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. 2012 Mar 2;287(10):7675-82.
doi: 10.1074/jbc.M111.335174. Epub 2011 Dec 29.

Glutathione peroxidase 4 is required for maturation of photoreceptor cells

Takashi Ueta  1 , Tatsuya Inoue, Takahisa Furukawa, Yasuhiro Tamaki, Yasuhito Nakagawa, Hirotaka Imai, Yasuo Yanagi
Affiliations

Glutathione peroxidase 4 is required for maturation of photoreceptor cells

Takashi Ueta et al. J Biol Chem. .

Abstract

Oxidative stress is implicated in the pathologies of photoreceptor cells, and the protective role of antioxidant enzymes for photoreceptor cells have been well understood. However, their essentiality has remained unknown. In this study we generated photoreceptor-specific conditional knock-out (CKO) mice of glutathione peroxidase 4 (GPx4) and showed the critical role of GPx4 for photoreceptor cells. In the wild-type retina the dominant GPx4 expression was in the mitochondria, indicating the mitochondrial variant was the major GPx4 in the retina. In the GPx4-CKO mice, although photoreceptor cells developed and differentiated into rod and cone cells by P12, they rapidly underwent drastic degeneration and completely disappeared by P21. The photoreceptor cell death in the GPx4-CKO mice was associated with the nuclear translocation of apoptosis-inducing factor (AIF) and TUNEL-positive cells. Photoreceptor cells before undergoing apoptosis (P11) exhibited decreased mitochondrial biomass, decreased number of connecting cilia, as well as disorganized structure of outer segments. These findings indicate that GPx4 is a critical antioxidant enzyme for the maturation and survival of photoreceptor cells.

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Figures

FIGURE 1.
FIGURE 1.
GPx4 expression in the wild-type mouse retina. A, lysates of the retina, retinal pigment epithelium (RPE)/choroid, and brain were immunoblotted for GPx4 and β-actin. GPx4 protein level in the retina was higher than that in RPE/choroid and brain. B–E, immunohistochemistry of the retina for GPx4 (green) at P1 and 8-week-old mice. GPx4 was abundantly expressed in the retina, especially in the inner segments (IS) of photoreceptor cells while there was no detectable expression of GPx4 protein in the outer segments (OS) depicted by autofluorescence. B and D, negative controls. GCL (ganglion cell layer), INL (inner nuclear layer), ONL (outer nuclear layer), NL (neuroblastic layer). Scale bar, 50 μm.
FIGURE 2.
FIGURE 2.
Mitochondrial GPx4 is the major variant of GPx4 in the retina. A, SOD1, a cytosolic antioxidant enzymes was expressed more dominantly in OS than in IS. B, SOD2, a mitochondrial antioxidant enzyme was expressed in IS, not in OS. C–E, GPx4 and prohibitin, a mitochondrial marker, co-localized in the retina. F, in contrast, in RPE GPx4 and prohibitin did not co-localize. Scale bar, 50 μm.
FIGURE 3.
FIGURE 3.
Generation of the photoreceptor-specific CKO mice of GPx4. A–D, immunohistochemistry of the retina for GPx4. A, at P1 the CKO mouse did not express GPx4 in the outer area of NL. B, at P12 photoreceptor cells of the CKO mice comprised ONL, but GPx4 was not expressed. C and D, the control mice expressed GPx4 in photoreceptor cells both at P1 and P12. E and F, consistent with the deletion of GPx4 in photoreceptor cells, significant amount of acrolein, a marker of lipid peroxidation, was found in IS of photoreceptor cells in the CKO mice at P12 compared with the control. Scale bar, 50 μm.
FIGURE 4.
FIGURE 4.
Phenotype comparison between the CKO and control mice by hematoxylin-eosin staining. A and C, massive retinal degeneration was seen in the central and peripheral retina of the CKO mice, but not in the retina of the control mice. B and D, sequential observations revealed that at P12, there was no apparent difference between the retina of the CKO and control mice. However, at P17, ONL of the CKO mice progressively thinned and by P21 completely disappeared. E, there is a clear statistical significance in this process (mean ± S.E.; n = 3; ****, p < 0.0001, by ANOVA, followed by Turkey's post hoc test). Scale bar, 500 μm (C) and 50 μm (D).
FIGURE 5.
FIGURE 5.
The photoreceptor cell loss in the CKO mice started in cone photoreceptor cells, followed by rod cells. Rod and cone photoreceptor cells were labeled with anti-rhodopsin antibody (red) and PNA (light blue), respectively. A and D, at P12 photoreceptor cells in the retina of the CKO and control mice presented a similar staining pattern for rod and cone cells. B and E, however, PNA-labeled cone cells disappeared by P15 in the CKO mice while rhodopsin localized abnormally in ONL. C and F, cone or rod photoreceptor cells were absent in the CKO mouse retina by P21. Normal maturation and OS formation was observed in the CKO mouse retina. Scale bar, 50 μm.
FIGURE 6.
FIGURE 6.
TUNEL-positive cells and nuclear translocation of AIF in photoreceptor cells of the CKO mice. A–C, significant number of TUNEL-positive photoreceptor cells were observed in the CKO mouse retina at P17, but not in the control mouse retina. The TUNEL-positive cells appeared after P12. D–G, nuclear translocation of AIF was observed in photoreceptor cells of the CKO mice at P15, but not in the control mice (mean ± S.E.; n = 4; ns, not significant; ***, p < 0.001 by two-tailed t test for CKO versus control mice). Scale bar, 50 μm (B) and 10 μm (G).
FIGURE 7.
FIGURE 7.
Decreased mitochondrial biomass in photoreceptor cells of the CKO mice at P11. A–C, in IS of photoreceptor cells of the CKO mice, mitochondrial area on TEM was significantly smaller than in IS of the control mice (mean ± S.E.; n = 4; ***, p < 0.001 by two-tailed t test). D, in the whole retina, mitochondrial DNA (mtDNA) replication was decreased in the CKO mice in comparison to the control mice (mean ± S.E.; n = 3; *, p < 0.05, by two-tailed t test). E, a paradoxical increase in Tfam mRNA in the whole retina of the CKO mice, suggesting a compensatory aspect of Tfam against the mitochondrial decrease by GPx4 deficit (mean ± S.E.; n = 3; **, p < 0.01 by two-tailed t test). Scale bar, 1 μm.
FIGURE 8.
FIGURE 8.
The decreased number of connecting cilia and disorganized OS in photoreceptor cells of the CKO mice. A and C, in these overview, OS layer (indicated by white arrows) existed in the CKO mice but profoundly thinner than OS of the control mice. B, D, E, the number of connecting cilia at the junction of IS and OS was significantly smaller in the CKO mice than in the control mice. The number of cilia was counted at the IS/OS junction with 50 μm span, shown as the boxed area in A and C. (mean ± S.E.; n = 3; ns, not significant; ***, p < 0.001 by two-tailed t test). F, OS of the CKO mice was disorganized and consists of cell membranes that branched and formed a large web-like network structure without apparent disc rim. Intra- and inter- OS membrane spaces were abnormally wide. G, OS of the control mice was composed of a pile of double membrane discs. Scale bar, 10 μm (C), 1 μm (D), and 0.5 μm (G).
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