Role of unfolded protein response and COL8A2 in Fuchs corneal dystrophy

Fuchs内皮角膜营养不良(FECD)是最常见和临床相关的遗传性疾病 2007年，美国人口中的角膜疾病是导致角膜移植的第三大原因。FECD结果 由于角膜内表面排列的角膜内皮细胞的慢性丢失并维持 角膜透明所需的生理性水合作用。在某些情况下，FECD是由错义突变引起的 III型胶原(COL8A2)基因。由于以下事实，人们对FECD的发病机制知之甚少 只有在角膜移植时获得的终末期角膜才可用于研究。有限的工作表明 晚期FECD角膜的内皮细胞正在发生凋亡，尽管导致 人们对细胞凋亡知之甚少。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。 我们的初步工作包括：1)确定了未折叠蛋白反应(UPR)的标记。 FECD患者的角膜内皮。正确的蛋白质折叠对细胞的正常功能至关重要。这个 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通过选择性的转录改变来缓解未折叠蛋白质的压力 还有翻译。如果蛋白质折叠需求和能力仍然不平衡，UPR会诱导细胞经历 细胞凋亡。此外，我们还建立了两个不同COL8A2基因靶向的敲入小鼠系 已知的导致人类FECD的突变。初步结果显示基底内有病理改变 与人类FECD角膜高度相似的角膜内皮细胞膜。 这一应用的总体假设是：COL8A2的角膜内皮细胞表达 突变激活未折叠的蛋白反应，导致内皮细胞死亡。 具有相关病理表型的COL8A2 FECD敲入小鼠的研究提供了一个宝贵的机会 验证这一假设如下：Aim 1将评估COL8A2突变小鼠的角膜内皮细胞凋亡 并将更详细地描述角膜内皮和内皮基底区的病理变化 可与人类FECD角膜相媲美的薄膜。AIM 2将评估角膜和培养的UPR活性 COL8A2突变小鼠角膜内皮细胞系。目标3将评估紫外线的协同效应(a 已知的UPR诱导者和角膜内皮环境应激源)对UPR激活的影响 COL8A2突变小鼠角膜和培养的角膜内皮细胞系。 本提案旨在利用COL8A2 FECD突变小鼠和角膜内皮细胞系建立 作为主要细胞应激途径的UPR与角膜内皮细胞凋亡之间的新机制联系 由COL8A2的FECD突变引起。建立这种联系将是一项重大进展 在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中。

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.