Impact of protein O-GlcNAcylation in retina: mechanisms regulating mRNA translation and Identification of novel therapeutic targets for diabetic retinopathy
Open Access
- Author:
- Dierschke, Sadie Katherine
- Graduate Program:
- Biomedical Sciences
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- March 03, 2020
- Committee Members:
- Michael Don Dennis, Dissertation Advisor/Co-Advisor
Michael Don Dennis, Committee Chair/Co-Chair
Alistair J Barber, Committee Member
Scot R Kimball, Committee Member
Amy Christine Arnold, Outside Member
David James Degraff, Committee Chair/Co-Chair
Ralph Lauren Keil, Program Head/Chair - Keywords:
- retina
diabetes
O-linked N-acetylglucosamine
eukaryotic translation initiation
Angiotenins-(1-7)
oxidative stress - Abstract:
- Diabetes promotes the post-translational modification of proteins by O-linked addition of N-acetylglucosamine (O-GlcNAcylation) to serine and threonine residues of proteins and thereby contributes to diabetic complications, including diabetic retinopathy (DR) which is the leading cause of blindness in working age adults. 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) by 4E-BP1 is enhanced. However, the functional consequence of this enhanced interaction in retina is unknown. The overarching goals of this dissertation were to determine if O-GlcNAc-mediated sequestration of eIF4E by 4E-BP1 affects gene expression in the retina and to identify the mechanism by which retinal protein O-GlcNAcylation is attenuated. To achieve our goal, we assessed retinal gene expression in mice administered thiamet G (TMG), an inhibitor of O-GlcNAcase (OGA), via ribosome profiling. The principal effect of TMG on retinal gene expression was observed in mRNAs undergoing translation, as <1% of mRNAs were altered in total abundance. In retina, the effect of OGA inhibition on translation of specific mitochondrial proteins was dependent on 4E-BP1/2. The retina of diabetic wild-type mice exhibited increased reactive oxygen species levels, an effect not observed in diabetic 4E-BP1/2-deficient mice. From our ribosome profiling dataset, we identified CD40 as a gene candidate in rodent retina subject to enhanced translational efficiency upon TMG administration. Hyperglycemia increases CD40 expression in Müller glia, which drives expression of other inflammatory molecules. The mechanisms responsible for this increase are unknown. To test the hypothesis that diabetes promotes translation of the mRNA encoding CD40 in Müller glia, we assessed CD40 mRNA translation in whole retina, Müller cells in situ, and Müller cells in vitro. We found that O-GlcNAcase inhibition increases CD40 mRNA translation, and a similar effect was observed in retina from diabetic mice. However, 4E-BP1/2 deletion prevented diabetes-induced translation of the CD40 mRNA. In cells in culture, 4E-BP1/2 deletion prevented O-GlcNAcylation-induced CD40 mRNA translation, and expression of a 4E-BP1 variant that constitutively binds eIF4E promoted cap-independent CD40 mRNA translation as assessed by a bicistronic reporter. Collectively, these findings provide evidence for a mechanism whereby diabetes-induced O-GlcNAcylation promotes oxidative stress and contributes to inflammation in the retina by altering the selection of mRNAs for translation. Given the impact of enhanced retinal protein O-GlcNAcylation on oxidative stress and induction of inflammation, we wanted to understand how it may be attenuated. Pharmacological blockade of the renin angiotensin system (RAS) mediates beneficial outcomes in the retina of diabetic patients, likely through increased systemic production of the RAS effector peptide angiotensin-(1-7) (Ang1-7). Thus, we hypothesized Ang1-7 was capable of modulating protein O-GlcNAcylation. To test this hypothesis, mice fed a high fat diet were treated with the ACE inhibitor captopril or captopril plus an Ang1-7 Mas receptor antagonist. In the retina of mice fed a high fat diet, captopril attenuated protein O-GlcNAcylation in a manner dependent on Mas receptor activation. In Müller cells in culture, Ang1-7 or adenylate cyclase activation were sufficient to enhance 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. We further provide evidence that Ang1-7 acts to suppress O-GlcNAcylation by inhibition of O-GlcNAc Transferase (OGT) activity. This dissertation thus provides evidence to support a model wherein stimulating cAMP production in Müller cells to attenuate retinal protein O-GlcNAcylation and its downstream negative consequences may represent a novel therapeutic strategy to preserve vision in diabetic individuals.