Role of Alpha 2 Collagen VIII in Fuchs Corneal Dystrophy

描述(由申请人提供)：这项建议的总体目标是为首席研究员(PI)提供必要的经验和技能，以成为研究角膜疾病基本机制的独立研究员。这项建议的科学重点是更好地了解Fuchs内皮营养不良症(FED)中VIII型胶原突变导致角膜内皮细胞(CEC)丢失所导致的细胞和细胞外基质(ECM)蛋白异常。VIII型胶原(Col8)在上皮细胞的特殊基底膜--Descemet膜(DM)的形成中起着核心作用，编码III型胶原α2链(COLSA2)的基因突变在一些患者中引起FED。FED在人类身上经历了几十年的进步，占角膜移植手术的29%。疾病的早期阶段是无症状的，并且没有受到轻微影响的组织。因此，几乎没有关于导致FED的早期细胞和细胞外基质变化的信息。通过建立可靠的FED动物模型，以及对致病基因COL8A2突变对VIII型胶原功能的详细生化分析，可以大大加深对这些早期致病事件的了解。这一假设的基本假设是COL8A2基因突变导致细胞和生化异常，从而导致FED的CEC丢失。识别这些异常将为FED的发病机制提供重要的见解，这可能为这一重要的角膜疾病提供合理的治疗方案。针对这一假设，提出了两个具体的目标：目标1：通过基因打靶技术引入R155Q COL8A2突变，建立并鉴定FED小鼠模型。目的：研究已知的引起FED的COLSA2突变对甲型胶原与COL8A2相互作用的另一个COL8亚链形成同三聚体和异三聚体的影响。在拟议的研究和选定的教学活动过程中，PI将在发展眼病动物模型和细胞外基质蛋白质的生化分析方面获得宝贵的培训经验和指导。对于渴望开发研究角膜疾病基本机制的独立研究计划的PI来说，这些领域的专业知识被认为是无价的。

DESCRIPTION (provided by applicant): The overall goal of this proposal is to provide the principal investigator (PI) with the experiences and skills necessary to become an independent researcher studying basic mechanisms of corneal diseases. The scientific focus of this proposal is to gain a better understanding of the cellular and extracellular matrix (ECM) protein abnormalities caused by mutations in collagen VIII which lead to corneal endothelial cell (CEC) loss in Fuchs endothelial dystrophy (FED). Collagen VIII (COL8) plays a central role in the formation of Descemet membrane (DM), the specialized basement membrane of CECs, and mutations in the gene encoding the alpha2 chain of collagen VIII (COLSA2) cause FED in some patients. FED progresses over decades in humans and accounts for up to 29% of corneal transplants. Early stages of the disease are asymptomatic, and mildly affected tissues are not available. Thus, virtually no information exists about the early cellular and ECM changes leading to FED. Understanding these early pathogenic events could be increased greatly through the development of a reliable animal model of FED as well as detailed biochemical analysis of pathogenic COL8A2 mutations on collagen VIII function. The underlying hypothesis of this proposal is that mutations in the COL8A2 gene produce cellular and biochemical abnormalities which cause CEC loss in FED. Identifying these abnormalities should provide important insights into the pathogenesis of FED, which may suggest rational therapies for this important corneal disease. To address this hypothesis, two specific aims are proposed: Aim 1: To develop and characterize a mouse model of FED by introducing the R155Q COL8A2 mutation using gene targeting techniques. Aim 2: To investigate the effects of COLSA2 mutations known to cause FED on homotrimer and heterotrimer formation with alpha1 collagen VIII, the other COL8 subchain interacting with COL8A2 in vivo. In the course of the proposed research and selected didactic activities, the PI will gain invaluable training experience and mentoring in developing animal models of ocular disease and biochemical analyis of ECM proteins. Expertise in these areas is deemed invaluable for the PI who aspires to develop an independent research program studying basic mechanisms of corneal diseases.