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Downregulation of Jun N-terminal kinase (JNK) signaling inhibits cell migration in diverse model systems. In Drosophila pupal development, attenuated JNK signaling in the thoracic dorsal epithelium leads to defective midline closure, resulting in cleft thorax. Here we report that concomitant expression of the Ciona intestinalis alternative oxidase (AOX) was able to compensate for JNK pathway downregulation, substantially correcting the cleft thorax phenotype. AOX expression also promoted wound-healing behavior and single-cell migration in immortalized mouse embryonic fibroblasts (iMEFs), counteracting the effect of JNK pathway inhibition. However, AOX was not able to rescue developmental phenotypes resulting from knockdown of the AP-1 transcription factor, the canonical target of JNK, nor its targets and had no effect on AP-1-dependent transcription. The migration of AOX-expressing iMEFs in the wound-healing assay was differentially stimulated by antimycin A, which redirects respiratory electron flow through AOX, altering the balance between mitochondrial ATP and heat production. Since other treatments affecting mitochondrial ATP did not stimulate wound healing, we propose increased mitochondrial heat production as the most likely primary mechanism of action of AOX in promoting cell migration in these various contexts.

Activated macrophages undergo metabolic reprogramming, which drives their pro-inflammatory phenotype, but the mechanistic basis for this remains obscure. Here, we demonstrate that upon lipopolysaccharide (LPS) stimulation, macrophages shift from producing ATP by oxidative phosphorylation to glycolysis while also increasing succinate levels. We show that increased mitochondrial oxidation of succinate via succinate dehydrogenase (SDH) and an elevation of mitochondrial membrane potential combine to drive mitochondrial reactive oxygen species (ROS) production. RNA sequencing reveals that this combination induces a pro-inflammatory gene expression profile, while an inhibitor of succinate oxidation, dimethyl malonate (DMM), promotes an anti-inflammatory outcome. Blocking ROS production with rotenone by uncoupling mitochondria or by expressing the alternative oxidase (AOX) inhibits this inflammatory phenotype, with AOX protecting mice from LPS lethality. The metabolic alterations that occur upon activation of macrophages therefore repurpose mitochondria from ATP synthesis to ROS production in order to promote a pro-inflammatory state.

Alternative oxidases (AOXs) bypass respiratory complexes III and IV by transferring electrons from coenzyme Q directly to O2. They have therefore been proposed as a potential therapeutic tool for mitochondrial diseases. We crossed the severely myopathic skeletal muscle-specific COX15 knockout (KO) mouse with an AOX-transgenic mouse. Surprisingly, the double KO-AOX mutants had decreased lifespan and a substantial worsening of the myopathy compared with KO alone. Decreased ROS production in KO-AOX versus KO mice led to impaired AMPK/PGC-1α signaling and PAX7/MYOD-dependent muscle regeneration, blunting compensatory responses. Importantly, the antioxidant N-acetylcysteine had a similar effect, decreasing the lifespan of KO mice. Our findings have major implications for understanding pathogenic mechanisms in mitochondrial diseases and for the design of therapies, highlighting the benefits of ROS signaling and the potential hazards of antioxidant treatment.

Mitochondria are vital cellular organelles that produce the majority of the energy required by cells and are therefore represented as "the power house of the cell". Cellular energy, in the form of adenosine triphosphate (ATP), is generated through oxidative phosphorylation (OXPHOS), which is energized by the redox reactions of the electron transport chain (ETC) in mitochondria. Mitochondria are essential for maintaining metabolic homeostasis, fatty-acid metabolism, nucleotide synthesis and cellular redox balance, in addition to producing ATP. Given the multitude of mitochondrial functions, failure of any of them can lead to mitochondrial dysfunction that can have drastic consequences. It can lead to cell loss, organ failure and even death. Mitochondrial disorders are commonly associated with debilitating childhood onset diseases with no known cure. Current therapeutic approaches are largey symptomatic, such as dietary modification for diabetes, anti-convulsant drugs to control seizures, physical exercise for hypotonia, pacemaker implants for cardiac rhythm abnormalities and cochlear implants for sensorineural deafness. However, these are not a permanent solution or cure for severe disorders caused by genetic mutations or irreversible physical damage. Cardiomyopathy, failure of the heart muscle, is one of the most common symptoms of mitochondrial disorders, while cardiomyopathies almost always are associated with mitochondrial dysfunction. In severe forms of cardiomyopathy, the ultimate treatment option of heart transplantation is invasive and carries high risk, and the waiting list for a functional healthy heart is increasingly outstripping availability. I have investigated the use of a mitochondrial enzyme, the alternative oxidase (AOX), which is found in plants and lower eukaryotes, as well as in primitive metazoans but not in mammals as a new approach to better understand the underlying molecular mechanisms of diseases involving mitochondrial dysfunction. On a broader perspective, this would also aid in the development of a possible treatment for diseases associated with mitochondrial dysfunction. If catalytically engaged, AOX branches the mitochondrial ETC by oxidizing ubiquinol, which accumulates in the reduced form if the cytochrome pathway is impaired. By doing so it reduces oxygen directly to water, which is typically carried out by cytochrome oxidase, and thus maintains redox balance by recycling NADH and FADH2, two by-products of the Krebs cycle and upstream metabolism. Focusing on different types of cardiomyopathy, my strategy has been to introduce the AOX gene from the tunicate Ciona intestinalis, a close relative of vertebrates, into mouse models of disease, using genomic manipulation. I have studied and characterized two transgenic mouse models expressing C. intestinalis AOX. The first was designed to express AOX constitutively and ubiquitously. I verified this at the RNA and protein level revealing high-level expression in all tissues tested, including the heart, with the exception of the adult brain. The mice did not show any observable phenotype, making them nearly indistinguishable from their wild-type littermates under non-stressed conditions. Heart function, as measured by ejection fraction and left ventricular mass, treadmill performance and grip strength were all normal in one-year-old AOX-expressing mice. Weight was indistinguishable from that of wild-type littermates on chow or high-fat diet; although on ketogenic diet AOX-expressing mice showed a slightly mitigated weight gain, at least when caged in same-sex groups. A second AOX transgenic line was characterized, in which AOX expression was designed to be activatable by Cre-loxP-mediated removal of a STOP cassette (SNAP coding sequence, pA signal) located downstream of a CAG promoter and upstream of the AOX coding sequence. After verification of the transgenic insertion by PCR and Southern blotting, test activation by breeding to mice ubiquitously expressing Cre recombinase under the control of a β-actin promoter resulted in successful activation, based on ubiquitous expression of AOX and concomitant loss of SNAP expression. This allowed me to test the tissue-specificity of AOX-mediated modification of pathological phenotypes in mouse models of cardiomyopathy. Two different mouse models of cardiomyopathy were investigated. In the first, inflammatory cardiomyopathy was induced by overexpressing monocyte chemo-attractant protein 1 (MCP1) in the cardiomyocytes, which leads to loss of cardiac function commencing during early adulthood (12 weeks of age). Previously published data showed mitochondrial structural damage, decrease in ATP levels; and suggested induced oxidative stress as a pathological mechanism. Although the majority of oxidative stress in the cardiomyocytes of Mcp1 mice is induced by the infiltrating monocytes, via NADPH oxidase pathway, the contribution of mitochondria to the production of reactive oxygen species (ROS) is not well understood. I hypothesized that mitochondrial ROS production, especially via a defective or overloaded cytochrome pathway in the mitochondrial respiratory chain, might play a key role in the underlying molecular mechanisms. Therefore we wanted to test whether AOX could mitigate it, as it has been shown to alleviate oxidative stress under conditions where the cytochrome pathway is dysfunctional. I found that expressing AOX in this model preserved cardiac ejection fraction at 12 weeks of age but not at later time points. At the molecular level, I determined that mitochondrial complex I-linked respiration was severely affected in Mcp1-overexpressing mice irrespective of AOX expression. However, AOX preserved complex II mediated respiration. In theory, this should maintain mitochondrial redox homeostasis and limit oxidative stress and damage within mitochondria, but at the expense of ATP production and mitochondrial-ROS signaling. Ultrastructural analysis revealed mitochondrial damage in the cardiac tissue of 12 week-old Mcp1-overexpressing mice, which was alleviated by AOX expression. Despite this, AOX had no effect on the survival of Mcp1-overexpressing mice, whilst cardiomyocyte-specific AOX expression actually resulted in earlier death than Mcp1 alone. Autophagic markers were mildly elevated in all cases, and metabolic changes consistent with OXPHOS dysfunction were essentially unaffected by AOX expression in the Mcp1 mouse model. Overall, I conclude that AOX is insufficient to block lethal damage to mitochondria and/or other cellular components in this inflammatory cardiomyopathy model, despite transient beneficial effects. Previously, West et al (2011) and Mills et al (2016) reported on the significance of mitochondrial ROS in inducing macrophage activity in a LPS-induced inflammatory mouse model. Additionally, Mills et al (2016) also showed AOX expression prevented LPS-induced lethality in mice, where succinate-dependent ROS production in the ETC was reported to be the underlying cause for the observed severe phenotype. However, since AOX was unable to prevent lethality in Mcp1 mice, it is indicative that the ROS production in this inflammatory model might not be dependent on the ETC. The second model I studied was of Cox-deficient mitochondrial cardiomyopathy, induced by cardiomyocyte-specific knockout of Cox10, an essential enzyme of heme a biosynthesis. Cox10 knockout in the heart was lethal within the first days or weeks of life, and concomitant AOX expression had hardly any effect on this. Mice with heterozygous knockout of Cox10 survived for months but eventually succumbed to heart failure. However, this phenotype was produced by the cardiomyocyte-specific Cre expression alone, and was again exacerbated by AOX activation, despite transient improvements in cardiac performance in young mice. In conclusion, AOX can serve as a valuable tool to study disease mechanisms where mitochondrial dysfunction is proposed to play a key role in the pathophysiology. Although AOX did not rescue the disease models I studied, it did shed light on underlying molecular mechanisms. Considering the fact that AOX showed a partial, if transient, functional rescue in both of the heart failure models investigated, it might yet be relevant as a potential therapeutic option. Further research is necessary to fully understand the therapeutic potential of AOX, either by itself or in combination with other treatments that address different pathological processes, as well as the apparently negative effects of AOX itself during disease progression. Mitokondriot ovat elintärkeitä soluorganelleja, jotka tuottavat suurimman osan solujen tarvitsemasta energiasta ja ovat välttämättömiä ylläpitämään useita aineenvaihduntareittejä. Siten mitokondrioiden toimintahäiriöt voivat usein johtaa solujen menetykseen, elinten vajaatoimintaan ja jopa kuolemaan. Mitokondriaaliset sairaudet liittyvät yleensä vakaviin lapsuuden aikaisiin sairauksiin, joille ei ole tunnettuja parannuskeinoja. Kardiomyopatia, sydänlihaksen vajaatoiminta, on yksi yleisimmistä mitokondriohäiriöiden oireista, ja se on yksi suurimmista kuolinsyistä maailmassa. Ymmärtääkseni paremmin sellaisten sairauksien taustalla olevia molekyylimekanismeja, joihin liittyy mitokondrioiden toimintahäiriöitä, etenkin hengityskompleksien III ja / tai IV aiheuttamia toimintahäiriöitä, olen tutkinut mitokondriaalista entsyymiä, vaihtoehtoista oksidaasia (AOX), joka löytyy kasveista ja alemmista eukaryooteista, mutta ei nisäkkäistä. Katalyyttisesti aktivoituneena AOX haaroittaa mitokondriaalisen hengitysketjun ohittaen hengityskompleksit III ja IV ja redusoiden hapen suoraan vedeksi, minkä tyypillisesti suorittaa hengityskompleksi IV. Keskityin tutkimaan AOX-ilmentymisen vaikutusta erityyppisissä kardiomyopatioissa. Tätä varten strategiani on ollut siirtää AOX-geeni Ciona intestinalis -vaippaeläimestä kardiomyopatiahiirimalleihin geenimanipulaatiolla. Olen tutkinut ja karakterisoinut kahta siirtogeenistä hiirimallia, jotka ekspressoivat C. intestinalis AOX-geeniä. Ensimmäinen malli oli suunniteltu ekspressoimaan AOX:ia jatkuvasti ja kaikkialla, ja toinen AOX-siirtogeeninen linja suunniteltiin aktivoitavaksi tietyissä kudoksissa Cre-loxP-välitteisellä geenimanipulaatiolla. Molemmissa hiirimalleissa AOX-ekspressio varmistettiin RNA- ja proteiinitasoilla. Hiirillä ei ollut havaittavaa fenotyyppiä, mikä teki niiden erottamisen villityyppistä poikuetovereistaan lähes mahdottomaksi stressittömissä olosuhteissa. Ensimmäisessä mallissa normaali tai korkearasvainen ruokavalio ei erottanut siirtogeenisiä eläimiä villityyppisistä eläimistä, tosin ketogeenisellä ruokavaliolla AOX:ia ekspressoivat hiiret osoittivat hieman lievempää painonnousua kuin villityyppiset eläimet, ainakin ollessaan samaa sukupuolta olevien joukossa. Kahta erilaista kardiomyopatiahiirimallia tutkittiin. Ensimmäisessä indusoitiin tulehduksellinen kardiomyopatia MCP1 (monocyte chemo-attractant protein 1) yliekspression avulla sydänlihassoluissa, mikä johtaa sydämen toiminnan menettämiseen, joka alkaa varhaisessa aikuisiässä (12 viikon ikäisenä). Aikaisempien julkaisujen tutkimustulokset ovat osoittaneet mitokondrioiden rakennevaurioita, ATP-tasojen laskua, ja viittaavat indusoituun oksidatiiviseen stressiin patologisena mekanismina. Hypoteesin mukaan mitokondriaalisten reaktiivisten happiradikaalien (ROS) tuotannolla voisi olla avainrooli taustalla olevissa molekyylimekanismeissa. Siksi halusin testata, voisiko AOX lieventää sitä, koska sen on osoitettu lievittävän oksidatiivista stressiä olosuhteissa, joissa hengityskomplekseissa III ja / tai IV on toimintahäiriöitä. Havaitsin, että AOX:n ilmentäminen tässä mallissa säilytti sydämen ejektiofraktion 12 viikon iässä, mutta ei myöhemmin. Molekyylitasolla määrittelin, että mitokondriaalisen kompleksin I-linkittynyt hengitys vaikutti voimakkaasti MCP1:tä yliekspressoivissa hiirissä riippumatta AOX-ekspressiosta. AOX kuitenkin säilytti kompleksin II välittämän hengityksen. Teoriassa tämän pitäisi ylläpitää mitokondrioiden redox-homeostaasia ja rajoittaa oksidatiivista stressiä ja vaurioita mitokondrioissa, mutta ATP-tuotannon ja mitokondrion ROS-signaloinnin kustannuksella. Tästä huolimatta AOX:lla ei ollut vaikutusta MCP1:tä yliekspressoivien hiirten selviytymiseen, kun taas sydänsolujen spesifinen AOX-ekspressio johti tosiasiallisesti aikaisempaan kuolemaan kuin pelkkä MCP1. Kaiken kaikkiaan päättelen, että AOX ei riitä estämään mitokondrioiden ja/tai muiden solukomponenttien tappavia vaurioita tässä tulehduksellisessa kardiomyopatian mallissa, huolimatta ohimenevistä hyödyllisistä vaikutuksista. Nämä havainnot osoittavat, että ROS-tuotanto tässä tulehduksellisessa mallissa ei ehkä ole riippuvainen ETC:stä. Toinen tutkimani malli oli Cox-vajaatoimintainen mitokondriaalinen kardiomyopatia, jonka aiheuttaa kardiomyosyyttispesifinen Cox10:n puute, hemibiosynteesin välttämätön entsyymi. Cox10:n geenin poisto sydämestä oli tappavaa elämän ensimmäisinä päivinä tai viikkoina, ja samanaikaisella AOX-ekspressiolla ei ollut juuri mitään vaikutusta tähän. Hiiret, joilla oli heterotsygoottinen Cox10-geenin poisto, selvisivät kuukausia, mutta lopulta menehtyivät sydämen vajaatoimintaan. Tämä fenotyyppi tuotettiin kuitenkin pelkästään kardiomyosyyttispesifisellä Cre-ilmentymällä, ja AOX-aktivaatio pahensi sitä uudelleen, huolimatta sydämen suorituskyvyn ohimenevistä parannuksista nuorilla hiirillä. Yhteenvetona voidaan todeta, että AOX voi toimia arvokkaana välineenä tutkittaessa sairauden mekanismeja, joissa mitokondrioiden toimintahäiriöiden uskotaan olevan avainasemassa patofysiologiassa. Vaikka AOX ei toiminut pelastuskeinona tutkimissani sairausmalleissa, se valotti taustalla olevia molekyylimekanismeja. Kun otetaan huomioon, että AOX osoitti olevansa osittainen, joskin ohimenevä, toiminnallinen pelastuskeino molemmissa tutkituissa sydämen vajaatoimintamalleissa, se saattaa vielä tulevaisuudessa olla potentiaalinen terapeuttinen vaihtoehto. Jatkotutkimukset ovat välttämättömiä, jotta voidaan täysin ymmärtää AOX:n terapeuttinen potentiaali, joko itsenäisesti tai yhdessä muiden patologisiin prosesseihin kohdistuvien hoitomenetelmien kanssa, mutta yhtä lailla myös AOX:n nähtävästi negatiiviset vaikutukset sairauden etenemisen aikana.