Targeting the Etiology of Diabetic Retinopathy

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PROJECT SUMMARY Diabetic retinopathy is the leading cause of blindness in working age Americans, affecting more than a third of the ~20 million individuals with diabetes. The project proposed for this research supplement to enhance diversity is designed to complement the aims of the parent R01 by examining the impact of diabetes on degradation of the stress response protein REDD1 (regulated in development and DNA damage 1) in the retina. Our laboratory has demonstrated that REDD1 expression is increased in the retina of diabetic mice, and is necessary for diabetes-induced retinal pathology and functional deficits in vision. Moreover, siRNA-mediated REDD1 knockdown improves best-corrected visual acuity in patients with diabetic macular edema. Thus, identification of the molecular events responsible for diabetes-induced REDD1 expression in the retina is of high clinical significance. In studies conducted as part of the Parental Grant, we recently found evidence that a post-transcriptional mechanism was potentially responsible for the increase in REDD1 protein in the retina of diabetic mice. Specifically, in the retina of streptozotocin-induced diabetic mice, REDD1 protein expression was increased in the absence of a change in REDD1 mRNA abundance. The central hypothesis of this supplement is that diabetes-induced oxidative stress promotes REDD1 interaction with TXNIP (thioredoxin interacting protein) in a manner that prevents the ubiquitination and subsequent proteasomal degradation of REDD1. To test the hypothesis, the applicant will pursue an experimental protocol involving model systems ranging from intact mice to retinal cell culture. Aim 1 will use cutting-edge molecular techniques (e.g. SNAP- tagging) to evaluate the impact of diabetes on retinal REDD1 protein degradation. Aim 2 will examine the impact of TXNIP on retinal REDD1 protein turnover in the context of diabetes. The specific mechanism(s) responsible for the increase in REDD1 that is associated with the diabetes-induced decline in visual function has not been previously established, and thus represents a significant gap in our understanding of the molecular events that cause visual dysfunction in diabetic retinopathy. Overall, the studies supported by this supplement will greatly enhance the parent R01 by exploring why hyperglycemic conditions promote retinal REDD1 protein content. The proposed project represents a focused thesis project that provides two key training opportunities for a promising pre-doctoral student. First, the applicant will develop the technical expertise to assess the impact of diabetes on retinal pathophysiology using optical coherence tomography, electroretinograms, and virtual optomotry. The applicant will also develop skills necessary to manipulate retinal gene expression in vivo using AAV.