Role of unfolded protein response and COL8A2 in Fuchs corneal dystrophy

描述（由申请人提供）：Fuchs内皮性角膜营养不良（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是由角膜内皮细胞的慢性丧失引起的，角膜内皮细胞排列在角膜内表面，维持角膜清晰度所必需的生理水合作用。在某些情况下，FECD是由α - 2胶原VIII （COL8A2）基因的错义突变引起的。由于只有在角膜移植中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，凋亡发生在晚期FECD角膜内皮细胞中，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)FECD患者角膜内皮中未折叠蛋白反应（UPR）标记物的鉴定。适当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质引起。最初，UPR通过转录和翻译的选择性改变来减轻未折叠蛋白的压力。如果蛋白质折叠需求和容量保持不平衡，UPR诱导细胞发生凋亡。此外，2)我们已经开发了两种基因靶向敲入小鼠系，它们具有不同的COL8A2突变，已知会导致人类FECD。初步结果显示，角膜内皮基底膜的病理变化与人FECD角膜的病理变化高度相似。这项应用的总体假设是“角膜内皮细胞COL8A2突变的表达激活了未折叠蛋白反应，导致内皮细胞死亡。”具有相关病理表型的COL8A2 FECD敲入小鼠的可用性为验证以下假设提供了宝贵的机会：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角膜相当的角膜内皮和内皮基底膜的病理变化。目的2将评估COL8A2突变小鼠角膜和培养的角膜内皮细胞系中UPR的激活情况。目的3将评估紫外线（一种已知的UPR诱导性因子和角膜内皮的环境应激源）对COL8A2突变小鼠角膜和培养的角膜内皮细胞系中UPR激活的协同效应。本研究旨在利用COL8A2 FECD突变小鼠和角膜内皮细胞系，在COL8A2 FECD突变引起的UPR（主要细胞应激途径）和角膜内皮细胞凋亡之间建立新的机制联系。建立这种联系将代表着美国国家眼科研究所提高对遗传性角膜疾病的认识和治疗的目标取得了重大进展。

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.