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。在这种致盲疾病的发病机制中， 视网膜线粒体功能障碍，电子传递链(ETC)受损，超氧化物 水平升高，虽然复合体III的活性受到抑制，但复合体I保持不变。线粒体 它们有自己的小DNA(MtDNA)，它缺乏保护性的组蛋白，但被包装成 为其生物发生提供一些保护和协助。糖尿病损害线粒体DNA，损害其生物发生，以及 下调线粒体DNA编码的细胞色素B(复合体III的细胞色素B)的基因表达。基因表达是 也受长非编码RNA(LncRNAs)调控，即含有200个核苷酸且无开放阅读的RNAs 框架用于翻译，但它们可以与DNA或RNA结合，或者可以作为支架来促进相互作用 蛋白质。虽然大多数lncRNAs是由核DNA编码的，但mtDNA也编码三个 络合物I的LncRNAs、LncND5和LncND6以及络合物III的LncCytB。初步数据显示，在 高血糖环境，而LncCytB下调，LncND5和LncND6保持不变，以及 核团减少，线粒体DNA对核酸酶消化的敏感性增加。基于这些，我们的中央 假说是糖尿病患者的LncCytB下调会损害线粒体DNA类核并减弱细胞色素B 转录，破坏线粒体DNA和电子传输链系统，以及受损的线粒体 导致视网膜病变的发生。 目标1将研究LncCytB在类核形成中的作用，该假说预测 糖尿病患者中的LncCytB会损害核样物质，破坏线粒体DNA的完整性，减少其拷贝数。目标2将 研究LncCytB在ETC调节中的作用，并将检验以下假设 LncCytB降低细胞色素B的转录，从而抑制复合体III的活性，并损害ETC “系统”。目标3将研究高血糖下调LnCytB的机制，并将 研究线粒体靶向核糖核酸酶P蛋白1在调控线粒体LncCytB中的作用。 该计划将使用体外(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。我们的总目标是 是确定糖尿病视网膜病变发病机制中涉及的新的调控机制，特别是在 线粒体动态平衡中mtDNA编码的lncRNA水平。可检验的中心假设是 创新，并具有显著的翻译影响，因为成功完成我们的研究将提供强大的 LncCytB作为预防这种视力发展/进展的潜在治疗靶点的背景- 有威胁的疾病。

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.