Deletion of the Akt/mTORC1 Repressor REDD1 Prevents Visual Dysfunction in a Rodent Model of Type 1 Diabetes

Abstract

Diabetes-induced visual dysfunction is associated with significant neuroretinal cell death. The current study was designed to investigate the role of the Protein Regulated in Development and DNA Damage Response 1 (REDD1) in diabetes-induced retinal cell death and visual dysfunction. We recently demonstrated that REDD1 protein expression was elevated in response to hyperglycemia in the retina of diabetic rodents. REDD1 is an important regulator of Akt and mammalian target of rapamycin and as such plays a key role in neuronal function and survival. In R28 retinal cells in culture, hyperglycemic conditions enhanced REDD1 protein expression concomitant with caspase activation and cell death. By contrast, in REDD1-deficient R28 cells, neither hyperglycemic conditions nor the absence of insulin in culture medium were sufficient to promote cell death. In the retinas of streptozotocin-induced diabetic mice, retinal apoptosis was dramatically elevated compared with nondiabetic controls, whereas no difference was observed in diabetic and nondiabetic REDD1-deficient mice. Electroretinogram abnormalities observed in b-wave and oscillatory potentials of diabetic wild-type mice were also absent in REDD1-deficient mice. Moreover, diabetic wild-type mice exhibited functional deficiencies in visual acuity and contrast sensitivity, whereas diabetic REDD1-deficient mice had no visual dysfunction. The results support a role for REDD1 in diabetes-induced retinal neurodegeneration.

Figure 1

Deletion of REDD1 protects against hyperglycemia-induced retinal cell death. R28 cells were maintained in DMEM containing 5 mmol/L glucose and supplemented with 10% FBS. A and B: Cells were serum deprived for 24 h in medium containing 5 or 30 mmol/L glucose plus the presence or absence of insulin (+Ins). C: Wild-type and REDD1 CRISPR R28 cells were serum deprived for 24 h in medium containing 5 or 30 mmol/L glucose plus the presence or absence of insulin. D–H: Wild-type and REDD1 CRISPR R28 cells were exposed to medium containing 10% FBS and 5 or 30 mmol/L glucose for 0–24 h. As a positive control for the induction of REDD1 expression, cells were treated with 2 μg/mL tunicamycin (TM) for 4 h. Relative cell death was assessed by ELISA for cytoplasmic nucleosomes. Expression of REDD1, Akt, FOXO1, GAPDH, caspase-3 cleavage, and phosphorylation of Akt and FOXO1 were assessed by Western blotting. Protein molecular mass in kDa is indicated at the left of the blots. Gel loading was assessed by protein stain. Akt activity in cell lysates was assessed by ELISA using a synthetic peptide substrate. Quantification of Western blots is presented in Supplementary Fig. 2. Values are means ± SE for two independent experiments (n = 9–10). Statistical significance is denoted by the presence of different letters above each scatter plot on the graphs. Scatter plots with different letters are statistically different, P < 0.05.

Figure 2

Constitutively nuclear FOXO1 prevents the protective effect of REDD1 deletion. Wild-type and REDD1 CRISPR R28 cells were maintained in DMEM containing 5 mmol/L glucose and supplemented with 10% FBS. Cells were transfected with empty vector (EV) or FOXO1n. Cells were exposed to medium containing 10% FBS and 5 or 30 mmol/L glucose for 24 h. Expression of cleaved caspase-3, phosphorylation of Akt, FLAG-tagged FOXO1n, and GAPDH were assessed by Western blotting. Protein molecular mass in kDa is indicated at the left of the blots. Gel loading was assessed by protein stain. Values are means ± SE for two independent experiments (n = 4). Statistical significance is denoted by the presence of different letters above each scatter plot on the graphs. Scatter plots with different letters are statistically different, P < 0.05.

Figure 3

REDD1 ablation prevents diabetes-induced retinal cell death. Retinas were isolated from diabetic (D) and nondiabetic (ND) wild-type and REDD1-knockout (KO) mice 4 weeks after STZ administration. A: Expression of REDD1 in retinal lysates was assessed by Western blotting. Gel loading was assessed by protein stain. B: Akt activity in retinal lysates was assessed by ELISA by using a synthetic peptide substrate. C: FOXO1 phosphorylation was assessed by Western blotting. D: Retinal lysates were assayed by ELISA for the presence of nucleosomal fragments in the cytoplasm. Values are means ± SE for two independent experiments (n = 5–7). *P < 0.05 vs. ND and #P < 0.05 vs. wild-type. Protein molecular mass in kDa is indicated at the left of blots. nq, no quantification of REDD1 expression was performed on retinal lysates from REDD1-deficient mice.

Figure 4

REDD1 ablation reduces the attenuation of b-wave amplitudes in response to diabetes. At 4 weeks after STZ administration, scotopic ERG responses were recorded from the eyes of diabetic and nondiabetic wild-type and REDD1-knockout (KO) mice at increasing stimulus intensities. A: Representative ERG response elicited from −1.0 cd-s/m² log flash intensity. ERG a-wave amplitudes are plotted against the stimulus flash intensity for nondiabetic and diabetic wild-type (B) and REDD1-deficient mice (C). The corresponding implicit times are shown for a-waves of nondiabetic and diabetic wild-type (D) and REDD1-deficient mice (E). ERG b-wave amplitudes are plotted against the flash intensity of stimulus luminance for nondiabetic and diabetic wild-type (F) and REDD1-deficient mice (G). H: Mean difference in b-wave amplitudes of nondiabetic and diabetic mice across flash intensities. The corresponding implicit times are shown for b-waves of nondiabetic and diabetic wild-type (I) and REDD1-deficient mice (J). Values are means ± SE for two independent experiments (n = 8). *P < 0.05 vs. wild-type. Graphs in C, E, G, and J share the y-axis with B, D, F, and I, respectively.

Figure 5

REDD1 ablation prevents attenuated OP amplitudes in response to diabetes. At 4 weeks after STZ administration, scotopic ERG responses were recorded from diabetic and nondiabetic wild-type and REDD1-knockout (KO) mice after overnight dark adaptation. A high-pass filter was used to extract OPs from raw ERG recordings. A: Representative OPs elicited from −1.0 cd-s/m² log flash intensity. Summed OP amplitudes are plotted against the stimulus flash intensity for nondiabetic and diabetic wild-type (B) and REDD1-KO mice (C). D: Comparison of the mean difference in summed OP amplitudes for wild-type and REDD1-KO mice. The corresponding implicit times of nondiabetic and diabetic wild-type (E) and REDD1-deficient mice (F) are shown. G: Comparison of the mean difference in summed OP implicit times for wild-type and REDD1-KO mice between nondiabetic and diabetic mice. Values are means ± SE for two independent experiments (n = 8). *P < 0.05 vs. wild-type. Graphs in C and F share the y-axis with B and E, respectively.

Figure 6

Diabetes-induced visual dysfunction is absent in REDD1-deficient mice. Visual function was assessed by virtual optomotor testing after 4 (A and B) and 8 weeks (C and D) of diabetes. SF (A and C) and CS (B and D) thresholds and were obtained in nondiabetic (ND) and diabetic (D) wild-type and REDD1-knockout (KO) mice. The CS threshold is expressed as the reciprocal value of the CS score. Values are means ± SE for two independent experiments (n = 5–7). *P < 0.05 vs. control and #P < 0.05 vs. wild-type. Graphs in C and D share the y-axis labels with A and B, respectively. c/d, cycles/degree.