Hyperglycemia-induced translational control of gene expression in the retina

描述（由申请人提供）：糖尿病视网膜病变是美国工作年龄段失明的主要原因，每年在美国有超过12，000例新发病例。增殖性糖尿病视网膜病变的原则性治疗包括激光介导的消融术，这无法改变疾病的分子病理学，因此，近一半的患者需要进一步治疗。因此，我们的总体目标是在分子水平上确定新的干预目标，这将导致开发创新的非破坏性疗法，解决糖尿病视网膜病变的病因治疗，而不是效果。这种疾病的发病机制是由高血糖症和胰岛素介导的信号传导减少的组合引起的，这通过诱导视网膜中的结构和生理变化而导致糖尿病神经血管并发症。本申请中提出的研究是创新的，因为它代表了一种完全不同的方法， 解决糖尿病视网膜病变的分子基础，即高血糖诱导的基因表达翻译控制的改变。中心假设是，将O-连接的N-乙酰葡糖胺（O-GlcNAc化）添加到翻译起始因子的丝氨酸或苏氨酸残基介导从帽依赖性mRNA翻译转变为帽非依赖性mRNA翻译，导致基因表达模式改变，导致糖尿病视网膜病变的病理生理学。在糖尿病诱导的高血糖症条件下，通过氨基己糖生物合成途径的葡萄糖通量升高和mRNA帽结合复合物的关键组分（包括eIF 4 E结合蛋白1、eIF 4G、eIF 4A和poly（A）结合蛋白）的O-GlcNAc化的发现支持了这一假设。此外，在此我们提供了初步证据，高血糖症有利于翻译的mRNA与内部核糖体进入网站，如那些编码关键血管生长因子，在某种程度上，这是依赖于破坏eIF 4F复合物组装。在指导阶段，PI将从Gerald哈特博士的实验室获得用于鉴定控制mRNA翻译的蛋白质中的O-GlcNAc化位点的方法的技术专业知识。一旦修饰位点被确定，高血糖症损害eIF 4F复合物组装的机制将被确定。指导阶段还将为候选人提供时间，以接受托马斯加德纳博士的指导，以评估防止eIF 4 F复合物组装的破坏是否足以抑制小鼠模型中该疾病发病机制的早期临床前阶段 糖尿病关于结果，预计该项目不仅将扩展PI的技能和分析系统，而且还将确定将糖尿病相关的代谢异常与视网膜中血管生长因子表达增强联系起来的新机制。这些机制的识别是重要的，因为它预计将验证新的目标，旨在解决糖尿病视网膜病变的分子基础和促进健康的视力的预防和/或治疗干预措施的发展。

DESCRIPTION (provided by applicant): 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.