Retinopathy continues to develop in diabetic patients long after termination of hyperglycemia, and the benefits of intensive control during early stages of the disease persist beyond its institution, suggesting a `metabolic memory' phenomenon. Re-institution of good glycemic control in diabetic rats fails to reverse increase in retinal oxidativ stress and mitochondria remain swollen with damaged (mtDNA) and transcription, and the electron transport chain (ETC) continues to be dysfunctional. DNA methylation, a robust epigenetic modification facilitated by DNA methyltransferases (Dnmts), plays an important role in regulating gene transcription. Retinal Dnmts are activated in diabetes, and nuclear DNA hypermethylation is implicated in the impaired mtDNA replication. Our preliminary data show that Dnmt1 is increased in the retinal mitochondria and the D-loop region of the mtDNA, the region with essential transcription and replication elements, is hypermethylated. Reversal of hyperglycemia does not prevent increase in Dnmt, and mtDNA remains hypermethylated. Based on these, our overall hypothesis is that `due to increased Dnmt, (a) mtDNA is hypermethylated and its transcription is decreased, and (b) nDNA-encoded genes, important in mitochondria homeostasis, are compromised. Cessation of hyperglycemia does not reverse DNA hypermethylation, and mitochondria continue to be damaged, contributing to the resistance of incipient diabetic retinopathy to arrest'. The hypothesis will be tested methodically by evaluating methylation of both mtDNA and nuclear DNA, and will be addressed in three specific aims.
Aim will investigate the role of mtDNA methylation in the continued damage of mitochondria in the progression of diabetic retinopathy, and will test the hypothesis that `hypermethylation of mtDNA impairs its transcription and ETC becomes dysfunctional; termination of hyperglycemia fails to reverse hypermethylation. Since majority of the proteins required for mitochondrial homeostasis are encoded by nuclear DNA, aim 2 will examine the role of nuclear DNA methylation in the continued mitochondrial damage, and the hypothesis is that `due to increased nuclear Dnmt, mitochondrial genomic stability and structure/ function remain compromised, further fueling into the mitochondrial damage'.
In aim 3, the effect of direct inhibition of Dnmt in the resistance of diabetic retinopathy to halt after reversal of hyperglycemi will be investigated, and the hypothesis predicts that `direct inhibition of Dnmt during normal glycemia, that has followed hyperglycemia, will inhibit continued DNA methylation (mtDNA and nDNA), and the progression of retinopathy'. These studies are based on compelling data generated using valid in vitro and in vivo models. The central hypothesis will be tested in isolated cells using siRNAs and pharmacological inhibitors, and in vitro findings will be validated in in vivo models using retinal microvessels from rats and genetically manipulated mice and also in retinal microvessels from human donors with diabetic retinopathy. Our novel epigenetic approach is anticipated to yield fresh insights into the failure of diabetic retinopathy to arrest after hyperglycemia is terminated, and is expected to demonstrate a critical role of DNA methylation of both mtDNA and nuclear DNA in mitochondrial homeostasis. This should help reveal novel targets for therapies to inhibit DNA methylation and prevent mitochondrial damage, and will offer patients an opportunity to supplement their best possible glycemic control with adjunct therapies to prevent/retard the progression of this sight-threatening complication of diabetes.

Public Health Relevance

Diabetic retinopathy is a slow progressing disease, and it is the most frequent cause of blindness among young adults. Clinical and experimental studies have shown that the progression of this devastating disease does not halt after re-institution of intensive glycemic control in diabetic patients. The exact molecular and cellular mechanisms underlying this metabolic memory phenomenon associated with its continued progression remains elusive. This application is focused on understanding the mechanism responsible for continued damage of retinal mtDNA in the progression of diabetic retinopathy after hyperglycemic insult is terminated. We will test the role of DNA methylation in the continued mitochondrial damage, and the central hypothesis is that due to increased DNA methylation, mtDNA transcription is compromised, impairing the electron transport chain system, and also transcription of the enzymes responsible for mitochondrial homeostasis (damage of mitochondrial structure/function and DNA) is decreased. Cessation of hyperglycemia does not provide any benefit to DNA methylation, and mitochondria remain damaged, contributing to the resistance of incipient diabetic retinopathy to arrest. The application will use biochemical, molecular and epigenetic techniques, and represents the first attempt to examine the potential role of DNA methylation (mtDNA and nuclear DNA-Mlh1 & Mfn2) in the metabolic memory phenomenon. Data from this study are expected to identify novel drug targets for halting the progression of this blinding disease in diabetic patients.

National Institute of Health (NIH)
National Eye Institute (NEI)
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Special Emphasis Panel (DPVS)
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Shen, Grace L
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Wayne State University
Schools of Medicine
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Kowluru, Renu A; Mishra, Manish (2018) Therapeutic targets for altering mitochondrial dysfunction associated with diabetic retinopathy. Expert Opin Ther Targets 22:233-245
Mishra, Manish; Duraisamy, Arul J; Kowluru, Renu A (2018) Sirt1: A Guardian of the Development of Diabetic Retinopathy. Diabetes 67:745-754
Duraisamy, Arul J; Mishra, Manish; Kowluru, Renu A (2017) Crosstalk Between Histone and DNA Methylation in Regulation of Retinal Matrix Metalloproteinase-9 in Diabetes. Invest Ophthalmol Vis Sci 58:6440-6448
Devi, Takhellambam Swornalata; Somayajulu, Mallika; Kowluru, Renu Anjan et al. (2017) TXNIP regulates mitophagy in retinal Müller cells under high-glucose conditions: implications for diabetic retinopathy. Cell Death Dis 8:e2777
Mishra, Manish; Kowluru, Renu A (2017) Role of PARP-1 as a novel transcriptional regulator of MMP-9 in diabetic retinopathy. Biochim Biophys Acta Mol Basis Dis 1863:1761-1769
Kowluru, Renu A; Shan, Yang (2017) Role of oxidative stress in epigenetic modification of MMP-9 promoter in the development of diabetic retinopathy. Graefes Arch Clin Exp Ophthalmol 255:955-962
Kowluru, Renu A; Mishra, Manish (2017) Epigenetic regulation of redox signaling in diabetic retinopathy: Role of Nrf2. Free Radic Biol Med 103:155-164
Mishra, Manish; Lillvis, John; Seyoum, Berhane et al. (2016) Peripheral Blood Mitochondrial DNA Damage as a Potential Noninvasive Biomarker of Diabetic Retinopathy. Invest Ophthalmol Vis Sci 57:4035-44
Kowluru, Renu A; Shan, Yang; Mishra, Manish (2016) Dynamic DNA methylation of matrix metalloproteinase-9 in the development of diabetic retinopathy. Lab Invest 96:1040-9
Mishra, Manish; Flaga, Jadwiga; Kowluru, Renu A (2016) Molecular Mechanism of Transcriptional Regulation of Matrix Metalloproteinase-9 in Diabetic Retinopathy. J Cell Physiol 231:1709-18

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