Diabetic Retinopathy, Mitochondria Damage and Long Non-coding RNAs - 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.
