Broad AOX expression in a genetically tractable mouse model does not disturb normal physiology

RESEARCH ARTICLE B oad AOX exp ession in a gene ically ac able mouse model does no dis u b no mal physiology Ma en Szibo 1,2,3, *, P a een K. Dhandapani 1,2, *, E ic Du ou 2 , Ki a M. Holms o m 1,2 , Yuan Zhuang 1 , Isabelle Salwig 3 , Ilka Wi ig 4,5,6 , Juliana Heidle 4 , Zem i a Giza ullina 7 , Timu Gainu dino 7 , Ge man Mouse Clinic Conso ium § , Helmu Fuchs 8 , Vale ie Gailus-Du ne 8 , Ma in H abe de Angelis 8,9,10 , Ja in Nandania 11 , Vidya Velagapudi 11 , As id Wie elmann 3 , Pie e Rus in 12 , F ank N. Gelle ich 7,13 , Howa d T. Jacobs 1,2,‡,¶ and Thomas B aun 3,‡ ABSTRACT Plan s and many lowe o ganisms, bu no mammals, exp ess al e na i e oxidases (AOXs) ha b anch he mi ochond ial espi a o y chain, ans e ing elec ons di ec ly om ubiquinol o oxygen wi hou p o on pumping. Thus, hey main ain elec on low unde condi ions when he classical espi a o y chain is impai ed, limi ing excess p oduc ion o oxygen adicals and suppo ing edox and me abolic homeos asis. AOX om Ciona in es inalis has been used o s udy and mi iga e mi ochond ial impai men s in mammalian cell lines, D osophila disease models and, mos ecen ly, in he mouse, whe e mul iple len i ec o -AOX ansgenes con e ed subs an ial exp ession in speci ic issues. He e, we desc ibe a gene ically ac able mouse model in which Ciona AOX has been a ge ed o he Rosa26 locus o ubiqui ous exp ession. The AOX Rosa26 mouse exhibi ed only sub le pheno ypic e ec s on espi a o y complex o ma ion, oxygen consump ion o he global me abolome, and showed an essen ially no mal physiology. AOX con e ed obus esis ance o inhibi o s o he espi a o y chain in o ganello; mo eo e , animals exposed o a sys emically applied LD50 dose o cyanide did no succumb. The AOX Rosa26 mouse is a use ul ool o in es iga e espi a o y con ol mechanisms and o deciphe mi ochond ial disease ae iology in i o. KEY WORDS: Mi ochond ia, Mi ochond ial disease, Respi a o y chain, Al e na i e oxidase INTRODUCTION The mi ochond ial sys em o oxida i e phospho yla ion (OXPHOS) comp ises ou mul isubuni complexes suppo ing s epwise espi a o y elec on low om p ima y elec on accep o s o oxygen, and a i h complex (ATP syn hase) ha uses he p o on g adien he eby gene a ed ac oss he inne mi ochond ial memb ane o syn hesize ATP. In many lowe o ganism and plan s, al e na i e oxidases (AOXs) a e exp essed ha b anch he mi ochond ial espi a o y chain, hus ans e ing elec ons di ec ly om ubiquinol o oxygen in a non-p o on-mo i e manne . AOXs a e absen in mammals (Young e al., 2013) (Fig. 1A). Thei main physiological ole is o main ain elec on low unde condi ions when he classical espi a o y chain is impai ed, limi ing excess p oduc ion o oxygen adicals and suppo ing edox and me abolic homeos asis. Because AOX is also ound in some in e eb a e phyla (McDonald e al., 2009), we ha e p oposed ha i s exp ession in commonly s udied animal models could be used o elucida e he pa hophysiology unde lying mi ochond ial OXPHOS diso de s, p o iding a a ional basis o i s e en ual implemen a ion in he apeu ic applica ions (Rus in and Jacobs, 2009; El-Khou y e al., 2014). In ea lie s udies, AOX om he unica e Ciona in es inalis,a sis e g oup o he e eb a es, was shown o be exp essible and ca aly ically ac i e in human cells (Hakkaa e al., 2006). I was ound o alle ia e he dele e ious consequences o oxic o pa hological inhibi ion o he downs eam po ion o he mi ochond ial espi a o y chain (Hakkaa e al., 2006; Dassa e al., 2009), speci ically OXPHOS complexes III (cIII) and IV (cIV), which AOX bypasses. A cDNA encoding Ciona AOX was subsequen ly shown o be ubiqui ously exp essible in D osophila, wi hou elici ing any ha m ul pheno ypic e ec s (Fe nandez-Ayala e al., 2009). In he ly, AOX exp ession was able o compensa e a ange o pa hological pheno ypes a he whole-o ganism le el, including le hali y caused by OXPHOS poisons such as an imycin A o cyanide (Fe nandez-Ayala e al., 2009), locomo o dis u bance o neu odegene a ion caused by cIV knockdown (Kemppainen e al., 2014) o o he causes o neu odegene a ion mimicking Pa kinson’s (Fe nandez-Ayala e al., 2009; Humph ey e al., 2012) o Alzheime ’s (El-Khou y e al., 2016) diseases. The po en ial o using AOX o s udy mi ochond ial pa hophysiology a he whole-o ganism le el in mammals has been demons a ed using len i ec o ansduc ion, c ea ing a ansgenic mouse exp essing Ciona AOX in mul iple issues (El-Khou y e al., 2013). No ably, ha m ul pheno ypes we e again no seen, despi e widesp ead ansgene exp ession. Howe e , he me hodological issues a ising om he na u e o ha model ha e p ecluded i s widesp ead use. On inse ion o AOX ansgenes a mul iple genomic si es in he model, none o hem indi idually con e ed exp ession a a high le el o in all issues. Thus, he model could no be combined Recei ed 14 Sep embe 2016; Accep ed 30 No embe 2016 1 Ins i u e o Bio echnology, FI-00014 Uni e si y o Helsinki, Finland. 2 BioMediTech and Tampe e Uni e si y Hospi al, FI-33014 Uni e si y o Tampe e, Finland. 3 Max Planck Ins i u e o Hea and Lung Resea ch, Ca diac De elopmen and Remodelling (Depa men I), Bad Nauheim D-61231, Ge many. 4 Func ional P o eomics, SFB 815 Co e Uni , Facul y o Medicine, Goe he-Uni e si y, F ank u am Main D-60590, Ge many. 5 Ge man Cen e o Ca dio ascula Resea ch (DZHK), Pa ne si e RheinMain, F ank u , Ge many. 6 Clus e o Excellence “Mac omolecula Complexes”, Goe he-Uni e si y, F ank u am Main D-60590, Ge many. 7 Leibniz Ins i u e o Neu obiology, Magdebu g D-39118, Ge many. 8 Ge man Mouse Clinic, Ins i u e o Expe imen al Gene ics, Helmhol z Zen um Mu nchen, Ge man Resea ch Cen e o En i onmen al Heal h GmbH, Ingols aed e Lands asse 1, Neuhe be g 85764, Ge many. 9 Chai o Expe imen al Gene ics, Cen e o Li e and Food Sciences Weihens ephan, TU Munich, Emil-E lenmeye -Fo um 2, F eising-Weihens ephan 85350, Ge many. 10 Membe o Ge man Cen e o Diabe es Resea ch (DZD), Ingols aed e Lands asse 1, Neuhe be g 85764, Ge many. 11 Ins i u e o Molecula Medicine Finland, FI-00014 Uni e si y o Helsinki, Finland. 12 INSERM UMR 1141 and Uni e si ePa is 7, Ho pi al Robe Deb e, Pa is 75019, F ance. 13 Depa men o Neu ology, O o- on-Gue icke-Uni e si y, Magdebu g D-39120, Ge many. *These au ho s con ibu ed equally o his wo k. ‡ These au ho s sha e senio au ho ship. § A ull lis o conso ium membe s appea s in Supplemen a y in o ma ion ¶ Au ho o co espondence (howa d.jacobs@helsinki. i) T.G., 0000-0003-1723-1780; H.T.J., 0000-0003-1895-6003 This is an Open Access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion License (h p://c ea i ecommons.o g/licenses/by/3.0), which pe mi s un es ic ed use, dis ibu ion and ep oduc ion in any medium p o ided ha he o iginal wo k is p ope ly a ibu ed. 163 © 2017. Published by The Company o Biologis s L d | Disease Models & Mechanisms (2017) 10, 163-171 doi:10.1242/dmm.027839 Disease Models & Mechanisms wi h gene ic disease models o o he mouse mu an s, could no be p ac ically ans e ed in o o he s ain backg ounds, and i s long- e m main enance was essen ially impossible. He e, we epo he c ea ion o a gene ically ac able ansgenic mouse ha ubiqui ously exp esses a single copy o Ciona AOX a subs an ial le els, a e a ge ed inse ion in o he Rosa26 locus. The Rosa26 knock-in ga e ise o a unc ional enzyme, which con e ed esis ance o espi a o y poisons. Su p isingly, comp ehensi e pheno yping e ealed only mino , biologically inconsequen ial e ec s o AOX exp ession in he AOX Rosa26 mouse. The new model o e s g ea p omise as a ool o elucida ing he mechanisms o mi ochond ial pa hology and cha ing heway owa ds u u e he apies. RESULTS Cons uc ion o AOX Rosa26 mice To c ea e a gene ically ac able mouse model ubiqui ously exp essing Ciona AOX, we used gene a ge ing in o he ubiqui ously ac i e Rosa26 locus (Hi oshi e al., 1991) in mouse emb yonic s em cells (ESC) (So iano, 1999; S ini as e al., 2001). P e ious au ho s ha e epo ed no de ec able pa hological al e a ions a ising om inse ions a his locus (F ied ich and So iano, 1991; Zamb owicz e al., 1997), and ansgene exp ession seems o be s able (Zamb owicz e al., 1997). To boos exp ession om he Rosa26 locus, we inco po a ed he syn he ic CAG enhance -p omo e in o he cons uc (Fig. 1B; Fig. S1), which enhances exp ession se e al- old (Nyabi e al., 2009; Chen e al., 2011). A e e i ica ion o he inse ion in ESCs by Sou he n blo ing (Fig. S1B,C), a chime ic line was es ablished ia blas ocys injec ion, wi h subsequen elimina ion o he posi i e-selec able (neomycin esis ance) casse e (Fig. S1A,B) by Flp ecombina ion in i o, ollowing ge m-line ansmission. Founde s we e backc ossed o e mo e han se en gene a ions o s ain C57Bl/6J, wi h ansgene p esence checked a each s ep by PCR (Fig. S1D). The a e o ansmission o he AOX ansgene om he e ozygous pa en s o ei he sex did no signi ican ly de ia e om 50% (Fig. 1C), no was he e any signi ican pa en -o -o igin e ec on li e size (Fig. 1C). The p ogeny sex a io was also una ec ed by he AOX ansgene (Fig. 1D). AOX is ubiqui ously exp essed in he AOX Rosa26 mouse No he n blo ing (Fig. 1E) con i med widesp ead, hough somewha une en, exp ession wi h highes AOX mRNA le els in hea and skele al muscle, bu lowe exp ession in b ain, aking accoun o he Fig. 1. Cons uc ion and cha ac e iza ion o AOX Rosa26 mice. (A) Schema ic diag am o he mi ochond ial OXPHOS sys em, showing he i e s anda d OXPHOS complexes (I-V), he di usible elec on ca ie s ubiquinone (Q) and cy och ome c (c), and he passage o elec ons and p o ons esul ing ul ima ely in he syn hesis o ATP om ADP and ino ganic phospha e (Pi). The addi ional p esence o AOX, whe he supplied ansgenically o in o ganisms na u ally endowed wi h i , p o ides an al e na i e ou e o he eoxida ion o ubiquinol by molecula oxygen, wi hou p o on pumping. (B) Schema ic diag am o inse ed Rosa26-AOX exp ession cons uc , ollowing emo al o addi ional elemen s (i.e. DTA nega i e selec able ma ke upon a ge ed in eg a ion, and neomycin esis ance casse e ollowing FRT-media ed excision in i o). Remaining elemen s a e he CAG p omo e , AOX coding sequence and β-globin in on and poly(A) addi ion signal (βGi+pA). Fo ull de ails see Fig. S1A. (C) T ansmission a e o AOX ansgenes (based on PCR) and li e sizes, acco ding o sex o AOX-hemizygous pa en . T ansmission a es om male (n=93, 12 c osses) and om emale (n=43, 6 c osses) we e no signi ican ly di e en om each o he (S uden ’s - es , P>0.05, mean±s.d.) o om Mendelian expec a ion o 50% (chi-squa ed es ). Li e sizes p oduced by AOX-hemizygous males and emales also showed no signi ican di e ence (S uden ’s - es , P>0.05). (D) Sex (% o males) o ansgenic and wild- ype p ogeny o hemizygous AOX Rosa26 mice (n=136, 18 c osses), again showing no signi ican di e ences (S uden ’s - es , P>0.05, mean±s.d.). (E) No he n blo showing AOX exp ession in RNA (10 μg) om issues o one-yea -old, male, hemizygous AOX Rosa26 mice and wild- ype (w ) li e ma e con ols: He, hea ; Lu, lung; Li, li e ; B , b ain; Ki, kidney; Sp, spleen; Te, es is; Sk, skele al muscle. The blo was ep obed o A p5b mRNA as well as mi ochond ial 12S and cy osolic 18S RNAs as loading con ols. RNA molecula weigh s we e ex apola ed om RNA mig a ion in he e hidium b omide-s ained gel. 164 RESEARCH ARTICLE Disease Models & Mechanisms (2017) 10, 163-171 doi:10.1242/dmm.027839 Disease Models & Mechanisms loading con ols. A he p o ein le el, exp ession seemed mo e uni o m, bu was again highes in hea , skele al muscle and panc eas, and lowes in b ain (Fig. 2A; Fig. S2). B ain exp ession was highes in newbo n mice (Fig. S2C), bu declined subs an ially by one mon h o age (Fig. S2C). As expec ed, AOX exp ession was highe in homozygo es compa ed wi h he e ozygous animals (Fig. S2D). The enzyme was ound o be associa ed wi h he memb ane ac ion o isola ed mi ochond ia a e ca bona e ex ac ion (Fig. S2E), albei less igh ly bound han some in eg al memb ane p o eins o he OXPHOS complexes, such as subuni 1 o cIV (M co1). In each issue es ed, he exp ession o ep esen a i e subuni s o he i e OXPHOS complexes was essen ially una ec ed by AOX exp ession (Fig. 2A; Fig. S2A). Mo eo e , he o e all s uc u e o he espi a o y memb ane, speci ically i s o ganiza ion in o supe complexes, was simila ly unal e ed, based on blue-na i e elec opho esis (BNE) ollowed by in-gel his ochemis y o hea mi ochond ia (Fig. 2B), and on BNE combined wi h wes e n blo s o OXPHOS subuni s, o eigh di e en issues (Fig. S2F). In BNE gels, AOX i sel mig a ed mainly a he size o a dime and as mul ime s he eo (Fig. S2F,G), a he han associa ing speci ically wi h any o he espi a o y complex. In each issue es ed, he mobili y o he espi a o y chain complexes de ec ed by BNE was iden ical o ha in con ols (Fig. S2F). P incipal componen analysis o me aboli e le els in skele al muscle (Fig. 2C) and hea (Fig. S2H) showed no consis en e ec o AOX exp ession, no did any o 100 indi idual me aboli es analyzed show any signi ican di e ence (Tables S1, S2). AOX is unc ional in AOX Rosa26 mice We conduc ed espi ome y o de e mine whe he AOX is enzyma ically unc ional in he AOX Rosa26 knock-in mice. Mi ochond ia om six issues (Fig. 3) we e es ed in a s anda d p o ocol o oxygen consump ion in he p esence o complex I-, II- and IV-linked subs a es, successi ely using inhibi o s o cI ( o enone), cIII (an imycin A), AOX (n-p opyl galla e) and cIV (cyanide o azide). The e we e no signi ican di e ences when oxygen consump ion was compa ed wi h ha om mi ochond ia o wild- ype li e ma es, excep o subs a e oxida ion in he p esence o an imycin A (i.e. media ed by AOX), which was signi ican o all issues es ed excep b ain, whe e exp ession was low. Mi ochond ia om issues o AOX Rosa26 mice o he han b ain showed an imycin A- esis an (AOX-dependen ) oxygen consump ion be ween 30% and 70% o he uninhibi ed le el d i en by succina e (Fig. 3A), simila also o p elimina y measu emen s in he ounde mouse (Fig. S3A). In hea mi ochond ia om AOX-exp essing compa ed wi h con ol mice, an imycin A- and azide- esis an subs a e oxida ion was e iden ac oss a wide ange o d ug concen a ions (Fig. S3B). Compa ed wi h li e ma e con ols, mi ochond ial ROS p oduc ion d i en by succina e was g ea ly dec eased (Fig. 3B). In e es ingly, his was only signi ican in he absence o o enone, implica ing AOX in p o iding Fig. 2. AOX Rosa26 mice show b oad AOX exp ession and no mal me abolism. (A) Wes e n blo s o 20 μg o al p o ein ex ac s om he indica ed issues (He, hea ; Tm, high muscle; Mm, masse e muscle; Pa, panc eas; Lu, lung; Li, li e ; Ki, kidney; Sp, spleen; Ce, ce eb um; Cb, ce ebellum; Sg, sali a y gland; Te, es is) o 54-week-old male hemizygous AOX Rosa26 (+) and wild- ype li e ma e con ol (–) mice, p obed o AOX and o ep esen a i e subuni s o he i e OXPHOS complexes (see Ma e ials and Me hods, p o ein molecula weigh s ex apola ed om ma ke s). Fo Ponceau S s aining o he memb anes see Fig. S2B. See also Fig. S2A,C,D. (B) BNE gels o mi ochond ial memb ane p o eins om hemizygous AOX Rosa26 (+) and wild- ype li e ma e con ol (–) mice, s ained wi h Coomassie Blue (CB) o p obed by in-gel his ochemis y o he indica ed OXPHOS complexes. * deno es he mig a ion o he espec i e monome ic complexes. Assignmen o mi ochond ial complexes (I, cI; III 2 , dime ic cIII; IV, cIV; V, cV; S 1 , espi a o y supe complexes con aining cI, dime ic cIII and one copy o cIV) is based on p o ein molecula weigh s ex apola ed om he mig a ion o he complexes om bo ine hea mi ochond ia, whose subuni composi ion is known. (C) P incipal componen analysis o me abolome da a om skele al muscle o hemizygous AOX Rosa26 ( ed ci cles) and wild- ype li e ma e con ol mice (g een ci cles). The wo se s o analysed da a o e lap, apa om wo mino ou lie s om he con ol g oup. See also Fig. S2H. 165 RESEARCH ARTICLE Disease Models & Mechanisms (2017) 10, 163-171 doi:10.1242/dmm.027839 Disease Models & Mechanisms an al e na i e pa hway o succina e oxida ion o he han e e se elec on anspo h ough cI. AOX Rosa26 mice exhibi no mal physiology The high le el o AOX exp ession, capable o eplacing a la ge ac ion o elec on low when cIII/IV is inhibi ed, aised he ques ion o po en ially dele e ious consequences unde no mal physiological condi ions. Su p isingly, AOX Rosa26 mice o bo hsexes we e simila in size o li e ma e con ols and gained weigh no mally du ing de elopmen (Fig. 4A). Muscle and hea unc ions showed no signi ican di e ences om li e ma e con ols, based on s anda d assays o g ip s eng h (Fig. 4B), eadmill pe o mance (Fig. 4B), ca diac ejec ion ac ion (Fig. 4C) and le en icula mass (LVM; Fig. 4C), conduc ed on mice o di e en ages. To complemen hese da a we implemen ed a comp ehensi e pheno yping, using he esou ces o he Ge man Mouse Clinic (h ps://www.mouseclinic.de, sea ch ‘phenomap’; he ea e e e ed o as ‘GMC Phenomap’). This analysis co e ed me abolic, beha iou al, mo phological, immunological, ca diac and neu ological pa ame e s, amongs o he s. None o he pa ame e s es ed showed subs an ial o sys ema ic de ia ions om li e ma e con ols. AOX con e s p o ec ion agains an LD50 dose o sys emically deli e ed cyanide Despi e he absence o any meaning ul pheno ype unde s anda d (non-s ess ul) physiological condi ions, we easoned ha he ubiqui ous exp ession (Fig. 2) o unc ional AOX (Fig. 3) should con e whole-o ganism esis ance o a espi a o y poison a ge ing cIII o cIV. Sample coho s o emale mice we e hus es ed o hei esponse o sys emically adminis e ed po assium cyanide a ∼LD50 (Yamamo o, 1995), wi h e alua ion o su i al a e 1, 24 and 48 h. All i e AOX Rosa26 ansgenic mice es ed su i ed he ea men , whe eas h ee o six li e ma e con ols succumbed as expec ed (Fig. 5). Al hough he sample sizes a e small, hence indica i e a he han de ini i e, he esul is consis en wi h p o ec ion agains cyanide a he whole-o ganism le el. DISCUSSION In his s udy we success ully enginee ed mice o s able, ubiqui ous exp ession o Ciona AOX, ia a single-copy inse ion in o he Rosa26 locus, con olled by he syn he ic CAG p omo e . AOX p o ein was widely exp essed and enzyma ically unc ional when es ed in he p esence o an imycin A in o ganello. AOX exp ession p oduced negligible pheno ypic e ec s unde s anda d physiological condi ions, bu seemed able o p o ec mice om he le hal e ec s o injec ed cyanide. The AOX Rosa26 mouse p o ides a gene ically ac able ool o analyzing he pa hophysiology o a wide spec um o diseases p oposed o be linked o mi ochond ial espi a o y dys unc ion. The AOX Rosa26 mouse is a gene ically ac able model The ‘Mi AOX’ ansgenic mice, p e iously gene a ed by len i ec o ansduc ion (El-Khou y e al., 2013), p o ided a p elimina y indica ion ha widesp ead Ciona AOX exp ession in he mouse is no ha m ul. Howe e , owing o he mul i-copy na u e o he inse ed ansgene a di e en genomic si es, as well as a ying exp ession le els, Mi AOX mice we e no sui able o s udies using gene ic disease models. To a oid hese p oblems, we c ea ed a e ised model con aining a single inse ion o AOX cDNA a he Rosa26 locus on ch omosome 6. We demons a ed (Fig. 1) ha he Fig. 3. AOX is enzyma ically unc ional in mi ochond ia om AOX Rosa26 mice. (A) Respi ome y (oxygen consump ion in he indica ed uni s) om isola ed mi ochond ia p epa ed om he indica ed issues o hemizygous AOX Rosa26 mice and wild- ype li e ma e con ols, as shown; means±s.d. o h ee biological eplica es in each case. No e he di e en scales. 1 (Mal) – o enone-sensi i e oxida ion o mala e in he p esence o glu ama e, py u a e and ADP; 2 (Succ) – an imycin A- plus n-p opyl galla e-sensi i e succina e oxida ion; 3 (AOX) – a e o n-p opyl galla e-sensi i e, an imycin A-insensi i e succina e oxida ion; 4 (TMPD) – a e o asco ba e- educed TMPD oxida ion. Fo u he de ails see Ma e ials and Me hods. See Fig. S3C o espi a o y con ol a io in hese samples. (B) ROS p oduc ion, measu ed as H 2 O 2 ou pu , om hea mi ochond ia o AOX Rosa26 mice and wild- ype li e ma es, as indica ed, d i en by he indica ed subs a es o inhibi o s (succ, succina e; o , o enone; py , py u a e; mal, mala e). # P<0.05 o *P<0.001 be ween gi en pai s o con ol and AOX Rosa26 alues, S uden ’s - es , means±s.d. No e ha i is no possible o e i y ha his e ec depends on he enzyma ic ac i i y o AOX, because he AOX inhibi o , n-p opyl galla e, is i sel a po en an ioxidan . 166 RESEARCH ARTICLE Disease Models & Mechanisms (2017) 10, 163-171 doi:10.1242/dmm.027839 Disease Models & Mechanisms in oduced AOX gene is s ably ansmi ed in a Mendelian manne , emains ac i e beyond a leas se en gene a ions o backc ossing o s ain C57Bl/6J, shows no pa en -o -o igin o sex-speci ic le hali y, and is widely exp essed. The AOX ansgene can, in p inciple, be ans e ed o any desi ed s ain backg ound sui able o combina ion wi h a gi en gene ic model o disease, al hough ou cu en analysis was con ined o he C57Bl/6J gene ic backg ound. We expec ha he AOX Rosa26 mouse will become a e sa ile model o s udying he na u e o mi ochond ial in ol emen in disease-like pheno ypes. AOX seems ine unde s anda d physiological condi ions Al hough AOX was enzyma ically unc ional in he p esence o an imycin A in o ganello, ou da a indica e ha he me azoan enzyme is unc ionally ine unde s anda d physiological condi ions, as sugges ed p e iously (Hakkaa e al., 2006; Fe nandez-Ayala e al., 2009; El-Khou y e al., 2013). Se e al lines o e idence suppo his conclusion: (1) any subs an ial con ibu ion by he non-p o on-mo i e AOX o espi a o y elec on low should mani es in a signi ican ly dec eased espi a o y con ol a io in espi ome ic measu emen s in o ganello. Howe e , we did no obse e any signi ican al e a ion in ou issue su ey a leas o cI-linked subs a es and wi hin he de ec ion limi s o he me hod applied (Fig. S3C). (2) Ine icien mobiliza ion o nu i ional esou ces by AOX-exp essing mice should al e me abolic pa ame e s in i o. Howe e , he e we e no di e ences in whole body weigh (Fig. 4A), a o lean body mass de e mined by nuclea magne ic esonance (NMR) (see GMC Phenomap), o physiological pa ame e s de e mined by indi ec calo ime y, including ood in ake, body empe a u e, oxygen/CO 2 exchange o ac i i y (see GMC Phenomap). (3) No signi ican di e ences in hea pe o mance we e de ec ed by elec o- o echoca diog aphy, o MRI (Fig. 4C; GMC Phenomap), al hough he hea is he mos ene gy-demanding issue and showed he highes AOX exp ession. Low AOX exp ession in b ain The ela i ely low exp ession o he AOX ansgene in adul b ain (Fig. 1E, Fig. 2A, Fig. S2A,D) is somewha puzzling, gi en p e ious epo s. The CAG p omo e has p e iously been used o d i e ansgene exp ession a a high le el in he mouse b ain du ing de elopmen (Liu e al., 2014) as well as in he adul (Ida- Hosonuma e al., 2002; Kim e al., 2013), and he Rosa26 locus e icien ly d i es exp ession in he b ain (Bana es e al., 2005; Hi z e al., 2007; Delaunay e al., 2009). Mo eo e , we also achie ed subs an ial exp ession in he b ains o Mi AOX ansgenic mice (El-Khou y e al., 2013), using he same CAG p omo e . Fig. 4. AOX Rosa26 mice exhibi no mal physiology. (A) Mean weigh ±s.d. o hemizygous AOX Rosa26 and wild- ype li e ma e con ol mice o he sexes indica ed, du ing pos -na al de elopmen , n≥8 o each sex and geno ype analysed. (B) Muscle pa ame e s o male hemizygous AOX Rosa26 and wild- ype li e ma e con ol mice o he ages indica ed; means±s.d. Fo each g oup analysed n≥4 (g ip s eng h) o n≥6 ( eadmill). (C) Ca diac pa ame e s, as indica ed, o hemizygous AOX Rosa26 and wild- ype li e ma e con ol mice o he sex and ages indica ed; means±s.d., n≥4 o each g oup analysed. All da a ob ained by echoca diog aphy excep ejec ion ac ion a 10 w o age, which used MRI, n≥5. The e we e no signi ican di e ences be ween AOX Rosa26 and wild- ype alues o any pa ame e measu ed (S uden ’s - es , P>0.05). Fig. 5. Sampled AOX Rosa26 mice a e p o ec ed agains cyanide oxici y in i o.Su i al cu es o samples o hemizygous emale AOX Rosa26 mice (n=5) and wild- ype con ols (n=6) ea ed sys emically wi h KCN as desc ibed in Ma e ials and Me hods. No e ha he expe imen would need o be conduc ed on a much g ea e scale o gene a e ully eliable s a is ics, bu is p ecluded by e hical conside a ions. 167 RESEARCH ARTICLE Disease Models & Mechanisms (2017) 10, 163-171 doi:10.1242/dmm.027839 Disease Models & Mechanisms In iguingly, neona al AOX Rosa26 b ains exp essed subs an ially mo e AOX a he p o ein le el han adul s (Fig. S2C), indica ing ha he ansgene can be ac i e, bu appa en ly egula ed, in neu al cells. A p esen we do no ha e a con incing explana ion o hese anomalies. Howe e , he ela i ely low exp ession o AOX in he adul b ain seemed su icien o p o ec agains he le hali y o sys emically deli e ed cyanide (Fig. 5), which can c oss he blood- b ain ba ie and has majo oxic e ec s in he cen al ne ous sys em (Yamamo o and Tang, 1996; Rei e e al., 2010; Zhang e al., 2015). I will be in e es ing o explo e in g ea e dep h he speci ic physiological e ec s o his dose o cyanide and how hese e ec s a e modi ied by AOX exp ession. Lack o me abolic dis u bance esul ing om AOX exp ession The lack o any disce nible, dele e ious pheno ype a he whole- o ganism le el a ising om AOX exp ession (Fig. 4; supplemen a y Da a), mi o s he lack o biochemical dis u bance in he AOX Rosa26 mouse. This was he case e en in hea (Fig. 2A,B) and skele al muscle (Fig. 2A) issues showing high le els o AOX exp ession. The highly p o einaceous inne mi ochond ial memb ane is o ganized in o di e en subcompa men s wi h dis inc s uc u es, p o ein composi ion and biochemical unc ions (Vogel e al., 2006). In pa icula , he sup amolecula o ganiza ion o he OXPHOS sys em in supe complexes is gene ally conside ed o maximize he e iciency o elec on low (Acín-Pé ez e al., 2008; Chaban e al., 2014). We obse ed no s uc u al (Fig. 2B) o unc ional (Fig. 3A) dis u bance o he endogenous espi a o y memb ane upon AOX exp ession, which migh e lec na u al p ope ies o Ciona AOX, enabling i o eside in he mi ochond ia o i s pa en species. T ansgenic AOX seems o o m homome ic complexes, a he han associa ing wi h (and po en ially dis up ing) o he OXPHOS complexes. The elec opho e ic mobili y o he s anda d OXPHOS complexes was indis inguishable om ha in con ols, in all issues es ed (Fig. 2B; Fig. S2F,G). Ou indings imply ha hese mul ime s a e hemsel es benign, al hough i emains unknown whe he AOX is s uc u ally a ayed in a simila manne in i s na u al con ex in Ciona. We eason ha , by emaining uncomplexed wi h o he espi a o y chain componen s, he enzyme would be unc ionally adap ed o ac as a sink o elec ons ans e ed om di usible quinols in he inne mi ochond ial memb ane. Quinone educ ion migh a ise om he ope a ion o di e se dehyd ogenases, including, o example, cII, elec on ans e ing la op o ein dehyd ogenase, he mi ochond ial iso o m o glyce ol-3-phospha e dehyd ogenase and dihyd oo o a e dehyd ogenase. Unde no mal physiological condi ions, di usible quinols would be e icien ly mopped up by (dime ic) cIII, whe he alone o a ached o cIV as a supe complex. As in plan s (Hoe nagel and Wiskich, 1998; Cas o-Gue e o e al., 2004), AOX would only become ac i e a high quinol concen a ions, e lec ing i s lowe a ini y o quinols han cIII. Thus, AOX would be b ough in o play only when quinols accumula e as a esul o inhibi ion o o e load o he s anda d espi a o y pa hway, as in e ed p e iously in human cells (Dassa e al., 2009). This hypo hesis is also consis en wi h ou obse a ion ha AOX exp ession d as ically dec eases mi ochond ial ROS p oduc ion by hea mi ochond ia in he p esence o high le els o succina e (Fig. 3B), which p omo es e e se elec on low h ough cI (Chouchani e al., 2014). Ou obse a ions sugges ha AOX migh ha e d ama ic consequences unde s ess, especially in hea and o he issues whe e i is highly exp essed. Tes s o his hypo hesis will also e eal whe he AOX could ha e bene icial oles in u u e he apies (El-Khou y e al., 2014). A i s demons a ion o he u ili y o AOX Rosa26 mice has ecen ly been published (Mills e al., 2016), in which AOX was shown o con e esis ance agains le hali y in models o bac e ial sepsis. Fu he ials, combining AOX Rosa26 wi h speci ic gene ic disease models, should e eal he ex en o which AOX can alle ia e he pa hophysiology o espi a o y chain dys unc ion. The AOX Rosa26 mouse model should ha e wide applica ions and is a ailable o he esea ch communi y, upon eques . MATERIALS AND METHODS Cons uc ion o a ge ing ec o S anda d cloning and ecombinee ing p ocedu es (Liu e al., 2003; Wa ming e al., 2005) we e used o assemble he pRosa26-Aox a ge ing ec o . B ie ly, PL451 was adap ed o se e as AOX en y ec o , by in eg a ing homology a ms o he mouse Rosa26 locus (PL451-Rosa26) ups eam and downs eam o he neomycin selec ion casse e. Ciona AOX (Hakkaa e al., 2006) was in eg a ed ups eam o he selec ion casse e, and used o co- ans o m ecombinee ing-compe en Esche ichia coli (EL250) wi h a Rosa26- a ge ing plasmid (pRosa26-DTA). Posi i e clones we e selec ed by kanamycin esis ance (pRosa26-Aox), e i ied by PCR and sequencing, and elec opo a ed in o 6.5 ESCs ollowing linea iza ion wi h SalI. A e nega i e (DTA, Dip he ia oxin A) and posi i e (G418) selec ion, homologous in eg a ion was e i ied by Sou he n blo ing (Koe sie e al., 1993) using gene-speci ic es ic ion enzymes and p obes o dis inguish he wild- ype and manipula ed alleles (see supplemen a y Ma e ials and Me hods and Fig. S1B,C o u he de ails). C ea ion o ansgenic mice ESC clones posi i e o in eg a ion we e injec ed in o blas ocys s and ans e ed o pseudop egnan mice. Chime ic males we e hen backc ossed on o he C57Bl/6J s ain backg ound o gene a e he e ozygous animals, and subsequen ly b ed wi h mice ubiqui ously exp essing FLP ecombinase (Rod íguez e al., 2000), in o de o dele e he neomycin selec ion casse e. Mice we e backc ossed (>7 gene a ions) o C57Bl/6J emales o ob ain a clean gene ic backg ound o all subsequen s udies. PCR geno yping o AOX Rosa26 mice C ude DNA o geno yping was ex ac ed om ea punches o ail cu s by s anda d me hods (p o einase K ea men , isop opanol p ecipi a ion and o e nigh esuspension in TE a 56°C). Mul iplex PCR geno yping was ca ied ou using p ime s Aox 317 s: 5′-GCGATGCAAGATGGAGGGTA-3′plus Aox 317 as: 5′-TGAATCCAACCGTGGTCTCG-3′ o AOX, and Rosa26_w s: 5′-GACCTCCATCGCGCACTCCG-3′plus Rosa26_w as: 5′- CTCCGAGGCGGATCACAAGC-3′ o he wild- ype Rosa26 locus, gi ing espec i e p oduc s o 317 and 523 bp. PCR eac ions o 20 μlcon ained 4 pmol o each p ime , DMSO a 2% and 0.2 μl DyNazyme II (The mo Fishe Scien i ic), wi h cycle pa ame e s o ini ial dena u a ion a 95°C o 5 min, hen 39 cycles o dena u a ion a 95°C o 20 s, annealing a 56°C o 30 s and ex ension a 72°C o 60 s, wi h inal ex ension s ep a 72°C o 10 min, ollowed by 1.5% aga ose gel elec opho esis. See Fig. S1D o example gel. RNA analysis RNA was p epa ed om dissec ed mouse issues by bead homogeniza ion in 700 μl (>10 olumes) o T izol eagen (Sigma). A e incuba ion o 5 min a oom empe a u e, samples we e gen ly ex ac ed wi h 0.2 olumes o chlo o o m and cen i uged a 12,000 g max o 15 min a 4°C. The uppe (aqueous) phase was decan ed and RNA eco e ed by isop opanol p ecipi a ion and cen i uga ion. Using s anda d p ocedu es (Samb ook e al., 1989), ai -d ied RNA pelle s we e esuspended in 20 μl RNase- ee wa e , ac iona ed on o maldehyde-aga ose gels, blo ed o Hybond-N+ memb ane (GE Heal hca e) in 10×SSC and hyb idized o end-labelled DNA oligonucleo ide p obes o AOX, mi ochond ial 12S and cy osolic 18S RNA, and A p5b mRNA, espec i ely 5′-CTTGACCCACTGTTTCTCA- TCTAGCCG-3′,5′-CATGGGCTACACCTTGACCT-3′,5′-TCGAACCC- TGATTCCCCGTCACCC-3′and 5′-GGTGAATATGACCATCTCCCAG- AACAAGC-3′. 168 RESEARCH ARTICLE Disease Models & Mechanisms (2017) 10, 163-171 doi:10.1242/dmm.027839 Disease Models & Mechanisms P o ein analysis Fo p o ein ex ac ion, small pieces o esh o ozen issue om dissec ed o gans we e placed in 500 μl o lysis bu e (50 mM T is/HCl, 150 mM NaCl, 1 mM EDTA, 1% T i on X-100, pH 7.4), con aining a dissol ed p o ease inhibi o cock ail able (Pie ce), in a 5 ml ube on ice. A e homogeniza ion using a POLYTRON PT 1200 E Manual Dispe se (Ecoline), samples we e incuba ed on ice o 30 min ollowed by cen i uga ion a 14,000 g max o 5 min a 4°C. Supe na an s we e sa ed and p o ein concen a ion was measu ed using B ad o d eagen (Bio-Rad) be o e dilu ion in o SDS-PAGE sample bu e o elec opho esis on SDS 12% polyac ylamide gels. A e semi-d y ans e o PROTRAN ni ocellulose memb anes (Pe kinElme ), wes e n blo s we e p obed using p ima y an ibodies o AOX [cus omized abbi an ibody, 21s Cen u y Biochemicals (Fe nandez-Ayala e al., 2009), 1:40,000 in T is-bu e ed saline (TBST) con aining 5% BSA] wi h seconda y an ibody pe oxidase- conjuga ed A iniPu e goa an i- abbi IgG (Jackson ImmunoResea ch, 111-035-144, 1:20,000). A e s ipping by wo 20 min washes wi h 100 mM β-me cap oe hanol, 2% SDS, 62.5 mM T is-HCl (pH 6.7), each ollowed by blocking wi h TBST con aining 5% milk o 30 min, blo s we e ep obed o ep esen a i e subuni s o he OXPHOS complexes, using To al OXPHOS Cock ail an ibody [Abcam, ab110413, 1:250; isualizing Sdhb (cII), Uqc c2 (cIII), M co1 (cIV) and A p5a (cV)], plus an an ibody agains complex 1 subuni Ndu s3 (Mi osciences, ab14711, 1:4000), bo h de ec ed wi h pe oxidase-conjuga ed A iniPu e goa an i-mouse IgG (Jackson ImmunoResea ch, 115-035-146, 1:1000) as seconda y an ibody. Chemiluminescen de ec ion used 20× LumiGLO Reagen and 20× Pe oxide om Cell Signaling Technology, acco ding o manu ac u e ’s ecommenda ions. En ichmen o mi ochond ial memb anes, solubilisa ion o mi ochond ial complexes and BNE we e ca ied ou as desc ibed (Wi ig e al., 2006; Heidle e al., 2013). Mi ochond ial complexes we e s ained wi h Coomassie Blue (Wi ig e al., 2006) and speci ic in-gel his ochemical s aining o cI, cIV, and cV was pe o med as desc ibed p e iously (Wi ig e al., 2007). Fo immunode ec ion, BNE-gels we e blo ed on o PVDF memb anes and p obed wi h an ibodies agains AOX (1:50,000) o mi ochond ial complexes (Mi oP o ile To al OXPHOS Roden WB An ibody Cock ail, Mi osciences, ab110413, 1:250) and cIV (1:1000; Heidle e al., 2013). Me abolomics Me aboli e analysis was conduc ed as desc ibed p e iously (Nikkanen e al., 2016), using skele al muscle om six hemizygous AOX Rosa26 and six wild- ype li e ma e con ol mice (8-week-old males, all culled a a single ime in he mo ning). B ie ly, a ge ed me abolomics was implemen ed by ul a- pe o mance liquid ch oma og aphy andem mass-spec ome y using a Wa e s XEVO-TQ-S mass spec ome e . Me aboli es ex ac ed wi h ace oni ile we e sepa a ed by hyd ophilic liquid-in e ac ion ch oma og aphy, hen analysed spec ome ically by mul iple eac ion moni o ing. Raw da a we e collec ed and analysed wi h Ta ge Lynx so wa e (Wa e s), and me aboli es quan i ied using in e nal s anda ds and calib a ion cu es. Fo ull de ails, see supplemen a y Ma e ials and Me hods. Bioene ge ic expe imen s Fo espi ome y o mi ochond ia om di e en issues, mice we e eu hanised by ce ical disloca ion and o gans we e dissec ed and collec ed in o ice-cold PBS. So issues we e ine chopped (1 mm 3 ) in ice-cold PBS and hand-homogenized in 3 ml e-suspension bu e [225 mM suc ose, 75 mM D-manni ol, 10 mM T is/HCl, 1 mM EGTA, 1 mg/ml bo ine se um albumin (BSA), pH 7.4], using a glass- e lon homogenize ( igh - i ing pes le). Ha d issues (hea , skele al muscle and kidney), chopped o a simila size, we e p e- ea ed wi h 3 ml (∼10 olumes) ice-cold ypsin-EDTA [500 μg/ml ypsin (Di co), 0.5 mM EDTA, 10 μg/ml phenol ed, pH 7.4] o 10 min, ollowed by blocking wi h 300 μl oe al bo ine se um (Gibco/Li e Technologies) and eco e y by low-speed cen i uga ion (40 g max ,1min,4° C) be o e homogeniza ion. Homogena es we e cen i uged a 1300 g max o 5 min a 4°C, a e which supe na an s we e collec ed and e-cen i uged a 17,000 g max o 15 min a 4°C. The mi ochond ial pelle was esuspended, acco ding o i s size, in 75-250 μl ice-cold MiR05 bu e [0.5 mM EGTA, 3 mM MgCl 2 , 60 mM lac obionic acid (Ald ich, bu e ed o pH 7.0 wi h 5 M KOH), 20 mM au ine (Sigma), 10 mM KH 2 PO 4 , 20 mM HEPES/KOH, 110 mM suc ose and 1 g/l a y-acid ee BSA (Sigma), pH 7.2 a oom empe a u e] and s o ed on ice un il espi ome y. Mi ochond ial p o ein con en was assayed using B ad o d eagen (Bio-Rad). Respi ome y, using an O2K oxyg aph (O obo os), was conduc ed in MiR05 bu e in a 2 ml chambe , o which was added 50 o 100 µg o mi ochond ia acco ding o he issue. Subs a es and inhibi o s we e added in he ollowing o de : (1) 5 mM sodium py u a e+5 mM sodium glu ama e+5 mM sodium mala e, (2) 4 mM ADP, (3) 150 nM o enone (Sigma), (4) 17 mM sodium succina e, (5) 22.5 ng/ml an imycin A (Sigma), (6) 200 µM n-p opyl galla e (nPG, Sigma), (7) 0.5 mM N,N,N′,N′- e ame hyl-p-phenylenediamine (TMPD, Sigma) +2 mM sodium L-asco ba e, (8) 100 mM NaN 3 o 1 mM KCN. The lux alues [pmol/(s×ml)] ob ained om he ace we e no malized o he amoun o mi ochond ial p o ein. Fo measu emen s o ROS p oduc ion, mouse hea mi ochond ia we e isola ed essen ially as desc ibed (Mela and Sei z, 1979), wi h mino modi ica ions: issue was minced in 225 mM manni ol, 20 mM MOPS, 75 mM suc ose, 1 mM EGTA, 0.5 mM di hio h e ol, pH 7.4 and hand-homogenized in 10 ml/g issue o he same bu e con aining 0.05% Naga se (Sigma). A e addi ion o 30 ml o he o iginal bu e , he homogena e was cen i uged a 2000 g max o 4 min a 4°C. The supe na an was passed h ough cheeseclo h and e-cen i uged a 12,000 g max o 10 min. The esul ing pelle was esuspended in 225 mM manni ol, 20 mM MOPS, 75 mM suc ose, 0.1 mM EGTA, 75 mM KCl, pH 7.4. Mi ochond ial p o ein con en was de e mined using he bicinchoninic acid assay (Wiechelman e al., 1988), wi h BSA as s anda d. ROS p oduc ion unde condi ions used o espi ome y was measu ed luo ime ically using 5 µM Amplex Red (Hyd ogen Pe oxide Assay Ki , The mo Fishe Scien i ic) and 3 uni s/ml ho se adish pe oxidase a 30°C, using a Ca y Eclipse luo ime e (Va ian) wi h exci a ion a 560 nm and de ec ion a 590 nm (Zhou e al., 1997). Mouse pheno yping Mouse body weigh was measu ed using a small elec onic balance sui able o oden s. G ip s eng h was measu ed using he BIO-GS3 appa a us (Bioseb). Mice we e placed on he pla o m un il all ou limbs we e engaged on he g id, and hen pulled o measu e he o ce gene a ed. The mean o h ee measu emen s was no malised o body weigh (g/g) o each animal es ed. All animals we e ained o h ee successi e days be o e he ac ual expe imen . Endu ance unning was measu ed as p e iously (Ya suga and Suomalainen, 2012), as he un ime on a s anda d unning bel (Exe - 6M T eadmill, Columbus Ins umen s), se o each a speed o 6.5 m/min in s eps o 0.5 m/min e e y 3 min. A s ay o mo e han 5 s on he elec i ied mo i a ion g id (0.5 mA cu en ) was conside ed as he end poin o each es . Ca diac pa ame e s (ejec ion ac ion, le en icula mass) we e de e mined by echoca diog aphy (Ve o 2100 sys em, FujiFilm VisualSonics Inc.) o , whe e indica ed in igu e legends, by magne ic esonance imaging (MRI), pe o med essen ially as desc ibed elsewhe e (Zieba e al., 2008). MRI da a we e analysed using Osi iX Imaging So wa e (h p://www.osi ix- iewe .com/index.h ml). Comp ehensi e pheno yping by he Ge man Mouse Clinic (GMC) was conduc ed using he p o ocols desc ibed and e e enced a h ps://www.mouseclinic.de (sea ch ‘phenomap’). In all es s, mouse geno ypes we e blinded o he expe imen e and e i ied subsequen ly. Sys emic adminis a ion o cyanide The p ocedu e was implemen ed unde con ac by Lu ia Scien i ic Indus ies, He zliya, Is ael ( esponsible scien is D I is Maimon). Mice, whose geno ypes we e blinded o he expe imen e , we e anes he ized wi h 3% iso lu ane in an induc ion chambe , a e which anaes hesia was main ained by 2% iso lu ane using a nose cone. Co e empe a u e was kep a 36.5°C using a hea ing pad. KCN was dissol ed in dis illed wa e a 10 mg/ml and deli e ed by IP injec ion o he mice a 8.5 mg/kg. Animals we e obse ed o 48 h o he onse o dea h, de ined as apnea wi hou u he espi a o y e o o mo emen o palpable ca diac pulsa ion. E hical pe mi s All mouse b eeding and expe imen s we e app o ed by he na ional e hical commi ee in Finland, unde pe mi s ESAVI/8766/04.10.07/2015 and ESAVI/ 169 RESEARCH ARTICLE Disease Models & Mechanisms (2017) 10, 163-171 doi:10.1242/dmm.027839 Disease Models & Mechanisms 2954/04.10.07/2015. Mouse expe imen s conduc ed unde con ac by Lu ia Scien i ic Indus ies we e app o ed by IACUC unde assu ance 7433J45, 07/22/2015. Main enance o mice in Magdebu g was in acco d wi h p ocedu es speci ied by he Animal Heal hand Ca e Commi eeso he O o- on-Gue icke Uni e si y, Magdebu g, and o he S a e o Sachsen-Anhal , Ge many. Image p ocessing Images we e op imized o b igh ness and con as and c opped o cla i y. No o he manipula ions such as gamma co ec ions we e made, no was any ele an in o ma ion excluded by c opping. Full, o iginal gel images a e a ailable on eques . Acknowledgemen s We hank Maa i Myo ha nen, Sonja K u ge , Susanne K eu ze , Bi gi Spi znagel and Jana Meis e knech o excellen echnical assis ance, Ch is ophe Ca oll, Giuseppe Cannino, Luca Gio dano, Riikka Ki ela and Uwe Rich e o echnical ad ice and help, Anu Suomalainen and B endan Ba e sby, o use ul discussions, and T oy Fai h ull o c i ical eading o he manusc ip . Compe ing in e es s The au ho s decla e no compe ing o inancial in e es s. Au ho con ibu ions H.T.J., T.B., P.R. and M.S. join ly concei ed he p ojec and de eloped he s a egy o c ea e he AOX Rosa26 mouse. M.S. supe ised and coo dina ed he labo a o y wo k, which was conduc ed by M.S., P.K.D., E.D., K.M.H., Y.Z., I.S., I.W., J.H., Z.G., T.G., J.N., V.V., A.W. and F.N.G. H.F., V.G.-D. and M.H.d.A. implemen ed comp ehensi e pheno yping in collabo a ion wi h hei colleagues om he Ge man Mouse Clinic. H.T.J. compiled he manusc ip and igu es wi h assis ance om all au ho s. Funding This wo k was suppo ed by unding om he Eu opean Resea ch Council (Ad anced G an 232738 o H.T.J.), Academy o Finland (Suomen Aka emia) Te eyden Tu kimuksen Toimikun a (g an 272376); Tampe e Uni e si y (Tampe een Yliopis o) Hospi al Medical Resea ch Fund; he Sig id Juselius Founda ion (Sig id Juseliuksen Sa a  io ), Max-Planck-Gesellscha , Deu sche Fo schungsgemeinscha (DFG SFB 815 p ojec Z1 o I.W. and DFG SFB 1213 p ojec A2 o T.B.), he Clus e o Excellence “Mac omolecula Complexes”, Goe he- Uni e si a  F ank u am Main, Ge many ( o I.W.), he Clus e o Excellence “Ca diopulmona y Sys em”( o T.B.), he Ge man Cen e o Lung Resea ch (DLZ), and he Uni e si ies o Helsinki and Tampe e (Helsingin Yliopis o and Tampe een Yliopis o). This wo k has also been unded by he Ge man Fede al Minis y o Educa ion and Resea ch (Bundesminis e ium u  Bildung und Fo schung) o he Ge man Mouse Clinic (In a on ie g an 01KX1012). 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