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. 2021 Jan-Jun:296:100187.
doi: 10.1074/jbc.RA120.015779. Epub 2020 Dec 20.

Ferroptosis drives photoreceptor degeneration in mice with defects in all-trans-retinal clearance

Chao Chen  1 , Jingmeng Chen  2 , Yan Wang  3 , Zuguo Liu  1 , Yalin Wu  4
Affiliations

Ferroptosis drives photoreceptor degeneration in mice with defects in all-trans-retinal clearance

Chao Chen et al. J Biol Chem. 2021 Jan-Jun.

Abstract

The death of photoreceptor cells in dry age-related macular degeneration (AMD) and autosomal recessive Stargardt disease (STGD1) is closely associated with disruption in all-trans-retinal (atRAL) clearance in neural retina. In this study, we reveal that the overload of atRAL leads to photoreceptor degeneration through activating ferroptosis, a nonapoptotic form of cell death. Ferroptosis of photoreceptor cells induced by atRAL resulted from increased ferrous ion (Fe2+), elevated ACSL4 expression, system Xc- inhibition, and mitochondrial destruction. Fe2+ overload, tripeptide glutathione (GSH) depletion, and damaged mitochondria in photoreceptor cells exposed to atRAL provoked reactive oxygen species (ROS) production, which, together with ACSL4 activation, promoted lipid peroxidation and thereby evoked ferroptotic cell death. Moreover, exposure of photoreceptor cells to atRAL activated COX2, a well-accepted biomarker for ferroptosis onset. In addition to GSH supplement, inhibiting either Fe2+ by deferoxamine mesylate salt (DFO) or lipid peroxidation with ferrostatin-1 (Fer-1) protected photoreceptor cells from ferroptosis caused by atRAL. Abca4-/-Rdh8-/- mice exhibiting defects in atRAL clearance is an animal model for dry AMD and STGD1. We observed that ferroptosis was indeed present in neural retina of Abca4-/-Rdh8-/- mice after light exposure. More importantly, photoreceptor atrophy and ferroptosis in light-exposed Abca4-/-Rdh8-/- mice were effectively alleviated by intraperitoneally injected Fer-1, a selective inhibitor of ferroptosis. Our study suggests that ferroptosis is one of the important pathways of photoreceptor cell death in retinopathies arising from excess atRAL accumulation and should be pursued as a novel target for protection against dry AMD and STGD1.

Keywords: Stargardt disease; all-trans-retinal; cell death; ferroptosis; iron metabolism; lipid peroxidation; macular degeneration; oxidative stress; photoreceptor.

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Conflict of interest statement

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

Figures

Figure 1
Figure 1
atRAL induces ferroptosis in 661W photoreceptor cells.A, cell viability, 3 and 6 h after incubating 661W photoreceptor cells with serial concentrations of atRAL (2.5, 5, 10, and 20 μM), was probed by MTS assay. B, cellular morphology, 3 and 6 h after incubating 661W photoreceptor cells with atRAL at concentrations of 2.5, 5, 10, and 20 μM, was imaged by a Leica DMi8 inverted microscope. Scale bars, 20 μm. C, qRT-PCR analysis of Ptgs2 and Acsl4 mRNA levels in 661W photoreceptor cells treated with 5-μM atRAL for 3 and 6 h. D–G, western blot analysis of COX2 and ACSL4 in 661W photoreceptor cells exposed to 5-μM atRAL for 3 and 6 h. Protein levels of COX2 and ACSL4 were normalized to those of GAPDH and presented as fold changes relative to vehicle (DMSO) controls. H, cytotoxicity was examined by MTS assay. 661W photoreceptor cells were pretreated with iron chelating agent DFO (50, 100, and 200 μM) for 2 h, followed by incubation with or without 5-μM atRAL for 6 h, respectively. Cells treated with atRAL or vehicle (DMSO) alone served as controls. I, cell viability was assessed by MTS assay. 661W photoreceptor cells were preincubated with Fer-1 (10 and 20 μM) for 2 h, followed by treatment with or without 5-μM atRAL for 6 h, respectively. Control cells were treated with atRAL or vehicle (DMSO) alone. Molecular mass markers (kDa) were indicated to the right of immunoblots. n.s., not significant.
Figure 2
Figure 2
atRAL disrupts iron homeostasis in 661W photoreceptor cells.A, intracellular Fe2+, 3 and 6 h after incubating 661W photoreceptor cells with 5-μM atRAL, was visualized by FeRhoNox-1 staining coupled with confocal microscopy. Nuclei were stained blue with Hoechst 33342. Scale bars, 20 μm. B, quantification of fluorescence intensity of Fe2+ specifically detected by FeRhoNox-1 in 661W photoreceptor cells treated with 5-μM atRAL for 3 and 6 h. C, qRT-PCR analysis of iron homeostasis-related genes in 661W photoreceptor cells exposed to 5-μM atRAL for 3 and 6 h. Control cells were treated with vehicle (DMSO) alone for 6 h. n.s., not significant.
Figure 3
Figure 3
atRAL causes lipid peroxidation in 661W photoreceptor cells.A, lipid peroxidation, 3 and 6 h after exposure of 661W photoreceptor cells to 5-μM atRAL, was examined by an Olympus FV1000 confocal fluorescence microscope using Click-iT lipid peroxidation imaging kit. Nuclei were stained with DAPI (blue). Scale bars, 20 μm. B, flow cytometry coupled with C11-BODIPY staining was used to determine levels of lipid peroxides in 661W photoreceptor cells treated with 5-μM atRAL for 6 h. Cells incubated for 6 h with 100-μM cumene hydroperoxide (CH), a compound that stimulates lipid peroxidation, served as a positive control. Control cells were treated with vehicle (DMSO) alone for 6 h.
Figure 4
Figure 4
GSH depletion promotes atRAL-induced ferroptosis in 661W photoreceptor cells.A, GSH levels, 3 and 6 h after incubating 661W photoreceptor cells with 5-μM atRAL, were determined using a GSH assay kit and shown as the percentage of GSH content in control cells. B, mRNA levels of Slc7a11, 3 and 6 h after incubating 661W photoreceptor cells with 5-μM atRAL, were determined using qRT-PCR. C, western blot analysis of SLC7A11 in 661W photoreceptor cells exposed to 5-μM atRAL for 3 and 6 h. D, protein levels of SLC7A11 were normalized to those of GAPDH, and presented as fold changes relative to vehicle (DMSO) controls. E, cell viability was probed by MTS assay. 661W photoreceptor cells were pretreated with 2 or 4-mM GSH for 1 h, followed by incubation with or without 5-μM atRAL for 6 h, respectively. F, intracellular ROS generation was visualized using the fluorescent probe H2DCFDA by fluorescence microscopy. 661W photoreceptor cells were pretreated with 4-mM GSH for 1 h and incubated with or without 5-μM atRAL for 6 h. Nuclei were stained with Hoechst 33342 (blue). Scale bars, 20 μm. G, qRT-PCR analysis of mRNA levels of Slc7a11, Ptgs2, and Acsl4 in lysates of 661W photoreceptor cells incubated with 5-μM atRAL for 6 h in the presence or absence of 4-mM GSH. Note that cells were pretreated with GSH for 1 h. H, western blot analysis of SLC7A11, COX2, and ACSL4 in lysates of 661W photoreceptor cells incubated with 5-μM atRAL for 6 h with or without 4-mM GSH. Note that cells were pretreated with GSH for 1 h. I, protein levels of SLC7A11, COX2, and ACSL4 were normalized to those of GAPDH, and presented as fold changes relative to vehicle (DMSO) controls. Cells treated with atRAL, GSH, or vehicle (DMSO) alone served as controls. n.s., not significant.
Figure 5
Figure 5
atRAL causes mitochondrial dyshomeostasis in 661W photoreceptor cells.A, TEM images of mitochondria in 661W photoreceptor cells treated with 5-μM atRAL for 3 and 6 h. Scale bars, 800 nm (upper panels) and 200 nm (lower panels). B, percentage of shrunken/destructed mitochondria in 661W photoreceptor cells exposed to 5-μM atRAL for 3 and 6 h. C, mitochondria in 661W photoreceptor cells exposed to 5-μM atRAL for 3 and 6 h were examined by confocal microscopy after cell staining with MitoTracker Red CMXRos. Cells incubated with vehicle (DMSO) alone for 6 h served as a control. Scale bars, 20 μm.
Figure 6
Figure 6
Iron-chelating agent DFO relieves atRAL-induced ferroptosis in 661W photoreceptor cells.A, cytotoxicity was evaluated by LDH release assay. 661W photoreceptor cells were pretreated with 200-μM DFO for 2 h and then incubated for 6 h with 5-μM atRAL. B, cell viability was examined by MTS assay. 661W photoreceptor cells were pretreated with 200-μM DFO for 2 h and then incubated with 20-μM atRAL for 6 h. C, confocal analysis of intracellular Fe2+ with the fluorescent probe FeRhoNox-1. 661W photoreceptor cells were pretreated with 200-μM DFO for 2 h and then incubated for 6 h with 5-μM atRAL. Nuclei were stained with Hoechst 33342 (blue). Scale bars, 20 μm. D, quantification of fluorescence intensity of Fe2+ specifically detected by FeRhoNox-1 in 661W photoreceptor cells treated with 5-μM atRAL for 6 h in the presence of 200-μM DFO. Note that cells were pretreated with DFO for 2 h. E, total iron levels were analyzed by ICP-MS. 661W photoreceptor cells were pretreated with 200-μM DFO for 2 h and then incubated for 6 h with 5-μM atRAL. F, the generation of intracellular ROS was visualized using the fluorescent probe H2DCFDA by fluorescence microscopy. 661W photoreceptor cells were pretreated with 200-μM DFO for 2 h and then incubated with 5-μM atRAL for 6 h. Nuclei were stained blue with Hoechst 33342. Scale bars, 20 μm. G, qRT-PCR analysis of mRNA levels of Ptgs2 and Acsl4 in 661W photoreceptor cells treated with 5-μM atRAL for 6 h in the presence of 200-μM DFO. Note that cells were pretreated with DFO for 2 h. H, immunoblot analysis of COX2 and ACSL4 in 661W photoreceptor cells exposed to 5-μM atRAL for 6 h with 200-μM DFO. Note that cells were pretreated with DFO for 2 h. I, protein levels of COX2 and ACSL4 were normalized to those of GAPDH, and presented as fold changes relative to vehicle (DMSO) controls. Cells treated with atRAL, DFO, or vehicle (DMSO) alone served as controls.
Figure 7
Figure 7
Ferroptosis inhibitor Fer-1 mitigates atRAL-induced ferroptosis in 661W photoreceptor cells.A, cytotoxicity was examined by LDH release assay. 661W photoreceptor cells were pretreated with 20-μM Fer-1 for 2 h and then incubated for 6 h with 5-μM atRAL. B, cell viability was determined by MTS assay. 661W photoreceptor cells were pretreated with 20-μM Fer-1 for 2 h and then incubated with 20-μM atRAL for 6 h. C, the production of intracellular ROS was visualized by use of the fluorescent probe H2DCFDA and fluorescence microscopy. 661W photoreceptor cells were pretreated with 20-μM Fer-1 for 2 h and then incubated with 5-μM atRAL for 6 h. Nuclei were stained blue with Hoechst 33342. Scale bars, 20 μm. D, lipid peroxidation was measured with Click-iT lipid peroxidation imaging kit and imaged on the confocal fluorescence microscope. 661W photoreceptor cells were pretreated with 20-μM Fer-1 for 2 h and then incubated with 5-μM atRAL for 6 h. Nuclei were stained blue by DAPI. Scale bars, 20 μm. E, levels of lipid peroxides in 661W photoreceptor cells treated with 5-μM atRAL for 6 h with 20-μM Fer-1 were assessed by C11-BODIPY staining coupled with flow cytometry. Note that cells were pretreated with Fer-1 for 2 h. F, qRT-PCR analysis of mRNA levels of Ptgs2 and Acsl4 in 661W photoreceptor cells exposed to 5-μM atRAL for 6 h in the presence of 20-μM Fer-1. Note that cells were pretreated with Fer-1 for 2 h. G, western blot analysis of COX2 and ACSL4 in 661W photoreceptor cells treated with 5-μM atRAL for 6 h with 20-μM Fer-1. Note that cells were pretreated with Fer-1 for 2 h. H, protein levels of COX2 and ACSL4 were normalized to those of GAPDH, and presented as fold changes relative to vehicle (DMSO) controls. Cells treated with atRAL, Fer-1, or vehicle (DMSO) alone served as controls.
Figure 8
Figure 8
Ferroptosis involves light-induced photoreceptor degeneration in Abca4−/−Rdh8−/−mice.A, schematic illustration of the experimental procedure. Four-week-old C57BL/6J and Abca4−/−Rdh8−/− mice were dark adapted and irradiated as we previously described (19). Eyeballs were harvested at day 5 after light exposure. B, histological examination of mouse retina was performed by using H&E staining. Square brackets in yellow indicate whole mouse retina. GCL, ganglion cell layer; INL, inner nuclear layer; IPL, inner plexiform layer; IS, inner segment; ONL, outer nuclear layer; OPL, outer plexiform layer; OS, outer segment; RPE, retinal pigment epithelium. Scale bars, 20 μm. C, thickness of whole retina, ONL, or IS+OS was quantified by Leica Application Suite X microscope software, and presented as a percentage of that measured in control C57BL/6J mice, respectively. D, lipid peroxidation in mouse retina was evaluated by immunofluorescence staining of lipid peroxidation marker acrolein. Nuclei were stained with DAPI (blue). Scale bars, 20 μm. E, GSH levels in mouse retina were determined by the GSH assay kit and expressed as a percentage of that detected in control C57BL/6J mice. F, western blot analysis of COX2, ACSL4, and GPX4 in lysates of mouse neural retina. G, protein levels of COX2, ACSL4, and GPX4 were normalized to those of GAPDH and presented as fold changes relative to control C57BL/6J mice. n.s., not significant.
Figure 9
Figure 9
Fer-1, a selective inhibitor of ferroptosis, alleviates light-induced photoreceptor degeneration and ferroptosis in Abca4−/−Rdh8−/−mice.A, schematic diagram of the experimental protocol. Abca4−/−Rdh8−/− mice aged 4 weeks were adapted in the dark for 48 h and then intraperitoneally injected with Fer-1 or vehicle (DMSO) (4 mg/kg body weight). After 1 h, pupils of mice were dilated with 1% tropicamide, and the mice were irradiated by 10,000-lx LED light for 2 h, followed by once-daily treatment with Fer-1 or vehicle (DMSO) (4 mg/kg body weight) in the dark for 4 days. Eyeballs, 5 days after light exposure, were harvested for subsequent analyses. Control Abca4−/−Rdh8−/− mice were administered intraperitoneally Fer-1 (4 mg/kg body weight) or vehicle (DMSO) in the dark without exposure to light. B, H&E staining for analysis of the morphology of mouse retina, Scale bars, 20 μM. C, thickness of whole retina, ONL, or IS+OS was quantified by Leica Application Suite X microscope software and shown as a percentage of that measured in vehicle (DMSO)-treated control Abca4−/−Rdh8−/− mice. D, lipid peroxidation in mouse retina was determined by immunofluorescence staining of lipid peroxidation marker acrolein. Nuclei were stained blue with DAPI. Scale bars, 20 μm. E, western blot analysis of COX2, ACSL4, and GPX4 in lysates of mouse neural retina. F, protein levels of COX2, ACSL4, and GPX4 were normalized to those of GAPDH and expressed as fold changes relative to vehicle (DMSO)-treated control Abca4−/−Rdh8−/− mice.
Figure 10
Figure 10
Proposed mechanisms of photoreceptor ferroptosis induced by atRAL. In photoreceptor cells, atRAL interrupts system Xc− to elicit a significant decrease of GSH levels. GSH depletion inactivates GPX4 and produces ROS, which promotes lipid peroxidation and thereby induces ferroptosis. Mitochondrial ROS generated by atRAL (19) may also participate in lipid peroxidation. Damage to mitochondria inflicted by atRAL may directly evoke ferroptotic cell death. Besides increasing protein levels of ACSL4, atRAL elevates intracellular Fe2+ levels through perturbation of iron homeostasis and then provokes ROS production via Fenton reaction. Both ROS induced by overload of Fe2+ and activation of ACSL4 incite lipid peroxidation that triggers ferroptosis. In addition, atRAL stimulates a remarkable increase in protein levels of COX2 encoded by Ptgs2 gene. Up-regulation of COX2 is a hallmarker of ferroptosis.
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