Molecular, metabolic, and therapeutic aspects of respiratory complex III deficiency : Bcs1l mutant mice as an experimental model

Mitochondrial disorders are rare diseases but collectively the most frequent group of inborn errors of metabolism. These disorders are genetically and phenotypically heterogenous and can manifest in any organ of the body with onset at any age. Mitochondrial functions are also diverse with the ATP production via the oxidative phosphorylation (OXPHOS) being the most notable. At the center of the OXPHOS machinery is the respiratory complex III (CIII, cytochrome bc1 complex). CIII deficiency in GRACILE syndrome belonging to the Finnish disease heritage causes a neonatal-lethal hepatorenal disease. The primary cause of GRACILE syndrome is a c.A232G (p.S78G) mutation in the BCS1L gene, which encodes a translocase required for Rieske Fe-S protein (RISP, UQCRFS1) incorporation into CIII. Homozygous Bcs1lp.S78G mice bearing the GRACILE syndrome mutation recapitulate the human syndrome, but unlike the patients they have a short asymptomatic period and relatively longer lifespan giving a window for therapeutic interventions. In this thesis project, we studied two potential therapies aiming to improve dysfunctional mitochondria in Bcs1lp.S78G mice: ketogenic diet and NAD+ repletion. We also utilized an alternative oxidase (AOX) transgene to bypass the electron-transfer blockade at CIII. Ketogenic diets are low-carbohydrate high-fat diets causing nutritional ketosis. They have been proposed to induce a beneficial starvation-like adaptive mitochondrial response involving increased mitochondrial biogenesis. Bcs1lp.S78G mice tolerated the carbohydrate restriction of ketogenic diet, were able to utilize dietary fat as the main energy source and developed ketosis. Ketogenic diet attenuated the hepatic CIII assembly defect, increased CIII activity and corrected mitochondrial structural aberrations. Our results suggested that these changes were not due to increased mitochondrial biogenesis. In line with the improved CIII function, Bcs1l mutant mice showed attenuated hepatopathy as shown by delayed liver fibrosis, inhibited stellate cell activation and hepatic progenitor cell response, decreased cell death and plasma liver enzyme activities. Liver transcriptomics and subsequent histochemical analyses suggested altered macrophage activation and a normalizing effect by ketogenic diet. In the second study, we characterized NAD+ metabolism in Bcs1lp.S78G mice. We found transcriptionally repressed NAD+ de novo biosynthesis and decreased hepatic NAD+ concentration. Changes in NAD+ consuming processes did not explain the decreased NAD+ levels. Aiming to replete the NAD+ levels, we fed the Bcs1lp.S78G mice a NAD+ precursor nicotinamide riboside (NR). In contrast to previous studies on mitochondrial myopathy models and mouse models with secondary mitochondrial dysfunctions, the hepatic NAD+ depletion of Bcs1lp.S78G mice was refractory to NR supplementation and the disease progression was unaltered. Cellular NAD+ levels regulate mitochondrial functions via sirtuin deacetylases, which are the main targets of NAD+ repletion therapies. Investigation of the upstream effectors of sirtuins showed that a starvation-like metabolic state of Bcs1lp.S78G mice is linked to AMP kinase and cAMP signaling, which likely counterbalances the repressive effect of decreased NAD+ levels on the activity of SIRT1 and SIRT3. In the third study, we introduced Ciona intestinalis AOX transgene into the Bcs1lp.S78G mice. AOXs are non-mammalian enzymes that can bypass a blockade of the CIII-CIV segment of the respiratory electron transfer. The AOX-expressing Bcs1lp.S78G mice were viable, and their CIII-deficiency stimulated AOX-mediated respiration in isolated mitochondria. AOX expression tripled the median lifespan of Bcs1lp.S78G mice from 200 to 600 days. The extension of the lifespan was predominantly due to the complete prevention of late-onset cardiomyopathy. The effects of AOX were tissue specific. In the heart of Bcs1lp.S78G mice, it preserved normal tissue structure and function, mitochondrial morphology, respiratory electron transfer, and wild-type-like transcriptome. In contrast, AOX only minimally affected the late-stage liver disease. Whereas, in the kidneys, AOX normalized an atrophic kidney phenotype and some histological lesions but it did not normalize kidney function or cause global normalization of transcriptome changes. Our results suggest tissue-specific thresholds of CIII deficiency for in vivo AOX-mediated respiration in CIII deficiency. Moreover, our study demonstrates the value of AOX as a research tool to dissect the pathogenesis of CIII deficiency. During our investigations, we observed approximately 5-fold difference in the lifespan of the Bcs1lp.S78G mice on two closely related congenic backgrounds. In the fourth study, we tracked the difference to a spontaneous homoplasmic mitochondrial DNA (mtDNA) variant (mt-Cybp.D254N) in an isolated congenic Lund University mouse colony. The variant changes a highly conserved negative amino acid residue in the only mtDNA-encoded subunit of CIII, cytochrome b (MT-CYB). A crossbreeding experiment utilizing the maternal inheritance of mtDNA verified the novel variant as the determinant of the survival difference. Functional studies showed that the variant exacerbated complex III deficiency in all assessed tissues. In otherwise wild-type mice, it also decreased cardiac CIII activity, caused a slight disturbance in mitochondrial bioenergetics, and decreased whole-body energy expenditure. Molecular dynamics simulations and their verification in isolated mutagenized Rhodobacter capsulatus cytochrome bc1 complex showed that the mt-Cybp.D254N variant restricts the mobility of RISP head domain movement. In summary, these studies provided novel mechanistic and therapeutic insights into CIII deficiency at genetic, molecular, and metabolic level. The results highlight the importance of knowing the underlying tissue-specific pathology and metabolic adaptations when designing therapies for mitochondrial diseases. The genetic epistasis between Bcs1lp.S78G and mt-Cybp.D254N also highlights the role of mitochondrial DNA background as a modifier of mitochondrial disease phenotypes.Mitokondriotaudit ovat harvinaissairauksia, jotka kuitenkin ryhmänä muodostavat yleisimmän synnynnäisten aineenvaihduntahäiriöiden joukon. Mitokondriotautien mutaatioiden ja oireiden kirjo on erittäin laaja. Mitokondriotauti voi ilmetä lähes missä elimessä ja missä iässä tahansa. Mitokondriot ovat soluelimiä, joilla on lukuisia välttämättömiä tehtäviä. Näistä tunnetuin on ATP:n tuotanto oksidatiivisen fosforylaation avulla. Yksi keskeisistä entsyymeistä oksidatiivisessa fosforylaatiossa on hengitysketjun kompleksi III (sytokromi bc1 -kompleksi). Suomalaiseen tautiperintöön kuuluvassa GRACILE-oireyhtymässä kompleksi III:n puutos aiheuttaa vastasyntyneiden kuolemaan johtavan maksa- ja munuaistaudin. Oireyhtymän aiheuttaa homotsygoottinen c.A232G-pistemutaatio (proteiinissa Ser78Gly) BCS1L-geenissä. BCS1L-proteiini on välttämätön Rieske rauta-rikki -proteiinin (RISP, UQCRFS1) kuljetukselle kompleksi III:een. Myös geneettisesti muokatuilla hiirillä homotsygoottinen Bcs1lp.S78G mutaatio aiheuttaa GRACILE-oireyhtymän kaltaisen taudin. Potilaisten poiketen mutanttihiirillä on lyhyt syntymän jälkeinen oireeton jakso ennen taudin puhkeamista, mikä mahdollistaa hoitokokeiden aloittamisen jo ennen taudin puhkeamista. Tämän väitöskirjan tutkimuksissa tutkimme kahta mahdollista hoitoa mitokondrioiden toiminnan kohentamiseksi GRACILE-oireyhtymän hiirimallissa: ketogeenistä ruokavaliota ja solujen NAD+-määrän (nikotiiniamidiadeniinidinukelotidi) lisäämistä. Tutkimme myös kompleksi III:n puutoksen ohittamista vaihtoehtoista oksidaasia (AOX, alternative oxidase) ilmentävän siirtogeenin avulla. Ketogeeniset ruokavaliot sisältävät erittäin niukasti hiilihydraatteja ja runsaasti rasvaa, mikä aiheuttaa ketoosin eli ketoaineiden lisääntymisen veressä. Ketogeenisen ruokavalion on esitetty käynnistävän osittaisen paastovasteen, johon liittyy mitokondrioiden toiminnan tehostuminen ja niiden lisääntynyt uudistuotanto. Koska Bcs1lp.S78G-mutanttihiiret sopeutuivat ketogeeniseen ruokavalioon, ne ilmeisesti pystyivät hyödyntämään tehokkaasti rasvahappoja ja ketoaineita pääasiallisena energianlähteenä. Ketogeenisellä ruokavaliolla kompleksi III:een sitoutuneen RISP:n määrä lisääntyi mutanttihiiriillä, kuten myös kompleksi III:n aktiivisuus maksan mitokondrioissa. Nämä muutokset näkyivät myös maksan mitokondrioiden rakenteen normalisoitumisena. Tuloksemme viittasivat siihen, että mitokondrioiden toiminnan ja rakenteen parantuminen ei liittynyt lisääntyneeseen mitokondrioiden uudistuotantoon. Ketogeenisen ruokavalio hidasti merkittävästi maksataudin etenemistä, mikä näkyi muun muassa vähentyneenä stellaatti- ja ovaalisolujen aktivoitumisena, sekä vähentyneenä maksan sidekudostumisena. Myös maksaentsyymien aktiivisuudet plasmassa alenivat mutanttihiirillä. Maksan transkriptomiikka ja immunohistokemialliset värjäykset viittasivat siihen, että ketogeeninen ruokavalio vaikutti makrofagien toimintaan. Väitöskirjan toisessa osatyössä tutkimme Bcs1lp.S78G-hiirten NAD+-aineenvaihduntaa. Havaitsimme, että mutanttihiirillä NAD+-tuotannosta vastaavien geenien ilmentyminen oli vähentynyt kuten myös maksan NAD+-pitoisuus. Muutokset NAD+:tä kuluttavissa aineenvaihduntaprosesseissa eivät selittäneet NAD+-pitoisuuden vähenemää. Syötimme Bcs1lp.S78G-hiirille NAD+:n esiastetta nikotiiniamidiribosidiä (NR) korjataksemme puutoksen. Poiketen aiemmista tutkimuksista, joissa NR:llä on ollut parantava vaikutus mitokondriaalisten lihastautien hiirimalleissa, NR ei vaikuttanut Bcs1lp.S78G-hiirten taudinkuvaan. NR ei myöskään vaikuttanut hoidon päätepisteessä mitattuun maksan NAD+-pitoisuuteen. Solujen NAD+-määrää kohentavien hoitojen yleinen tavoite on tehostaa NAD+:sta riippuvaisten deasetylaasien, sirtuiinien, aktiivisuutta. Tutkimme sirtuiinien NAD+:sta riippumattomia säätelymekanismeja ja havaitsimme, että mutanttihiirten nälkiintymistila aktivoi AMP-kinaasia ja cAMP-signalointia, jotka tunnetusti aktivoivat sirtuiini 1:tä ja 3:sta. Nämä muutokset oletettavasti kompensoivat vähentyneen NAD+-määrän mahdollisia haitallisia vaikutuksia. Kolmannessa osatyössä siirsimme Ciona intestinalis -vaippaeläimen AOX:ia koodaavan geenin Bcs1lp.S78G-hiiriin. AOX:it ovat nisäkkäiltä puuttuvia mitokondrioiden entsyymejä, jotka voivat ohittaa soluhengityksen tukoksen kompleksi III:n tai IV:n kohdalla. AOX ei ollut haitallinen Bcs1lp.S78G-hiirille ja kompleksi III:n toimintavajaus sai sen aktivoitumaan eristetyissä hiiren mitokondrioissa. Bcs1lp.S78G-hiirille alkoi kehittyä 150 elinpäivän jälkeen sydänlihassairaus, mihin ne kuolivat keskimäärin 200 päivän iässä. AOX:ia ilmentäville mutanttihiirille ei kehittynyt lainkaan sydäntautia, ja niiden mediaanielinikä oli 600 päivää. AOX esti täysin sydäntautiin liittyvän sydämen laajentuman ja toiminnan heikkenemisen. Sydämen mitokondrioissa AOX korjasi soluhengityksen sekä mitokondrioiden rakenteen muutokset. Vastaava pelastavaa vaikutusta AOX:illa ei ollut maksatautiin 150-200 päivän ikäisillä Bcs1lp.S78G-hiirillä. Munuaisissa taas AOX osittain korjasi tai esti kudosrakenteen muutoksia, mutta vaikutti vain vähäisesti tämän elimen toimintaan. Tuloksemme osoittivat, että AOX:n vaikutukset ovat kudoksesta riippuvaisia kompleksi III:n puutoksessa ja että se on hyödyllinen työkaluksi kompleksi III:n tautimekanismien tutkimuksessa. Tutkimustemme alkuvaiheessa huomasimme noin viisinkertaisen eron Bcs1lp.S78G-hiirten eliniässä kahden eri hiirikannan välillä. Väitöskirjan neljännessä osatyössä löysimme koko genomin sekvensoinnilla homoplasmiseen yhden nukleotidin muutokseen Lundin yliopiston hiirikannan mitokondrio-DNA:ssa. Muutos vaihtaa negatiivisesti varautuneen aspartaatin neutraaliiin asparagiiniin (D254N) sytokromi b -proteiinissa (MT-CYB). MT-CYB on kompleksi III:n alayksiköistä ainoa, jonka geeni sijaitsee mitokondrio-DNA:ssa. Käyttäen hyväksi mitokondrio-DNA:n maternaalista periytymistä osoitimme mt-Cybp.D254N-variantin aiheuttavan Bcs1lp.S78G-hiirten varhaisen kuoleman n. kuukauden iässä. Kuten oletimme mt-Cybp.D254N pahensi Bcs1lp.S78G-hiirten kompleksi III:n puutosta kaikissa tutkimissamme kudoksissa. Yksinäänkin mt-Cybp.D254N vähensi sydämessä kompleksi III:n aktiivisuutta, aiheutti pienen häiriön hengitysketjun toiminnassa ja pienensi hiirten energiankulutusta. Kompleksi III:n rakenteen tietokonemallinnukset ja biofysikaaliset tutkimukset Rhodobacter capsulatus -bakteerin sytokromi bc1-kompleksissa osoittivat, että D254N-aminohappomuutos häiritsee RISP:n elektroneja siirtävän domeenin liikettä kompleksi III:ssa. Tämän väitöskirjan tutkimukset tuottivat uutta tietoa kompleksi III:n puutoksen tautimekanismeista ja mahdollisista hoidoista. Tuloksemme osoittavat, että on tärkeää tuntea mitokondriotaudin kudoskohtaisia ja vaihtelevia aineenvaihduntamuutoksia suunniteltaessa hoitoja. Bcs1lp.S78G-mutaation ja mt-Cybp.D254N-variantin yhteisvaikutus puolestaan osoitti, että geneettinen tausta voi vaikuttaa odottamattomalla tavalla mitokondriotaudin kulkuun

The mitochondrial coenzyme Q junction and complex III : biochemistry and pathophysiology

Coenzyme Q (CoQ, ubiquinone) is the electron-carrying lipid in the mitochondrial electron transport system (ETS). In mammals, it serves as the electron acceptor for nine mitochondrial inner membrane dehydrogenases. These include the NADH dehydrogenase (complex I, CI) and succinate dehydrogenase (complex II, CII) but also several others that are often omitted in the context of respiratory enzymes: dihydroorotate dehydrogenase, choline dehydrogenase, electron-transferring flavoprotein dehydrogenase, mitochondrial glycerol-3-phosphate dehydrogenase, proline dehydrogenases 1 and 2, and sulfide:quinone oxidoreductase. The metabolic pathways these enzymes are involved in range from amino acid and fatty acid oxidation to nucleotide biosynthesis, methylation, and hydrogen sulfide detoxification, among many others. The CoQ-linked metabolism depends on CoQ reoxidation by the mitochondrial complex III (cytochrome bc(1) complex, CIII). However, the literature is surprisingly limited as for the role of the CoQ-linked metabolism in the pathogenesis of human diseases of oxidative phosphorylation (OXPHOS), in which the CoQ homeostasis is directly or indirectly affected. In this review, we give an introduction to CIII function, and an overview of the pathological consequences of CIII dysfunction in humans and mice and of the CoQ-dependent metabolic processes potentially affected in these pathological states. Finally, we discuss some experimental tools to dissect the various aspects of compromised CoQ oxidation.Peer reviewe

MYC—an emerging player in mitochondrial diseases

The mitochondrion is a major hub of cellular metabolism and involved directly or indirectly in almost all biological processes of the cell. In mitochondrial diseases, compromised respiratory electron transfer and oxidative phosphorylation (OXPHOS) lead to compensatory rewiring of metabolism with resemblance to the Warburg-like metabolic state of cancer cells. The transcription factor MYC (or c-MYC) is a major regulator of metabolic rewiring in cancer, stimulating glycolysis, nucleotide biosynthesis, and glutamine utilization, which are known or predicted to be affected also in mitochondrial diseases. Albeit not widely acknowledged thus far, several cell and mouse models of mitochondrial disease show upregulation of MYC and/or its typical transcriptional signatures. Moreover, gene expression and metabolite-level changes associated with mitochondrial integrated stress response (mt-ISR) show remarkable overlap with those of MYC overexpression. In addition to being a metabolic regulator, MYC promotes cellular proliferation and modifies the cell cycle kinetics and, especially at high expression levels, promotes replication stress and genomic instability, and sensitizes cells to apoptosis. Because cell proliferation requires energy and doubling of the cellular biomass, replicating cells should be particularly sensitive to defective OXPHOS. On the other hand, OXPHOS-defective replicating cells are predicted to be especially vulnerable to high levels of MYC as it facilitates evasion of metabolic checkpoints and accelerates cell cycle progression. Indeed, a few recent studies demonstrate cell cycle defects and nuclear DNA damage in OXPHOS deficiency. Here, we give an overview of key mitochondria-dependent metabolic pathways known to be regulated by MYC, review the current literature on MYC expression in mitochondrial diseases, and speculate how its upregulation may be triggered by OXPHOS deficiency and what implications this has for the pathogenesis of these diseases

A sensitive assay for dNTPs based on long synthetic oligonucleotides, EvaGreen dye and inhibitor-resistant high-fidelity DNA polymerase

Deoxyribonucleoside triphosphates (dNTPs) are vital for the biosynthesis and repair of DNA. Their cellular concentration peaks during the S phase of the cell cycle. In non-proliferating cells, dNTP concentrations are low, making their reliable quantification from tissue samples of heterogeneous cellular composition challenging. Partly because of this, the current knowledge related to the regulation of and disturbances in cellular dNTP concentrations derive mostly from cell culture experiments with little corroboration at the tissue or organismal level. Here, we fill the methodological gap by presenting a simple non-radioactive microplate assay for the quantification of dNTPs with a minimum requirement of 4-12 mg of biopsy material. In contrast to published assays, this assay is based on long synthetic single-stranded DNA templates (50-200 nucleotides), an inhibitor-resistant high-fidelity DNA polymerase, and the double-stranded-DNA-binding EvaGreen dye. The assay quantified reliably less than 50 fmol of each of the four dNTPs and discriminated well against ribonucleotides. Additionally, thermostable RNAse HII-mediated nicking of the reaction products and a subsequent shift in their melting temperature allowed near-complete elimination of the interfering ribonucleotide signal, if present. Importantly, the assay allowed measurement of minute dNTP concentrations in mouse liver, heart and skeletal muscle.Peer reviewe

A spontaneous mitonuclear epistasis converging on Rieske Fe-S protein exacerbates complex III deficiency in mice

We previously observed an unexpected fivefold (35 vs. 200 days) difference in the survival of respiratory chain complex III (CIII) deficient Bcs1/(p.S78G) mice between two congenic backgrounds. Here, we identify a spontaneous homoplasmic mtDNA variant (m.G14904A, mt-Cyb(p.D254N)), affecting the CIII subunit cytochrome b (MT-CYB), in the background with short survival. We utilize maternal inheritance of mtDNA to confirm this as the causative variant and show that it further decreases the low CIII activity in Bcs1/(p.S78G) tissues to below survival threshold by 35 days of age. Molecular dynamics simulations predict D254N to restrict the flexibility of MT-CYB ef loop, potentially affecting RISP dynamics. In Rhodobacter cytochrome bc(1) complex the equivalent substitution causes a kinetics defect with longer occupancy of RISP head domain towards the quinol oxidation site. These findings represent a unique case of spontaneous mitonuclear epistasis and highlight the role of mtDNA variation as modifier of mitochondrial disease phenotypes.Peer reviewe

Ketogenic diet attenuates hepatopathy in mouse model of respiratory chain complex III deficiency caused by a Bcs1l mutation

Mitochondrial disorders are among the most prevalent inborn errors of metabolism but largely lack treatments and have poor outcomes. High-fat, low-carbohydrate ketogenic diets (KDs) have shown beneficial effects in mouse models of mitochondrial myopathies, with induction of mitochondrial biogenesis as the suggested main mechanism. We fed KD to mice with respiratory chain complex III (CIII) deficiency and progressive hepatopathy due to mutated BCS1L, a CIII assembly factor. The mutant mice became persistently ketotic and tolerated the KD for up to 11 weeks. Liver disease progression was attenuated by KD as shown by delayed fibrosis, reduced cell death, inhibition of hepatic progenitor cell response and stellate cell activation, and normalization of liver enzyme activities. Despite no clear signs of increased mitochondrial biogenesis in the liver, CIII assembly and activity were improved and mitochondrial morphology in hepatocytes normalized. Induction of hepatic glutathione transferase genes and elevated total glutathione level were normalized by KD. Histological findings and transcriptome changes indicated modulation of liver macrophage populations by the mutation and the diet. These results reveal a striking beneficial hepatic response to KD in mice with mitochondrial hepatopathy and warrant further investigations of dietary modification in the management of these conditions in patients.Peer reviewe

Alternative oxidase-mediated respiration prevents lethal mitochondrial cardiomyopathy

Alternative oxidase (AOX) is a non-mammalian enzyme that can bypass blockade of the complex III-IV segment of the respiratory chain (RC). We crossed a Ciona intestinalis AOX transgene into RC complex III (cIII)-deficient Bcs1l(p.S78G) knock-in mice, displaying multiple visceral manifestations and premature death. The homozygotes expressing AOX were viable, and their median survival was extended from 210 to 590 days due to permanent prevention of lethal cardiomyopathy. AOX also prevented renal tubular atrophy and cerebral astrogliosis, but not liver disease, growth restriction, or lipodystrophy, suggesting distinct tissue-specific pathogenetic mechanisms. Assessment of reactive oxygen species (ROS) production and damage suggested that ROS were not instrumental in the rescue. Cardiac mitochondrial ultrastructure, mitochondrial respiration, and pathological transcriptome and metabolome alterations were essentially normalized by AOX, showing that the restored electron flow upstream of cIII was sufficient to prevent cardiac energetic crisis and detrimental decompensation. These findings demonstrate the value of AOX, both as a mechanistic tool and a potential therapeutic strategy, for cIII deficiencies.Peer reviewe

Respiratory chain complex III deficiency due to mutated BCS1L : a novel phenotype with encephalomyopathy, partially phenocopied in a Bcs1l mutant mouse model

Background: Mitochondrial diseases due to defective respiratory chain complex III (CIII) are relatively uncommon. The assembly of the eleven-subunit CIII is completed by the insertion of the Rieske iron-sulfur protein, a process for which BCS1L protein is indispensable. Mutations in the BCS1L gene constitute the most common diagnosed cause of CIII deficiency, and the phenotypic spectrum arising from mutations in this gene is wide. Results: A case of CIII deficiency was investigated in depth to assess respiratory chain function and assembly, and brain, skeletal muscle and liver histology. Exome sequencing was performed to search for the causative mutation(s). The patient's platelets and muscle mitochondria showed respiration defects and defective assembly of CIII was detected in fibroblast mitochondria. The patient was compound heterozygous for two novel mutations in BCS1L, c.306A > T and c.399delA. In the cerebral cortex a specific pattern of astrogliosis and widespread loss of microglia was observed. Further analysis showed loss of Kupffer cells in the liver. These changes were not found in infants suffering from GRACILE syndrome, the most severe BCS1L-related disorder causing early postnatal mortality, but were partially corroborated in a knock-in mouse model of BCS1L deficiency. Conclusions: We describe two novel compound heterozygous mutations in BCS1L causing CIII deficiency. The pathogenicity of one of the mutations was unexpected and points to the importance of combining next generation sequencing with a biochemical approach when investigating these patients. We further show novel manifestations in brain, skeletal muscle and liver, including abnormality in specialized resident macrophages (microglia and Kupffer cells). These novel phenotypes forward our understanding of CIII deficiencies caused by BCS1L mutations.Peer reviewe

Planetary well-being

Tensions between the well-being of present humans, future humans, and nonhuman nature manifest in social protests and political and academic debates over the future of Earth. The increasing consumption of natural resources no longer increases, let alone equalises, human well-being, but has led to the current ecological crisis and harms both human and nonhuman well-being. While the crisis has been acknowledged, the existing conceptual frameworks are in some respects ill-equipped to address the crisis in a way that would link the resolving of the crisis with the pivotal aim of promoting equal well-being. The shortcomings of the existing concepts in this respect relate to anthropocentric normative orientation, methodological individualism that disregards process dynamics and precludes integrating the considerations of human and nonhuman well-being, and the lack of multiscalar considerations of well-being. This work derives and proposes the concept of planetary well-being to address the aforementioned conceptual issues, to recognise the moral considerability of both human and nonhuman well-being, and to promote transdisciplinary, cross-cultural discourse for addressing the crisis and for promoting societal and cultural transformation. Conceptually, planetary well-being shifts focus on well-being from individuals to processes, Earth system and ecosystem processes, that underlie all well-being. Planetary well-being is a state where the integrity of Earth system and ecosystem processes remains unimpaired to a degree that species and populations can persist to the future and organisms have the opportunity to achieve well-being. After grounding and introducing planetary well-being, this work shortly discusses how the concept can be operationalised and reflects upon its potential as a bridging concept between different worldviews.</p

Ketogeenisen ruokavalion vaikutus maksasairauteen GRACILE-mitokondriotaudin hiirimallissa

GRACILE syndrome (Fellman disease) is a neonatal mitochondrial disorder with hepatopathy belonging to Finnish disease heritage. The GRACILE acronym comes from the most important clinical features of the disorder: fetal growth restriction, aminoaciduria, cholestasis (with steatosis and fibrosis), hepatic iron overload, lactic acidosis, and early death during infancy. The syndrome is caused by mutated (homoyzygous c.232A>G) BCS1L, a nuclear gene encoding a respiratory chain complex III assembly factor. BCS1L mutations compromise Rieske iron-sulfur protein incorporation into complex III, which leads to respiratory chain deficiency. Bcs1l c.232A>G knock-in C57BL/6 mouse model mimics the human disorder, and the homozygous mice (GRACILE mice) display growth restriction, progressive liver disease and a short life span. For a vast majority of mitochondrial disorders no effective treatments currently exist. One promising treatment strategy is to increase mitochondrial biogenesis with the idea that respiratory chain deficiency can be compensated by boosting mitochondrial function. In mouse models of mitochondrial myopathies induction of mitochondrial biogenesis with a transgene approach, pharmaceutical intervention or ketogenic diet has alleviated the disease. It has been hypothesized that ketogenic diet activates energy deprivation signals of the cells, which are the most important regulators of mitochondrial biogenesis. So far ketogenic diet has not been studied in animal models of mitochondrial hepatopathies. The objective of this master’s thesis was to study effect of ketogenic diet on hepatopathy of GRACILE mouse model. From weaning on, wild-type and homozygous mutant mice (n = 11 - 14 per group) were fed standard chow or ketogenic diet (90,5 E% fat) for three weeks after which the mice were sacrificed and tissues collected for analysis. The effect of ketogenic diet was evaluated with following methods: liver histology (H&E, Sirius Red, Oil Red O, PAS and semiquantitative scoring), liver triglyceride content, mitochondrial DNA copy number (qPCR), expression of selected genes (qPCR) and respiratory chain proteins (Western blot). The mice tolerated the ketogenic diet, and the diet did not affect weight gain. Livers of wild-type mice on ketogenic diet were considered normal except for slight fat accumulation. In GRACILE mice ketogenic diet ameliorated some aspects of the liver disease (less signs of inflammation and fibrosis) but increased microvesicular steatosis. Mitochondrial DNA copy number was unchanged among groups. Ketogenic diet did not affect gene or protein expression of selected respiratory chain components. The GRACILE mutation increased gene expression of Pgc1a, a gene encoding a master regulator of mitochondrial biogenesis, as well as protein and mRNA levels of respiratory chain complex IV subunits. The ketogenic diet had a clear effect on liver phenotype in the mutant mice, but a longer follow-up time is needed to show the effect on disease progression. The beneficial action of ketogenic diet on liver histology might not be related to increased number of mitochondria or respiratory chain proteins, and the mechanism behind the effect need to be elucidated in further investigations.GRACILE-oireyhtymä (Fellmanin tauti) on suomalaiseen tautiperintöön kuuluva mitokondriotauti. Sen pääasialliset oireet ovat sikiöaikainen kasvuhäiriö, aminohappovirtsaisuus (proksimaalinen tubulopatia), maksasairaus (rasvamaksa, kolestaasi, raudan kertyminen ja fibroosi), maitohappoasidoosi sekä kuolema vastasyntyneenä. Oireyhtymän aiheuttaa BCS1L-geenin pistemutaatio (c.232A>G) homotsygoottisena. BCS1L koodaa hengitysketjun kompleksi III:n kokoajaproteiinia, jonka toistaiseksi ainoa tunnettu tehtävä on siirtää katalyyttisesti tärkeä Rieske rauta-rikki proteiini kompleksi III:een. Myös taudin hiirimallissa (Bcs1l:n c.232A>G knock-in-C57BL/6) oireina ovat kasvuhäiriö, maksasairas ja varhainen kuolema. Suurimpaan osaan mitokondriotaudeista ei ole tehokkaita hoitoja. Eräänä lupaavana hoitokeinona pidetään mito-kondrioiden uudismuodostuksen lisäämistä. Oletuksena on, että hengitysketjupuutosta voidaan kompensoida lisäämällä mitokondrioiden määrää ja tehostamalla niiden toimintaa. Eräissä mitokondrioperäisten lihassairauksien hiirimalleissa siirtogeenin avulla, lääkkein tai ketogeenisellä ruokavaliolla aiheutettu mitokondrioiden uudismuodostus hidastaa taudin etenemistä. Ketogeenisen ruokavalion oletetaan aktivoivan solujen energianpuutossignaaleja, jotka ovat solujen tärkeimpiä mitokondrioiden uudistuotannon säätelijöitä. Mitokondrioperäisten maksasairauksien eläinmalleissa ketogeenistä ruokavaliota ei ole aiemmin tutkittu. Tämän pro gradu -työn tarkoituksena oli tutkia ketogeenisen ruokavalion maksavaikutuksia GRACILE-oireyhtymän hiirimallissa. Villityypin hiirille tai mutaation suhteen homotsygoottisille hiirille (GRACILE-hiiri) annettiin vieroituksen jälkeen joko tavanomaista jyrsijöiden ruokaa tai ketogeenistä ruokaa (rasvaa 90,5 E%) noin kolmen viikon ajan, minkä jälkeen hiiret lopetettiin (n = 11 - 14 / ryhmä) ja kudosnäytteet kerättiin. Vaikutusta tutkittiin seuraavin menetelmin: maksahistologia (H&E, Sirius Red, Oil Red O, PAS ja semikvantitatiivinen pisteytys), maksojen triglyseridimääritys; valikoitujen geenien ilmentyminen ja mitokondrio-DNA:n kopioluku (qPCR), hengitysketjukomponenttien määrät (Western blot). Hiiret sietivät ketogeenistä ruokaa, eikä ruokavaliolla ollut vaikutusta hiirten painoon. Villityypin hiirillä ketogeeninen ruokavalio ei aiheuttanut merkittäviä histologisia muutoksia maksoihin lievää maksan rasvapitoisuuden lisääntymistä lukuun ottamatta. GRACILE-hiirillä ketogeeninen ruokavalio vaikutti maksojen histologiaan osin myönteisesti (vähemmän merkkejä fibroosista ja tulehduksesta), osin haitallisesti (enemmän mikrovesikulaarista rasvakertymää). Mitokondrio-DNA:n kopioluvussa ei ollut eroja ryhmien välillä. Ketogeeninen ruokavalio ei vaikuttanut tutkittujen hengitysketjukomponenttien mRNA- tai proteiinimääriin. Sen sijaan GRACILE-mutaatio lisäsi mitokondrioiden uudistuotantoa aktivoivan PGC1?-koaktivaattorin mRNA-määrää ja hengitysketjun kompleksi IV:n alayksikön mRNA- ja proteiinimäärää. Ketogeeninen ruokavalio vaikutti selvästi GRACILE-hiirten maksojen histologiseen ilmiasuun, mutta tarvitaan pidempi seuranta-aika, jotta voidaan nähdä ketogeenisen ruokavalion vaikutus taudin etenemiseen. Ruokavalion myönteinen vaikutus maksasairauteen ei todennäköisesti johdu lisääntyneestä mitokondrioiden tai hengitysketjuproteiinien määrästä, ja sen mekanismia selvitetään jatkotöissä