Faulty homocysteine recycling in diabetic retinopathy

Background: Although hyperglycemia is the main instigator in the development of diabetic retinopathy, elevated circulating levels of a non-protein amino acid, homocysteine, are also associated with an increased risk of retinopathy. Homocysteine is recycled back to methionine by methylenetetrahydrofolate reductase (MTHFR) and/or transsulfurated by cystathionine beta-synthase (CBS) to form cysteine. CBS and other transsulfuration enzyme cystathionine-gamma-lyase (CSE), through desulfuration, generates H2S. Methionine cycle also regulates DNA methylation, an epigenetic modification associated with the gene suppression. The aim of this study was to investigate homocysteine and its metabolism in diabetic retinopathy. Methods: Homocysteine and H2S levels were analyzed in the retina, and CBS, CSE and MTHFR in the retinal microvasculature from human donors with established diabetic retinopathy. Mitochondrial damage was evaluated in retinal microvessels by quantifying enzymes responsible for maintaining mitochondrial dynamics (fission-fusion-mitophagy). DNA methylation status of CBS and MTHFR promoters was examined using methylated DNA immunoprecipitation technique. The direct effect of homocysteine on mitochondrial damage was confirmed in human retinal endothelial cells (HRECs) incubated with 100 mu ML-homocysteine. Results: Compared to age-matched nondiabetic control human donors, retina from donors with established diabetic retinopathy had similar to 3-fold higher homocysteine levels and similar to 50% lower H2S levels. The enzymes important for both transsulfuration and remethylation of homocysteine including CBS, CSE and MTHFR, were 40-60% lower in the retinal microvasculature from diabetic retinopathy donors. While the mitochondrial fission protein, dynamin related protein 1, and mitophagy markers optineurin and microtubule-associated protein 1A/1B-light chain 3 (LC3), were upregulated, the fusion protein mitofusin 2 was downregulated. In the same retinal microvessel preparations from donors with diabetic retinopathy, DNA at the promoters of CBS and MTHFR were hypermethylated. Incubation of HRECs with homocysteine increased reactive oxygen species and decreased transcripts of mtDNA-encoded CYTB. Conclusions: Compromised transsulfuration and remethylation processes play an important role in the poor removal of retinal homocysteine in diabetic patients. Thus, regulation of their homocysteine levels should ameliorate retinal mitochondrial damage, and by regulating DNA methylation status of the enzymes responsible for homocysteine transsulfuration and remethylation, should prevent excess accumulation of homocysteine.