Hyperglycemia-induced translational control of gene expression in the retina

描述(申请人提供)：糖尿病视网膜病变是美国工作年龄失明的主要原因，每年在美国有超过12000例新病例。基于证据的增殖性糖尿病视网膜病变的治疗原则包括激光介导性消融，但未能改变疾病的分子病理，因此，近一半的患者需要未来的治疗。因此，我们的总体目标是在分子水平上确定新的干预目标，以开发创新的、非破坏性的治疗方法，解决糖尿病视网膜病变的原因而不是影响。这种疾病的发病机制是由高血糖和胰岛素介导的信号转导减少共同引起的，后者通过诱导视网膜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 阐述糖尿病视网膜病变的分子基础，即高血糖引起的基因表达翻译控制的改变。中心假说是，翻译起始因子的丝氨酸或苏氨酸残基上加入O-连接的N-乙酰氨基葡萄糖(O-GlcNacylation)，可以介导从帽依赖到帽非依赖性的mRNA翻译，导致基因表达模式的改变，从而参与糖尿病视网膜病变的病理生理。在糖尿病诱导的高血糖条件下，通过己糖胺生物合成途径和信使核糖帽结合复合体的关键成分，包括eIF4E结合蛋白1、eIF4G、eIF4A和聚(A)结合蛋白的O-GlcN酰化，葡萄糖流量增加的发现支持了这一假说。此外，在这里，我们提供了初步的证据，表明高血糖有利于具有内部核糖体进入位点的mRNAs的翻译，例如那些编码关键血管生长因子的mRNAs，其方式依赖于eIF4F复合体组装的破坏。在指导阶段，PI将从Gerald Hart博士的实验室获得用于识别控制mRNA翻译的蛋白质中的O-GlcN酰化位点的方法方面的技术专业知识。一旦修饰的位点被确定，高血糖损害eIF4F复合体组装的机制将被确定。指导阶段还将为候选人提供时间接受Thomas Gardner博士的指导，以评估防止eIF4F复合体组装的破坏是否足以抑制这种疾病在小鼠模型中的早期临床前阶段的发病 糖尿病的症状。在结果方面，该项目预计不仅将扩展PI的技能和分析系统，还将确定将与糖尿病相关的代谢异常与视网膜血管生长因子表达增强联系起来的新机制。这种机制的确定具有重要意义，因为它有望验证旨在解决糖尿病视网膜病变的分子基础和促进健康视力的预防和/或治疗干预措施开发的新目标。

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.