The Role Of Regulated In Development and DNA Damage Response 1 (REDD1) in Diabetes-Induced Retinal Pathology and Visual Dysfunction
Restricted (Penn State Only)
- Author:
- Miller, William
- Graduate Program:
- Biomedical Sciences
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- December 06, 2021
- Committee Members:
- Alistair Barber, Outside Unit & Field Member
Jeffrey Sundstrom, Major Field Member
Scot Kimball, Major Field Member
David Waning, Major Field Member
Michael Dennis, Chair & Dissertation Advisor
Ralph Keil, Program Head/Chair - Keywords:
- antioxidants
diabetes
oxidative stress
retina
vision
glycogen glycogen synthase kinase 3 (GSK-3)
hyperglycemia
nuclear factor 2 (erythroid-derived 2-like factor) (NFE2L2) (Nrf2)
oxidative stress; post-translational modification (PTM)
reactive oxygen species (ROS)
retinopathy
chaperone mediated autophagy (CMA) - Abstract:
- Diabetic Retinopathy (DR) is a major cause of visual dysfunction, yet much remains unknown regarding the specific molecular events that contribute to diabetes-induced retinal pathophysiology. Herein, we explored a role for the stress response protein regulated in development and DNA damage 1 (REDD1) in the development of diabetes-induced oxidative stress and functional defects in vision. It is well established that REDD1 is associated with the development of oxidative stress in a number of cell types and tissues and that REDD1 protein content and oxidative stress are both increased in the retina of diabetic mice. However, prior to the work detailed in this dissertation, little was known surrounding the mechanism by which the diabetic metabolic environment promotes retinal REDD1 accumulation and if that increase in REDD1 content contributes to the development of oxidative stress and retinal defects. The overarching goal of this dissertation was to test the hypothesis that diabetes-induced REDD1 contributes to the development of retinal pathology by exacerbating oxidative stress. To test the hypothesis, we first assessed the role of REDD1 in mitochondrial reactive oxygen species (ROS) production. In the retina of streptozotocin (STZ)-induced diabetic mice, REDD1 protein expression and ROS levels were increased. In rat retinal R28 cell cultures, hyperglycemic conditions enhanced REDD1 protein expression, ROS levels, and the mitochondrial membrane potential. However, similar effects were not observed in the retina of diabetic mice or R28 cells lacking REDD1. In the retina of diabetic mice and cells exposed to hyperglycemic conditions, the antioxidant N-acetyl-L-cysteine (NAC) normalized ROS levels and prevented an increase in REDD1 expression. Diabetic mice treated with NAC also exhibited improved contrast sensitivity as compared to diabetic controls. Hydrogen peroxide (H2O2) addition to culture medium increased REDD1 expression in R28 cells and attenuated Akt/Glycogen Synthase Kinase 3 (GSK3) phosphorylation in a REDD1-dependent manner. In REDD1-deficient R28 cells exposed to hyperglycemic conditions, expression of a dominant negative Akt or constitutively active GSK3 increased the mitochondrial membrane potential and promoted ROS levels. The findings provided new insight into the mechanism(s) whereby diabetes-induced hyperglycemia causes oxidative stress and visual dysfunction. Specifically, the findings are consistent with a model wherein hyperglycemia-induced REDD1 activated a ROS-generating feedback loop that included Akt/GSK3. The transcription factor Nuclear factor erythroid 2-related factor 2 (Nrf2) plays a critical role in preventing oxidative stress by promoting the expression of antioxidant genes. The retinal antioxidant response to diabetes is insufficient to prevent oxidative stress, due at least in part to blunted activation of Nrf2. In a second series of studies, we tested the hypothesis that diabetes-induced REDD1 promotes oxidative stress in the retina by suppressing Nrf2 activity. We found that REDD1 ablation enhanced Nrf2 DNA-binding activity in the retina and that the suppressive effect of STZ-diabetes on Nrf2 activity was absent in the retina of REDD1-deficient mice compared with wild-type (WT). In human MIO-M1 retinal Müller cell cultures, REDD1 deletion prevented oxidative stress in response to hyperglycemic conditions, and this protective effect required Nrf2. REDD1 suppressed Nrf2 stability by promoting its proteasomal degradation independently of Nrf2's interaction with Kelch-like ECH-associated protein 1 (Keap1), but REDD1-mediated Nrf2 degradation required GSK3 activity and Ser-351/Ser-356 of Nrf2. Diabetes diminished inhibitory phosphorylation of the beta isoform of GSK3 (GSK3β) at Ser-9 in the retina of WT mice but not in REDD1-deficient mice. Pharmacological inhibition of GSK3 enhanced retinal Nrf2 activity and prevented oxidative stress in the retina of diabetic mice. The findings support a model wherein hyperglycemia-induced REDD1 blunts the Nrf2 antioxidant response to diabetes by activating GSK3, which, in turn, phosphorylates Nrf2 to promote its degradation. Finally, we investigated the hypothesis that diabetes promotes REDD1 expression by reducing the rate of REDD1 degradation. In the retina of STZ-diabetic mice, REDD1 protein content was increased in the absence of a change in REDD1 mRNA. In human MIO-M1 retinal Müller cells, the rate of REDD1 degradation was reduced upon exposure to hyperglycemic conditions or the oxidant H2O2. Antioxidant addition to culture medium prevented both the increase in oxidative stress and the reduced rate of REDD1 degradation in cells exposed to hyperglycemic conditions. Surprisingly, the suppressive effect of oxidative stress on REDD1 degradation was independent of the ubiquitin-proteasome pathway. Rather, chemical or genetic activation of chaperone-mediated autophagy (CMA) caused REDD1 localization to lysosomes and degradation of the protein. Moreover, exposure to hyperglycemic conditions prevented the suppressive effect of CMA activation on REDD1 expression. Overall, the findings support a model wherein diabetes promotes retinal REDD1 content by preventing the protein from being degraded by CMA.