Retinal Protein O-GlcNAcylation and the Ocular Renin-angiotensin System: Signaling Cross-roads in Diabetic Retinopathy.

It is well established that diabetes and its associated hyperglycemia negatively impact retinal function, yet we know little about the role played by augmented flux through the Hexosamine Biosynthetic Pathway (HBP). This offshoot of the glycolytic pathway produces UDP-Nacetyl- glucosamine, which serves as the substrate for post-translational O-linked modification of proteins in a process referred to as O-GlcNAcylation. HBP flux and subsequent protein O-GlcNAcylation serve as nutrient sensors, enabling cells to integrate metabolic information to appropriately modulate fundamental cellular processes including gene expression. Here we summarize the impact of diabetes on retinal physiology, highlighting recent studies that explore the role of O-GlcNAcylation- induced variation in mRNA translation in retinal dysfunction and the pathogenesis of Diabetic Retinopathy (DR). Augmented O-GlcNAcylation results in wide variation in the selection of mRNAs for translation, in part, due to O-GlcNAcylation of the translational repressor 4E-BP1. Recent studies demonstrate that 4E-BP1 plays a critical role in regulating O-GlcNAcylation-induced changes in the translation of the mRNAs encoding Vascular Endothelial Growth Factor (VEGF), a number of important mitochondrial proteins, and CD40, a key costimulatory molecule involved in diabetes-induced retinal inflammation. Remarkably, 4E-BP1/2 ablation delays the onset of diabetes- induced visual dysfunction in mice. Thus, pharmacological interventions to prevent the impact of O-GlcNAcylation on 4E-BP1 may represent promising therapeutics to address the development and progression of DR. In this regard, we discuss the potential interplay between retinal O-GlcNAcylation and the ocular renin-angiotensin system as a potential therapeutic target of future interventions.

Diabetes enhances translation of Cd40 mRNA in murine retinal Müller glia via a 4E-BP1/2-dependent mechanism.

Activation of the immune costimulatory molecule cluster of differentiation 40 (CD40) in Müller glia has been implicated in the initiation of diabetes-induced retinal inflammation. Results from previous studies support that CD40 protein expression is elevated in Müller glia of diabetic mice; however, the mechanisms responsible for this increase have not been explored. Here, we evaluated the hypothesis that diabetes augments translation of the Cd40 mRNA. Mice receiving thiamet G (TMG), an inhibitor of the O-GlcNAc hydrolase O-GlcNAcase, exhibited enhanced retinal protein O-GlcNAcylation and increased Cd40 mRNA translation. TMG administration also promoted Cd40 mRNA association with Müller cell-specific ribosomes isolated from the retina of RiboTag mice. Similar effects on O-GlcNAcylation and Cd40 mRNA translation were also observed in the retina of a mouse model of type 1 diabetes. In cultured cells, TMG promoted sequestration of the cap-binding protein eIF4E (eukaryotic translation in initiation factor 4E) by 4E-BP1 (eIF4E-binding protein 1) and enhanced cap-independent Cd40 mRNA translation as assessed by a bicistronic reporter that contained the 5'-UTR of the Cd40 mRNA. Ablation of 4E-BP1/2 prevented the increase in Cd40 mRNA translation in TMG-exposed cells, and expression of a 4E-BP1 variant that constitutively sequesters eIF4E promoted reporter activity. Extending on the cell culture results, we found that in contrast to WT mice, diabetic 4E-BP1/2-deficient mice did not exhibit enhanced retinal Cd40 mRNA translation and failed to up-regulate expression of the inflammatory marker nitric-oxide synthase 2. These findings support a model wherein diabetes-induced O-GlcNAcylation of 4E-BP1 promotes Cd40 mRNA translation in Müller glia.

Angiotensin-(1-7) Attenuates Protein O-GlcNAcylation in the Retina by EPAC/Rap1-Dependent Inhibition of O-GlcNAc Transferase.

Purpose: O-GlcNAcylation of cellular proteins contributes to the pathophysiology of diabetes and evidence supports a role for augmented O-GlcNAcylation in diabetic retinopathy. The aim of this study was to investigate the impact of the renin-angiotensin system on retinal protein O-GlcNAcylation. Methods: Mice fed a high-fat diet were treated chronically with the angiotensin-converting enzyme inhibitor captopril or captopril plus the angiotensin-(1-7) Mas receptor antagonist A779. Western blotting and quantitative polymerase chain reaction were used to analyze retinal homogenates. Similar analyses were performed on lysates from human MIO-M1 retinal Müller cell cultures exposed to media supplemented with angiotensin-(1-7). Culture conditions were manipulated to influence the hexosamine biosynthetic pathway and/or signaling downstream of the Mas receptor. Results: In the retina of mice fed a high-fat diet, captopril attenuated protein O-GlcNAcylation in a manner dependent on Mas receptor activation. In MIO-M1 cells, angiotensin-(1-7) or adenylate cyclase activation were sufficient to enhance cyclic AMP (cAMP) levels and inhibit O-GlcNAcylation. The repressive effect of cAMP on O-GlcNAcylation was dependent on exchange protein activated by cAMP (EPAC), but not protein kinase A, and was recapitulated by a constitutively active variant of the small GTPase Rap1. We provide evidence that cAMP and angiotensin-(1-7) act to suppress O-GlcNAcylation by inhibition of O-GlcNAc transferase (OGT) activity. In cells exposed to an O-GlcNAcase inhibitor or hyperglycemic culture conditions, mitochondrial superoxide levels were elevated; however, angiotensin-(1-7) signaling prevented the effect. Conclusions: Angiotensin-(1-7) inhibits retinal protein O-GlcNAcylation via an EPAC/Rap1/OGT signaling axis.

O-GlcNAcylation alters the selection of mRNAs for translation and promotes 4E-BP1-dependent mitochondrial dysfunction in the retina.

Diabetes promotes the posttranslational modification of proteins by O-linked addition of GlcNAc (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 less than 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 the retina, the effect of O-GlcNAcase inhibition on translation of specific mitochondrial proteins, including superoxide dismutase 2 (SOD2), depended on 4E-BP1/2. O-GlcNAcylation enhanced cellular respiration and promoted mitochondrial superoxide levels in WT cells, and 4E-BP1/2 deletion prevented O-GlcNAcylation-induced mitochondrial superoxide in cells in culture and in the retina. The retina of diabetic WT 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.

Consumption of a high fat diet promotes protein O-GlcNAcylation in mouse retina via NR4A1-dependent GFAT2 expression.

The incidence of type 2 diabetes, the most common cause of diabetic retinopathy (DR), is rapidly on the rise in developed countries due to overconsumption of calorie rich diets. Using an animal model of diet-induced obesity/pre-diabetes, we evaluated the impact of a diet high in saturated fat (HFD) on O-GlcNAcylation of retinal proteins, as dysregulated O-GlcNAcylation contributes to diabetic complications and evidence supports a role in DR. Protein O-GlcNAcylation was increased in the retina of mice fed a HFD as compared to littermates receiving control chow. Similarly, O-GlcNAcylation was elevated in retinal Müller cells in culture exposed to the saturated fatty acid palmitate or the ceramide analog Cer6. One potential mechanism responsible for elevated O-GlcNAcylation is increased flux through the hexosamine biosynthetic pathway (HBP). Indeed, inhibition of the pathway's rate-limiting enzyme glutamine-fructose-6-phosphate amidotransferase (GFAT) prevented Cer6-induced O-GlcNAcylation. Importantly, expression of the mRNA encoding GFAT2, but not GFAT1 was elevated in both the retina of mice fed a HFD and in retinal cells in culture exposed to palmitate or Cer6. Notably, expression of nuclear receptor subfamily 4 group A member 1 (NR4A1) was increased in the retina of mice fed a HFD and NR4A1 expression was sufficient to promote GFAT2 mRNA expression and O-GlcNAcylation in retinal cells in culture. Whereas palmitate or Cer6 addition to culture medium enhanced NR4A1 and GFAT2 expression, chemical inhibition of NR4A1 transactivation repressed Cer6-induced GFAT2 mRNA expression. Overall, the results support a model wherein HFD increases retinal protein O-GlcNAcylation by promoting NR4A1-dependent GFAT2 expression.

Deletion of the stress-response protein REDD1 promotes ceramide-induced retinal cell death and JNK activation.

The role of dyslipidemia in the development of retinal dysfunction remains poorly understood. Using an animal model of diet-induced obesity/pre-type 2 diabetes, we investigated molecular defects in the retina arising from consumption of a diet high in saturated fats and sugars ( i.e., a Western diet). We found that feeding mice a Western diet increased the abundance of retinal sphingolipids, attenuated protein kinase B (Akt) phosphorylation, enhanced JNK activation, and increased retinal cell death. When we used palmitate or C6-ceramide (Cer) to assess sphingolipid-mediated signaling in cultured murine and human cells, we observed similar effects on Akt, JNK, and cell death. Furthermore, both Western diet and C6-Cer exposure enhanced expression of the stress-response protein regulated in development and DNA damage response 1 (REDD1) and loss of REDD1 increased C6-Cer-induced JNK activation and cell death. Exogenous REDD1 expression repressed JNK-mediated phosphorylation in cultured cells. We found that thioredoxin-interacting protein (TXNIP) expression was elevated in REDD1-deficient cell lines and C6-Cer promoted TXNIP expression in both wild-type and REDD1-deficient cells. Likewise, TXNIP knockdown attenuated JNK activation and caspase 3 cleavage after either C6-Cer exposure or REDD1 deletion. The results support a model wherein Cer-induced REDD1 expression attenuates TXNIP-dependent JNK activation and retinal cell death.-Dai, W., Miller, W. P., Toro, A. L., Black, A. J., Dierschke, S. K., Feehan, R. P., Kimball, S. R., Dennis, M. D. Deletion of the stress-response protein REDD1 promotes ceramide-induced retinal cell death and JNK activation.