Targeting the Etiology of Diabetic Retinopathy

项目摘要 糖尿病视网膜病变是美国工作年龄段失明的主要原因， 约2000万糖尿病患者。该项目提出的这项研究补充，以提高 多样性旨在通过检查糖尿病对以下方面的影响来补充母体R 01的目标： 应激反应蛋白REDD 1（在发育和DNA损伤1中受调节）在细胞中的降解 视网膜。我们的实验室已经证明，糖尿病小鼠视网膜中的REDD 1表达增加， 是糖尿病引起的视网膜病变和视觉功能缺陷所必需的。此外，siRNA介导的 REDD 1基因敲除可改善糖尿病性黄斑水肿患者的最佳矫正视力因此，在本发明中， 鉴定负责糖尿病诱导的REDD 1在视网膜中表达的分子事件， 临床意义高。在作为父母补助金的一部分进行的研究中，我们最近发现证据表明， 转录后机制可能是负责增加REDD 1蛋白在视网膜中， 糖尿病小鼠具体来说，在链脲佐菌素诱导的糖尿病小鼠的视网膜中，REDD 1蛋白表达 在REDD 1 mRNA丰度没有变化的情况下，这个问题的核心假设是 补充的是糖尿病诱导的氧化应激促进REDD 1与TXNIP（硫氧还蛋白）的相互作用 相互作用蛋白），以防止泛素化和随后的蛋白酶体降解的方式， REDD 1.为了验证这一假设，申请人将采用一种涉及模型系统的实验方案 范围从完整的小鼠到视网膜细胞培养物。Aim 1将使用尖端分子技术（例如SNAP- 标记）来评估糖尿病对视网膜REDD 1蛋白降解的影响。目标2将审查 TXNIP对糖尿病背景下视网膜REDD 1蛋白周转的影响。具体机制 负责REDD 1的增加，这与糖尿病引起的视觉功能下降有关 以前没有建立，因此代表了我们理解的重大差距。 导致糖尿病视网膜病变中视觉功能障碍的分子事件。总的来说，这项研究支持的研究 补充将大大提高父母R 01探索为什么高血糖条件促进视网膜 REDD 1蛋白质含量。建议的项目代表了一个重点论文项目，提供了两个关键 为有前途的博士预科生提供培训机会。首先，申请人将开发技术 使用光学相干断层扫描技术评估糖尿病对视网膜病理生理学的影响的专业知识， 视网膜电图和虚拟验光。申请人还将发展必要的技能，操作视网膜 使用AAV的体内基因表达。

PROJECT SUMMARY Diabetic retinopathy is the leading cause of blindness in working age Americans, affecting more than a third of the ~20 million individuals with diabetes. The project proposed for this research supplement to enhance diversity is designed to complement the aims of the parent R01 by examining the impact of diabetes on degradation of the stress response protein REDD1 (regulated in development and DNA damage 1) in the retina. Our laboratory has demonstrated that REDD1 expression is increased in the retina of diabetic mice, and is necessary for diabetes-induced retinal pathology and functional deficits in vision. Moreover, siRNA-mediated REDD1 knockdown improves best-corrected visual acuity in patients with diabetic macular edema. Thus, identification of the molecular events responsible for diabetes-induced REDD1 expression in the retina is of high clinical significance. In studies conducted as part of the Parental Grant, we recently found evidence that a post-transcriptional mechanism was potentially responsible for the increase in REDD1 protein in the retina of diabetic mice. Specifically, in the retina of streptozotocin-induced diabetic mice, REDD1 protein expression was increased in the absence of a change in REDD1 mRNA abundance. The central hypothesis of this supplement is that diabetes-induced oxidative stress promotes REDD1 interaction with TXNIP (thioredoxin interacting protein) in a manner that prevents the ubiquitination and subsequent proteasomal degradation of REDD1. To test the hypothesis, the applicant will pursue an experimental protocol involving model systems ranging from intact mice to retinal cell culture. Aim 1 will use cutting-edge molecular techniques (e.g. SNAP- tagging) to evaluate the impact of diabetes on retinal REDD1 protein degradation. Aim 2 will examine the impact of TXNIP on retinal REDD1 protein turnover in the context of diabetes. The specific mechanism(s) responsible for the increase in REDD1 that is associated with the diabetes-induced decline in visual function has not been previously established, and thus represents a significant gap in our understanding of the molecular events that cause visual dysfunction in diabetic retinopathy. Overall, the studies supported by this supplement will greatly enhance the parent R01 by exploring why hyperglycemic conditions promote retinal REDD1 protein content. The proposed project represents a focused thesis project that provides two key training opportunities for a promising pre-doctoral student. First, the applicant will develop the technical expertise to assess the impact of diabetes on retinal pathophysiology using optical coherence tomography, electroretinograms, and virtual optomotry. The applicant will also develop skills necessary to manipulate retinal gene expression in vivo using AAV.