Diabetic retinopathy and damage to mitochondrial structure and transport machinery.

Diabetic retinopathy remains the leading cause of blindness in young adults. Research to understand how the disease develops has explored several hyperglycemia-initiated pathways, but the molecular mechanism remains elusive. In diabetes, retinal mitochondria become dysfunctional, their DNA (mtDNA) is damaged, and capillary cell apoptosis is increased, and these abnormalities are observed before histopathology can be detected, suggesting a major role of mitochondria in the development of diabetic retinopathy.1–3 Mitochondria are highly dynamic organelles; depending on the energy demand, they can branch, retract, and increase the number of cristae; change their shape and fuse; or increase in size.4,5 Their function is regulated by cycle of fusion and fission, and alterations in membrane structure and morphology are regarded as significant components of mitochondrial dysfunction.6 Fission of mitochondria is generally related with apoptosis and fusion with inhibition of apoptosis.7,8 These fission (e.g., dynamin 1-like protein, Dnm1l/Drp1) and fusion (e.g., mitofusin 2, Mfn2; Mfn1) proteins are GTPases and are energy dependent.6 How diabetes affects mitochondrial fusion and fission proteins in the retina is not clear. Mitochondria are equipped with approximately 1500 proteins, but only 13 proteins are encoded by mtDNA. Thus, most proteins, encoded by nuclear DNA, have a mitochondrial leading sequence in their preproteins.9 These proteins are imported into the mitochondria via the translocase of the outer and inner membrane (TOM and TIM, respectively) complexes. The TOM complex has a central component, Tom40, with three preprotein receptors: Tom20, Tom22, and Tom70.9 Tom34 is on the outer membrane and is mostly a cytosolic chaperon-binding mature preprotein.10,11 In the matrix side of the inner membrane, Tim44 serves as a docking protein for heat shock protein 70 (Hsp70).12 Our recent work has shown that, in diabetes, the enzymes involved in repairing damaged retinal mtDNA, do not reach the mitochondria, despite the increase in their mRNA levels,3 suggesting impaired mitochondrial membrane structure and transport machinery. Good glycemic control, if instituted in the early stage of diabetes, prevents the development of retinopathy, but if reinstituted after a period of poor glycemic control, fails to halt its progression, suggesting a “metabolic memory” phenomenon. Our studies have demonstrated that the reversal of hyperglycemia fails to prevent mtDNA damage and the repair machinery remains subnormal.3 The purpose of this study was to investigate whether the retinal mitochondrial dysfunction observed in diabetes correlates with the ultrastructure of the mitochondria and the genes associated with mitochondrial membrane structure, including the fusion–fission and transport proteins. Further, we investigated whether these changes are reversed when the hyperglycemic insult is terminated. The effect of diabetes on the ultrastructure of the mitochondria was evaluated in the microvascular region of the retina by transmission electron microscope (TEM). Retinas from rats maintained in various glycemic controls were analyzed with a rat mitochondrial PCR array for mitochondrial biogenesis and structure–function. The PCR array results were validated by quantifying the mRNA and protein levels of the key genes by real-time PCR and Western blot analysis, respectively. The key proteins involved in maintaining mitochondrial structure and transport machinery were also confirmed in the retina from donors with diabetic retinopathy.