要的细胞应激途径。

DESCRIPTION (provided by applicant): Fuchs endothelial corneal dystrophy (FECD) is the most common and clinically relevant inherited corneal disease in the US population and the third leading cause of corneal transplants in 2007. FECD results from chronic loss of corneal endothelial cells which line the inner surface of the cornea and maintain physiologic hydration necessary for corneal clarity. In some cases, FECD is caused by missense mutations in the alpha 2 collagen VIII (COL8A2) gene. Very little is known about the pathogenesis of FECD due to the fact that only end stage corneas obtained at corneal transplant are available for study. Limited work indicates that apoptosis is occurring in endothelial cells from advanced FECD corneas, although the mechanisms leading to apoptosis are poorly understood. Thus, animal and cell culture models would be invaluable tools to elucidate early events leading to endothelial cell apoptosis in FECD. Our preliminary work includes: 1) identification of markers for the unfolded protein response (UPR) in the endothelium of FECD patient corneas. Proper protein folding is critical for normal function of the cell. The UPR is a highly conserved, broad ranging cellular response induced by an excess of misfolded proteins. Initially, the UPR functions to relieve stress of unfolded proteins through selective alterations in transcription and translation. If protein folding demand and capacity remain unbalanced, the UPR induces cells to undergo apoptosis. In addition, 2) we have developed two gene targeted knock-in mouse lines with different COL8A2 mutations known to cause FECD in humans. Preliminary results show pathologic changes in the basement membrane of the corneal endothelium which are highly similar to those seen in human FECD corneas. The overall hypothesis of this application is that "corneal endothelial cell expression of COL8A2 mutations activates the unfolded protein response and results in endothelial cell death." The availability of COL8A2 FECD knock-in mice with a relevant pathologic phenotype provides an invaluable opportunity to test this hypothesis as follows: Aim 1 will assess COL8A2 mutant mice for corneal endothelial cell apoptosis and will characterize in greater detail pathologic changes in corneal endothelium and the endothelial basement membrane comparable to human FECD corneas. Aim 2 will assess for UPR activation in corneas and cultured corneal endothelial cell lines from COL8A2 mutant mice. Aim 3 will assess for synergistic effects of UV light (a known inducer of the UPR and environmental stressor for the corneal endothelium) on UPR activation in corneas and cultured corneal endothelial cell lines from COL8A2 mutant mice. This proposal seeks to use COL8A2 FECD mutant mice and corneal endothelial cell lines to establish a novel mechanistic link between the UPR, a major cell stress pathway, and corneal endothelial cell apoptosis resulting from FECD mutations in COL8A2. The establishment of such a link would represent a major advance in the National Eye Institute's goals of improving knowledge and treatment of inherited corneal diseases. PUBLIC HEALTH RELEVANCE: Fuchs endothelial corneal dystrophy (FECD) is a common disease which causes loss of vision from decreased corneal clarity due to death of corneal endothelial cells lining the inner layer of the cornea (Borboli 2002). FECD affects approximately 0.6% of the US population (Krachmer 1978) and is the third leading cause of corneal transplant surgery in the US (Van Meter 2007). The purpose of this study is to investigate the role of the unfolded protein response, a major cell stress pathway, in causing endothelial cell death in FECD.