LAUSR

Purpose Diabetes dysfunctions retinal mitochondria and increases base pair mismatches in mitochondrial DNA (mtDNA), and damaged mtDNA impairs the electron transport chain system. A mismatch repair system actively repairs the uncomplimentary base pairs, but in diabetes, the enzyme responsible for cutting the base-mismatches, MutL homolog 1 (Mlh1), is also compromised, which further adds to the damage of mtDNA. In diabetes, retinal DNA methylation machinery is also altered, and the activity of DNA methyl transferases (Dnmts) is significantly increased. Since Mlh1 promoter has over 50 CpG sites, making it a good target for DNA methylation, our aim was to investigate the role of DNA methylation in mtDNA damage in the development of diabetic retinopathy. Methods Retinal microvessels from streptozotocin-induced diabetic mice, with or without Dnmt inhibitor 5-azacytidine (Aza, 2.5mg/kg, intraperitoneal), were analyzed for methylated cytosine (5mC) levels using immuno-capture method and Mlh1 promoter- specific primers. Base-mismatch was quantified by digesting mtDNA amplicons using mismatch specific surveyor endonuclease, followed by DNA fragmentation analysis. Similar experiments were performed in retinal microvessels prepared from human donors with documented diabetic retinopathy. Results Compared to normal mice, retinal microvessels from diabetic mice had decreased Mlh1 expression and increased Dnmt1 and 5mC levels at Mlh1 promoter. In the same mice, base-mismatches in their mtDNA were also increased. Administration of Aza ameliorated diabetes-induced decrease in Mlh1 expression, and prevented increase in Dnmt1 and DNA methylation of Mlh1 promoter. Similarly, compared to age-matched non diabetic human donors, retinal microvessels from donors with established diabetic retinopathy had decreased levels of Mlh1 and increased levels of Dnmt1 and 5mC at Mlh1 promoter. Conclusions Thus, DNA methylation of Mlh1 promoter has a major role in impaired mtDNA damage. Therapies targeted to halt DNA methylation could have potential to prevent/halt mtDNA damage, and the development of diabetic retinopathy.