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Eႇects of Alternative Oxidase on Drosophila Under Environmental

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Eႇects of Alternative Oxidase on Drosophila Under Environmental 6,1$6$$5, (ႇHFWVRI$OWHUQDWLYH 2[LGDVHRQDrosophila XQGHU(QYLURQPHQWDO6WUHVV 7DPSHUH8QLYHUVLW\'LVVHUWDWLRQV 7DPSHUH8QLYHUVLW\'LVVHUWDWLRQV 6,1$6$$5, (IIHFWVRI$OWHUQDWLYH 2[LGDVHRQDrosophila XQGHU(QYLURQPHQWDO6WUHVV $&$'(0,&',66(57$7,21 7REHSUHVHQWHGZLWKWKHSHUPLVVLRQRI WKH)DFXOW\RI0HGLFLQHDQG+HDOWK7HFKQRORJ\ RI7DPSHUH8QLYHUVLW\ IRUSXEOLFGLVFXVVLRQLQWKHDXGLWRULXP) RIWKH$UYREXLOGLQJ$UYR<OS|QNDWX7DPSHUH RQ2FWREHUDWR¶FORFN $&$'(0,&',66(57$7,21 7DPSHUH8QLYHUVLW\)DFXOW\RI0HGLFLQHDQG+HDOWK7HFKQRORJ\ )LQODQG Responsible 3URIHVVRU+RZDUG7-DFREV supervisor 7DPSHUH8QLYHUVLW\ and Custos )LQODQG Pre-examiners 3URIHVVRU:LOOLDP-%DOODUG 'RFHQW(LMD3LULQHQ 8QLYHUVLW\RI1HZ6RXWK:DOHV 8QLYHUVLW\RI+HOVLQNL $XVWUDOLD )LQODQG Opponent $VVRFLDWH3URIHVVRU9LOOH+LHWDNDQJDV 8QLYHUVLW\RI+HOVLQNL )LQODQG 7KHRULJLQDOLW\RIWKLVWKHVLVKDVEHHQFKHFNHGXVLQJWKH7XUQLWLQ2ULJLQDOLW\&KHFN VHUYLFH &RS\ULJKWDXWKRU &RYHUGHVLJQ5RLKX,QF ,6%1 SULQW ,6%1 SGI ,661 SULQW ,661 SGI KWWSXUQIL851,6%1 3XQD0XVWD2\±<OLRSLVWRSDLQR 7DPSHUH To Pulla iii iv ABSTRACT 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 v 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 a therapeutic treatment. vi Tiivistelmä 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 vii 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. viii TABLE OF CONTENTS LIST OF ORIGINAL COMMUNICATIONS ......................................................................... xii ABBREVIATION .......................................................................................................................... xiii 1 ......... INTRODUCTION ............................................................................................................. 15 2 ......... REVIEW OF LITERATURE ........................................................................................... 17 2.1 Mitochondria ............................................................................................................ 17 2.1.1 Origin & evolution ............................................................................................ 18 2.1.2 Oxidative phosphorylation (OXPHOS) ........................................................ 20 2.1.2.1...... The core respiratory chain (RC) ....................................... 21 2.1.2.2...... Alternative respiratory chain enzymes ............................ 23 2.1.2.2.1... Alternative oxidase (AOX) ............................................... 24 2.1.2.2.2... Alternative NADH dehydrogenase (NDH2)................. 25 2.2 Taxonomic distribution of AOX .......................................................................... 26 2.2.1 AOX in plants .................................................................................................... 26 2.2.2 AOX in bacteria, fungi and protists ............................................................... 28 2.2.3 AOX in animals ................................................................................................. 29 2.2.3.1...... Ciona AOX .......................................................................... 31 2.3 Roles of the Mitochondria ..................................................................................... 32 2.3.1 Metabolism: Tricarboxylic acid (TCA) cycle ................................................. 32 2.3.1.1...... TCA cycle intermediates in metabolism ......................... 34 2.3.1.1.1... Citrate/Malate ..................................................................... 34 2.3.2 Signaling
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