2018-Ingold-需要GPX4的硒利用来防止氢过氧化物诱导的Ferroptosis.pdf

ArticleSelenium Utilization by GPX4 Is Required to PreventHydroperoxide-Induced FerroptosisGraphical AbstractHighlightsdSelenium-containing GPX4 is necessary for full viabilityof micedThe GPX4-Cys variant is highly susceptible tohydroperoxide-induced inactivationdHydroperoxide induces ferroptosis in Gpx4cys/cyscellsdGPX4-Cys bypasses the requirement of selenoproteins forcell viabilityAuthorsIrina Ingold,Carsten Berndt,Sabine Schmitt,.,Hans Zischka,Jose Pedro Friedmann Angeli,Marcus ConradCorrespondencemarcus.conradhelmholtz-muenchen.deIn BriefThe trace element selenium protects acritical population of interneurons fromferroptotic cell death.Ingold et al.,2018,Cell 172,409422January 25,2018 2017 Elsevier Inc.https:/doi.org/10.1016/j.cell.2017.11.048ArticleSelenium Utilization by GPX4 Is Requiredto Prevent Hydroperoxide-Induced FerroptosisIrina Ingold,1Carsten Berndt,2Sabine Schmitt,3Sebastian Doll,1Gereon Poschmann,4Katalin Buday,1Antonella Roveri,5Xiaoxiao Peng,6Florencio Porto Freitas,1Tobias Seibt,7Lisa Mehr,1Michaela Aichler,8Axel Walch,8Daniel Lamp,9,10Martin Jastroch,9,10Sayuri Miyamoto,11Wolfgang Wurst,1,12,13Fulvio Ursini,5Elias S.J.Arne r,14Noelia Fradejas-Villar,15Ulrich Schweizer,15Hans Zischka,3,16Jose Pedro Friedmann Angeli,1,17and Marcus Conrad1,18,*1Helmholtz Zentrum Mu nchen,Institute of Developmental Genetics,85764 Neuherberg,Germany2Heinrich-Heine University,Department of Neurology,Medical Faculty,40255 Du sseldorf,Germany3Institute of Toxicology and Environmental Hygiene,Technical University of Munich,80802 Munich,Germany4Heinrich-Heine University,Molecular Proteomics Laboratory,Biomedical Research Center(BMFZ),40225 Du sseldorf,Germany5Department of Molecular Medicine,University of Padova,Padova,Italy6CVMD Translational Medicine Unit,Early Clinical Development,IMED Biotech Unit,AstraZeneca,Gothenburg,Sweden7Department of Nephrology,Medizinische Klinik und Poliklinik IV,Klinikum der Universita t Mu nchen,80336 Mu nchen,Germany8Helmholtz Zentrum Mu nchen,Research Unit of Analytical Pathology,85764 Neuherberg,Germany9Helmholtz Zentrum Mu nchen,Helmholtz Diabetes Center and German Diabetes Center(DZD),85764 Neuherberg,Germany10Helmholtz Zentrum Mu nchen,Institute for Diabetes and Obesity,85748 Garching,Germany11Departamento de Bioqu mica,Instituto de Qu mica,Universidade de Sa o Paulo,Sa o Paulo,Brazil12German Center for Neurodegenerative Diseases(DZNE),81377 Munich,Germany13Technische Universita t Mu nchen-Weihenstephan,Chair of Developmental Genetics,c/o Helmholtz Zentrum Mu nchen,85764 Neuherberg,Germany14Division of Biochemistry,Department of Medical Biochemistry and Biophysics,Karolinska Institutet,171 77 Stockholm,Sweden15Rheinische Friedrich-Wilhelms-University Bonn,Institute for Biochemistry and Molecular Biology,53115 Bonn,Germany16Helmholtz Zentrum Mu nchen,Institute of Molecular Toxicology and Pharmacology,85764 Neuherberg,Germany17Present address:Rudolf Virchow Center for Experimental Biomedicine,University of Wu rzburg,97080 Wu rzburg,Germany18Lead Contact*Correspondence:marcus.conradhelmholtz-muenchen.dehttps:/doi.org/10.1016/j.cell.2017.11.048SUMMARYSelenoproteins are rare proteins amongall kingdomsof life containing the 21stamino acid,selenocysteine.Selenocysteine resembles cysteine,differing onlyby the substitution of selenium for sulfur.Yetthe actual advantage of selenolate-versus thiolate-based catalysis has remained enigmatic,as most oftheknownselenoproteinsalsoexistascysteine-con-taining homologs.Here,we demonstrate that sele-nolate-based catalysis of the essential mammalianselenoprotein GPX4 is unexpectedly dispensablefor normal embryogenesis.Yet the survival of a spe-cific type of interneurons emerges to exclusivelydepend onselenocysteine-containing GPX4,therebypreventing fatal epileptic seizures.Mechanistically,selenocysteine utilization by GPX4 confers exqui-siteresistancetoirreversibleoveroxidationascells expressing a cysteine variant are highly sensi-tive toward peroxide-induced ferroptosis.Remark-ably,concomitant deletion of all selenoproteins inGpx4cys/cyscells revealed that selenoproteins aredispensable for cell viability provided partial GPX4activity is retained.Conclusively,200 years after itsdiscovery,a specific and indispensable role for sele-nium is provided.INTRODUCTIONThe trace element selenium(Se)was discovered two centuriesago(in1817)bytheSwedishscientistJo nsJacobBerzelius(Ber-zelius,1818).Inbiological systems,Seexerts its essential role asthe 21stamino acid,selenocysteine(Sec).Sec incorporation,atthe opalcodon UGA,isa highlycomplexand energetically costlyprocess(Hatfield et al.,2014).Despite the complexity for Secusage,it is recognized that Se in form of Sec is indispensablefor mammalian life(Bo sl et al.,1997).This is supported by theembryonic lethal phenotype of mice deficient for the Sec-spe-cific tRNA gene Trsp(nuclear encoded tRNA selenocysteine 2anticodon TCA)(Bo sl et al.,1997).Because Sec differs fromcysteine(Cys)only by the replacement of sulfur for Se andbecause Cys incorporation presents a canonical translationalinsertion,the actual biological advantage of selenolate-basedover thiolate-based catalysis has remained elusive.Remarkably,while some organisms like higher plants and fungi use the readilyavailable sulfur to express Cys-homologs,mammals,fish,birds,nematodes,and bacteria still maintain the energetically costlyand inefficient process of selenoprotein expression(Lobanovet al.,2007).Model studies in mice deficient for individual selenoproteinshave indicated that the only protein closely mimicking loss ofTrsp is glutathione peroxidase 4(GPX4).Constitutive deletionof Gpx4 causes embryonic death almost at the same develop-mental stage as Trsp knockout mice(Yant et al.,2003).More-over,tissue-specific knockout approaches unveiled that loss ofCell 172,409422,January 25,2018 2017 Elsevier Inc.409Figure 1.Gpx4cys/cysMice Develop Normally but Fail to Survive the Pre-weaning Age(A)Genetargeting strategy forthetargeted conversion ofSectoCys inGpx4.Intheupper line,thewild-type(WT)allele ofGpx4 withthecritical exon3highlightedin red is shown.In the lower part,the targeting vector used to generate the point mutation in exon 3,where the UGA codon(marked with an asterisk)is located,isshown.For homologous recombination in F1 embryonic stem(ES)cells,the neomycin phosphotransferase gene(neo)and thymidine kinase gene(TK)were usedas positive and negative selection marker,respectively.BS,pBluescript vector backbone.(B)ES cell with homologous recombination(HR)of the targeting construct were identified by long range PCR spanning the 30arm.Germline transmission(GT)ofthe targeted allele was confirmed by PCR from ear punch DNA(one representative clone out of 25 is shown).(C)Sequencing of the region covering the critical exon 3 confirmed the targeted mutation in the active site of Gpx4(UGA/UGC)in mice heterozygous andhomozygous for the targeted Gpx4 allele(for each genotype one representative chromatogram is shown).(D)GPX4-specificactivitywasundetectable intissuesderived fromGpx4cys/cysanimals usingPCOOHassubstrate(datarepresent meanSDofn=3tissuespergenotype;statistical analysis was conducted using two-tailed t test*p 0.01).(E)Gpx4cys/cysanimals were normal in appearance but tended to loose body weight between P14P16(P=postnatal day)(data represent mean SD of n=3animals per genotype).(legend continued on next page)410Cell 172,409422,January 25,2018Gpx4 alone often phenocopied the effects induced by condi-tional Trsp deletion as demonstrated for certain neurons andepidermis(Sengupta et al.,2010;Wirth et al.,2010,2014).Inthe present work,we took advantage of this specific character-istic of GPX4 and challenged the relevance and importance ofselenolate-versus thiolate-based catalysis by generating micewith targeted mutation of the active site Sec to Cys.Datapresented herein provide hitherto unrecognized and intriguinginsights into the requirement for Se utilization in mice and estab-lish an essential function for GPX4-Sec-based catalysis in sup-pressing peroxide-induced ferroptosis.RESULTSSelenolate-Based GPX4 Catalysis Is Dispensable forNormal Embryogenesis but Essential for the Survival ofParvalbumin-Positive Interneurons and Prevention ofSeizuresThegenerationofmicewithatargetedmutationofthecatalyticallyactive Sec to Cys of GPX4 is depicted in Figures 1A1C.Upongermline transmission,breeding of heterozygous Gpx4wt/cysmice was setup to examine whether homozygous Gpx4cys/cysmice are viable.Unexpectedly,homozygous Gpx4cys/cysmicedeveloped normally and were born at the expected Mendelianratio(28%)(Table 1).Thisisinstark contrast to systemic Gpx4?/?or Gpx4ser/ser(enzymatically inactive)mice,which were bothshown to die in utero as early as E7.5(E,embryonic day)(Brutschet al.,2015;Ingold et al.,2015).Next,GPX4-specific activity indifferent tissues derived from wild-type(WT)and Gpx4cys/cyspups was measured using phosphatidylcholine hydroperoxide(PCOOH)as substrate.Yet,we were unable to detect anyGPX4-specific activity in kidney and brain extracts of Gpx4cys/cysanimals(Figure 1D).Although Gpx4cys/cysmice were born normally(Table 1),ho-mozygous mice appeared to lose body weight by P14P16(P,postnatal day)(Figure 1E);by P18 all animals had to be sacri-ficed(Figure 1F).Gpx4cys/cysanimals showed severe sponta-neous seizures or were hyperexcitable(see Movie S1).Becauseparvalbumin-positive(PV+)GABAergic interneurons are impor-tant regulators of cortical network excitability(Mihaly et al.,1997;Schwaller et al.,2004)and mature between P8 and P16,we asked whether seizures are caused by a lack of thesespecialized neurons.In fact,staining for parvalbumin(PV)showed a marked decrease of PV+cells in the cortex ofGpx4cys/cysmice(Figures 1G and S1A),whereas the number ofcalbindin-and calretinin-positive neurons was unaltered(Fig-ure S1B).Along with the decrease of PV+interneurons,anincreased number of TUNEL(terminal deoxynucleotidyl trans-ferase dUTP nick end labeling)-positive cells was detectable inthe cortex of Gpx4cys/cysmice(Figures 1G and S1A).Cell deathcoincided with an increased immunohistological staining of glialfibrillary acidic protein and ionized calcium binding adaptormolecule 1,suggestive of reactive astrogliosis and neuroinflam-mation,respectively(Figure 1G).Hence,GABAergic PV+inter-neurons emerge to exclusivelydepend on Se-containingGPX4,which we identify here to be the limiting factor for survivalof mice on a mixed C57BL/6J x 129S6SvEv genetic background.During backcrossing Gpx4wt/cysmice on a C57BL/6J back-ground for more than 78 generations and subsequent inter-cross of heterozygous Gpx4wt/cysmice,we failed to obtain viablehomozygous mutant animals.A ratio of 74%for Gpx4wt/cysand26%for Gpx4wt/wtsuggested embryonic death of homozygousGpx4cys/cysembryos.Histopathological analysis of embryos iso-lated at different times of gestation revealed that embryos diedbetween E11.5 and E12.5(Figure S1C;Table S1).Overall,mutant embryos showed severe malformations of the brain,growth retardation,hemorrhages,and generalized paleness(Figures S1C and S1D).Crossbreeding of Gpx4wt/cysmice(on a C57BL/6J background)with 129S6SvEv WT mice and sub-sequent intercross of F1 Gpx4wt/cysmice allowed to regain theinitial phenotype,indicating that the background has a strongimpact on the severity of the phenotype of Gpx4cys/cysembryos.Thiolate-Based GPX4 Catalysis Permits Survival ofAdult MiceTo bypass early embryonic death or pre-weaning lethality ofGpx4cys/cysmice and to address whether the Cys variant ofGPX4(GPX4-Cys)is able to sustain viability in adult mice,wecross-bred Gpx4wt/cysmice(and Gpx4wt/seras controls)(Ingoldet al.,2015)with mice harboring loxP-flanked(floxed)Gpx4alleles and transgenic for tamoxifen(TAM)-inducible CreERT2(Friedmann Angeli et al.,2014)(Figure 2A).TAM injection resultsin whole body deletion of the floxed Gpx4 allele(except inbrain),while still expressing the Cys or the serine(Ser)variantof GPX4.TAM injection caused loss of GPX4 expression inGpx4flox/wt;Rosa26_CreERT2animalstosomeextent(agene-dosageeffect has been previously reported)(Friedmann Angeli et al.,2014;Yant et al.,2003),whereas expression of mutant GPX4(F)Gpx4cys/cysanimalsdiedduetosuddendeathorhadtobesacrificedastheysufferedfromseverespontaneousepilepticseizures(seeMovieS1).Theyfailedtosurvive beyond the third week after birth(Kaplan Meyer:statistical survival analysis was conducted using Mantel-Cox test*p 0.0001,n=13 Gpx4wt/wt,39 Gpx4wt/cys or 23 Gpx4cys/cys animals).(G)Immunohistological analysis of brain obtained from Gpx4cys/cysmice and Gpx4wt/wtlittermates at the age of 16 days after birth(P16)revealed the presence ofterminal deoxynucleotidyl transferase dUTP nick end labeling(TUNEL)-positive cells in cortex of homozygous mutant mice,which were absent in WT samples.Whileparvalbumin-positive(PV+)interneuronsweredramaticallydecreased inGpx4cys/cysanimals,theyshowedanincreaseinglialfibrillary acidic protein(GFAP)and ionized calcium-binding adaptor molecule 1(IBAI)staining,indicating reactiveastrogliosis and microglia activation,respectively.One representativestainingis shown of 5 brain tissues per genotype.Scale bar,10 mm.See also Figure S1A.Table 1.Genotyping of Mice Obtained from HeterozygousGpx4cys/wtBreedingswt/wtwt/cyscys/cysTotal25(20.8%)61(50.8%)34(28.3%)120Homozygous pups of heterozygous Gpx4cys/wtbreeding were born at theexpected Mendelian ratio.Cell 172,409422,January 25,2018411was maintained in both induced Gpx4flox/ser;Rosa26_CreERT2andGpx4flox/cys;Rosa26_CreERT2mice(Figure 2B).Survival analysisrevealed that induced Gpx4flox/ser;Rosa26_CreERT2mice phenocop-ied Gpx4flox/flox;Rosa26_CreERT2mice leading to mouse lethality?11 days after TAM administration(Friedmann Angeli et al.,2014)(Figure 2C).By stark contrast,Gpx4flox/cys;Rosa26_CreERT2mice survived the entire observation period of 40 dayslike Gpx4flox/wt;Rosa26_CreERT2control mice without showingany signs of kidney damage(Figures 2C,2D,and S2).Gpx4flox/ser;Rosa26_CreERT2animals,however,died of acute renalfailure(ARF)and presented the same histopathological pheno-typeinkidneyasreportedforinducibleGpx4nullmice(Figure2D)(Friedmann Angeli et al.,2014).These findings demonstrate thatSecofGPX4canbesubstitutedbyCysinadultanimals,whereasSec appears to be essential during specific developmentalevents independent of the genetic background.Figure 2.Survival of Adult Animals Requires Just the Cys Variant of GPX4(A)Breeding scheme describing the mating steps of Gpx4wt/cysor Gpx4wt/serwith a mouse strain expressing a loxP-flanked Gpx4 allele and atamoxifen(TAM)-inducible Cre recombinase under the control of the Rosa26 locus(Gpx4fl/fl;Rosa26_CreERT2),further referred to as Gpx4flox/ser;Rosa26_CreERT2,Gpx4flox/cys;Rosa26_CreERT2,and Gpx4flox/wt;Rosa26_CreERT2mice.(B)Analysis of GPX4 expression in kidney tissues derived from Gpx4flox/ser;Rosa26_CreERT2,Gpx4flox/cys;Rosa26_CreERT2,and Gpx4flox/wt;Rosa26_CreERT2animals re-vealed decreased protein levels in Gpx4flox/wt;Ros