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OPA1 and MFN2 are two critical mitochondrial membrane proteins required for mitochondrial fusion. OPA1 mutations account for approximately 60% of cases of autosomal-dominant optic atrophy (DOA) and up to 20% of mutational carriers develop a more severe multi-systemic neurological phenotype (DOA+) in addition to visual failure. MFN2 mutations result in Charcot-Marie-Tooth disease type 2A (CMT-2A) and in a subgroup of patients, the peripheral neuropathy is complicated by optic atrophy, highlighting a degree of phenotypic overlap with DOA+. POLG1 encodes the catalytic subunit of DNA polymerase gamma (POLG) and POLG1- related diseases are clinically highly heterogeneous, ranging from early-onset Alpers- Huttenlocher syndrome to late-onset isolated chronic progressive external ophthalmoplegia. OPA1, MFN2 and POLG1 mutations all result in disturbed mitochondrial DNA (mtDNA) maintenance, with both quantitative (depletion) and qualitative (point mutations and deletions) mtDNA abnormalities having been identified in patient tissue samples. In this PhD project, the disease mechanisms underpinning these nuclear mitochondrial disorders have been studied further. OPA1 and MFN2 mutations were found to result in significant mtDNA proliferation as a likely compensatory mechanism to impaired mitochondrial oxidative phosphorylation. Using a previously-validated repopulation assay, mtDNA replication in cultured POLG1-mutant fibroblasts was severely depressed following a period of ethidium bromide-induced mtDNA depletion. A similar observation was made with OPA1-mutant fibroblasts, but this effect was not as marked as for POLG1-mutant fibroblasts. Significant reorganisation of the mitochondrial network was also apparent for both groups of mutant fibroblasts. Although neuromyelitis optica (NMO) shares some clinical features with DOA, genetic variations within OPA1 are not associated with the risk of developing NMO. Finally, research into DOA and other mitochondrial optic neuropathies have been severely restricted by the lack of the access to retinal ganglion cells (RGCs), precluding direct studies to be performed on the cell type which is preferentially affected in this group of disorders. To circumvent this limitation, human induced pluripotent stem cell (hiPSC) lines have been generated from patient-derived fibroblasts harbouring confirmed OPA1 mutations. The future differentiation of these induced pluripotent stem cell lines into RGCs will hopefully provide a powerful and versatile tool for disease modelling and the development of targeted therapeutic strategies.

Mitochondria are cellular organelles that are present in all nucleated eukaryotic cells and contain their own genome (mtDNA). Mitochondrial DNA encodes beside 13 polypeptides and 22 tRNAs, 2 ribosomal RNAs. All mtDNA encoded proteins are synthesized within the mitochondrial matrix and essential parts of intra membrane multi-enzyme complexes, involved in oxidative phosphorylation. Mitochondrial protein synthesis is therefore essential for life. This process, however, is still poorly understood. Around 100 mitoribosomal proteins, initiation and elongations factors are involved in mitochondrial translation but the exact mechanisms of how the mitochondrial 55S monosome and the constituted subunits assemble remain obscure. In an attempt to identify factors that play a role in mitoribosome assembly, proteomic analyses of affinity purified complexes using mtRRF were performed (Rorbach et al., 2008). One identified protein was ERAL1, a KH domain containing GTPase with sequence similarity to Era, a eubacterial protein involved in maturation of the 16SrRNA. SiRNA mediated ERAL1 depletion experiments in human cell lines were established and used to investigate the molecular function of the protein. As data in this thesis show, ERAL1 is a mitochondrial protein and is essential for mammalian cells. ERAL1 acts in mitochondria as a 12S-rRNA chaperone via binding at a conserved stem loop structure close to the 3’ terminus of the 12S-rRNA and loss affects the assembly of the small mitoribosomal subunit. Depletion of ERAL1 causes its major growth phenotype, partly by inducing apoptosis. Thus the mitochondrial oxidative phosphorylation machinery is not affected during ERAL1 depletion. ERAL1 is therefore an essential protein in eukaryotic cells, involved in 12S-rRNA maintenance.

ROS production is proportional to the increase in mitochondrial respiration and electron chain activity. Numerous factors (including radiations, oxygen shortage, carcinogens and inflammation) can give rise to ROS, ultimately leading to ROS- mediated genomic instability and cancer. Under normal conditions, the balance between ROS levels are tightly controlled by an inducible antioxidant program that responds to cellular stressor, including enzymes such as superoxide dismutase, catalase, and those involved in glutathione metabolism. Some components of the ROS scavenging pathway are linked to mitochondrial oxidative metabolism by the PGC-l coactivators that enable cells to maintain normal redox status in response to changing oxidative capacity. The increase in mitochondrial number stimulated by these proteins could cause an increase in the production of ROS. Therefore, if ROS production were proportional to the increase of electron transport activity stimulated by the PGC-l proteins, these molecules would ultimately drive ROS levels higher. However, PGC-la is able to upgrade aerobic energy metabolism in tissues with high aerobic demand promoting ROS formation on one hand and ROS scavenging .systems on the other. Since the role of PGC-l ß in this process is completely unknown and ROS production is .commonly linked to mitochondrial dysfunctions that, in turn, are linked with several diseases, such as intestinal cancer and several metabolic diseases including steatohepatitis, we decided to investigate whether PGC-l ß could be involved in the mitochondrial homeostasis and ROS generation in two different organs, the intestine and the liver.

The Mitochondrial Carrier Family encloses a group of transmembrane proteins that transport metabolites across the mitochondrial inner membrane. The ADP/ATP carrier is the most widely studied MCF protein. It catalyzes the counter exchange of ADP for ATP in the mitochondrion of all eukaryotes. In the genome of the kinetoplastid parasite Trypanosoma brucei, three putative ADP/ATP carrier sequences (MCP5, MCP15 and MCP16) and one GDP/GTP (MCP13) entries were analyzed by sequence analyses and phylogenetic reconstruction. AACs phylogenetic reconstruction proved a strong association with yeast, funghi and plant clades, whilst separates from those AACs and from metazoans. MCP13 groups with GGCs, seems to be present only on lower eukaryotes and do not seem to present any homologues in metazoans. Gene deletion studies were performed to assess the roles of MCP5, MCP15, MCP16 and 13. A conditional double knockout cell line, with an inducible myc-tagged rescue copy was constructed for MCP5, which proves the essentiality of the protein for the parasite. Growth curves of the mutant cell line proved a growth defect phenotype in various carbon sources conditions. Mitochondrial ATP production assays were performed in the mutant cell line, in presence and absence of the inducible protein, using permeabilized cells with digitonin that confirmed the ADP/ATP transport activity of the carrier. For invitro activity assays, the carriers were cloned and expressed in Escherichia coli and Spodoptera frugiperda, solubilised and reconstituted into liposomes. Unfortunately, the reconstitution was unsuccessful and the conditions and methodologies are discussed.

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All eukaryotic cells possess mitochondrial DNA (mtDNA), which is maternally inherited through the oocyte, its replication being regulated by nuclear-encoded replication factors. It was hypothesised that mtDNA replication is highly regulated in oocytes, pre-implantation embryos and embryonic stem cells (ESCs) and that this may be disrupted following nuclear transfer (NT). MtDNA copy number decreased between 2-cell and 8-cell staged porcine embryos and increased between the morula and expanded blastocyst stages, coinciding with increased expression of mtDNA replication factors. Competent porcine oocytes replicated their mtDNA prior to and during in vitro maturation to produce and maintain the 100000 mtDNA copies required for fertilisation. Those oocytes in which mtDNA replication was delayed had reduced developmental ability. Expression of pluripotency-associated genes decreased as murine ESCs differentiated into embryoid bodies, although expression of mtDNA replication factors did not increase until the stage equivalent to organogenesis. Cross-species NT embryos in which the donor cell-derived mtDNA was replicated produced decreased developmental outcomes compared to those in which no mtDNA replication took place. Disruption of the strict regulation of mtDNA replication that occurs during early embryogenesis, as is likely following NT, may therefore contribute to the reduced developmental ability of embryos produced using such techniques.

La dynamique et la distribution mitochondriale sont essentielles pour l’homéostasie énergétique cellulaire. CLUH est une protéine indispensable à la distribution mitochondriale, dont la déplétion provoque une agrégation mitochondriale périnucléaire. Afin de comprendre le rôle de CLUH dans le métabolisme cellulaire, nous avons généré des cellules knockout CLUH par la méthode CRISPR-cas9. Nos résultats montrent que l’agrégation mitochondriale est associée à la diminution de la taille cellulaire et à la réduction quantitative des complexes de la chaîne respiratoire, menant ainsi à des défauts de la phosphorylation oxydative. Cette déficience énergétique est due à la perturbation de la traduction mitochondriale, et provoque un shift métabolique vers la glycolyse. Le profil métabolique des cellules KO montre un dysfonctionnement du cycle de Krebs et une altération de l’oxydation des acides gras. Dans ce sens, nous avons déterminé une fonction cruciale de CLUH dans le couplage de la distribution mitochondriale au contrôle de l’état cellulaire énergétique et métabolique. Pour approfondir l’analyse de la fonction de CLUH, nous avons effectué une étude de prédiction des domaines fonctionnels in silico, et avons identifié cinq domaines évolutivement conservés au sein de la séquence primaire de CLUH. De plus, nous démontrons que CLUH oligomérise en tétramères et en octomères, qui sont déstabilisés par l’expression ectopique de formes tronquées de CLUH dépourvues des domaines Clu-Nou TPR, par un effet dominant négatif. En résumé, nos résultats montrent l’importance de CLUH dans le maintien de l’homéostasie métabolique cellulaire, et une régulation potentielle de ses fonctions par oligomérisation. / Mitochondrial dynamics and distribution are critical insupplying ATP in response to energy demands. CLUHis a highly conserved protein involved in mitochondrial distribution, whose dysfunction leads to mitochondrial clustering around the nucleus. To gain insight into the role of CLUH in cellular metabolism, we generated CLUH knockout cells using CRISPR/Cas9. We show that mitochondrial clustering is associated with a smaller cell size and with decreased abundance of respiratory complexes, resulting in OXPHOS defects. This energetic impairment was found to be due to the alteration of mitochondrial translation, leading to a metabolic shift towards glucose dependency. Metabolomic profiling by mass spectrometry disclosed a dysfunctional Krebs cycle and an alteration of fatty acidoxidation. Thus, we established a clear function of CLUH in coupling mitochondrial distribution to the control of cellular energetic and metabolic status. To further analyze CLUH function, we performed in silico the prediction of the functional domains of this protein, disclosing 5 evolutionary conserved domains within the CLUH primary sequence. We reveal an oligomerization of CLUH into tetramers and octamers, and show a dominant negative effect associated to the expression of CLUH truncated forms missing Clu-N or TPR domains. Taken together, our studies reveal the importance of CLUH in maintaining cellular metabolism homeostasis and the potential regulation of its function through oligomerization.

La mitochondrie est le siège principal de la production d’énergie sous forme d’ATP en conditions aérobies, mais aussi d’espèces réactives dérivées de l’oxygène (ROS). La fonction mitochondriale est étroitement liée à la production de ROS puisque ces derniers, selon leur taux, peuvent altérer ou optimiser le rendement énergétique. La plasticité structurale et fonctionnelle de la mitochondrie est essentielle au maintien de l’homéostasie dans toute situation qui nécessite des ajustements métaboliques comme l’exercice physique. Les mécanismes adaptatifs de la fonction mitochondriale et des ROS lors de l’entrainement sont encore loin d’être élucidés ainsi que l’impact du genre sur ces réponses. Dans cette perspective, deux modèles animaux (rat Wistar et anguille européenne) ont été choisis. Les effets d’un entrainement en endurance de même intensité (70% de la vitesse maximale aérobie de course ou de nage) ont été étudiés chez le rat Wistar et l’anguille européenne argentée. Cette dernière est une espèce endurante capable d’effectuer une migration de reproduction de 6000 km et caractérisée par un dimorphisme sexuel de taille. Des mesures in vitro de la consommation d’oxygène, la production radicalaire et d’ATP ont été effectuées simultanément à partir de fibres perméabilisées de cœur et de muscle squelettique. La vulnérabilité ou résistance de la fonction mitochondriale à l’exposition à un système générateur de ROS (mimant un stress oxydant) a également été étudiée. Avant entrainement, chez le rat Wistar, la femelle présente une fonction mitochondriale plus efficiente énergétiquement et plus résistante aux ROS, alors que chez l’anguille, ce profil métabolique et radicalaire est plutôt observé chez le mâle. Après entrainement, quelle que soit l’espèce, la meilleure performance physique observée s’accompagne de modifications métaboliques et radicalaires différentes selon le genre et l’espèce. Chez le rat, l’amélioration de la fonction mitochondriale se traduit différemment selon le sexe. Chez le mâle, l’entrainement induit une amélioration du rendement énergétique via un meilleur couplage entre oxydation et phosphorylation et/ou une meilleure utilisation des électrons au niveau de la chaine respiratoire. Chez la femelle, l’augmentation de la production d’ATP serait liée à l’augmentation de la consommation d’oxygène mitochondriale. Comme chez le rat, l’entraînement induit globalement chez l’anguille une amélioration du rendement énergétique et de la résistance de la fonction mitochondriale aux ROS, mais uniquement chez le mâle. L’ensemble de ces résultats montre des réponses métaboliques et radicalaires dépendantes du genre. Quelle que soit l’espèce, l’entrainement semble être chez le mâle plus bénéfique que chez la femelle en termes d’efficacité énergétique mitochondriale et de résistance de la fonction mitochondriale à un stress oxydant. Dans le contexte de la migration de l’anguille, ces adaptations permettraient au mâle, largement plus petit que la femelle, une efficacité de nage supérieure, permettant leur synchronisation d’arrivée sur le lieu de reproduction. Les similitudes interspécifiques de réponse à l’entraînement selon le genre confortent l’intérêt d’utilisation du modèle poisson dans le champ de la physiologie de l’exercice. / Mitochondrion is the main site of aerobic energy (ATP) and reactive oxygen species (ROS) productions. Mitochondrial function is closely linked to ROS, which, according their rate, can alter or optimize energy efficiency. Structural and functional plasticity of mitochondria is essential to maintain homeostasis in any situation that requires metabolic adjustments as physical exercise. The adaptive mechanisms of mitochondrial function and ROS during training and the impact of gender on these responses are still far from being solved. In this perspective, two animal models (Wistar rat and European eel) were chosen.The effects of endurance training of the same intensity (70% of maximal aerobic speed running or swimming) were studied in Wistar rat and silver European eel. The latter is an enduring species capable of performing a spawning migration of 6000 km and characterized by sexual dimorphism in size. In vitro measurements of oxygen consumption, free radical and ATP productions were carried out simultaneously from heart and skeletal muscle permeabilized fibers. The vulnerability or resistance of the mitochondrial function to a ROS generating system exposure (mimicking oxidative stress) was also studied.Before training, in rat, female has a mitochondrial function energetically more efficient and more resistant to ROS, whereas in eel, this metabolic and radical profile is observed rather in male. After training, whatever the species, the improved physical performance observed is associated with various metabolic and radical changes which depending on gender and species. In rats, the improving of mitochondrial function translates differently according to gender. In male, training induces improvement in energy efficiency through a better coupling between oxidation and phosphorylation and/or better use of electrons at the respiratory chain level. In female, increasing in ATP production may be related to the increase in mitochondrial oxygen consumption. As in rats, training induces globally in eel an improvement in energy efficiency and resistance of mitochondrial function to ROS, but only in male. All these results show metabolic and radical responses depending on gender. Whatever the species, training seems to be most beneficial in males than in females in terms of mitochondrial energy efficiency and resistance of mitochondrial function to oxidative stress. In the context of eel migration, these adaptations allow to male, largely smaller than female, a higher swim efficiency, allowing their synchronization on breeding site. Interspecific similarities in training response by gender confirm the interest of fish model’s using in the field of exercise physiology.