Hyperglycemia-induced translational control of gene expression in the retina

项目概要/摘要 糖尿病视网膜病变是工作年龄美国人失明的主要原因，占超过 美国每年新增病例 12,000 例。增殖性疾病的循证治疗原则 糖尿病视网膜病变涉及激光介导的消融，但无法改变视网膜的分子病理学 疾病，因此，近一半的患者需要未来的治疗。因此，我们的总体目标是确定新的 分子水平干预的目标将导致创新、非破坏性的发展 治疗方法针对的是糖尿病视网膜病变的病因，而不是其影响。发病机制 这种疾病是由高血糖和胰岛素介导的信号传导减少共同引起的， 这通过诱导结构和生理导致糖尿病神经血管并发症 视网膜的变化。本申请中提出的研究具有创新性，因为它代表了 解决糖尿病视网膜病变的分子基础的完全不同的方法，即高血糖引起的 基因表达翻译控制的改变。中心假设是添加 O- 将 N-乙酰葡糖胺（O-GlcNAcylation）连接至翻译起始因子的丝氨酸或苏氨酸残基 介导从帽依赖型到帽独立型 mRNA 翻译的转变，导致基因改变 有助于糖尿病视网膜病变病理生理学的表达模式。假设得到支持 通过发现葡萄糖通过己糖胺生物合成途径和 O-GlcNAc 酰化的通量增加 mRNA 帽结合复合物的关键成分，包括 eIF4E 结合蛋白 1、eIF4G、eIF4A 和 聚（A）结合蛋白，在糖尿病引起的高血糖条件下。此外，在此我们提供 初步证据表明高血糖有利于具有内部核糖体进入位点的 mRNA 的翻译， 例如那些编码关键血管生长因子的因子，其方式依赖于 eIF4F 的破坏 复杂的装配。在指导阶段，PI将从实验室获得技术专业知识 Gerald Hart 博士介绍用于识别控制 mRNA 的蛋白质中 O-GlcNAcylation 位点的方法 翻译。一旦修饰位点被确定，高血糖的机制 损害 eIF4F 复杂的装配将被定义。指导阶段还将为候选人提供时间 接受 Thomas Gardner 博士的指导，评估是否可以防止 eIF4F 复杂组装的破坏 足以抑制糖尿病小鼠模型中该疾病发病机制的早期临床前阶段。 就成果而言，该项目不仅有望扩展 PI 的技能和分析系统， 但还将确定将与糖尿病相关的代谢异常与 视网膜中血管生长因子的表达增强。识别此类机制具有重要意义 因为它有望验证开发预防和/或治疗药物的新目标 旨在解决糖尿病视网膜病变的分子基础并促进健康视力的干预措施。

Project Summary/Abstract Diabetic retinopathy is the leading cause of blindness in working age Americans, accounting for more than 12,000 new cases in the United States each year. The principle evidenced based treatment for proliferative diabetic retinopathy involves laser-mediated ablation, which fails to alter the molecular pathology of the disease, and as such, nearly half of patients require future treatments. Thus, our overall goal is to identify new targets for intervention at the molecular level that will lead to development of innovative, nondestructive therapies that address treatment of the cause of diabetic retinopathy, rather than the effect. The pathogenesis of this disease is caused by a combination of hyperglycemia and a reduction in insulin mediated signaling, which results in diabetic neurovascular complications through the induction of structural and physiological changes in the retina. The research proposed in this application is innovative, because it represents an entirely different approach to address the molecular basis of diabetic retinopathy, i.e. hyperglycemia-induced alterations in the translational control of gene expression. The central hypothesis is that the addition of O- linked N-Acetylglucosamine (O-GlcNAcylation) to serine or threonine residues of translation initiation factors mediates a shift from cap-dependent to cap-independent mRNA translation, resulting in an altered gene expression pattern that contributes to the pathophysiology of diabetic retinopathy. The hypothesis is supported by findings of elevated flux of glucose through the hexosamine biosynthetic pathway and O-GlcNAcylation of key components of the mRNA cap-binding complex, including eIF4E binding protein 1, eIF4G, eIF4A, and poly(A)-binding protein, under conditions of diabetes-induced hyperglycemia. Furthermore, herein we provide preliminary evidence that hyperglycemia favors the translation of mRNAs with internal ribosome entry sites, such as those encoding key vascular growth factors, in a manner that is dependent on the disruption of eIF4F complex assembly. During the mentored phase, the PI will acquire technical expertise from the laboratory of Dr. Gerald Hart on the methodology used to identify O-GlcNAcylation sites in proteins that control mRNA translation. Once the modified sites have been identified, the mechanisms through which hyperglycemia impairs eIF4F complex assembly will be defined. The mentored phase will also provide time for the candidate to receive guidance from Dr. Thomas Gardner to evaluate if preventing disruption of eIF4F complex assembly is sufficient to inhibit early preclinical phases of the pathogenesis of this disease in a mouse model of diabetes. With respect to outcomes, this project is expected to not only expand the PI's skills and systems of analysis, but will also identify novel mechanisms that link the metabolic abnormalities associated with diabetes to enhanced vascular growth factor expression in the retina. Identification of such mechanisms is significant because it is expected to validate new targets for the development of preventive and/or therapeutic interventions aimed at addressing the molecular basis of diabetic retinopathy and promoting healthy vision.