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Aim: Hyperglycemia damages mitochondria and downregulates transcription of mtDNA-encoded genes and the long noncoding RNA LncCytB, causing mitochondrial genomic instability. The genes encoded by mtDNA are transcribed as large polycistronic transcripts, and the 5′ ends of precursor tRNAs are processed by mitochondrial-targeted ribonuclease P (MRPPs). Our aim was to investigate the role of MRPP1 in the downregulation of LncCytB in diabetic retinopathy. Methods: Using human retinal endothelial cells incubated in 20 mM D-glucose for 96 h, the gene expression and mitochondrial localization (immunofluorescence) of MRPP1 and the interaction between MRPP1 and LncCytB (determined by RNA-FISH and RNA immunoprecipitation) were quantified. The results were confirmed in retinal microvessels from streptozotocin-induced diabetic mice and from human donors with documented diabetic retinopathy. Results: Compared to normal glucose, high glucose decreased mRNA and mitochondrial localization of MRPP1 and its interaction with LncCytB. While MRPP1 overexpression prevented glucose-induced decrease in MRPP1–LncCytB interaction, LncCytB expression and mitochondrial damage (reduction in protective nucleoids in mtDNA), MRPP1-siRNA further worsened them. Similar results were obtained from retinal microvessels from diabetic mice and from human donors with diabetic retinopathy. Conclusions: Downregulation of MRPP1 in diabetes suppresses LncCytB transcription, resulting in mitochondrial functional and genomic instability, ultimately leading to the development of diabetic retinopathy. Thus, preventing MRPP1 downregulation has the potential to inhibit retinopathy and prevent the fear of vision loss in diabetic patients. [ABSTRACT FROM AUTHOR]

Mitochondria experience genomic and functional instability in diabetes, and mitochondrial dysfunction has a critical role in the development of diabetic retinopathy. Diabetes also alters expressions of many long noncoding RNAs (LncRNAs), the RNAs with >200 nucleotides and no open reading frame. LncRNAs are mainly encoded by the nuclear genome, but mtDNA also encodes three LncRNAs. Our goal was to investigate the effect of hyperglycemia on mtDNA-encoded LncRNA cytochrome B (LncCytB) in mtDNA stability in diabetic retinopathy. Retinal endothelial cells, transfected with LncCytB-overexpressing plasmids or siRNA, incubated in 5 mmol/L d-glucose (normal glucose [NG]) or 20 mmol/L d-glucose (high glucose [HG]) for 4 days, were analyzed for LncCytB expression by strand-specific PCR and its mitochondrial localization by RNA fluorescence in situ hybridization. Damage-sensitive mtDNA regions were examined by micrococcal nuclease (MNase) digestion sequencing and LncCytB occupancy at mtDNA by chromatin isolation by RNA purification. Protective nucleoids in mtDNA were analyzed by SYBR Green-MitoTracker Red staining and confirmed in isolated mitochondria by flow cytometry. Compared with NG, HG downregulated LncCytB by >50% but had no significant effect on the other mtDNA-encoded LncRNAs. mtDNA packaging was impaired, MNase sensitivity was increased, and LncCytB occupancy at mtDNA was decreased. While LncCytB overexpression ameliorated mtDNA damage and decrease in nucleoids and copy numbers, LncCytB-siRNA exacerbated damage and further reduced nucleoids. Retinal microvessels from streptozotocin-induced diabetic mice and human donors with diabetic retinopathy presented a similar decrease in LncCytB and mtDNA nucleoids. Thus, LncCytB has a major role in maintaining mitochondrial genomic stability, and its downregulation in the hyperglycemic milieu contributes to increased vulnerability of mtDNA to damage. ARTICLE HIGHLIGHTS: In the pathogenesis of diabetic retinopathy, an mtDNA-encoded long noncoding RNA, long noncoding cytochrome B (LncCytB), is downregulated. Downregulation of LncCytB results in the reduced number of protective nucleoids, and the mtDNA becomes vulnerable to the damage. The damaged mtDNA compromises the electron transport chain system, increasing superoxide levels, and the vicious cycle of free radicals continues to self-propagate. Preventing the downregulation of LncCytB in diabetes will interfere in the self-perpetuating vicious cycle of free radicals and should inhibit the development of diabetic retinopathy. [ABSTRACT FROM AUTHOR]