Kresge Eye Institute, Wayne State University, Detroit, MI, 48201, USA.
Mol Neurobiol. 2019 Jan;56(1):88-101. doi: 10.1007/s12035-018-1086-9. Epub 2018 Apr 21.
In the development of diabetic retinopathy, retinal mitochondria are dysfunctional, and mitochondrial DNA (mtDNA) is damaged with increased base mismatches and hypermethylated cytosines. DNA methylation is also a potential source of mutation, and in diabetes, the noncoding region, the displacement loop (D-loop), experiences more methylation and base mismatches than other regions of the mtDNA. Our aim was to investigate a possible crosstalk between mtDNA methylation and base mismatches in the development of diabetic retinopathy. The effect of inhibition of Dnmts (by 5-aza-2'-deoxycytidine or Dnmt1-siRNA) on glucose-induced mtDNA base mismatches was investigated in human retinal endothelial cells by surveyor endonuclease digestion and validated by Sanger sequencing. The role of deamination factors on increased base mismatches was determined in the cells genetically modulated for mitochondrial superoxide dismutase (Sod2) or cytidine-deaminase (APOBEC3A). The results were confirmed in an in vivo model using retinal microvasculature from diabetic mice overexpressing Sod2. Inhibition of DNA methylation, or regulation of cytosine deamination, significantly inhibited an increase in base mismatches at the D-loop and prevented mitochondrial dysfunction. Overexpression of Sod2 in mice also prevented diabetes-induced D-loop hypermethylation and increase in base mismatches. The crosstalk between DNA methylation and base mismatches continued even after termination of hyperglycemia, suggesting its role in the metabolic memory phenomenon associated with the progression of diabetic retinopathy. Inhibition of DNA methylation limits the availability of methylated cytosine for deamination, suggesting a crosstalk between DNA methylation and base mismatches. Thus, regulation of DNA methylation, or its deamination, should impede the development of diabetic retinopathy by preventing formation of base mismatches and mitochondrial dysfunction.
在糖尿病性视网膜病变的发展过程中，视网膜线粒体功能失调，线粒体 DNA（mtDNA）受损，碱基错配增加，胞嘧啶超甲基化。DNA 甲基化也是突变的潜在来源，在糖尿病中，非编码区、移位环（D-loop）比 mtDNA 的其他区域经历更多的甲基化和碱基错配。我们的目的是研究 mtDNA 甲基化和碱基错配在糖尿病性视网膜病变发展过程中的可能相互作用。通过 Surveyor 内切酶消化研究了抑制 Dnmts（通过 5-aza-2'-脱氧胞苷或 Dnmt1-siRNA）对人视网膜内皮细胞中葡萄糖诱导的 mtDNA 碱基错配的影响，并通过 Sanger 测序进行了验证。通过遗传修饰线粒体超氧化物歧化酶（Sod2）或胞嘧啶脱氨酶（APOBEC3A）的细胞，确定脱氨酶因子在增加碱基错配中的作用。在使用过表达 Sod2 的糖尿病小鼠的视网膜微血管体内模型中验证了这些结果。抑制 DNA 甲基化或调节胞嘧啶脱氨作用可显著抑制 D-loop 碱基错配的增加，并防止线粒体功能障碍。Sod2 在小鼠中的过表达也可防止糖尿病诱导的 D-loop 高甲基化和碱基错配增加。即使在高血糖终止后，DNA 甲基化和碱基错配之间的相互作用仍在继续，这表明其在与糖尿病性视网膜病变进展相关的代谢记忆现象中发挥作用。抑制 DNA 甲基化限制了用于脱氨的甲基化胞嘧啶的可用性，这表明 DNA 甲基化和碱基错配之间存在相互作用。因此，通过防止碱基错配和线粒体功能障碍的形成，调节 DNA 甲基化或其脱氨作用可能会阻碍糖尿病性视网膜病变的发展。
