Epigenetic Modifications Compromise Mitochondrial DNA Quality Control in the Development of Diabetic Retinopathy

Abstract

PURPOSE. Diabetes causes dysfunction in the retinal mitochondria and increases base mismatches in their DNA (mtDNA). The enzyme responsible for repairing the base mismatches, MutL homolog 1 (Mlh1), is compromised. Diabetes also favors many epigenetic modifications and activates DNA methylation machinery, and Mlh1 has a CpG-rich promoter. Our aim is to identify the molecular mechanism responsible for impaired mtDNA mismatch repair in the pathogenesis of diabetic retinopathy. METHODS. Human retinal endothelial cells, incubated in 20 mM glucose, were analyzed for mitochondrial localization of Mlh1 by an immunofluorescence technique, Mlh1 promoter DNA methylation by the methylated DNA capture method, and the binding of Dnmt1 and transcriptional factor Sp1 by chromatin immunoprecipitation. The results were confirmed in retinal microvessels from streptozotocin-induced diabetic mice, with or without Dnmt inhibitors, and from human donors with diabetic retinopathy. RESULTS. Compared with cells in 5 mM glucose, high glucose decreased Mlh1 mitochondrial localization, and its promoter DNA was hypermethylated with increased Dnmt-1 binding and decreased Sp1 binding. Dnmt inhibitors attenuated Mlh1 promoter hypermethylation and prevented a decrease in its gene transcripts and an increase in mtDNA mismatches. The administration of Dnmt inhibitors in mice ameliorated a diabetes-induced increase in Mlh1 promoter hypermethylation and a decrease in its gene transcripts. Similar decreases in Mlh1 gene transcripts and its promoter DNA hypermethylation were observed in human donors. CONCLUSIONS. Thus, as a result of the epigenetic modifications of the Mlh1 promoter, its transcription is decreased, and decreased mitochondrial accumulation fails to repair mtDNA mismatches. Therapies targeted to halt DNA methylation have the potential to prevent/halt mtDNA damage and the development of diabetic retinopathy.

Epigenetics and Mitochondrial Stability in the Metabolic Memory Phenomenon Associated with Continued Progression of Diabetic Retinopathy

“…The number of sequence variants is increased in the retinal mtDNA in diabetes, and Mlh1 expression is decreased 17,22 . Our recent work has shown that due to hypermethylation of the promoter DNA of Mfn2 and Mlh1, their gene transcripts are downregulated, mtDNA mismatches are increased and mitochondrial function is compromised 13,14 . Here, we clearly demonstrate that the institution of intensive control fails to provide any benefit to DNA hypermethylation of both Mfn2 and Mlh1 promoters, and mtDNA transcription remains impaired.…”

“…Retinal endothelial cells. Human retinal endothelial cells (Cell system, Kirkland, WA) from 7-8 th passage were incubated in NG or HG media containing 1% fetal calf serum, 9% Nu-serum and 1 µg/mL endothelial growth supplement for 4 days 13,14 . Cell incubated in high glucose were divided into two groups, cells in group 1 remained in high glucose for eight days, in the absence or presence of 1 µM 5-aza-2′-deoxycytidine (Aza; Sigma-Aldrich Corp, St. Louis, MO, USA), respectively.…”

“…Methyl cytosine and Dnmt1 binding. Using MethyLamp Methylated DNA capture (MeDIP) Kit (EPIGENTEK, Farmingdale, NY), 5mC was immunoprecipitated in the sonicated total genomic DNA 13,14,22 . Enriched 5mC fractions were quantified by qPCR using gene and species-specific primers (Table 2), and input DNA was used as an internal control.…”

“…no. ab92312, Abcam, Cambridge, MA, USA; 1:100 dilution), as previously described 13,14 . CoxIV was used as a mitochondrial marker; Cat.…”

“…TI-2000, Vector Laboratories, Burlingame, CA); each at 1:500 dilution. Immuno-labelled cells were mounted using DAPI-containing (blue) Vectashield mounting medium (Vector Laboratories), and were examined under ZEISS (Carles Zeiss, Inc., Chicago, IL, USA) at 40X objective magnification with the Apotome module 13,14 . The images were calibrated with the ZEISS proinbuilt software package and modules, and the Pearson's correlation coefficient was calculated using the colocalization software module 13 .…”