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

Abstract

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; yet, 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 eukaryotic translation in initiation factor 4E (eIF4E) by eIF4E-binding protein 1 (4E-BP1) 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 wild-type mice, diabetic 4E-BP1/2-deficient mice did not exhibit enhanced retinal Cd40 mRNA translation and failed to upregulate 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.

Feedback Regulation of O-GlcNAc Transferase through Translation Control to Maintain Intracellular O-GlcNAc Homeostasis

“…Therefore, it is expected that the levels of a plethora of proteins would be affected by O -GlcNAcylation in a 4E-BP1-dependent manner. Examples of such proteins include superoxide dismutase 2 (SOD2) and Cd40 [ 44 , 45 ]. In mouse retina, TMG treatment inhibits the translation of SOD2 in a 4E-BP-dependent manner; the functional significance of this regulation is evidenced by the increased oxidative stress in the retina of diabetic mice which may be attributed to altered translation of SOD [ 45 ].…”

“…In mouse retina, TMG treatment inhibits the translation of SOD2 in a 4E-BP-dependent manner; the functional significance of this regulation is evidenced by the increased oxidative stress in the retina of diabetic mice which may be attributed to altered translation of SOD [ 45 ]. Conversely, because Cd40 mRNA undergoes cap-independent translation, TMG treatment causes an increase in association of Cd40 mRNA with ribosomes and enhancement of Cd40 translation in Muller glia cells in mouse retina [ 44 ]. Our study establishes OGT as another example regulated by O -GlcNAcylation in a 4E-BP1-dependent way.…”

Regulations of Retinal Inflammation: Focusing on Müller Glia

“…Intravitreal administration of a cell-penetrating CD40-TRAF2,3 blocking peptide impaired CXCL1 upregulation in endothelial and MG, diminished inflammation and neuronal loss after ischemia/reperfusion, as mentioned above ( Portillo et al, 2021 ). CD40 expression in MG can be increased by diabetes ( Dierschke et al, 2020 ), which amplifies inflammation and induces death of retinal endothelial cells, an event key to the development of capillary degeneration and retinal ischemia ( Subauste, 2019 ).…”

Müller Glial Expression of REDD1 Is Required for Retinal Neurodegeneration and Visual Dysfunction in Diabetic Mice

“…In REDD1-mgKO mice, Cre expression is directed to retinal Müller glia by the Pdgfra (platelet-derived growth factor receptor, α polypeptide) promoter ( 21 ). Pdgfra-Cre; HA-Rpl22 mice were generated as previously described ( 22 ). REDD1 fl/fl and REDD1-mgKO littermates were administered streptozotocin (STZ) or sodium citrate buffer for 5 consecutive days to induce diabetes.…”