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

2021; Elsevier BV; Volume: 296; Linguagem: Inglês

10.1074/jbc.ra120.015779

1083-351X

Chao Chen, Jingmeng Chen, Yan Wang, Zuguo Liu, Yalin Wu,

Mitochondrial Function and Pathology

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. 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. Rhodopsin, a primary photoreceptor molecule in vision, consists of apoprotein opsin and 11-cis-retinal (11-cis-RAL) chromophore (1Palczewski K. 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ATP-binding cassette transporter 4 (ABCA4) is a retina-specific ABC transporter that involves in shuttling atRAL from POS discs to the cytosol (12Sun H. Nathans J. Stargardt's ABCR is localized to the disc membrane of retinal rod outer segments.Nat. Genet. 1997; 17: 15-16Crossref PubMed Scopus (212) Google Scholar, 13Weng J. Mata N.L. Azarian S.M. Tzekov R.T. Birch D.G. Travis G.H. Insights into the function of Rim protein in photoreceptors and etiology of Stargardt's disease from the phenotype in abcr knockout mice.Cell. 1999; 98: 13-23Abstract Full Text Full Text PDF PubMed Scopus (723) Google Scholar, 14Quazi F. Lenevich S. Molday R.S. ABCA4 is an N-retinylidene-phosphatidylethanolamine and phosphatidylethanolamine importer.Nat. Commun. 2012; 3: 925Crossref PubMed Scopus (174) Google Scholar) where all-trans-retinol dehydrogenase 8 (RDH8) reduces atRAL into all-trans-retinol (15Chen C. Thompson D.A. Koutalos Y. 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A. 2014; 111: E1428-E1437Crossref PubMed Scopus (39) Google Scholar). These lines of evidence imply a direct relationship between atRAL toxicity and photoreceptor atrophy. Masutomi and coworkers (17Masutomi K. Chen C. Nakatani K. Koutalos Y. All-trans retinal mediates light-induced oxidation in single living rod photoreceptors.Photochem. Photobiol. 2012; 88: 1356-1361Crossref PubMed Scopus (20) Google Scholar) disclose that atRAL evokes photoinduced oxidation in rod photoreceptors. Previous studies also show that atRAL causes cell death in a murine photoreceptor cell line (661W) (18Sawada O. Perusek L. Kohno H. Howell S.J. Maeda A. Matsuyama S. Maeda T. All-trans-retinal induces Bax activation via DNA damage to mediate retinal cell apoptosis.Exp. Eye Res. 2014; 123: 27-36Crossref PubMed Scopus (37) Google Scholar). Most recently, we introduce that activation of c-Jun N-terminal kinase (JNK) promotes photoreceptor apoptosis induced by atRAL, and blocking JNK significantly mitigates photoreceptor atrophy and apoptosis in Abca4−/−Rdh8−/− mice subjected to light exposure (19Liao C. Cai B. Feng Y. Chen J. Wu Y. Zhuang J. Liu Z. Wu Y. Activation of JNK signaling promotes all-trans-retinal-induced photoreceptor apoptosis in mice.J. Biol. Chem. 2020; 295: 6958-6971Abstract Full Text Full Text PDF PubMed Scopus (6) Google Scholar). These findings suggest that patients with dry AMD and STGD1 may benefit from the suppression of photoreceptor apoptosis. However, we cannot exclude the possibility that nonapoptotic processes involve the death of photoreceptor cells in dry AMD and STGD1. In 2012, Stockwell and coworkers reported ferroptosis, a unique form of nonapoptotic cell death (20Dixon S.J. Lemberg K.M. Lamprecht M.R. Skouta R. Zaitsev E.M. Gleason C.E. Patel D.N. Bauer A.J. Cantley A.M. Yang W.S. Morrison B. Stockwell B.R. Ferroptosis: an iron-dependent form of nonapoptotic cell death.Cell. 2012; 149: 1060-1072Abstract Full Text Full Text PDF PubMed Scopus (5632) Google Scholar). Ferroptosis, different from apoptosis, necrosis, and autophagic cell death based on morphological, biochemical, and genetical criteria (20Dixon S.J. Lemberg K.M. Lamprecht M.R. Skouta R. Zaitsev E.M. Gleason C.E. Patel D.N. Bauer A.J. Cantley A.M. Yang W.S. Morrison B. Stockwell B.R. Ferroptosis: an iron-dependent form of nonapoptotic cell death.Cell. 2012; 149: 1060-1072Abstract Full Text Full Text PDF PubMed Scopus (5632) Google Scholar), features lipid peroxidation and depends on iron and lipid-based reactive oxygen species (lipid ROS) (20Dixon S.J. Lemberg K.M. Lamprecht M.R. Skouta R. Zaitsev E.M. Gleason C.E. Patel D.N. Bauer A.J. Cantley A.M. Yang W.S. Morrison B. Stockwell B.R. Ferroptosis: an iron-dependent form of nonapoptotic cell death.Cell. 2012; 149: 1060-1072Abstract Full Text Full Text PDF PubMed Scopus (5632) Google Scholar, 21Yang W.S. Stockwell B.R. Ferroptosis: death by lipid peroxidation.Trends Cell Biol. 2016; 26: 165-176Abstract Full Text Full Text PDF PubMed Scopus (1238) Google Scholar, 22Dixon S.J. Stockwell B.R. The role of iron and reactive oxygen species in cell death.Nat. Chem. Biol. 2014; 10: 9-17Crossref PubMed Scopus (1281) Google Scholar). Several lines of investigation have identified prostaglandin-endoperoxide synthase 2 (Ptgs2) and its gene product cyclooxygenase 2 (COX2) as downstream molecular markers of ferroptosis (23Yang W.S. SriRamaratnam R. Welsch M.E. Shimada K. Skouta R. Viswanathan V.S. Cheah J.H. Clemons P.A. Shamji A.F. Clish C.B. Brown L.M. Girotti A.W. Cornish V.W. Schreiber S.L. Stockwell B.R. Regulation of ferroptotic cancer cell death by GPX4.Cell. 2014; 156: 317-331Abstract Full Text Full Text PDF PubMed Scopus (2749) Google Scholar, 24Li Q. Han X. Lan X. Gao Y. Wan J. Durham F. Cheng T. Yang J. Wang Z. Jiang C. Ying M. Koehler R.C. Stockwell B.R. Wang J. Inhibition of neuronal ferroptosis protects hemorrhagic brain.JCI Insight. 2017; 2e90777Crossref PubMed Scopus (368) Google Scholar). Acyl-CoA synthetase long-chain family member 4 (ACSL4) is a convertor of free fatty acids into fatty CoA esters (25Grevengoed T.J. Klett E.L. Coleman R.A. Acyl-CoA metabolism and partitioning.Annu. Rev. Nutr. 2014; 34: 1-30Crossref PubMed Scopus (228) Google Scholar). The induction of ACSL4 contributes significantly to ferroptosis execution by catalyzing the formation of polyunsaturated fatty acid (PUFA)-containing phospholipids required for the production of lipid peroxidation products (26Yuan H. Li X. Zhang X. Kang R. Tang D. Identification of ACSL4 as a biomarker and contributor of ferroptosis.Biochem. Biophys. Res. Commun. 2016; 478: 1338-1343Crossref PubMed Scopus (423) Google Scholar, 27Doll S. Proneth B. Tyurina Y.Y. Panzilius E. Kobayashi S. Ingold I. Irmler M. Beckers J. Aichler M. Walch A. Prokisch H. Trümbach D. Mao G. Qu F. Bayir H. et al.ACSL4 dictates ferroptosis sensitivity by shaping cellular lipid composition.Nat. Chem. Biol. 2017; 13: 91-98Crossref PubMed Scopus (1284) Google Scholar). System Xc− is a cystine–glutamate antiporter that affords sufficient levels of cystine used for the synthesis of antioxidant tripeptide glutathione (GSH) in nearly all living cells (28Ishii T. Sugita Y. Bannai S. Regulation of glutathione levels in mouse spleen lymphocytes by transport of cysteine.J. Cell. Physiol. 1987; 133: 330-336Crossref PubMed Scopus (136) Google Scholar). Inhibition of system Xc− activity by erastin in cancer cells triggers ferroptosis and results in a compensatory transcriptional upregulation of solute carrier family 7 membrane 11 (SLC7A11), a key component of system Xc− (20Dixon S.J. Lemberg K.M. Lamprecht M.R. Skouta R. Zaitsev E.M. Gleason C.E. Patel D.N. Bauer A.J. Cantley A.M. Yang W.S. Morrison B. Stockwell B.R. Ferroptosis: an iron-dependent form of nonapoptotic cell death.Cell. 2012; 149: 1060-1072Abstract Full Text Full Text PDF PubMed Scopus (5632) Google Scholar, 29Lo M. Ling V. Wang Y.Z. Gout P.W. The xc-cystine/glutamate antiporter: a mediator of pancreatic cancer growth with a role in drug resistance.Br. J. Cancer. 2008; 99: 464-472Crossref PubMed Scopus (145) Google Scholar). In addition, ferroptotic cancer cells exhibit smaller than normal mitochondria with increased membrane density and collapsed mitochondrial cristae (20Dixon S.J. Lemberg K.M. Lamprecht M.R. Skouta R. Zaitsev E.M. Gleason C.E. Patel D.N. Bauer A.J. 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Ding X.Q. Saadi A. Agarwal N. Naash M.I. Al-Ubaidi M.R. Expression of cone-photoreceptor-specific antigens in a cell line derived from retinal tumors in transgenic mice.Invest. Ophthalmol. Vis. Sci. 2004; 45: 764-768Crossref PubMed Scopus (253) Google Scholar). The results of the MTS assay shown in Figure 1A demonstrated that atRAL diminished the viability of 661W photoreceptor cells in a concentration- and time-dependent manner. Treating 661 W photoreceptor cells with atRAL for 3 and 6 h at a concentration of 5 μM gave rise to significant decreases in cell viability of approximately 26.3 and 39.8%, respectively. When exposed to atRAL for 3 and 6 h at concentrations starting from 2.5 μM, 661W photoreceptor cells exhibited altered morphology, which is characterized by rounding, shrinkage, and cytoplasmic rupture (Fig. 1B). On the basis of MTS data and morphological images, atRAL at the concentration of 5 μM was used to treat 661W photoreceptor cells for 3 or 6 h in subsequent experiments. Evidence from our recent research has indicated that 5-μM atRAL triggers apoptosis of 661W photoreceptor cells after 6 h of treatment (19Liao C. Cai B. Feng Y. Chen J. Wu Y. Zhuang J. Liu Z. Wu Y. Activation of JNK signaling promotes all-trans-retinal-induced photoreceptor apoptosis in mice.J. Biol. Chem. 2020; 295: 6958-6971Abstract Full Text Full Text PDF PubMed Scopus (6) Google Scholar). However, to further ascertain whether atRAL-loaded photoreceptor cells suffer from nonapoptotic forms of cell death, we used quantitative reverse transcription–polymerase chain (qRT-PCR) and western blotting to analyze the expression of recognized ferroptosis-related genes and proteins, respectively. Interestingly, 5-μM atRAL significantly and time-dependently increased mRNA levels of Ptgs2 and Acsl4 in 661W photoreceptor cells (Fig. 1C). Immunoblot analysis also indicated that 5-μM atRAL elicited a significant time-dependent elevation in protein levels of COX2 encoded by Ptgs2 gene in lysates of 661W photoreceptor cells (Fig. 1, D–E). By contrast, protein expression of ACSL4 in lysates of atRAL-treated 661W photoreceptor cells showed a trend of increase from 3 to 6 h, but manifested statistical significance at 6 h (Fig. 1, F–G). Deferoxamine mesylate salt (DFO) is a ferrous ion (Fe2+) chelator that relieves ferroptotic cell death (20Dixon S.J. Lemberg K.M. Lamprecht M.R. Skouta R. Zaitsev E.M. Gleason C.E. Patel D.N. Bauer A.J. Cantley A.M. Yang W.S. Morrison B. Stockwell B.R. Ferroptosis: an iron-dependent form of nonapoptotic cell death.Cell. 2012; 149: 1060-1072Abstract Full Text Full Text PDF PubMed Scopus (5632) Google Scholar). The results of MTS assay showed that treatment with DFO at concentrations of 50, 100, and 200 μM concentration-dependently and effectively protected 661W photoreceptor cells from ferroptosis caused by 5-μM atRAL (Fig. 1H). More importantly, treatment with 10- and 20-μM ferrostatin-1 (Fer-1), a selective ferroptosis inhibitor (38Skouta R. Dixon S.J. Wang J. Dunn D.E. Orman M. Shimada K. Rosenberg P.A. Lo D.C. Weinberg J.M. Linkermann A. Stockwell B.R. Ferrostatins inhibit oxidative lipid damage and cell death in diverse disease models.J. Am. Chem. Soc. 2014; 136: 4551-4556Crossref PubMed Scopus (551) Google Scholar, 39Miotto G. Rossetto M. Di P. Orian L. Venerando R. Roveri A. Vučković A. Bosello T. Zaccarin M. Zennaro L. Maiorino M. Toppo S. Ursini F. Cozza G. 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Imaging of Fe2+ using FeRhoNox-1 showed that 5-μM atRAL dramatically enhanced intracellular Fe2+ levels in 661W photoreceptor cells at 3 to 6 h of exposure (Fig. 2, A–B). The mechanisms by which cellular iron homeostasis is modulated have been well elucidated (41Yanatori I. Kishi F. DMT1 and iron transport.Free Radic. Biol. Med. 2019; 133: 55-63Crossref PubMed Scopus (99) Google Scholar, 42Shu W. Dunaief J.L. Potential treatment of retinal diseases with iron chelators.Pharmaceuticals (Basel). 2018; 11: 112Crossref Scopus (24) Google Scholar). Transferrin (TF) captures ferric iron (Fe3+) and binds to transferrin receptor (TFRC). Cellular uptake of iron, which maintains intracellular iron pool, is accomplished by TFRC–mediated endocytosis of the complex into specialized endosomes where Fe3+ is reduced to Fe2+ by six-transmembrane epithelial antigen of prostate 3 (STEAP3). Divalent metal transporter 1 (DMT1) serves to deliver Fe2+ from the endosomes to cytoplasm where some of Fe2+ is stored in ferritin including ferritin heavy chain 1 (FTH1) and ferritin light chain 1 (FTL1). Instead, ferritinophagy modulated by a cargo protein called nuclear receptor coactivator 4 (NOCA4) leads to ferritin degradation and thereby releases Fe2+. Ferroportin (FPN) moves Fe2+ out of the cells in collaboration with ferroxidases ceruloplasmin (CP) or hephaestin (HEPH). Iron-responsive element binding protein 2 (IREB2) is an RNA binding protein that regulates transcript stability (43Coon K.D. Siegel A.M. Yee S.J. Dunckley T.L. Mueller C. Nagra R.M. Tourtellotte W.W. Reiman E.M. Papassotiropoulos A. Petersen F.F. Stephan D.A. Kirsch W.M. Preliminary demonstration of an allelic association of the IREB2 gene with Alzheimer's disease.J. Alzheimers Dis. 2006; 9: 225-233Crossref PubMed Scopus (19) Google Scholar, 44Hentze M.W. Rouault T.A. Harford J.B. Klausner R.D. Oxidation-reduction and the molecular mechanism of a regulatory RNA-protein interaction.Science. 1989; 244: 357-359Crossref PubMed Scopus (293) Google Scholar). A previous study has revealed that knockdown of IREB2 expression by RNA interference can restrict erastin-induced ferroptosis (20Dixon S.J. Lemberg K.M. Lamprecht M.R. Skouta R. Zaitsev E.M. Gleason C.E. Patel D.N. Bauer A.J. Cantley A.M. Yang W.S. Morrison B. Stockwell B.R. Ferroptosis: an iron-dependent form of nonapoptotic cell death.Cell. 2012; 149: 1060-1072Abstract Full Text Full Text PDF PubMed Scopus (5632) Google Scholar). In the current investigation, mRNA levels of iron homeostasis-related genes in atRAL-loaded 661W photoreceptor cells were examined by qRT-PCR. We observed that treatment with 5-μM atRAL significantly upregulated mRNA levels of Tf, Tfrc, Steap3, Dmt1, Ireb2, Fth1, Ftl1, Ncoa4, and Cp, but clearly downregulated the expression of Heph and Fpn genes (Fig. 2C). These results imply that atRAL disrupts iron homeostasis and thereby increases Fe2+, which may contribute to photoreceptor ferroptosis. One of critical ferroptosis hallmarks is lipid peroxidation (20Dixon S.J. Lemberg K.M. Lamprecht M.R. Skouta R. Zaitsev E.M. Gleason C.E. Patel D.N. Bauer A.J. Cantley A.M. Yang W.S. Morrison B. Stockwell B.R. Ferroptosis: an iron-dependent form of nonapoptotic cell death.Cell. 2012; 149: 1060-1072Abstract Full Text Full Text PDF PubMed Scopus (5632) Google Scholar, 21Yang W.S. Stockwell B.R. Ferroptosis: death by lipid peroxidation.Trends Cell Biol. 2016; 26: 165-176Abstract Full Text Full Text PDF PubMed Scopus (1238) Google Scholar, 45Conrad M. Kagan V.E. Bayir H. Pagnussat G.C. Head B. Traber M.G. Stockwell B.R. Regulation of lipid peroxidation and ferroptosis in diverse species.Genes Dev. 2018; 32: 602-619Crossref PubMed Scopus (232) Google Scholar). 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Moreover, we employed C11-BODIPY staining and flow cytometry to quantify lipid peroxidation in 661W photoreceptor cells exposed to 5-μM atRAL for 6 h and found that levels of lipid peroxides remarkably increased (Fig. 3B). Based on these findings, we conclude that atRAL provokes photoreceptor ferroptosis by accentuating lipid peroxidation. GSH is indispensable for physiologically defensive responses against oxidative stress (48Owen J.B. Butterfield D.A. Measurement of oxidized/reduced glutathione ratio.Methods Mol. Biol. 2010; 648: 269-277Crossref PubMed Scopus (232) Google Scholar), and its depletion is also regarded as a cause of ferroptosis onset (20Dixon S.J. Lemberg K.M. Lamprecht M.R. Skouta R. Zaitsev E.M. Gleason C.E. Patel D.N. Bauer A.J. Cantley A.M. Yang W.S. Morrison B. Stockwell B.R. Ferroptosis: an iron-dependent form of nonapoptotic cell death.Cell. 2012; 149: 1060-1072Abstract Full Text Full Text PDF PubMed Scopus (5632) Google Scholar, 49Tang H. Chen D. Li C. Zheng C. 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