Expression of Ciona intestinalis AOX causes male reproductive defects in Drosophila melanogaster

Background: Mitochondrial alternative respiratory-chain enzymes are phylogenetically widespread, and buffer stresses affecting oxidative phosphorylation in species that possess them. However, they have been lost in the evolutionary lineages leading to vertebrates and arthropods, raising the question as to what survival or reproductive disadvantages they confer. Recent interest in using them in therapy lends a biomedical dimension to this question. Methods: Here, we examined the impact of the expression of Ciona intestinalis alternative oxidase, AOX, on the reproductive success of Drosophila melanogaster males. Sperm-competition assays were performed between flies carrying three copies of a ubiquitously expressed AOX construct, driven by the a-tubulin promoter, and wild-type males of the same genetic background. Results: In sperm-competition assays, AOX conferred a substantial disadvantage, associated with decreased production of mature sperm. Sperm differentiation appeared to proceed until the last stages, but was spatially deranged, with spermatozoids retained in the testis instead of being released to the seminal vesicle. High AOX expression was detected in the outermost cell-layer of the testis sheath, which we hypothesize may disrupt a signal required for sperm maturation. Conclusions: AOX expression in Drosophila thus has effects that are deleterious to male reproductive function. Our results imply that AOX therapy must be developed with caution.Peer reviewe

Expression of Ciona intestinalis AOX causes male reproductive defects in Drosophila melanogaster

Background: Mitochondrial alternative respiratory-chain enzymes are phylogenetically widespread, and buffer stresses affecting oxidative phosphorylation in species that possess them. However, they have been lost in the evolutionary lineages leading to vertebrates and arthropods, raising the question as to what survival or reproductive disadvantages they confer. Recent interest in using them in therapy lends a biomedical dimension to this question. Methods: Here, we examined the impact of the expression of Ciona intestinalis alternative oxidase, AOX, on the reproductive success of Drosophila melanogaster males. Sperm-competition assays were performed between flies carrying three copies of a ubiquitously expressed AOX construct, driven by the a-tubulin promoter, and wild-type males of the same genetic background. Results: In sperm-competition assays, AOX conferred a substantial disadvantage, associated with decreased production of mature sperm. Sperm differentiation appeared to proceed until the last stages, but was spatially deranged, with spermatozoids retained in the testis instead of being released to the seminal vesicle. High AOX expression was detected in the outermost cell-layer of the testis sheath, which we hypothesize may disrupt a signal required for sperm maturation. Conclusions: AOX expression in Drosophila thus has effects that are deleterious to male reproductive function. Our results imply that AOX therapy must be developed with caution.Peer reviewe

The Most Recently Discovered Carbonic Anhydrase, CA XV, Is Expressed in the Thick Ascending Limb of Henle and in the Collecting Ducts of Mouse Kidney

BACKGROUND: Carbonic anhydrases (CAs) are key enzymes for physiological pH regulation, including the process of urine acidification. Previous studies have identified seven cytosolic or membrane-bound CA isozymes in the kidney. Recently, we showed by in situ hybridization that the mRNA for the most novel CA isozyme, CA XV, is present in the renal cortex. CA XV is a unique isozyme among mammalian CAs, because it has become a pseudogene in primates even though expressed in several other species. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, we raised a polyclonal antibody against recombinant mouse CA XV that was produced in a baculovirus/insect cell expression system, and the antibody was used for immunohistochemical analysis in different mouse tissues. Positive immunoreactions were found only in the kidney, where the enzyme showed a very limited distribution pattern. Parallel immunostaining experiments with several other anti-CA sera indicated that CA XV is mainly expressed in the thick ascending limb of Henle and collecting ducts, and the reactions were most prominent in the cortex and outer medulla. CONCLUSION/SIGNIFICANCE: Although other studies have proposed a role for CA XV in cell proliferation, its tightly limited distribution may point to a specialized function in the regulation of acid-base homeostasis

Alternative respiratory chain enzymes: Therapeutic potential and possible pitfalls

The alternative respiratory chain (aRC), comprising the alternative NADH dehydrogenases (NDX) and quinone oxidases (AOX), is found in microbes, fungi and plants, where it buffers stresses arising from restrictions on electron flow in the oxidative phosphorylation system. The aRC enzymes are also found in species belonging to most metazoan phyla, including some chordates and arthropods species, although not in vertebrates or in Drosophila. We postulated that the aRC enzymes might be deployed to alleviate pathological stresses arising from mitochondrial dysfunction in a wide variety of disease states. However, before such therapies can be contemplated, it is essential to understand the effects of aRC enzymes on cell metabolism and organismal physiology. Here we report and discuss new findings that shed light on the functions of the aRC enzymes in animals, and the unexpected benefits and detriments that they confer on model organisms. In Ciona intestinalis, the aRC is induced by hypoxia and by sulfide, but is unresponsive to other environmental stressors. When expressed in Drosophila, AOX results in impaired survival under restricted nutrition, in addition to the previously reported male reproductive anomalies. In contrast, it confers cold resistance to developing and adult flies, and counteracts cell signaling defects that underlie developmental dysmorphologies. The aRC enzymes may also influence lifespan and stress resistance more generally, by eliciting or interfering with hormetic mechanisms. In sum, their judicious use may lead to major benefits in medicine, but this will require a thorough characterization of their properties and physiological effects.Peer reviewe

Alternative respiratory chain enzymes: Therapeutic potential and possible pitfalls

The alternative respiratory chain (aRC), comprising the alternative NADH dehydrogenases (NDX) and quinone oxidases (AOX), is found in microbes, fungi and plants, where it buffers stresses arising from restrictions on electron flow in the oxidative phosphorylation system. The aRC enzymes are also found in species belonging to most metazoan phyla, including some chordates and arthropods species, although not in vertebrates or in Drosophila. We postulated that the aRC enzymes might be deployed to alleviate pathological stresses arising from mitochondrial dysfunction in a wide variety of disease states. However, before such therapies can be contemplated, it is essential to understand the effects of aRC enzymes on cell metabolism and organismal physiology. Here we report and discuss new findings that shed light on the functions of the aRC enzymes in animals, and the unexpected benefits and detriments that they confer on model organisms. In Ciona intestinalis, the aRC is induced by hypoxia and by sulfide, but is unresponsive to other environmental stressors. When expressed in Drosophila, AOX results in impaired survival under restricted nutrition, in addition to the previously reported male reproductive anomalies. In contrast, it confers cold resistance to developing and adult flies, and counteracts cell signaling defects that underlie developmental dysmorphologies. The aRC enzymes may also influence lifespan and stress resistance more generally, by eliciting or interfering with hormetic mechanisms. In sum, their judicious use may lead to major benefits in medicine, but this will require a thorough characterization of their properties and physiological effects.</p

Molecular pathways behind acquired obesity : Adipose tissue and skeletal muscle multiomics in monozygotic twin pairs discordant for BMI

Tissue-specific mechanisms prompting obesity-related development complications in humans remain unclear. We apply multiomics analyses of subcutaneous adipose tissue and skeletal muscle to examine the effects of acquired obesity among 49 BMI-discordant monozygotic twin pairs. Overall, adipose tissue appears to be more affected by excess body weight than skeletal muscle. In heavier co-twins, we observe a transcriptional pattern of downregulated mitochondrial pathways in both tissues and upregulated inflammatory pathways in adipose tissue. In adipose tissue, heavier co-twins exhibit lower creatine levels; in skeletal muscle, glycolysis- and redox stress-related protein and metabolite levels remain higher. Furthermore, metabolomics analyses in both tissues reveal that several proinflammatory lipids are higher and six of the same lipid derivatives are lower in acquired obesity. Finally, in adipose tissue, but not in skeletal muscle, mitochondrial downregulation and upregulated inflammation are associated with a fatty liver, insulin resistance, and dyslipidemia, suggesting that adipose tissue dominates in acquired obesity.Peer reviewe

Nicotinamide riboside improves muscle mitochondrial biogenesis, satellite cell differentiation, and gut microbiota in a twin study

Nicotinamide adenine dinucleotide (NAD(+)) precursor nicotinamide riboside (NR) has emerged as a promising compound to improve obesity-associated mitochondrial dysfunction and metabolic syndrome in mice. However, most short-term clinical trials conducted so far have not reported positive outcomes. Therefore, we aimed to determine whether long-term NR supplementation boosts mitochondrial biogenesis and metabolic health in humans. Twenty body mass index (BMI)- discordant monozygotic twin pairs were supplemented with an escalating dose of NR (250 to 1000 mg/day) for 5 months. NR improved systemic NAD(+) metabolism, muscle mitochondrial number, myoblast differentiation, and gut microbiota composition in both cotwins. NR also showed a capacity to modulate epigenetic control of gene expression in muscle and adipose tissue in both cotwins. However, NR did not ameliorate adiposity or metabolic health. Overall, our results suggest that NR acts as a potent modifier of NAD(+) metabolism, muscle mitochondrial biogenesis and stem cell function, gut microbiota, and DNA methylation in humans irrespective of BMI.Peer reviewe

Effects of Alternative Oxidase on Drosophila under Environmental Stress

Mitokondrio on ainutlaatuinen soluelin, joka toimii keskeisenä osana solun energiantuotantoa ja aineenvaihduntaa säädellen näin koko solun elämää ja kuolemaa. Häiriöt mitokondrioiden toiminnassa, kuten oksidatiivisen fosforylaation (OXPHOS) vajaatoiminta, heikentävät solun energiantuotantoa. Mitokondrion vaihtoehtoisen oksidaasin (AOX) kyky sivuuttaa kompleksit III ja IV OXPHOS - ketjussa on tehnyt entsyymistä potentiaalisen hoitomuodon sairauksiin, joissa mitokondrioiden toimintahäiriöitä esiintyy. AOX:n on todettu lisäävän aineenvaihdunnallista sopeutumiskykyä useissa eukaryooteissa, mutta entsyymi on kadonnut selkärankaisista ja useimmista niveljalkaisista evoluution myötä. Siitä huolimatta vaippaeläin Ciona intestinaliksen AOX on onnistuneesti siirretty useisiin mallieliöihin kuten viljeltyihin ihmissoluihin, banaanikärpäseen Drosophila melanogasteriin sekä viimeisimpänä hiireen. Normaaleissa laboratorio-olosuhteissa entsyymillä ei ole todettu olevan haitallisia vaikutuksia mallieliöiden kehitykseen tai elinkykyyn. Selvittääkseni paremmin mahdollisia syitä AOX:n katoamiselle selkärankaisista ja banaanikärpäsestä sekä sen mahdollisia rajoituksia terapeuttisena hoitona, altistin AOX-kärpäsiä haasteellisille ympäristötekijöille, joita eläin kohtaa luonnollisessa ympäristössään. Näihin lukeutui eläinten lisääntymiskyvyn testaaminen kilpailutilanteessa sekä kehittyminen ravitsemuksellisesti niukoissa olosuhteissa. AOX-kärpäskoiraiden lisääntymiskykyä testattiin parittamalla villityypin naaraita peräkkäin sekä AOX-koiraiden että villityypin koiraiden kanssa. Koeasetelma osoitti naaraiden suosivan villityypin koiraiden siittiöitä, myös tapauksissa, joissa AOX- koiras oli jälkimmäisenä paritteleva koiras, joka yleisesti syrjäyttää ensimmäisen koiraan siittiöt. Kivesten histologisessa tarkastelussa AOX-koiraiden spermatogeneesiprosessi osoittautui heikentyneeksi. AOX-kärpästen painonpudotus ja hidastunut kehitys standardiolosuhteissa herätti kysymyksen siitä, vaikuttaako AOX negatiivisesti kärpäsen energia- aineenvaihduntaan. Tutkin asiaa kasvattamalla kärpäsiä niukassa ravinnossa, joka koostui ainoastaan kahdesta ainesosasta, kuivahiivasta ja glukoosista. Ravinnon ollessa rajallisempaa kuoriutuvien AOX-kärpästen osuus laski merkittävästi ja peräti ~80 % kärpäsistä kuoli muodonmuutosvaiheessa, kun kontrollikärpäsistä puolestaan ~90 % kehittyi normaalisti. Pelkän hiivan, sakkaroosin tai monosakkaridien lisääminen ravintoon ei parantanut kehitystä mutta melassin, sokerituotannossa syntyvän ravintorikkaan sivutuotteen, lisäys palautti kuoriutuvien kärpästen määrän normaaliksi, mikä viittasi aineenvaihdunnalliseen epätasapainoon yksinkertaisen energianpuutteen sijaan. Sekä lisääntymis- että ravintokokeiden tulokset viittaavat AOX:n aktivoitumiseen tietyissä kudosten ja solujen kehitysvaiheissa kuten uudelleen organisoitumisessa ja solujen erilaistuessa muodonmuutoksessa tai spermatogeneesissä, ja mahdollisesti häiritsevän normaalia signalointia ja energia-aineenvaihduntaa. Nämä vaikutukset ja niiden parempi ymmärtäminen on syytä ottaa huomioon AOX:n mahdollisessa kehityksessä terapeuttiseksi hoitomuodoksi.The mitochondrion is a unique organelle with a central role in energy production and metabolic homeostasis while regulating the life and death of the entire cell. In mitochondrial dysfunction, deficiencies or abnormalities of the mitochondrial oxidative phosphorylation (OXPHOS) system impair cellular energy production. Due to its ability to bypass Complex III and IV of the OXPHOS system and divert electrons from ubiquinol to oxygen, the mitochondrial alternative oxidase (AOX) has drawn attention as a potential therapy for diseases where mitochondria are affected. AOX provides metabolic flexibility to organisms across the eukaryote kingdoms but has been lost in vertebrates and most arthropods in the course of evolution. The AOX from the tunicate Ciona intestinalis has been introduced into several model organisms such as cultured human cells, the fruit fly Drosophila melanogaster and most recently, mice. Characterization of these transgenic model systems under standard laboratory conditions revealed no obvious detrimental effects on the survival and fitness of these organisms. To shed light on the possible reasons behind the loss of AOX in vertebrates and Drosophila and on the potential drawbacks of implementing the enzyme for therapeutic purposes, I exposed AOX-expressing flies to environmental stressors which the animals encounter in the wild. This included testing reproductive fitness and nutritional requirements. The reproductive competence of AOX-expressing male flies was tested by successively mating wild-type females with AOX-expressing and wild-type males. The assay demonstrated a clear selective advantage of sperm from wild-type over AOX-expressing males, even when the AOX-expressing male was the second male to be mated, which usually competes out the sperm of the first male. Histological examination of the testis showed a spatially deranged spermatogenesis programme in the AOX-expressing males. The slight but significant weight loss and development delay that we regularly observe in AOX-expressing flies, led me to suspect a negative effect of the transgene on the energy metabolism of the flies. I investigated this further by rearing AOX flies on media in which one or more component of the standard laboratory diet was omitted. Diets restricted to two ingredients, dry yeast and glucose caused a decreased eclosion rate in AOX-expressing flies, ~80 % of them failing to complete metamorphosis, whereas most control flies (~90 %) developed normally. Rescue by dietary supplementation with treacle, a nutritionally complex by-product of sugar refinement, but not by yeast, sucrose or monosaccharides, pointed to an imbalance in metabolic homeostasis rather than a simple insufficiency of metabolic fuel. Altogether these findings indicate that AOX may be activated in specific developmental contexts involving tissue reorganization and cell differentiation, such as metamorphosis and spermatogenesis, thereby potentially interfering with developmental signaling and the efficient use of nutrients. These effects need to be better understood and taken into consideration for the development of AOX as atherapeutic treatment

Effects of Alternative Oxidase on Drosophila under Environmental Stress

Mitokondrio on ainutlaatuinen soluelin, joka toimii keskeisenä osana solun energiantuotantoa ja aineenvaihduntaa säädellen näin koko solun elämää ja kuolemaa. Häiriöt mitokondrioiden toiminnassa, kuten oksidatiivisen fosforylaation (OXPHOS) vajaatoiminta, heikentävät solun energiantuotantoa. Mitokondrion vaihtoehtoisen oksidaasin (AOX) kyky sivuuttaa kompleksit III ja IV OXPHOS - ketjussa on tehnyt entsyymistä potentiaalisen hoitomuodon sairauksiin, joissa mitokondrioiden toimintahäiriöitä esiintyy. AOX:n on todettu lisäävän aineenvaihdunnallista sopeutumiskykyä useissa eukaryooteissa, mutta entsyymi on kadonnut selkärankaisista ja useimmista niveljalkaisista evoluution myötä. Siitä huolimatta vaippaeläin Ciona intestinaliksen AOX on onnistuneesti siirretty useisiin mallieliöihin kuten viljeltyihin ihmissoluihin, banaanikärpäseen Drosophila melanogasteriin sekä viimeisimpänä hiireen. Normaaleissa laboratorio-olosuhteissa entsyymillä ei ole todettu olevan haitallisia vaikutuksia mallieliöiden kehitykseen tai elinkykyyn. Selvittääkseni paremmin mahdollisia syitä AOX:n katoamiselle selkärankaisista ja banaanikärpäsestä sekä sen mahdollisia rajoituksia terapeuttisena hoitona, altistin AOX-kärpäsiä haasteellisille ympäristötekijöille, joita eläin kohtaa luonnollisessa ympäristössään. Näihin lukeutui eläinten lisääntymiskyvyn testaaminen kilpailutilanteessa sekä kehittyminen ravitsemuksellisesti niukoissa olosuhteissa. AOX-kärpäskoiraiden lisääntymiskykyä testattiin parittamalla villityypin naaraita peräkkäin sekä AOX-koiraiden että villityypin koiraiden kanssa. Koeasetelma osoitti naaraiden suosivan villityypin koiraiden siittiöitä, myös tapauksissa, joissa AOX- koiras oli jälkimmäisenä paritteleva koiras, joka yleisesti syrjäyttää ensimmäisen koiraan siittiöt. Kivesten histologisessa tarkastelussa AOX-koiraiden spermatogeneesiprosessi osoittautui heikentyneeksi. AOX-kärpästen painonpudotus ja hidastunut kehitys standardiolosuhteissa herätti kysymyksen siitä, vaikuttaako AOX negatiivisesti kärpäsen energia- aineenvaihduntaan. Tutkin asiaa kasvattamalla kärpäsiä niukassa ravinnossa, joka koostui ainoastaan kahdesta ainesosasta, kuivahiivasta ja glukoosista. Ravinnon ollessa rajallisempaa kuoriutuvien AOX-kärpästen osuus laski merkittävästi ja peräti ~80 % kärpäsistä kuoli muodonmuutosvaiheessa, kun kontrollikärpäsistä puolestaan ~90 % kehittyi normaalisti. Pelkän hiivan, sakkaroosin tai monosakkaridien lisääminen ravintoon ei parantanut kehitystä mutta melassin, sokerituotannossa syntyvän ravintorikkaan sivutuotteen, lisäys palautti kuoriutuvien kärpästen määrän normaaliksi, mikä viittasi aineenvaihdunnalliseen epätasapainoon yksinkertaisen energianpuutteen sijaan. Sekä lisääntymis- että ravintokokeiden tulokset viittaavat AOX:n aktivoitumiseen tietyissä kudosten ja solujen kehitysvaiheissa kuten uudelleen organisoitumisessa ja solujen erilaistuessa muodonmuutoksessa tai spermatogeneesissä, ja mahdollisesti häiritsevän normaalia signalointia ja energia-aineenvaihduntaa. Nämä vaikutukset ja niiden parempi ymmärtäminen on syytä ottaa huomioon AOX:n mahdollisessa kehityksessä terapeuttiseksi hoitomuodoksi.The mitochondrion is a unique organelle with a central role in energy production and metabolic homeostasis while regulating the life and death of the entire cell. In mitochondrial dysfunction, deficiencies or abnormalities of the mitochondrial oxidative phosphorylation (OXPHOS) system impair cellular energy production. Due to its ability to bypass Complex III and IV of the OXPHOS system and divert electrons from ubiquinol to oxygen, the mitochondrial alternative oxidase (AOX) has drawn attention as a potential therapy for diseases where mitochondria are affected. AOX provides metabolic flexibility to organisms across the eukaryote kingdoms but has been lost in vertebrates and most arthropods in the course of evolution. The AOX from the tunicate Ciona intestinalis has been introduced into several model organisms such as cultured human cells, the fruit fly Drosophila melanogaster and most recently, mice. Characterization of these transgenic model systems under standard laboratory conditions revealed no obvious detrimental effects on the survival and fitness of these organisms. To shed light on the possible reasons behind the loss of AOX in vertebrates and Drosophila and on the potential drawbacks of implementing the enzyme for therapeutic purposes, I exposed AOX-expressing flies to environmental stressors which the animals encounter in the wild. This included testing reproductive fitness and nutritional requirements. The reproductive competence of AOX-expressing male flies was tested by successively mating wild-type females with AOX-expressing and wild-type males. The assay demonstrated a clear selective advantage of sperm from wild-type over AOX-expressing males, even when the AOX-expressing male was the second male to be mated, which usually competes out the sperm of the first male. Histological examination of the testis showed a spatially deranged spermatogenesis programme in the AOX-expressing males. The slight but significant weight loss and development delay that we regularly observe in AOX-expressing flies, led me to suspect a negative effect of the transgene on the energy metabolism of the flies. I investigated this further by rearing AOX flies on media in which one or more component of the standard laboratory diet was omitted. Diets restricted to two ingredients, dry yeast and glucose caused a decreased eclosion rate in AOX-expressing flies, ~80 % of them failing to complete metamorphosis, whereas most control flies (~90 %) developed normally. Rescue by dietary supplementation with treacle, a nutritionally complex by-product of sugar refinement, but not by yeast, sucrose or monosaccharides, pointed to an imbalance in metabolic homeostasis rather than a simple insufficiency of metabolic fuel. Altogether these findings indicate that AOX may be activated in specific developmental contexts involving tissue reorganization and cell differentiation, such as metamorphosis and spermatogenesis, thereby potentially interfering with developmental signaling and the efficient use of nutrients. These effects need to be better understood and taken into consideration for the development of AOX as atherapeutic treatment

Localization and expression of carbonic anhydrase isozyme XV in mouse tissues

Tutkimuksen tausta ja tavoitteet: Hiilihappoanhydraasi XV (CA XV) on a-hiilihappoanhydraasi-entsyymiperheen viimeisin jäsen. Kädellisissä sitä koodaavasta geenistä on tullut pseudogeeni ja sen fysiologinen merkitys muissa lajeissa on vielä tuntematon. Tutkimuksen tavoitteena oli selvittää CA XV:n sijainti hiiren kudoksissa ja selvittää sen mahdollisia kompensoivia ominaisuuksia muiden hiilihappoanhydraasien suhteen poistogeenisissä kudoksissa. Tutkimusmenetelmät: CA XV paikannettiin hiiren kudoksista immunohistokemian avulla ja sen sijaintia vertailtiin muiden isoentsyymien esiintymiseen. CA XV:n kompensoivaa merkitystä CA IV ja CA XIV poistogeenisissä kudoksissa tutkittiin immunohistokemian lisäksi myös geeni-ilmentymisen tasolla kvantitatiivisella reaaliaikaisella PCR:llä (qRT-PCR). Tutkimustulokset: CA XV:n esiintyminen hiiren kudoksissa on hiilihappoanhydraasiisoentsyymeistä rajoittuneinta. Entsyymiä esiintyy vain munuaisissa, jossa sitä havaittiin eniten kuorikerroksen alueella mutta myös jonkin verran ytimen ulommassa osassa. Munuaistiehyessä entsyymiä esiintyy Henlen lingon paksussa nousevassa osassa sekä kokoojatiehyessä. Vertailevat värjäykset osoittivat, että tämä esiintymiskaava on ainutlaatuinen munuaisissa esiintyvien isoentsyymien keskuudessa. Immunohistokemialla oli havaittavissa pieniä muutoksia CA XV:n esiintymisessä poistogeenisissä kudoksissa, mutta transkriptionaalisella tasolla CA IV tai CA XIV puuttumisella ei ollut vaikutusta Car15:n geenin ilmentymiseen. Johtopäätökset: Immunohistokemialla havaittu voimakas reaktio munuaisissa viittaa siihen, että CA XV:llä on oleellinen merkitys munuaisten fysiologiassa ja isoentsyymi tulisi ottaa huomioon tulevissa eläinmalleja hyödyntävissä inhibiittoritutkimuksissa