Diabetic retinopathy (DR) is clinically defined as a disease of the retinal microvasculature, and most research on its pathogenesis to date has focused on the vasculature itself. Recent advances in multifocal ERG demonstrate that neuro-retinal defects precede and even predict the development of DR. Thus, it is important to investigate the molecular events that contribute to early loss of retinal adaptation to the metabolic environment in diabetes.!Translation of mRNA is a major regulatory step in gene expression that is important for controlling the expression of vascular endothelial growth factor (VEGF), as well as other critical growth factors and cytokines in response to metabolic stress. Our central hypothesis is that a diabetes-induced shift in the selection of mRNAs for translation within Mller glia results in loss of retinal homeostasis and the eventual development of DR. Mller cells, the principal glial cell of the retina, are well recognized for the role they play in the production of homeostatic and trophic factors that support both the vasculature and neuronal layers of the retina. In diabetic patients, glial activation occurs prior to clinical manifestation of DR and likely serves as an adaptive response to mitigate tissue damage. However, prolonged changes in Mller glial protein expression become causative in the development of retinal complications. Specifically, Mller glia are the principal source of increased retinal VEGF expression in diabetes, as conditional Muller cell specific disruption of VEGF prevents elevated growth factor expression and reduces retinal vascular pathology. Our laboratory has shown that diabetes-induced activation of the translational repressor 4E-BP1 promotes retinal VEGF expression and the development of visual dysfunction in a model of type 1 diabetes. The objective here is to address a fundamental gap in our understanding of the molecular events that produce early changes in Mller cell specific protein expression. Using a newly developed RiboTag mouse model, wherein expression of an epitope-tagged ribosomal subunit is directed to Mller glia, the proposed studies will provide an unprecedented assessment of translationally active mRNAs in Mller glia within the intact retina. The proposed studies are designed to characterize defects in the selection of specific mRNAs for translation in two experimental models of diabetes: streptozotocin administration and high fat/high carbohydrate diet. In addition to identifying regulatory mechanisms for specific mRNAs that contribute to glial dysfunction, the proposed studies will also assess the development of retinal defects and visual deficits in the two experimental models following Mller-specific genetic manipulation of the stress response protein REDD1 or protein O- GlcNAcylation (i.e. two novel mechanisms for mediating specific changes in mRNA translation). The rationale is that once the molecular defects in translational control mechanisms in retinal Mller cells are known, the function/assembly of translation initiation factors can be manipulated pharmacologically, resulting in new therapeutics that address dysregulated expression of multiple growth factors and cytokines including VEGF. !
A deficit in our understanding of the early molecular defects that underlie the development of diabetic retinopathy represents a critical barrier to the design and implementation of improved therapeutics. The studies proposed herein will identify translational control mechanisms that produce alterations in the expression of angiogenic, neurotrophic, and homeostatic factors in the retina of experimental models of type 1 diabetes and pre-diabetes. The results from the proposed studies are expected to identify new targets for intervention at the level of gene expression and thus lead to the development of innovative therapies to prevent diabetic retinopathy. !
