Role of unfolded protein response and COL8A2 in Fuchs corneal dystrophy

Fuchs角膜内皮营养不良（FECD）是最常见和临床相关的遗传性角膜营养不良， 2007年，角膜疾病在美国人口中是角膜移植的第三大原因。FECD结果 由于角膜内皮细胞的慢性损失， 角膜透明所需的生理水合作用。在某些情况下，FECD是由以下基因的错义突变引起的： α 2胶原蛋白VIII（COL8A2）基因。关于FECD的发病机制知之甚少， 只有在角膜移植时获得的末期角膜可用于研究。有限的工作表明， 凋亡发生在来自晚期FECD角膜的内皮细胞中，尽管导致凋亡的机制不清楚。 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）鉴定未折叠蛋白反应（UPR）的标记物， FECD患者角膜的内皮。正确的蛋白质折叠对于细胞的正常功能至关重要。的 UPR是由过量错误折叠蛋白诱导的高度保守的广泛细胞反应。 最初，UPR的功能是通过选择性改变转录来缓解未折叠蛋白的应激 和翻译。如果蛋白质折叠的需求和能力仍然不平衡，UPR诱导细胞经历 凋亡此外，2）我们已经开发了两种具有不同COL8A2的基因靶向敲入小鼠品系 已知导致人类FECD的突变。初步结果显示， 角膜内皮细胞膜上的细胞膜，这与人FECD角膜中观察到的高度相似。 本申请的总体假设是“角膜内皮细胞表达COL8A2 突变激活未折叠蛋白反应并导致内皮细胞死亡。“可用性 具有相关病理表型的COL8A2 FECD基因敲入小鼠提供了宝贵的机会， 如下检验该假设：目的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的病理变化 膜与人FECD角膜相当。目的2将评估角膜和培养的角膜中的UPR活化 来自COL8A2突变小鼠的角膜内皮细胞系。目标3将评估紫外线的协同效应（a 已知的UPR诱导剂和角膜内皮的环境应激物）对UPR活化的影响。 角膜和来自COL8A2突变小鼠的培养角膜内皮细胞系。 该提议试图使用COL8A2 FECD突变小鼠和角膜内皮细胞系来建立一种用于治疗角膜内皮细胞疾病的方法。 UPR（一种主要的细胞应激途径）与角膜内皮细胞凋亡之间的新机制联系 由COL8A2中的FECD突变引起。建立这种联系将是一个重大进展 国家眼科研究所的目标是提高遗传性角膜疾病的知识和治疗。

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.