July 2018
Volume 59, Issue 9
ARVO Annual Meeting Abstract  |   July 2018
Link between Epigenetics and Poor Mitochondrial DNA Quality Control in the Development of Diabetic Retinopathy
Author Affiliations & Notes
  • Renu A. Kowluru
    Wayne State Univ, Detroit, Michigan, United States
  • Manish Mishra
    Wayne State Univ, Detroit, Michigan, United States
  • Lalit Singh
    Wayne State Univ, Detroit, Michigan, United States
  • Footnotes
    Commercial Relationships   Renu Kowluru, None; Manish Mishra, None; Lalit Singh, None
  • Footnotes
    Support  EY014370, EY017313 and EY022230
Investigative Ophthalmology & Visual Science July 2018, Vol.59, 3542. doi:
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      Renu A. Kowluru, Manish Mishra, Lalit Singh; Link between Epigenetics and Poor Mitochondrial DNA Quality Control in the Development of Diabetic Retinopathy. Invest. Ophthalmol. Vis. Sci. 2018;59(9):3542.

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      © ARVO (1962-2015); The Authors (2016-present)

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Purpose : In diabetes, retinal mitochondria are dysfunctional, and their DNA (mtDNA) is damaged with increased base-mismatches. Although mtDNA harbors both mutant and non-mutant DNA within the same cell, the drift towards increase in mutant mtDNA results in reduced energy capacity, and this threshold is lower in highly oxidative tissues. Mitochondria are also equipped with a mismatch repair system to repair uncomplimentary base pairs, but the repair enzyme, MutL homolog 1 (Mlh1, which assists in cutting the base-mismatches), is compromised in diabetes. Diabetes also favors many epigenetic modifications, and methylation of cytosine by DNA methyl transferases (Dnmts) is a relatively stable repressive epigenetic mark. The promoter of Mlh1 is rich in CpG dinucleotides with over 50 CpG sites. Our aim was to investigate the role of Mlh1 promoter DNA methylation in mtDNA damage in diabetes.

Methods : Human retinal endothelial cells, incubated in high glucose (20mM) or normal glucose (5mM) for four days, in the presence of chemical (1µM 5-Azacytidine, 1mM Valproic acid, 250µM zebularine) or molecular (Dnmt1-siRNA) inhibitor of Dnmts, were analyzed for methylated cytosine (5mC) levels. 5mC-enriched DNA was immuno-captured using MeDIP kit, and this was followed by q-PCR using Mlh1 promoter- specific primers. Base-mismatch was quantified by digesting mtDNA amplicons using mismatch specific surveyor endonuclease, followed by DNA gel electrophoresis. Similar experiments were performed in retinal microvessels from streptozotocin-induced diabetic mice receiving 5-Azacytidine (2.5mg/kg, intraperitoneal) or Dnmt1-siRNA (2µg/2µl, intravitreal).

Results : Compared to cells in 5mM glucose, high glucose increased 5mC levels at Mlh1 promoter DNA by over 2.5 fold, and decreased Mlh1 expression by ~2 fold. Inhibition of Dnmts ameliorated increase in DNA methylation and decrease in Mlh1 expression, and prevented increase in base-mismatches in mtDNA. Consistent with in vitro results, in mouse retinal microvessels, diabetes-induced increase in 5mC at Mlh1 promoter DNA was also attenuated by Dnmt inhibitors.

Conclusions : Thus, due to epigenetic modifications of Mlh1, repair of hyperglycemia-induced increase in base-mismatches in mtDNA is impaired. Therapies targeted to inhibit DNA methylation could have potential to prevent/halt mitochondrial damage, and the development of diabetic retinopathy.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.


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