Dysfunctions of the mitochondrial electron transport chain cause severe, currently untreatable, diseases in humans. A new study by Jain et al. (2019) reports increased oxygen levels in the brain of complex-I-deficient mice. Reducing the O2 levels by hypoxia, carbon monoxide, or anemia, improved the clinical phenotype and prolonged the lifespan of the mouse model.The authors’ work is supported by an MRC core grant to the Mitochondrial Biology Unit (MC_UU_00015/5)

Viscomi, Carlo

Zeviani, Massimo

English

Dysfunctions of the mitochondrial electron transport chain cause severe, currently untreatable, diseases in humans. A new study by Jain et al. (2019) reports increased oxygen levels in the brain of complex-I-deficient mice. Reducing the O2 levels by hypoxia, carbon monoxide, or anemia, improved the clinical phenotype and prolonged the lifespan of the mouse model

Viscomi C.

Zeviani M.

Archivio istituzionale della ricerca - Università di Padova

Original Citation:Breathe: Your Mitochondria Will Do the Rest… If They Are Healthy!Cell PressPublisher:Published version:DOI:Terms of use:Open Access(Article begins on next page)This article is made available under terms and conditions applicable to Open Access Guidelines, asdescribed at http://www.unipd.it/download/file/fid/55401 (Italian only)Availability:This version is available at: 11577/3321260 since: 2020-01-15T17:59:18Z10.1016/j.cmet.2019.09.004Università degli Studi di PadovaPadua Research Archive - Institutional RepositoryCell MetabolismPreviewsBreathe: Your Mitochondria Will Dothe Rest. If They Are Healthy!Carlo Viscomi1 and Massimo Zeviani1,2,*1MRC-Mitochondrial Biology Unit, Cambridge CB2 0XY, UK2Department of Neurosciences, University of Padova, Padova, Italy*Correspondence: mdz21@mrc-mbu.cam.ac.ukhttps://doi.org/10.1016/j.cmet.2019.09.004Dysfunctions of the mitochondrial electron transport chain cause severe, currently untreatable, diseases inhumans. A new study by Jain et al. (2019) reports increased oxygen levels in the brain of complex-I-deficientmice. Reducing the O2 levels by hypoxia, carbon monoxide, or anemia, improved the clinical phenotype andprolonged the lifespan of the mouse model.Oxygen is an extremely reactive elementwith a distinguished propensity to attractelectrons from other substrates, andbecause of its high electronegativity formschemical bonds with almost all other ele-ments. (Lane, 2002). Through respiration,operated by the electron transfer chain(ETC), almost all eukaryotes (by means oftheir endosymbiont mitochondria) andaerobic bacteria use oxygen as the ulti-mate ‘‘sink’’ of electrons, convertinghydrogen and oxygen atoms into harmlesswater. This controlled, stepwise combus-tion, not only allows the conversion of en-ergy, extracted from oxidation of nutrients,into the common energy currency of thecell, ATP, but also the neutralization ofthe highly toxic effects of free oxygen itself.In fact, free oxygen can give rise to highlyreactive oxygen species (ROS) and theirradicals, particularly the hydroxyl radical.These species react with proteins, nucleicacids, phospholipids, and more molecularspecies, damaging their structures and im-pairing their functions. Thus, like Janus,the Roman god of passages and changes,oxygen is a double-facedmolecule. No lifewould exist on earth without it, and yet it isa highly poisonous gas. The toxic effectsof an excess of oxygen, produced forinstance by breathing pure oxygen orgaseous mixes with high percentages ofO2, are well known, determining acute orchronic symptoms with serious, some-times fatal consequences on target or-gans, particularly the brain (Haldane,1922), the lungs, and the eyes. In eukary-otes, respiration is carried out within mito-chondria, semi-autonomous organellesoften referred to as the powerhouses ofthe cell because of their central role in en-ergy metabolism. The physiological pro-628 Cell Metabolism 30, October 1, 2019 ª 2duction of a low amount of ROS, anintrinsic process of normal mitochondrialrespiration, acts as a signal pathway regu-latingmitochondrial biogenesis and bioen-ergetic function through a retrograderesponse (Butow and Avadhani, 2004),dubbed as mito-hormesis. Absence ofthis regulatory mechanism can indeedworsen clinical conditions because ofimpaired ETC (Dogan et al., 2018).Dysfunctions of the mitochondrial ETCcause severe, currently untreatable dis-eases in humans (Gorman et al., 2016).Critically, the pathogenetic mechanismof these diseases is far fromclear. Defectsof the mitochondrial ETC are believed tocompromise the efficient utilization of ox-ygen in the electron flow mechanism, andthere is consequent backlogging inver-sion of electron flow along the ETC. Thiseffect amplifies the physiological leakageof electrons during respiration, causingROS and free oxygen radicals to increaseand consequent damage of biologicallyrelevant substrates (Murphy, 2016). This‘‘free radical’’ theory has been popular-ized by a huge amount of experimentalwork and publications and has promotedthe use of antioxidants as the most com-mon pharmacological treatment of mito-chondrial disease. On the other hand,absence of oxygen or its reduction belowa critical threshold impairs the electronflow of ETC and impedes respiration andenergy conversion by oxidative phos-phorylation. Severe hypoxia can be pro-duced by a number of mechanisms,including severely reduced or stop ofblood flow either locally (ischemia) or inthe whole organism (shock), severelyreduced or arrest of breathing, severeanemia, toxic impairment of blood-oxy-019 Elsevier Inc.gen-carrying haemoglobin, or by breath-ing air or gas mixtures with severelyreduced oxygen concentration (pO2).In higher eukaryotes, under normoxia,the key components of the hypoxicresponse, i.e., the hypoxia induciblefactors HIF1-a and HIF2-a, are rapidly hy-droxylated by the prolyl hydroxylases(PHD1–PHD3) becoming a substrate forthe von Hippel-Lindau factor (vHL), an E3ligase polyubiquitinating the HIF proteins,which are thus degraded by the protea-some (Samanta and Semenza, 2017).Contrariwise, hypoxia inhibits the activityof prolyl hydroxylases, determining refrac-toriness of HIF1-a and HIF2-a from beingdisposed by the vHL E3 ligase. As aconsequence, HIF transcription factorsaccumulate, activating the hypoxia geneticprogram, including promotion of angio-genesis and a vast repertoire of otheranti-hypoxic adaptive responses thatmight help the survival of hypoxic tissues.Considering the toxic effects of oxygenin conditions of failing mitochondrialrespiration, Jain et al., (2016), in Prof.Vamsi Mootha’s laboratory, publishedimportant results demonstrating that areduction in aerial oxygen partial pressure(pO2) to 11% (i.e., approximately half ofthe normal, sea level concentration of ox-ygen on Earth), could determine substan-tial improvement in a mouse model ofsevere ETC complex I (cI) deficiency dueto the lack of a cI structural subunit,NDUFS4. This improvement includedhighly significant prolongation of the life-span; substantial amelioration of the clin-ical symptoms, particularly those specificto the central nervous system; and reduc-tion of Leigh-disease-related lesions ofthe brain. Intermittent or more moderateCell MetabolismPreviewshypoxia was ineffective to rescue thephenotype (Ferrari et al., 2017). In thisnew chapter of this fascinating story,Jain et al., 2019 now add some relevantmechanistic information. They crossedthe Ndufs4/ mice with mice lackingPhd1–Phd3, or carrying a point mutationin the vHL, characterized by impairedHIF-related hypoxic response. They thenexposed the crosses to 11% oxygenand observed the persistence of the clin-ical and neuropathological improvement,therefore ruling out an essential role ofthe activation of the HIF program in deter-mining the effects of breathing low-con-centration oxygen.They also showed, by exploiting twoindependent methodologies, that thereduced respiration rate and oxygen con-sumption linked to cI deficiency actuallycauses, when breathing normal air, a hy-peroxic condition in critical tissues, whichseems to be a major damaging factor inthe brain in Ndufs4/ mice. They furtherreinforced the evidence that hypoxia cannormalize deleterious brain hyperoxia inNdufs4/ mice by inducing severe ane-mia-induced hypoxia by recurrent phle-botomy and low-iron diet or toxic hypoxiaby exposing animals to non-lethal con-centrations of carbon monoxide (CO),which irreversibly dislodges oxygen fromthe haeme centers of haemoglobin. Thesefindings strongly suggest that the reducedmitochondrial respiration in theNdufs4/mice in normal air leads to increased pO2and, possibly, ROS production, particu-larly in the brain. Hypoxia would thusimprove the phenotype by reducingexcessive local pO2 and ROS production.This is an important study that points toa new pathogenic mechanism for mito-chondrial diseases. As a consequence ofthe beneficial results of hypoxia, thera-peutic interventions, aimed at reducingpO2 levels in tissues, should be beneficialto patients affected by mitochondrial dis-ease. However, the findings by Jain andcolleagues have so far been limited to asingle mouse model of cI deficiency.Whether this mechanism holds true alsoin other models of cI-related disordersor, more generally, in other defects ofthe ETC is currently unknown. In addition,the methods used in this work to lowerpO2 are interesting from the experimentalpoint of view, but it is unlikely that they canbe transferred to the patients. Asmentioned above, CO is a dangerousgas with a rather narrow safety window;in addition, severe anemia would berather risky in severely sick children.Another conundrum that warrants addi-tional investigation is the role of the hyp-oxic program in rescuing the phenotypeof Ndufs4/ mice. Previous experimentsfrom the same authors had shown that thedeletion of the vHL factor improved thegrowth rate and glycolytic switch of anti-mycin-A-treated cells, a model of respira-tory chain dysfunction, and prolonged thelifespan of a vHL-less zebrafish modelexposed to antimycin A. On the same to-ken, FG4592, an inhibitor of PHD currentlyin clinical trial for anemia due to kidneydiseases, triggering HIF1-a stabilizationeven in normoxic conditions, was able tomarkedly increase the survival rate of theantimycin-treated zebrafish embryos. Allthese data suggest a genetic link betweenthe hypoxic response and the ameliora-tion of the phenotype, but the data pre-sented here argue against a direct roleof HIF-dependent pathways in vivo.Finally, the work by Jain et al. (2019)does not provide direct evidence thatROS are involved in the pathogenesis ofcI-related disorders, and indeed severalattempts to treat mitochondrial patientsof antioxidants have given erratic results.Unlike Janus, oxygen might well havemore than two faces; we are only nowstarting to understand the deep conse-quences of altered oxygen consumptionin mitochondrial dysfunction, and thiswill hopefully pave new avenues fortreatment.ACKNOWLEDGMENTSThe authors’ work is supported by an MRCcore grant to the Mitochondrial Biology Unit(MC_UU_00015/5).REFERENCESButow, R.A., and Avadhani, N.G. (2004).Mitochondrial signaling: the retrograde response.Mol. Cell 14, 1–15.Dogan, S.A., Cerutti, R., Benincá, C., Brea-Calvo,G., Jacobs, H.T., Zeviani, M., Szibor, M., andViscomi, C. (2018). Perturbed Redox SignalingExacerbates a Mitochondrial Myopathy. CellMetab. 28, 764–775.e5.Ferrari, M., Jain, I.H., Goldberger, O., Rezoagli, E.,Thoonen, R., Cheng, K.H., Sosnovik, D.E.,Scherrer-Crosbie, M., Mootha, V.K., and Zapol,W.M. (2017). Hypoxia treatment reverses neurode-generative disease in a mouse model of Leighsyndrome. Proc. Natl. Acad. Sci. USA 114,E4241–E4250.Gorman, G.S., Chinnery, P.F., DiMauro, S., Hirano,M., Koga, Y., McFarland, R., Suomalainen, A.,Thorburn, D.R., Zeviani, M., and Turnbull, D.M.(2016). Mitochondrial diseases. Nat. Rev. Dis.Primers 2, 16080.Haldane,J. (1922).Respiration (YaleUniversityPress).Jain, I.H., Zazzeron, L., Goli, R., Alexa, K.,Schatzman-Bone, S., Dhillon, H., Goldberger, O.,Peng, J., Shalem, O., Sanjana, N.E., et al. (2016).Hypoxia as a therapy for mitochondrial disease.Science 352, 54–61.Jain, I.H., Zazzeron, L., Goldberg, O., Marutani, E.,Wojtkiewicz, G.R., Ast, T., Wang, H., Schleifer, G.,et al. (2019). Leigh Syndrome Mouse Model CanBe Rescued by Interventions that Normalize BrainHyperoxia, but Not HIF Activation. Cell Metab.30, 1–9.Lane, N. (2002). Oxygen: the molecule that madethe world (Oxford University Press).Murphy, M.P. (2016). Understanding and prevent-ing mitochondrial oxidative damage. Biochem.Soc. Trans. 44, 1219–1226.Samanta, D., and Semenza, G.L. (2017).Maintenance of redox homeostasis by hypoxia-inducible factors. Redox Biol. 13, 331–335.Cell Metabolism 30, October 1, 2019 629

Breathe: Your Mitochondria Will Do the Rest... If They Are Healthy!

Apollo (Cambridge)

Title Breath: your mitochondria will do the rest…if they are healthy! Carlo Viscomi1, Massimo Zeviani1,2* 1MRC-Mitochondrial Biology Unit, Cambridge CB2 0XY, UK 2Department of Neurosciences, University of Padova, Padova, Italy   *Correspondence to:  Massimo Zeviani, MD, PhD  MRC MBU, Wellcome Trust/MRC Building Hills Road, Cambridge, CB2 0XY, UK FAX +44(0)1223 252715; Phone +44 (0)1223 252700 E-mail: mdz21@mrc-mbu.cam.ac.uk   Abstract Dysfunctions of the mitochondrial electron transport chain cause severe, currently untreatable, diseases in humans. A new study by Jain et al. reports increased oxygen levels in the brain of complex I-deficient mice. Reducing the O2 levels by hypoxia, carbon monoxide or anaemia, improved the clinical phenotype and prolong the lifespan of the mouse model.   Main text Oxygen is an extremely reactive element, with a distinguished propensity to attract electrons from other substrates, due to its high electronegativity, forming chemical bonds with almost all other elements. Through respiration, operated by the electron transfer chain (ETC), almost all eukaryotes (by means of their endosymbiont mitochondria) and aerobic bacteria use oxygen as the ultimate “sink” of electrons, converting hydrogen and oxygen atoms into harmless water. This controlled, stepwise combustion, not only allows the conversion of energy, extracted from oxidation of nutrients, into the common energy currency of the cell, ATP, but also the neutralization of the highly toxic effects of free oxygen itself. In fact, free oxygen can accept two electrons, one at a time, giving rise to highly reactive oxygen species (ROS), and its radicals, particularly the hydroxyl radical. These species react with proteins, nucleic acids, phospholipids, etc., damaging their structures and impairing their functions. Thus, like Janus, the Roman god of the passages and changes, oxygen is a double-face molecule. No life would exist on earth without it and yet it is a highly poisonous gas. The toxic effects of an excess of oxygen, produced for instance by breathing pure oxygen or gaseous mixes with high percentages of O2, is well known, determining acute or chronic symptoms, with serious, sometimes fatal consequences on target organs, particularly the brain (Haldane, 1922), the lung and the eye. In eukaryotes, respiration is carried out within mitochondria, semi-autonomous organelles often referred to as the powerhouses of the cell because of their central role in energy metabolism.  the physiological production of a low amount of ROS, an intrinsic process of normal mitochondrial respiration, acts as a signal pathway regulating mitochondrial biogenesis and bioenergetic function through a retrograde response (Butow and Avadhani, 2004), dubbed as mito-hormesis. Absence of this regulatory mechanism can indeed worsen clinical conditions due to impaired ETC (Dogan et al., 2018). Dysfunctions of the mitochondrial ETC cause severe, currently untreatable, diseases in humans (Gorman et al., 2016). Critically, the pathogenetic mechanism of these diseases is far from clear. Defects of the mitochondrial ETC are believed to compromise the efficient utilization of oxygen in the electron flow mechanism, with consequent backlogging inversion of electron flow along the ETC. This effect amplifies the physiological leakage of electrons during respiration, causing ROS and free oxygen radicals to increase, with consequent damage of biologically relevant substrates (Murphy, 2016). This “free radical” theory has been popularized by a huge amount of experimental work and publications and has promoted the use of antioxidants as the most common pharmacological treatment of mitochondrial disease. On the other hand, absence of oxygen, or its reduction below a critical threshold, impairs the electron flow of ETC and impedes respiration and energy conversion by oxidative phosphorylation. Severe hypoxia can be produced by a number of mechanisms, including severely reduced or stop of blood flow either locally (ischemia) or in the whole organism (shock), severely reduced or arrest of breathing, severe anaemia, toxic impairment of blood oxygen-carrying haemoglobin, or by breathing air or gas mixtures with severely reduced oxygen concentration (pO2).  In higher eukaryotes, under normoxia, the key components of the hypoxic response, i.e. the hypoxia inducible factors HIF1a and HIF2a, are rapidly hydroxylated by the prolyl hydroxylases (PHD1-3) becoming a substrate for the von Hippel-Lindau factor (vHL), an E3 ligase polyubiquitinating the HIF proteins, which are thus degraded by the proteasome (Samanta and Semenza, 2017). Contrariwise, hypoxia inhibits the activity of prolyl hydroxylases, determining refractoriness of HIF1a and HIF2a from being disposed by the vHL E3 ligase. As a consequence, HIF transcription factors accumulate, activating the “hypoxia” genetic program, including promotion of angiogenesis and a vast repertoire of other anti-hypoxic adaptive responses that may help the survival of hypoxic tissues. Considering the toxic effects of oxygen in conditions of failing mitochondrial respiration, in 2016 Jain et al., in Prof. Vamsi Mootha’s laboratory, published important results demonstrating that a reduction in aerial pO2 to 11% (i.e. approximately half of the concentration of normal, sea level concentration of oxygen on Earth), could determine substantial improvement in a mouse model of severe ETC complex I (cI) deficiency due to the lack of a cI structural subunit, NDUFS4  (Jain et al., 2016). This improvement included highly significant prolongation of the lifespan, substantial amelioration of the clinical symptoms, particularly those specific to the CNS, and reduction of Leigh-like disease-related lesions of the brain. Intermittent or more moderate hypoxia was ineffective to rescue the phenotype (Ferrari et al., 2017). In this new chapter of this fascinating story, Jain et al. add now some relevant mechanistic information (Jain et al., 2019). They crossed the Ndufs4-/- mice with mice lacking Phd1-3, or carrying a point mutation in the vHL, characterised by impaired HIF-related hypoxic response. They then exposed the crosses to 11% oxygen, and observed the persistence of the clinical and neuropathological improvement, therefore ruling out an essential role of the activation of the HIF program in determining the effects of breathing low-concentration oxygen.  They also showed, by exploiting two independent methodologies, that the reduced respiration rate and oxygen consumption linked to cI deficiency actually causes, when breathing normal air, a hyperoxic condition in critical tissues, which seems to be a major damaging factor in the brain in NDUFS4-/- mice. To further reinforce the evidence that hypoxia can normalize deleterious hyperoxia in NDUFS4-/- mice, they induced severe anaemia-induced hypoxia by recurrent phlebotomy and low-iron diet, or toxic hypoxia by exposing animals to non-lethal concentrations of CO (carbon monoxide), which irreversibly dislodges oxygen from the haeme centers of haemoglobin. These findings strongly suggest that the reduced mitochondrial respiration in the Ndufs4-/- mice in normal air leads to increased pO2 and, possibly, ROS production, in particularly in the brain. Hypoxia would thus improve the phenotype by reducing excessive local pO2 and ROS production.          This is an important study which points to a new pathogenic mechanism for mitochondrial diseases. As a consequence of the beneficial results of hypoxia, therapeutic interventions, aimed at reducing pO2 levels in tissues, should be beneficial to patients affected by mitochondrial disease. However, the findings by Jain and colleagues have so far been limited to a single mouse model of cI deficiency. Whether this mechanism holds true also in other models of cI-related disorders or, more generally, in other defects of the ETC is currently unknown. In addition, the methods used in this work to lower pO2 are interesting from the experimental point of view, but it is unlikely that they can be transferred to the patients. As mentioned above, CO is a dangerous gas with a rather narrow safety window; in addition,severe anaemia would be rather risky in severely sick children.  Another conundrum that warrants additional investigation is the role of the hypoxic program in rescuing the phenotype of Ndufs4-/- mice. Previous experiments from the same authors had shown that the deletion of the vHL factor improved the growth rate and glycolytic switch of antimycin A-treated cells, a model of respiratory chain dysfunction, and also prolonged the lifespan of a vHL-less zebrafish model exposed to antimycin A. By the same token, FG4592, an inhibitor of PHD currently in clinical trial for anaemia due to kidney diseases, triggering HIF1a stabilization even in normoxic conditions was able to markedly increase the survival rate of the antimycin-treated zebrafish embryos. All these data suggest a genetic link between the hypoxic response and the amelioration of the phenotype, but the data presented here argue against a direct role of HIF-dependent pathways in vivo.  Finally, the work by Jain et al. does not provide direct evidence that ROS are involved in the pathogenesis of cI-related disorders, and indeed several attempts to treat mitochondrial patients of antioxidants have given erratic results.  Unlike Janus, oxygen may well have more than two faces; we are only now starting to understand the deep consequences of altered oxygen consumption in mitochondrial dysfunction, and this will hopefully pave new avenues for treatment.    Acknowledgements The authors’ work is supported by an MRC core grant to the Mitochondrial Biology Unit (MC_UU_00015/5).    References Lane N. (2002). Oxygen: the molecule that made the world. Oxford University Press, Oxford, UK. Haldane J (1922). Respiration. Yale University Press, New Haven, US Dogan SA, Cerutti R, Benincá C, Brea-Calvo G, Jacobs HT, Zeviani M, Szibor M, Viscomi C. (2018) Perturbed Redox Signaling Exacerbates a Mitochondrial Myopathy. Cell Metabolism 28:764-775. Butow RA, Avadhani NG. (2004). Mitochondrial signaling: the retrograde response. Mol Cell. 14:1-15. Gorman, G.S., Chinnery, P.F., DiMauro, S., Hirano, M., Koga, Y., McFarland, R., Suomalainen, A., Thorburn, D.R., Zeviani, M., and Turnbull, D.M. (2016). Mitochondrial diseases. Nat Rev Dis Primers 2, 16080. Jain, I.H., Zazzeron, L., Goli, R., Alexa, K., Schatzman-Bone, S., Dhillon, H., Goldberger, O., Peng, J., Shalem, O., Sanjana, N.E., et al. (2016). Hypoxia as a therapy for mitochondrial disease. Science 352, 54-61. Ferrari, M., Jain, I.H., Goldberger, O., Rezoagli, E., Thoonen, R., Cheng, K.H., Sosnovik, D.E., Scherrer-Crosbie, M., Mootha, V.K., and Zapol, W.M. (2017). Hypoxia treatment reverses neurodegenerative disease in a mouse model of Leigh syndrome. Proceedings of the National Academy of Sciences of the United States of America 114, E4241-E4250. Murphy, M.P. (2016). Understanding and preventing mitochondrial oxidative damage. Biochemical Society transactions 44, 1219-1226. Samanta, D., and Semenza, G.L. (2017). Maintenance of redox homeostasis by hypoxia-inducible factors. Redox Biol 13, 331-335.  Jain, I.H., Zazzeron, L., Goldberg, O., Marutani, E., Wojtkiewicz, GR., Ast, T., Wang, H., Schleifer, G., et al. (2016). Leigh Syndrome Mouse Model Can Be Rescued by Interventions that Normalize Brain Hyperoxia, but Not HIF Activation. (2019). Cell Metabolism 30, 1-9 

Breathe: Your Mitochondria Will Do the Rest… If They Are Healthy!

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Breathe: Your Mitochondria Will Do the Rest… If They Are Healthy!

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Archivio istituzionale della ricerca - Università di Padova

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