Redox homeostasis is a delicate balancing act of maintaining appropriate levels of antioxidant defense mechanisms and reactive oxidizing oxygen and nitrogen species. Any disruption of this balance leads to oxidative stress, which is a key pathogenic factor in several ocular diseases. In this review, we present the current evidence for oxidative stress and mitochondrial dysfunction in conditions affecting both the anterior segment (e.g., dry eye disease, keratoconus, cataract) and posterior segment (age-related macular degeneration, proliferative vitreoretinopathy, diabetic retinopathy, glaucoma) of the human eye. We posit that further development of therapeutic interventions to promote pro-regenerative responses and maintenance of the redox balance may delay or prevent the progression of these major ocular pathologies. Continued efforts in this field will not only yield a better understanding of the molecular mechanisms underlying the pathogenesis of ocular diseases but also enable the identification of novel druggable redox targets and antioxidant therapies.

The transcription factor nuclear factor erythroid-2-related factor 2 (Nrf2) plays a critical role in reducing oxidative stress by promoting the expression of antioxidant genes. Both individuals with diabetes and preclinical diabetes models exhibit evidence of a defect in retinal Nrf2 activation. We recently demonstrated that increased expression of the stress response protein regulated in development and DNA damage 1 (REDD1) is necessary for the development of oxidative stress in the retina of streptozotocin-induced diabetic mice. In the present study, we tested the hypothesis that REDD1 suppresses the retinal antioxidant response to diabetes by repressing Nrf2 function. We found that REDD1 ablation enhances Nrf2 DNA-binding activity in the retina and that the suppressive effect of diabetes on Nrf2 activity is absent in the retina of REDD1-deficient mice compared with wild-type. In human MIO-M1 Müller cell cultures, REDD1 deletion prevented oxidative stress in response to hyperglycemic conditions, and this protective effect required Nrf2. REDD1 suppressed Nrf2 stability by promoting its proteasomal degradation independently of Nrf2’s interaction withKelch-like ECH-associated protein 1 (Keap1), but REDD1-mediated Nrf2 degradation required glycogen synthase kinase 3 (GSK3) activity and Ser-351/Ser-356 of Nrf2. Diabetes diminished inhibitory phosphorylation of GSK3β at Ser-9 in the retina of wild-type mice but not in REDD1-deficient mice. Pharmacological inhibition of GSK3 enhanced Nrf2 activity and prevented oxidative stress in the retina of diabetic mice. The findings support a model wherein hyperglycemia-induced REDD1 blunts the Nrf2 antioxidant response to diabetes by activating GSK3, which, in turn, phosphorylates Nrf2 to promote its degradation.

Diabetes-induced visual dysfunction is associated with significant neuroretinal cell death. The current study was designed to investigate the role of the Protein Regulated in Development and DNA Damage Response 1 (REDD1) in diabetes-induced retinal cell death and visual dysfunction. We recently demonstrated that REDD1 protein expression was elevated in response to hyperglycemia in the retina of diabetic rodents. REDD1 is an important regulator of Akt and mammalian target of rapamycin and as such plays a key role in neuronal function and survival. In R28 retinal cells in culture, hyperglycemic conditions enhanced REDD1 protein expression concomitant with caspase activation and cell death. By contrast, in REDD1-deficient R28 cells, neither hyperglycemic conditions nor the absence of insulin in culture medium were sufficient to promote cell death. In the retinas of streptozotocin-induced diabetic mice, retinal apoptosis was dramatically elevated compared with nondiabetic controls, whereas no difference was observed in diabetic and nondiabetic REDD1-deficient mice. Electroretinogram abnormalities observed in b-wave and oscillatory potentials of diabetic wild-type mice were also absent in REDD1-deficient mice. Moreover, diabetic wild-type mice exhibited functional deficiencies in visual acuity and contrast sensitivity, whereas diabetic REDD1-deficient mice had no visual dysfunction. The results support a role for REDD1 in diabetes-induced retinal neurodegeneration.

Purpose: The present study was designed to evaluate the role of the stress response protein REDD1 in diabetes-induced oxidative stress and retinal pathology. Methods: Wild-type and REDD1-deficient mice were administered streptozotocin to induce diabetes. Some mice received the antioxidant N-acetyl-l-cysteine (NAC). Visual function was assessed by virtual optometry. Retinas were analyzed by Western blotting. Reactive oxygen species (ROS) were assessed by 2,7-dichlorofluoroscein. Similar analyses were performed on R28 retinal cells in culture exposed to hyperglycemic conditions, NAC, and/or the exogenous ROS source hydrogen peroxide. Results: In the retina of diabetic mice, REDD1 expression and ROS were increased. In cells in culture, hyperglycemic conditions enhanced REDD1 expression, ROS levels, and the mitochondrial membrane potential. However, similar effects were not observed in the retina of diabetic mice or cells lacking REDD1. In the retina of diabetic mice and cells exposed to hyperglycemic conditions, NAC normalized ROS and prevented an increase in REDD1 expression. Diabetic mice receiving NAC also exhibited improved contrast sensitivity as compared to diabetic controls. Hydrogen peroxide addition to culture medium increased REDD1 expression and attenuated Akt/GSK3 phosphorylation in a REDD1-dependent manner. In REDD1-deficient cells exposed to hyperglycemic conditions, expression of a dominant negative Akt or constitutively active GSK3 increased the mitochondrial membrane potential and promoted ROS. Conclusions: The findings provide new insight into the mechanism whereby diabetes-induced hyperglycemia causes oxidative stress and visual dysfunction. Specifically, hyperglycemia-induced REDD1 activates a ROS-generating feedback loop that includes Akt/GSK3. Thus, therapeutic approaches targeting REDD1 expression and ROS may be beneficial for preventing diabetes-induced visual dysfunction.

Diabetes promotes the post-translational modification of proteins by O-linked addition of N-acetylglucosamine (O-GlcNAcylation) to Ser/Thr residues of proteins and thereby contributes to diabetic complications. In the retina of diabetic mice, the repressor of mRNA translation eIF4E-binding protein 1 (4E-BP1) is O-GlcNAcylated and sequestration of the cap-binding protein eukaryotic translation initiation factor (eIF4E) is enhanced. O-GlcNAcylation has also been detected on several eukaryotic translation initiation factors and ribosomal proteins. However, the functional consequence of this modification is unknown. Here, using ribosome profiling, we evaluated the effect of enhanced O-GlcNAcylation on retinal gene expression. Mice receiving thiamet G (TMG), an inhibitor of the O-GlcNAc hydrolase O-GlcNAcase, exhibited enhanced retinal protein O-GlcNAcylation. The principal effect of TMG on retinal gene expression was observed in ribosome-associated mRNAs (i.e., mRNAs undergoing translation), as <1% of mRNAs exhibited changes in abundance. Remarkably, ~19% of the transcriptome exhibited TMG-induced changes in ribosome occupancy, with 1912 mRNAs having reduced and 1683 mRNAs having increased translational rates. In retina, the effect of O-GlcNAcase inhibition on translation of specific mitochondrial proteins, including superoxide dismutase 2 (SOD2), was dependent on 4E-BP1/2. O-GlcNAcylation enhanced cellular respiration and promoted mitochondrial superoxide levels in wild-type cells, and 4E-BP1/2 deletion prevented O-GlcNAcylation-induced mitochondrial superoxide in both cells in culture and retina. The retina of diabetic wild-type mice exhibited increased reactive oxygen species levels, an effect not observed in diabetic 4E-BP1/2-deficient mice. These findings provide evidence for a mechanism whereby diabetes-induced O-GlcNAcylation promotes oxidative stress in the retina by altering the selection of mRNAs for translation.