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. 2022 Apr;298(4):101824.
doi: 10.1016/j.jbc.2022.101824. Epub 2022 Mar 11.

Mitochondrial glutathione peroxidase 4 is indispensable for photoreceptor development and survival in mice

Kunihiro Azuma  1 , Tomoko Koumura  2 , Ryo Iwamoto  2 , Masaki Matsuoka  2 , Ryo Terauchi  3 , Shu Yasuda  2 , Tomoyasu Shiraya  1 , Sumiko Watanabe  4 , Makoto Aihara  1 , Hirotaka Imai  5 , Takashi Ueta  6
Affiliations

Mitochondrial glutathione peroxidase 4 is indispensable for photoreceptor development and survival in mice

Kunihiro Azuma et al. J Biol Chem. 2022 Apr.

Abstract

Glutathione peroxidase 4 (GPx4) is known for its unique function in the direct detoxification of lipid peroxides in the cell membrane and as a key regulator of ferroptosis, a form of lipid peroxidation-induced nonapoptotic cell death. However, the cytosolic isoform of GPx4 is considered to play a major role in inhibiting ferroptosis in somatic cells, whereas the roles of the mitochondrial isoform of GPx4 (mGPx4) in cell survival are not yet clear. In the present study, we found that mGPx4 KO mice exhibit a cone-rod dystrophy-like phenotype in which loss of cone photoreceptors precedes loss of rod photoreceptors. Specifically, in mGPx4 KO mice, cone photoreceptors disappeared prior to their maturation, whereas rod photoreceptors persisted through maturation but gradually degenerated afterward. Mechanistically, we demonstrated that vitamin E supplementation significantly ameliorated photoreceptor loss in these mice. Furthermore, LC-MS showed a significant increase in peroxidized phosphatidylethanolamine esterified with docosahexaenoic acid in the retina of mGPx4 KO mice. We also observed shrunken and uniformly condensed nuclei as well as caspase-3 activation in mGPx4 KO photoreceptors, suggesting that apoptosis was prevalent. Taken together, our findings indicate that mGPx4 is essential for the maturation of cone photoreceptors but not for the maturation of rod photoreceptors, although it is still critical for the survival of rod photoreceptors after maturation. In conclusion, we reveal novel functions of mGPx4 in supporting development and survival of photoreceptors in vivo.

Keywords: cell death; development; glutathione peroxidase 4; lipid peroxidation; mitochondria; phospholipid; photoreceptor; retina.

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Conflict of interest statement

Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article.

Figures

Figure 1
Figure 1
Photoreceptor degeneration in mGPx4 KO mouse retina.A, Western blots of cytosolic (C) and mitochondrial (M) fractions of the retina from WT, control, and mGPx4 KO mice. TIM23 and SOD1 served as mitochondrial and cytosolic markers, respectively. mGPx4 was detected in samples from WT and control retina but not in samples from mGPx4 KO retina. B, quantification of cGPx4 levels in cytosol fraction, indicating retained cGPx4 levels in mGPx4 KO mouse retina (n = 4 per group). Analysis was performed using one-way ANOVA and the Tukey–Kramer post hoc test. C, GPx4 expression in the retina of control and mGPx4 KO mice. Higher GPx4 levels were observed in inner segments in control mouse where mitochondria are condensed, which was not observed in mGPx4 KO mice (consistent in three independent samples per group). D, color fundus photographs of control and mGPx4 KO mice. Pigmentation, retinal vascular narrowing, and optic atrophy were observed in the fundus of mGPx4 KO mice (consistent in three independent samples per group). E, mGPx4 KO mouse retina contained more acrolein derived from lipid peroxidation than control retina on Western blot analysis (n = 6 per group). F, hematoxylin–eosin staining showed thinner ONL in mGPx4 KO mouse at P14 and P30 (consistent in three independent samples per group). G, immunohistochemistry for rhodopsin-expressing rod and PNA-stained cone photoreceptors. In mGPx4 KO mouse retina, cone photoreceptors were not present at P7, P14, or P30. Some of the rod photoreceptors remained in existence at least until P30 (consistent in three independent samples per group). H, immunohistochemistry of RPE flatmounts using anti-ZO-1 antibody in control and mGPx4 KO mouse at P14. No apparent degeneration was observed in the RPE of mGPx4 KO mice (consistent in three independent samples per group). All scale bars are 25 μm except one in (D), which is 200 μm. ∗p < 0.05, ∗∗p < 0.01 (KO with vitamin E versus KO), #p < 0.05, ##p < 0.01 (KO versus control) using Student’s t test for two-group comparison and one-way ANOVA and the Tukey–Kramer post hoc test for three-group comparison. mGPx4, mitochondrial isoform of GPx4; ONL, outer nuclear layer; PNA, peanut agglutinin; RPE, retinal pigment epithelium; SOD1, super dismutase 1.
Figure 2
Figure 2
Vitamin E supplementation ameliorated retinal degeneration in mGPx4 KO mice.A, TUNEL staining of the retinas in control, mGPx4 KO, and vit E-supplemented mGPx4 KO mice at P14. B, ONL thickness at P14 in control, mGPx4 KO, and vit E-supplemented mGPx4 KO mice. The ONL of mGPx4 KO mouse retina was significantly thinner than control mouse retina, which was partially rescued by vit E supplementation (n = 4–5 in each group). C, a significantly larger number of TUNEL-positive photoreceptors were detected in mGPx4 KO mouse retina than in control at P14, which was partially rescued by vit E supplementation (n = 6 per group). D, immunohistochemistry for cone-specific marker s-opsin and cone arrestin. S-opsin expression was not detectable in mGPx4 KO mice at P7 and P14. Vit E supplementation mildly rescued the s-opsin expression at P7 but not at P14. Cone–arrestin expression was not observed in mGPx4 KO or vit E-supplemented mGPx4 KO mice at P14. E, quantification for the number of s-opsin-expressing outer segments of cone photoreceptors in control, mGPx4 KO, and vit E-supplemented mGPx4 KO mice (n = 8 per group). F, immunohistochemistry for cone precursor marker Rxr-γ. Rxr-γ expression levels were similar between control and mGPx4 KO mouse retina at P0 but diminished at P4 and P7 in mGPx4 KO mice. Rxr-γ expression was restored in vit E-supplemented mGPx4 KO mice at P4 but not at P7. G, quantification for the number of Rxr-γ-expressing cells at P0 and P4 (n = 12 per group). All scale bars represent 25 μm. ∗p < 0.05, ∗∗p < 0.01 (KO with vitamin E versus KO), #p < 0.05, ##p < 0.01 (KO versus control) using one-way ANOVA and the Tukey–Kramer post hoc test. mGPx4, mitochondrial isoform of GPx4; ONL, outer nuclear layer; vit E, vitamin E.
Figure 3
Figure 3
Functional abnormalities in mGPx4 KO mouse retina on electroretinogram.A, representative scotopic ERG in 7-week-old mice. B and C, in scotopic condition, the amplitude of both a-wave and b-wave was significantly diminished in mGPx4 KO mice, which was partially rescued by vit E supplementation (n = 3, 5, and 6 for control, mGPx4 KO, and vit E-supplemented mGPx4 KO). D, representative photopic ERG in 4-week-old mice. E and F, in photopic condition, the amplitude of both a-wave and b-wave was totally lost in mGPx4 KO mice, which could not be rescued by vit E supplementation (n = 6 for control and mGPx4 KO, n = 8 for vit E-supplemented mGPx4 KO). ∗p < 0.05, ∗∗p < 0.01 (KO with vitamin E versus KO), #p < 0.05, ##p < 0.01 (KO versus control) using one-way ANOVA and the Tukey–Kramer post hoc test. ERG, electroretinogram; mGPx4, mitochondrial isoform of GPx4; vit E, vitamin E.
Figure 4
Figure 4
Mitochondrial abnormalities in mGPx4 KO mouse photoreceptors.A, representative transmission electron microscopy (TEM) images of longitudinal sections of inner segments of photoreceptors at P17. Areas inside the enclosing frames in the left (the scale bars represent 2.5 μm) are shown magnified in the right (the scale bars represent 1 μm). Mitochondria were marked with red shadows. In photoreceptor inner segments of control mice, mitochondria of neighboring photoreceptors align with close association. In contrast, such physiological mitochondrial alignment was disrupted in mGPx4 KO mouse photoreceptors (representatives of three independent samples per group). B, the smaller number of mitochondria was observed in photoreceptor inner segments of mGPx4 KO mice compared with those of control mice on TEM (the number of mitochondria per 23.5 μm2 area of inner segments, n = 4 per group). C, area of each mitochondrion in photoreceptor inner segments was larger in mGPx4 KO compared with control mice on TEM (n = 3 per group, each value was the representative of >30 mitochondria). D, Western blot analysis indicated higher MFN2 expression in mGPx4 KO than in control mice (n = 6 per group). ∗p < 0.05, ∗∗p < 0.01 using Student’s t test. E, representative TEM images of transverse sections of inner segments of photoreceptors at P17 in control and mGPx4 KO mice. Areas inside the enclosing frames in the left (the scale bars represent 1 μm) are shown magnified in the right (the scale bars represent 0.2 μm). Mitochondrial fusions (red arrows) were observed in mGPx4 KO mice and not in control mice (consistent in three independent samples per group). MFN2, mitofusion 2; mGPx4, mitochondrial isoform of GPx4.
Figure 5
Figure 5
Apoptosis of photoreceptors in mGPx4 KO mice.A, in the ONL of P17 mGPx4 KO mouse retina, TEM disclosed the presence of uniformly dense and shrunk pyknotic nuclei (red arrows), which was not observed in control mouse retina. Areas inside the enclosing frames in the left (the scale bars represent 10 μm) are shown magnified in the right (the scale bars represent 2.5 μm). B, quantification of the apoptotic nuclei observed in TEM images in control and mGPx4 KO mice (n = 3 for control, n = 5 for mGPx4 KO; three views per sample). ∗p < 0.05 (KO versus control) using Student’s t test. C, immunohistochemistry for cleaved-caspase 3 confirmed caspase activation in photoreceptors of mGPx4 KO mice. Vit E treatment decreased the number of cleaved-caspase 3-positive cells in mGPx4 KO mice (the scale bar represent 25 μm). ∗∗p < 0.01 (KO with vit E versus KO), #p < 0.05, ##p < 0.01 (KO versus control) using one-way ANOVA and the Tukey–Kramer post hoc test. n = 3 per group and three views per sample. mGPx4, mitochondrial isoform of GPx4; ONL, outer nuclear layer; TEM, transmission electron microscopy; Vit E, vitamin E.
Figure 6
Figure 6
LC–MS analysis indicated increased phospholipid peroxidation of PE 22:6/22:6 in mGPx4 KO mice retina.A, PE 22:6/22:6 (DHA esterified at both sn-1 and sn-2 positions of PE) was significantly decreased in mGPx4 KO mice retina at P10 compared with control, which was blocked by vit E supplementation (n = 3 per group). B, peroxidized PE 22:6/22:6-OOH was significantly increased in mGPx4 KO mice at P10 compared with control, which was rescued by vit E supplementation (n = 3 per group). C, there was no significant difference among the three groups in hydroxy PE-PUFAs-OH (n = 3 per group). D–F, there was no significant change in the compositions of PC-PUFAs, PC-PUFAs-OOH, or PC-PUFAs-OH among control, mGPx4 KO, and vit E-supplemented mGPx4 KO mice (n = 3 per group). ∗p < 0.05, ∗∗p < 0.01 (KO with vitamin E versus KO), #p < 0.05 (KO versus control) using one-way ANOVA and the Tukey–Kramer post hoc test. DHA, docosahexaenoic acid; mGPx4, mitochondrial isoform of GPx4; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PUFA, polyunsaturated fatty acid; vit E, vitamin E.
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