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Michael D Dennis, Sadie K Dierschke, William P Miller; O-linked glycosylation of the translational repressor 4E-BP1 promotes mitochondrial dysfunction in retina. Invest. Ophthalmol. Vis. Sci. 2019;60(9):6426.
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Post-translational modification of proteins by O-linked N-Acetylglucosamine (O-GlcNAcylation) is enhanced by diabetes and contributes to the development of diabetic retinopathy (DR). In the retina of diabetic mice, the translational repressor 4E-BP1 is O-GlcNAcylated, concomitant with enhanced sequestration of the mRNA cap-binding protein eIF4E. 4E-BP1 binding to eIF4E regulates VEGF mRNA translation and plays an important role in diabetes-induced visual dysfunction. The purpose of this study was to examine the extent to which retinal gene expression is altered by O-GlcNAcylation.
Wild-type and 4E-BP-deficient mice were administered the O-GlcNAcase inhibitor thiamet G (TMG) to enhance retinal O-GlcNAcylation. Total mRNA abundances and ribosome-bound mRNAs undergoing translation in retina were analyzed by RNAseq and RiboSeq, respectively. Ingenuity Pathway Analysis was used to assess the biological impact of retinal gene expression changes. TMG-induced changes in mitochondrial superoxide in cells in culture and reactive oxygen species (ROS) in retina were evaluated by MitoSOX red and 2,7-dichlorofluoroscein, respectively.
The principal effect of O-GlcNAcylation on retinal gene expression was observed in mRNAs undergoing translation, as only 4 mRNAs exhibited changes in abundance. Whereas 413 mRNAs exhibited TMG-induced increase in translation, 567 mRNAs exhibited reduced translation. Pathway analysis of mRNAs exhibiting altered translation identified molecular networks linked to mitochondrial dysfunction and oxidative stress. Using polysome fractionation, we found that the effect of O-GlcNAcylation on translational of specific mitochondrial proteins in retina was dependent on 4E-BP expression. O-GlcNAcylation promoted mitochondrial superoxide levels in cells in culture and increased ROS in retina, and these effects were prevented by 4E-BP deletion. However, enhanced O-GlcNAcylation did not increase ROS in cells expressing the eIF4E-binding deficient 4E-BP1 Y54A variant. Moreover, expression of the constitutively active 4E-BP1 F113A variant was sufficient to increase ROS.
This study provides new evidence that O-GlcNAcylation alters mitochondrial function in retina and identifies a molecular network of translationally regulated mRNAs that potentially underlie dysfunctional mitochondrial respiration and superoxide production in DR.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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