Role of unfolded protein response and COL8A2 in Fuchs corneal dystrophy

未折叠蛋白反应和 COL8A2 在 Fuchs 角膜营养不良中的作用

• 批准号: 7987111
• 负责人: ALBERT S JUN
• 金额: $ 41万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-01 至 2015-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7987111
• 关键词: Affect; Age; Age-Months; Alleles; Amino Acids; Animals; Apoptosis; Basement membrane; Blindness; Caspase; Cell Culture Techniques; Cell Death; Cell Line; Cell Shape; Cell physiology; Cells; Cellular Morphology; Cellular Stress; Cessation of life; Chronic; Collagen; Cornea; Corneal Diseases; Corneal Endothelium; Corneal dystrophy; Data; Descemet' s membrane; Disease; Endothelial Cells; Endothelium; Event; Exhibits; Functional disorder; Gene Targeting; Genes; Genetic Transcription; Glutamine; Goals; Human; Hydration status; Immunofluorescence Immunologic; In Vitro; Induction of Apoptosis; Inherited; Keratoplasty; Knock-in Mouse; Knowledge; Laboratories; Leucine; Link; Lysine; Mediating; Missense Mutation; Modeling; Mus; Mutant Strains Mice; Mutation; National Eye Institute; Operative Surgical Procedures; PAWR protein; Pathogenesis; Pathologic; Pathway interactions; Patients; Phenotype; Physiological; Population; Positioning Attribute; Proteins; Research; Risk Factors; Role; Specimen; Staging; Staining method; Stains; Stress; Surface; Testing; Translations; Tryptophan; Tunicamycin; Ultraviolet Rays; Up-Regulation; Work; caspase 12; clinically relevant; density; environmental stressor; genetic risk factor; improved; in vivo; insight; meter; modifiable risk; mutant; novel; protein folding; protein misfolding; public health relevance; response; response marker; tool

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.

描述（由申请人提供）：Fuchs角膜内皮营养不良（FECD）是美国人群中最常见和临床相关的遗传性角膜疾病，是2007年角膜移植的第三大原因。FECD由角膜内皮细胞的慢性损失引起，角膜内皮细胞排列在角膜的内表面并维持角膜透明所需的生理水合作用。在某些情况下，FECD是由α 2胶原蛋白VIII（COL8A2）基因中的错义突变引起的。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。有限的工作表明，细胞凋亡发生在内皮细胞从先进的FECD角膜，虽然导致细胞凋亡的机制知之甚少。因此，动物和细胞培养模型将是非常宝贵的工具，以阐明早期事件导致内皮细胞凋亡的FECD。 我们的初步工作包括：1）鉴定FECD患者角膜内皮中未折叠蛋白反应（UPR）的标志物。正确的蛋白质折叠对于细胞的正常功能至关重要。UPR是由过量的错误折叠蛋白诱导的高度保守、广泛的细胞反应。最初，UPR的功能是通过选择性改变转录和翻译来缓解未折叠蛋白的应激。如果蛋白质折叠需求和能力保持不平衡，UPR诱导细胞进行凋亡。此外，2）我们已经开发了两种基因靶向敲入小鼠系，其具有已知在人类中引起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FECD角膜中观察到的高度相似。 本申请的总体假设是“COL8A2突变的角膜内皮细胞表达激活未折叠的蛋白反应并导致内皮细胞死亡。“具有相关病理表型的COL8A2 FECD基因敲入小鼠的可用性提供了一个宝贵的机会来测试这一假设，如下所示：目标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膜的病理变化，与人类FECD角膜相当。目的2将评估来自COL8A2突变小鼠的角膜和培养的角膜内皮细胞系中的UPR活化。目的3将评估UV光（已知的UPR诱导物和角膜内皮的环境应激物）对来自COL8A2突变小鼠的角膜和培养的角膜内皮细胞系中的UPR活化的协同效应。 该提案旨在使用COL8A2 FECD突变小鼠和角膜内皮细胞系来建立UPR（主要细胞应激途径）与COL8A2中FECD突变引起的角膜内皮细胞凋亡之间的新机制联系。这种联系的建立将代表国家眼科研究所在提高遗传性角膜疾病的知识和治疗目标方面的重大进展。 公共卫生关系：Fuchs内皮角膜营养不良（FECD）是一种常见疾病，由于角膜内层的角膜内皮细胞死亡导致角膜清晰度降低，导致视力丧失（Borboli 2002）。FECD影响约0.6%的美国人群（Krachmer 1978），是美国角膜移植手术的第三大原因（货车Meter 2007）。本研究的目的是探讨未折叠蛋白反应，一个主要的细胞应激途径，在导致内皮细胞死亡的FECD的作用。