Diabetic Retinopathy, Mitochondria Damage and Long Non-coding RNAs

摘要 视网膜病变是糖尿病最可怕的并发症之一。在这种致盲性疾病的发病机制中， 视网膜线粒体功能失调，电子传递链（ETC）受损，超氧化物 水平升高，而复合物III活性被抑制，复合物I保持不变。线粒体 有自己的小DNA（mtDNA），缺乏保护性组蛋白，但被包装成类核蛋白， 提供一些保护，并协助其生物合成。糖尿病损害线粒体DNA，损害其生物合成， 下调线粒体DNA编码的细胞色素B（复合物III的CYTB）的基因表达。基因表达 也受长链非编码RNA（LncRNA）调控，这些RNA具有>200个核苷酸且无开放阅读 翻译框架，但它们可以结合到DNA或RNA，或可以作为支架，以促进相互作用， proteins.虽然大多数LncRNA由核DNA编码，但mtDNA也编码三种LncRNA。 LncRNA，LncND 5和LncND 6用于复合物I，LncCytB用于复合物III。初步数据显示， 在高血糖环境中，LncCytB下调，LncND 5和LncND 6保持不变， 类核减少，mtDNA对核酸酶消化的敏感性增加。基于此，我们的中央 一种假说是糖尿病中的“LncCytB”下调损害mtDNA类核苷酸并减弱细胞色素B 转录，破坏线粒体DNA和电子传递链系统，以及受损的线粒体 导致视网膜病变的发展。 目的1将研究LncCytB在类核形成中的作用，该假说预测， 糖尿病中的LncCytB损害类核，破坏mtDNA的完整性并减少其拷贝数。目标2将 检查LncCytB在ETC调节中的作用，并将测试“下调ETC”的假设。 LncCytB降低CYTB的转录，从而抑制复合物III活性并损害ETC 系统”。目标3将研究高血糖症下调LnCytB的机制，并将 检查线粒体靶向RNA酶P蛋白1在线粒体中LncCytB调节中的作用。 该计划将采用体外（人视网膜内皮细胞）和体内（啮齿动物视网膜微血管） 糖尿病视网膜病变模型，并将利用充分优化的分子生物学方法。我们的总目标 目的是确定参与糖尿病视网膜病变发病机制的新的调节机制，特别是在 线粒体DNA编码的LncRNA在线粒体内稳态中的水平。可检验的中心假设是 创新，并具有重大的翻译影响，因为我们的研究成功完成将提供强大的 LncCytB作为潜在的治疗靶点来预防这种视力的发展/进展的背景- 威胁疾病

ABSTRACT Retinopathy is one of the most-feared complications of diabetes. In the pathogenesis of this blinding disease, retinal mitochondria become dysfunctional, the electron transport chain (ETC) is compromised, superoxide levels are elevated, and while complex III activity is inhibited, complex I remains unchanged. Mitochondria have their own small DNA (mtDNA), which lacks protective histones, but is packaged into nucleoids that provide some protection and assist in its biogenesis. Diabetes damages mtDNA, impairs its biogenesis, and downregulates gene expression of mtDNA-encoded cytochrome B (CYTB of complex III). Gene expression is also regulated by long noncoding RNAs (LncRNAs), the RNAs with >200 nucleotides and no open reading frame for translation, but they can bind to DNA or RNA, or can act as scaffolds to promote the interaction of proteins. Although majority of the LncRNAs are encoded by nuclear DNA, mtDNA also encodes three LncRNAs, LncND5 and LncND6 for complex I and LncCytB for complex III. Preliminary data show that in hyperglycemic milieu, while LncCytB is downregulated, LncND5 and LncND6 remain unchanged, and nucleoids are decreased and mtDNA sensitivity to nuclease digestion is increased. Based on these, our central hypothesis is that `LncCytB downregulation in diabetes impairs mtDNA nucleoids and attenuates cytochrome B transcription, damaging the mtDNA and the electron transport chain system, and the damaged mitochondria lead to the development of retinopathy'. Aim 1 will investigate the role of LncCytB in nucleoid formation, and the hypothesis predicts that `decrease in LncCytB in diabetes impairs nucleoids, damaging mtDNA integrity and reducing its copy numbers'. Aim 2 will examine the role of LncCytB in the regulation of the ETC, and will test the hypothesis that `downregulation of LncCytB decreases transcription of CYTB, which inhibits the complex III activity and compromises the ETC system'. Aim 3 will investigate the mechanism by which hyperglycemia downregulates LnCytB, and will examine the role of mitochondrial-targeted RNAse P protein 1 in regulation of LncCytB in the mitochondria. The plan will employ in vitro (human retinal endothelial cells) and in vivo (retinal microvessels from rodents) models of diabetic retinopathy, and will utilize fully optimized molecular biological approaches. Our overall goal is to identify novel regulatory mechanisms involved in the pathogenesis of diabetic retinopathy, specifically at the level of mtDNA-encoded LncRNA in mitochondrial homeostasis. The testable central hypothesis is innovative, and has significant translational impact as successful completion of our studies will provide strong background for LncCytB as a potential therapeutic target to prevent the development/ progression of this sight- threatening disease.