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. 2009 Apr;11(4):715-24.
doi: 10.1089/ars.2008.2171.

Increased expression of glutathione peroxidase 4 strongly protects retina from oxidative damage

Lili Lu  1 , Brain C Oveson, Young-Joon Jo, Thomas W Lauer, Shinichi Usui, Keiichi Komeima, Bing Xie, Peter A Campochiaro
Affiliations

Increased expression of glutathione peroxidase 4 strongly protects retina from oxidative damage

Lili Lu et al. Antioxid Redox Signal. 2009 Apr.

Abstract

Oxidative damage contributes to cone cell death in retinitis pigmentosa and death of rods, cones, and retinal pigmented epithelial (RPE) cells in age-related macular degeneration. In this study, we explored the strategy of overexpressing components of the endogenous antioxidant defense system to combat oxidative damage in RPE cells and retina. In transfected cultured RPE cells with increased expression of superoxide dismutase1 (SOD1) or SOD2, there was increased constitutive and stress-induced oxidative damage measured by the level of carbonyl adducts on proteins. In contrast, RPE cells with increased expression of glutathione peroxidase 1 (Gpx1) or Gpx4 did not show an increase in constitutive oxidative damage. An increase in Gpx4, and to a lesser extent Gpx1, reduced oxidative stress-induced RPE cell damage. Co-expression of Gpx4 with SOD1 or 2 partially reversed the deleterious effects of the SODs. Transgenic mice with inducible expression of Gpx4 in photoreceptors were generated, and in three models of oxidative damage-induced retinal degeneration, increased expression of Gpx4 provided strong protection of retinal structure and function. These data suggest that gene therapy approaches to augment the activity of Gpx4 in the retina and RPE should be considered in patients with retinitis pigmentosa or age-related macular degeneration.

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Figures

FIG. 1.
FIG. 1.
Increased oxidative damage and reduced viability in retinal pigmented epithelial (RPE) cells overexpressing superoxide dismutase 1 (SOD1) or SOD2. Untransfected ARPE 19 cells (control) or those transfected with empty plasmid or plasmid containing an expression construct for glutathione peroxidase 1 (Gpx1), Gpx4, SOD1, or SOD2 were scraped into lysis buffer 48 h after transfection. The bars represent the mean (±SEM) calculated from four experimental values. (A) Protein carbonyl content was measured by ELISA and was not significantly different in cells overexpressing Gpx1 or Gpx4 compared to control cells, but it was significantly elevated in cells overexpressing SOD1 or SOD2 (*p <0.01; **p < 0.05 for difference from untransfected cells and +p < 0.05 for difference from cells transfected with SOD1 or 2 alone by ANOVA with Dunnett's correction for multiple comparisons). (B) Cell viability was measured by MTT and was not different in cells overexpressing Gpx1 or Gpx4 compared to untransfected cells, but it was significantly reduced in cells overexpressing SOD2 or co-expressing SOD2 and Gpx1 (*p < 0.05 by ANOVA with Dunnett's correction). (C) Forty-eight hours after transfections, cell lysates (50 μg total protein) were run in immunoblots for SOD2 or Gpx4, and then stripped and reblotted for actin to control for loading differences. There were increased levels of SOD2 in cells transfected with the Sod2 expression construct or cells co-transfected with Sod2 and Gpx expression constructs (top row) with no difference in actin levels (second row). Immunoblots for Gpx4 showed increased levels of Gpx4 in cells transfected with the Gpx4 expression construct or co-transfected with Gpx4 and Sod2 expression constructs (third row) with no difference in actin levels ( fourth row).
FIG. 2.
FIG. 2.
Glutathione peroxidase 1 (Gpx1) and Gpx4 protect RPE cells from oxidative stress. Twenty-four hours after transfection with an expression construct for glutathione peroxidase 1 (Gpx1), Gpx4, SOD1, or SOD2, RPE cells were treated with 7 mM paraquat, 0.5 mM H2O2, or hyperoxia for 24 h. Untransfected RPE cells were treated in the same way to serve as controls. Cell lysates were used to measure protein carbonyl content by ELISA (A) and cell viability by MTT (B). The bars represent the mean (±SEM) calculated from four experimental values. (A) When exposed to oxidative stress, compared to control cells, those overexpressing Gpx1 or Gpx4 showed significantly less carbonyl content for two of three, or three of three types of stress, respectively. In contrast, cells overexpressing SOD1 or SOD2 showed significantly greater carbonyl content for two of three, or three of three types of stress, respectively. Cells co-expressing SOD1 and Gpx1 had carbonyl contents no different from control cells and those co-expressing SOD2 and Gpx1 had a significant increase in carbonyl content compared to controls for two of three types of stress. In contrast, cells co-expressing Gpx4 and SOD1 or SOD2 had significantly less carbonyl content than control cells for one of three types of stress. *p < 0.05 for decrease from control cells; +p < 0.05 for increase from control cells by ANOVA with Dunnett's correction for multiple comparisons. (B) Compared to control cells, those overexpressing Gpx1 or Gpx4 showed a significant increase in viability for two of three types of oxidative stress, whereas those overexpressing SOD1 or SOD2 had a significant reduction in viability for two of three, or one of three types of oxidative stress, respectively. Cells co-expressing Gpx1 with SOD1 or 2 showed reduced viability for one type of oxidative stress, while cells co-expressing Gpx4 with SOD1 or 2 showed increased viability for one type of oxidative stress. *p < 0.05 for increase from control cells; +p < 0.05 for decrease from control cells by ANOVA with Dunnett's correction for multiple comparisons.
FIG. 3.
FIG. 3.
Transgenic mice with doxycycline-inducible expression of glutathione peroxidase 4 (Gpx4). Tetracycline response element (TRE)/Gpx4 mice were generated as described in Methods and crossed with opsin/rtTA transgenic mice to generate Tet/opsin/Gpx4 double transgenic mice. Adult Tet/opsin/Gpx4 mice or littermates lacking one of the transgenes were given drinking water containing (+) or lacking (-) 2 mg/ml doxycycline. After 2 weeks, mice were euthanized, and retinal homogenates were assayed for protein concentration; samples containing 50 μg were run in immunoblots for Gpx4. The blots were stripped and reprobed for actin. There was an increase in Gpx4 in the retinas of Tet/opsin/Gpx4 mice treated with doxycycline.
FIG. 4.
FIG. 4.
Doxycycline-induced expression of Gpx4 in Tet/opsin/Gpx4 double transgenics reduces oxidative damage in the retina. Tet/opsin/Gpx4 double transgenic mice or littermates lacking one of the transgenes (controls) were given drinking water containing or lacking 2 mg/ml of doxycycline for 2 weeks and then assessed for effects of paraquat (A) or hyperoxia (B) on carbonyl content in the retina. (A) Mice were given an intravitreous injection of 1 μl of PBS containing 0.75 mM paraquat in one eye and 1 μl of PBS in the other eye, and after 24 h the protein carbonyl content in the retina was measured by ELISA. The bars represent the mean (±SEM) calculated from 6 mice in each group. For paraquat-injected eyes, the carbonyl content was significantly less (*p < 0.05 by ANOVA with Dunnett's correction) in the retinas of Tet/opsin/Gpx4 mice that received doxycycline compared to retinas of Tet/opsin/Gpx4 mice that did not receive doxycycline or retinas of control mice either treated with doxycycline or not (**p < 0.005). Paraquat-injected eyes had greater carbonyl content in the retina than fellow eyes-injected with PBS. (B) Mice were placed in 75% oxygen for 2 weeks and then carbonyl content was measured in the retina. The bars represent the mean (±SEM) calculated from 5 mice in each group. Retinal carbonyl content was significantly less in Tet/opsin/Gpx4 mice treated with doxycycline (*p < 0.05) compared to Tet/opsin/Gpx4 mice that did not receive doxycycline or control mice whether or not they received doxycycline.
FIG. 5.
FIG. 5.
Induced expression of Gpx4 reduces paraquat-induced thinning of the outer nuclear layer (ONL) of the retina. Tet/opsin/Gpx4 double transgenic mice received drinking water containing or lacking 2 mg/ml of doxycycline, and littermate control mice were given normal drinking water. After 1 week, the mice were given an intraocular injection of 1 μl of 0.75 mM paraquat in the left eye and 1 μl of PBS in right eye. After another 2 weeks of water containing or lacking doxycycline, the mice were euthanized and outer nuclear layer (ONL) thickness was measured as described in Methods. The bars represent the mean (±SEM) calculated from 5 mice in each group. Compared to identical regions of the retina in eyes of control mice injected with PBS (A), those from paraquat-injected eyes of doxycycline-treated Tet/opsin/Gpx4 mice appeared to have a slightly thinner ONL, and this was confirmed by image analysis (E, *p < 0.05, by ANOVA with Dunnett's correction for multiple comparisons), but significantly thicker than the ONL from paraquat-injected eyes of Tet/opsin/Gpx4 mice that were not treated with doxycycline (C, **p < 0.001) or paraquat-injected eyes of control mice (D, **p < 0.001).
FIG. 6.
FIG. 6.
Induced expression of Gpx4 reduces hyperoxia-induced thinning of the outer nuclear layer (ONL) of the retina. Tet/opsin/Gpx4 double transgenic mice were placed in 75% O2 and given drinking water containing or lacking 2 mg/ml of doxycycline. Littermate controls were also placed in 75% oxygen or left in room air. After 2 weeks, the mice were euthanized, 10 μm ocular frozen sections were stained with hematoxylin and eosin, and the ONL thickness was measured as described in Methods. The bars represent the mean (±SEM) calculated from 5 mice in each group. Compared to control mice that remained in room air (A, n = 5), the ONL of the same region of the retina from eyes of hyperoxia-treated Tet/opsin/Gpx4 mice (B, n = 5) appeared somewhat thinner which was confirmed by image analysis (E, *p < 0.05 by ANOVA with Dunnett's correction), but was significantly thicker than the ONL of hyperoxia-exposed Tet/opsin/Gpx4 mice that did not receive doxycycline (C, n = 5, **p <0.002) or hyperoxia-treated control mice (D, n = 5, **p < 0.002).
FIG. 7.
FIG. 7.
Induced expression of Gpx4 prevents loss of retinal function assessed by electroretinograms (ERGs) after intraocular injection of paraquat. Tet/opsin/Gpx4 double transgenic or littermate control mice were given water containing or lacking 2 mg/ml of doxycycline, and after 1 week received an intraocular injection of 1 μl of 0.75 mM paraquat in one eye and PBS in the fellow eye. Scotopic ERGs were performed at 2 and 8 days after injection. The points represent the mean (±SEM) calculated from 6 mice in each group. At 2 days after injection, all eyes injected with paraquat showed a significant reduction in a-wave (A) and b-wave (C) amplitude compared to eyes injected with PBS. However, at 8 days after injection, paraquat-injected eyes of Tet/opsin/Gpx4 mice that received doxycycline showed a-wave (B) and b-wave (D) amplitudes that were essentially identical to those of PBS-injected eyes, and significantly greater than all other paraquat-injected eyes.
FIG. 8.
FIG. 8.
Induced expression of Gpx4 prevents hyperoxia-induced loss of retinal function assessed by electroretinograms (ERGs). Tet/opsin/Gpx4 double transgenic or littermate control mice were given water containing or lacking 2 mg/ml of doxycycline, and after 1 week were placed in 75% oxygen. After another 1 (A and C) and 2 (B and D) weeks, scotopic ERGs (the points represent the mean ± SEM calculated from 6 mice in each group) showed that eyes of Tet/opsin/Gpx4 mice exposed to hyperoxia had significantly greater a-wave (A, B) and b-wave (C, D) amplitudes than Tet/opsin/Gpx4 that did not receive doxycycline or control mice that received water containing or lacking doxycycline.
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