Rozprawy doktorskie na temat „Mitochondrial diseases”

Sous le terme de maladies mitochondriales, on désigne des maladies multi-systémiques ou à expression tissu-spécifique dues à un déficit de la phosphorylation oxydative qui est assurée par le fonctionnement de 5 complexes protéiques enzymatiques (chaîne respiratoire) parmi lesquelles 13 sous-unités sont codées par le génome mitochondrial, les autres par le génome nucléaire. Ces pathologies recouvrent donc en pratique des maladies génétiques par mutation de l’ADN mitochondrial (ADNmt) mais aussi des maladies génétiques à hérédité mendélienne classique. Dans les cytopathies mitochondriales liées à des mutations de gènes nucléaires, il existe deux sortes de gènes (i) à effet direct correspondant à des gènes codant pour les sous-unités protéiques de la chaîne respiratoire ou leur assemblage, et (ii) à effet indirect correspondant à des gènes codant pour des protéines impliquées dans le maintien et la réplication de l'ADN mitochondrial. Des mutations dans cette dernière classe de gènes peuvent s'accompagner d'anomalies quantitatives ou qualitatives de l'ADNmt : déplétion de l'ADNmt (réduction majeure du nombre de molécules d'ADNmt) et délétions multiples.Après des dosages enzymatiques de l’activité des complexes respiratoires mitochondriaux chez les patients, le ou les types de complexes altérés sont connus. Un grand nombre de gènes mutés responsables de pathologies mitochondriales ont été identifiés, tous codant des constituants des différents complexes de la chaîne respiratoire. Ces dernières années, le groupe d’Agnès Rötig (Hôpital Necker, Paris) a identifié de nouveaux gènes grâce à une approche gènes candidats ou grâce à des tours de génome de familles consanguines de patients qui permettent de délimiter une région chromosomique portant la mutation à l’état homozygote. La validation de l’effet délétère de la mutation identifiée se fait en général en utilisant des organismes modèles d’étude comme les cellules humaines en culture ou bien la levure. Cependant, il reste un grand nombre de cas où la mutation n’a pas pu être identifiée, soit parce que le déficit de tel ou tel complexe ne met pas en jeu un des composants connus de ce complexe ou bien plusieurs complexes de la chaîne respiratoire sont déficitaires mettant en jeu, dans un grand nombre de cas, le maintien de l’ADN mitochondrial pour lequel peu de gènes sont connus.Au laboratoire d’Orsay, nous disposons de deux organismes modèles d’étude, la levure S. cerevisiae et le nématode C. elegans. La levure S. cerevisiae est l’organisme modèle de choix pour étudier les fonctions mitochondriales grâce à ses caractéristiques comme la respiration facultative, mais aussi et surtout par la puissance de sa génétique et le fait que les mitochondries peuvent être transformées. Cependant de par sa respiration facultative et sa division clonale, elle ne se prête pas facilement à des études sur la stabilité de l’ADNmt. En effet, S. cerevisiae perd très facilement son ADNmt après inactivation d’un grand nombre de gènes impliqués dans pratiquement toutes les voies de la biogenèse mitochondriale. Cette levure ne peut donc pas être utilisée de façon simple pour l’étude de la transmission de l’ADN mitochondrial. C’est pourquoi nous nous sommes alors intéressés à l’autre organisme modèle développé au laboratoire, le nématode C. elegans dont ses caractéristiques en font un excellent modèle complémentaire à la levure
Mitochondrial respiratory chain diseases of nuclear origin represent one of the major causes of metabolic disorders. These diseases are characterized by a huge clinical and genetic heterogeneity which is a major problem in identifying the disease causing gene. Although several gene mutations have already been found in some patients or families, the disease causing gene of the majority is yet to be determined. The overall structure and gene content of the mitochondrial genome and the proteins required for mtDNA transactions are largely conserved from yeast to human offering the opportunity to use animal models to understand the molecular basis of mitochondrial dysfunctions. To expand the number of human candidate genes of mitochondrial diseases involved in mtDNA maintenance, we have developed in this study, the nematode Caenorhabditis elegans as a model organism to identify new proteins involved in mtDNA maintenance by combining RNAi and ethidium bromide exposure. We have developed a large-scale screening method of genes required for mtDNA maintenance in the worm and initially indentified four new C. elegans genes (atad-3, dnj-10, polrmt, phi-37 and immt-1) involved in mtDNA stability. The human homologs of these genes (ATAD3, DNAJA3, POLRMT and ATP5A1) can be now considered as candidate genes for patients with quantitative mtDNA deficiencies. Using our screening design we have begun to screen all the C. elegans genes encoding mitochondrial proteins. Of the 721 estimated C. elegans mitochondrial genes homologous to human genes, we have tested 185 genes and found that 41 genes are required for the maintenance of the mitochondrial genome in post mitotic cells. These genes fall into three main functional categories of metabolism, protein synthesis and oxidative phosphorylation. Finally, in this study, we investigated the reversibility of mtDNA depletion with drugs to counteract POLG dificiency. Three molecules, Chlorhexidine, Resveratrol and Bezafibrate, have been tested to restore normal mtDNA content and worm life cycle. These experiments hold promise for future work using C. elegans as a pharmacological model for mitochondrial diseases.Altogether, the data generated in this work is a starting point for promising advances in the mitochondrial field, showing the relevance of the nematode as a model organism to study fundamental processes as well as human health research

Mitochondrial disorders are a group of highly invalidating human conditions due to defective oxidative phosphorylation, for which no effective treatment is nowadays available. In order to develop effective therapies for these disorders, I focused on an experimental approach based on the manipulation of mitochondrial morphology. Opa1 is a GTPase of the inner mitochondrial membrane involved in both mitochondrial fusion and cristae shaping. The role of OPA1 in mitodynamics has also a documented impact on controlling the assembly of the respiratory supercomplexes and respiratory proficiency. Based on these considerations, I tested whether Opa1 overexpression could mitigate the effects of a severe mitochondrial respiratory chain deficit in vivo. In this thesis, the effects of mild transgenic overexpression of Opa1 on two mouse models of defective mitochondrial bioenergetics, a constitutive knockout for Ndufs4 (Ndufs4-/-), encoding a structural component of complex I, and a muscle-specific conditional knockout for Cox15, (Cox15sm/sm), encoding a heme-a biosynthetic enzyme involved in complex IV hemylation and assembly are shown. Crossing of both models with an Opa1 transgenic mouse line (Opa1tg) was associated with clinical and biochemical improvement, but whilst the effect was limited in Ndufs4-/-::Opa1tg mice, the Cox15sm/sm::Opa1tg mice showed relevant amelioration of motor performance, remarkable prolongation of survival, and significant correction of mitochondrial respiration. This effect was associated with the increased amount of active cIV holocomplex and cIV-containing supercomplexes.

Ca2+ is one of the main second messengers of cells and, in particular the Ca2+ signaling in mitochondria is involved in different physiological processes spanning from cell metabolism, through the control of mitochondrial respiration and crucial metabolic enzymes, to the response in stress conditions. Despite the lack of a mechanistic understanding, it is well known that mitochondrial Ca2+ overload is the most important trigger for the opening of permeability transition pore responsible for apoptosis induction after several toxic challenges. On the contrary, the role of Ca2+ signaling in autophagy only recently started to emerge. Autophagy is a process of self-eating by which cellular organelles and proteins are sequestered and degraded in order to produce energy and amino acids in metabolic stress conditions, such as nutrient deprivation. It is not surprising that mitochondrial Ca2+ also plays an important role in the pathological alteration of cell physiology in different human disorders. In the present work we will consider, in particular, the involvement of mitochondrial Ca2+ homeostasis and its correlated metabolic processes in two models of human diseases: mitochondrial disorders and neurodegeneration. Mitochondrial disorders are a large group of heterogeneous diseases, commonly defined by a lack of cellular energy due to oxidative phosphorylation defects. We used skin primary fibroblasts derived from a patient with a complex I mutation in ND5 subunit, as a model of mitochondrial disorders. This system revealed an interesting correlation between the decrease in mitochondrial Ca2+ uptake and the increase in autophagic flux. In addition, our results suggest that this is due to a structural rearrangement of intracellular organelle architecture causing a loss of ER-mitochondria contact sites. Neurodegeneration is caused by selective and progressive death of specific neuronal subtypes. In order to understand the involvement of mitochondrial Ca2+ signaling in the pathogenesis of neurodegeneration, we developed an in vitro system of mouse primary cortical neurons and we optimized an in vivo model of microinjection in mouse brain regions. In particular, we studied the effect of an increased mitochondrial Ca2+ uptake, induced by the overexpression of mitochondrial Ca2+ uniporter (MCU, the main responsible of Ca2+ entry in mitochondrial matrix), on cell survival, in both primary cultures and in midbrain mouse area. We concluded that mitochondrial Ca2+ accumulation induces mitochondrial fragmentation and higher sensitivity to cell death in neurons both in vitro and in vivo
Il Ca2+ è uno dei principali secondi messaggeri cellulari, ed in particolare il segnale Ca2+ mitocondriale è implicato in vari processi fisiologici che spaziano dal metabolismo, attraverso il controllo della respirazione mitocondriale, alla risposta a condizioni di stress. Nonostante alcuni meccanismi d’azione non siano ancora stati chiariti, il ruolo del Ca2+ nell’attivazione del processo apoptotico è ampiamente riconosciuto e comprovato. Al contrario, il coinvolgimento del segnale Ca2+ in un altro importante processo, quale quello autofagico, ha cominciato ad emergere solo recentemente. Il ruolo del Ca2+ a livello fisiologico risulta dunque fondamentale all’interno della cellula e alterazioni nella sua regolazione hanno ripercussioni così profonde da indurre l'evolversi di differenti patologie umane. Nel presente lavoro verrà approfondito il ruolo del Ca2+ mitocondriale in particolar modo in due modelli di patologie umane: le malattie mitocondriali e la neurodegenerazione. Le malattie mitocondriali sono un gruppo molto eterogeneo di patologie, accomunate principalmente dalla perdita di funzionalità della catena respiratoria. Come modello di studio di queste patologie abbiamo scelto di utilizzare delle colture primarie di fibroblasti umani derivanti da pazienti con una specifica mutazione nel gene per la subunità ND5 del complesso I della catena respiratoria del DNA mitocondriale. L’utilizzo di questo modello sperimentale si è rivelato molto utile per l’identificazione di una interessante correlazione tra la diminuzione dell’uptake di Ca2+ mitocondriale e l’aumento del flusso autofagico in queste cellule. Inoltre, i nostri risultati suggeriscono che la causa del ridotto accumulo di Ca2+ mitocondriale è direttamente correlato con un riarrangiamento spaziale nella distribuzione di reticolo endoplasmatico e mitocondri, tale per cui i siti di contatti presenti tra questi due organelli diminuiscono nettamente. La neurodegenerazione è causata dalla selettiva e progressiva perdita di specifici tipi neuronali. Allo scopo di studiare il coinvolgimento del Ca2+ nella neurodegenerazione, abbiamo sviluppato un modello in vitro di neuroni primari di corteccia di topo, in cui abbiamo analizzato gli effetti della sovraespressione del canale per il Ca2+ mitocondriale, MCU (mitochondrial Ca2+ uniporter). Dai nostri dati possiamo concludere che la sovraespressione di MCU ha degli effetti dannosi per le cellule neuronali, tanto da indurne la morte. Inoltre, abbiamo dei risultati preliminari anche in un sistema in vivo, i quali confermano e consolidano i dati ottenuti in vitro. Nello specifico, abbiamo iniettato vettori adeno-virali esprimenti il canale del Ca2+ mitocondriale nel mesencefalo di topo, utilizzando la tecnica dell’iniezione stereotassica, ed anche in questo caso osserviamo l’induzione di morte cellulare e degenerazione neuronale

Myocardial ischemia, infarction, heart failure and arrhythmias are the manifestations of coronary artery disease. Reduction of ischemic damage is a major concern of cardiovascular biology research. As per recent studies, the mitochondrial ATP-sensitive potassium channel (mitoKATP) opening is believed to play key role in the physiology of cardioprotection, protection against ischemia-reperfusion injury or apoptosis. However, the structural information of mitoKATP is not precisely known. Elucidating the structural integrity and functioning of the mitoKATP is therefore a major goal of cardiovascular biology research. The known structure and function of the cell ATP-sensitive potassium channel (cellKATP) is functional in interpreting the structural and functional properties of mitoKATP. The primary goal of my research was to characterize the activity of mitoKATP in the isolated mitochondria from the control mouse heart. The mitoKATP activity, if preliminarily characterized in the control strains through the light scattering technique, then the structure of the channel could possibly be established and analyzed by means of the transgenic model and with the help of immunological techniques such as western blotting and immunoflorescence. With this experimental model it was possible to demonstrate that the mitoKATP activity in control mouse heart mitochondria is activated by potassium channel openers (KCOs) such as diazoxide and cromakalim and activators of mitoKATP such as PMA (phorbol12 myristate-13-acetate), and inhibited by KATP inhibitors such as glibenc1amide and 5-hydroxydecanoate (5 HD). It was evident that the KATP activity in mouse heart mitochondria was comparable to that exhibited by the rat heart mitochondria. The various selective and non-selective activators and inhibitors of the channel elicited their activity at a similar concentration used for the rat heart mitochondria. The results were reproducible in five independent experiments for each combination, further reinforcing the significance of existing channel activity in the mouse heart mitochondria.

Background: Mitochondrial diseases are one of the most common hereditary neuromuscular conditions. Late-onset presentations are common. Genotype-phenotype correlations are poor, making the diagnosis difficult and prognostication imprecise. Objectives: We aim to refine the diagnostic pathway for adult patients suspected to have a mitochondrial disease, to better understand their clinical presentations, and to review their management strategies. Methods: At the Neurogenetics Clinic, we saw over 270 patients with suspected mitochondrial disease, of whom 148 patients had “probable” or “definite” disease. To review their most common and distinctive features, we set up a database to collate data on their clinical and investigational findings. Patients presenting with symptoms consistent with POLG1 mutations were screened by direct whole gene sequencing. We report cases of diagnostic challenge and interest. We explored for new diagnostic biomarkers, and devised a prediagnostic screening scale. We audited the patients who required critical care admissions. Results: We identified common pitfalls in diagnosing mitochondrial disease in adult patients, and proposed a new diagnostic paradigm. Among our cohort with features suggestive of POLG-related syndromes, only 10% had pathogenic mutations. We explored the use of FGF-21 (elevated in patients with muscle-manifesting mitochondrial disease); and GDF-15 (indicative of mitochondrial diseases more broadly), and both had significantly greater sensitivity and specificity than traditional blood biomarkers of disease. We created the MitoScale which as a screening tool, has a high sensitivity for the disease. We retrospectively followed a group of critically ill adult patients, and identified the common precipitating events, prodromal symptoms and complications. Conclusion: The diagnostic pathway for patients with suspected mitochondrial disease is improving with next generation genetic sequencing techniques and new serum diagnostic biomarkers. These should be reflected in the revision of diagnostic criteria and screening algorithms. The evidence for patient prognostication and management remains rudimentary, but ongoing research into larger patient cohorts enabled by database networks will help to improve patients’ management outcome.

Darrerament s’han produït avanços importants que han contribuït al coneixement dels mecanismes de disfunció cel·lular i mort en la malaltia de Parkinson (MP) i en la malaltia de Huntington (MH). Ambdues malalties són trastorns del moviment que es caracteritzen per la pèrdua específica de neurones dels ganglis basals, les neurones dopaminèrgiques de la substància nigra (SN), en el cas de la MP i les neurones espinoses de l’estriat, en el cas de la MH. Malgrat les diferències, ambdues comparteixen processos patològics comuns com la presència de proteïnes malplegades, l’estrés oxidatiu i disfunció mitocondrial. La mitocòndria és la font d’energia principal en les cèl·lules eucariotes, però també és un orgànul dinàmic relacionat amb una gran quantitat de processos cel·lulars. La disrupció de la homeòstasis mitocondrial i la subseqüent disfunció mitocondrial juguen un paper important en la patofisiologia de les malalties neurodegeneratives. El manteniment de la integritat mitocondrial a través de diferents mecanismes de control és crític per a la superviviència neuronal. Aquesta tesi es centra en l’estudi dels mecanismes de control de qualitat mitocondrial en la MP i la MH, per tal d’entendre millor els mecanismes que duen a la mort cel·lular. En el primer capítol, he estudiat el transport de proteïnes a la mitocòndria en models in vitro i in vivo de la MP. In vitro, la inhibició del complexe I produeix una alteració del transport de proteïnes a la mitocòndria així com una disminució dels nivells de proteïnes OXPHOS, acumulació de proteïnes agregades i disminució dels nivells de chaperones mitocondrials. Per tal de restablir el transport de proteïnes mitocondrials es van sobreexpressar dos components clau del sistema de translocases: la translocasa de la membrana externa 20 (TOM20) i la translocasa de la membrana interna 23 (TIM23). La sobreexpressió in vitro de TOM20 i TIM23 va restaurar el transport de proteïnes mitocondrials i va alleugerar la disfunció mitocondrial i la mort cel·lular. La inhibició del complexe I en ratolins també dóna lloc a una alteració del transport de proteïnes mitocondrials i produeix neurodegeneració del sistema dopaminèrgic. La sobreexpressió de TIM23 va restaurar parcialment el transport de proteïnes i va protegir lleugerament les neurones dopaminèrgiques de la SN. En canvi, la sobreexpressió de TOM20 va ser incapaç de millorar el transport de proteïnes mitocondrials i, fins i tot, va exacerbar la mort cel·lular. Aquests resultats posen de relleu el paper de la disfunció del transport de proteïnes mitocondrials, en particular de dos dels seus components, en la patogènesis de la MP i suggereixen la necessitat de futurs estudis es centrin en altres elements d’aquest sistema. En el segon capítol, he estudiat el paper de la proteïna huntingtina en la mitofàgia i com la seva mutació, que dóna lloc a una expansió de glutamines, pot afectar a aquesta funció. Per a tal fi, he treballat en un model in vitro de cèl·lules estriatals ST-Q7 (control) i ST-Q111 (mutant). En condicions fisiològiques, la mitofàgia induïda no es troba mitjançada pel reclutament de parkin als mitocondris despolaritzats. La huntingtina mutada afecta la mitofàgia induïda a través de l’alteració de la seva funció de scaffold en diferents passos del procés de mitofàgia: (i) activació d’ULK1 a través de l’alliberament de mTORC1, (ii) formació del complexe Beclin 1-Vps15,(iii) interacció dels adaptadors de mitofàgia OPTN i NDP52 amb huntingtina i, (iv) amb LC3. Com a resultat, els mitocondris de les cèl·lules ST-Q111 estan més danyats i tenen una respiració mitocondrial deficient. Aquests resultats demostren la presència d’una alteració en la mitofàgia com un mecanisme lligat a la MH. En conclusió, el descobriment de noves dianes mitocondrials en la MP i MH emfatitza el paper important que juga el control de qualitat mitocondrial en la neurodegeneració.
In the past years, several important advances have expanded our understanding of the pathways that lead to cell dysfunction and death in Parkinson’s disease (PD) and Huntington’s disease (HD). Both diseases are movement disorders characterized by the loss of a specific subset of neurons within the basal ganglia, dopaminergic neurons in the substantia nigra pars compacta (SNpc), in the case of PD, and medium spiny neurons in the striatum, in the case of HD,. Despite distinct clinical and pathological features, these two neurodegenerative disorders share critical underlying pathogenic mechanisms such as the presence of misfolded and/or aggregated proteins, oxidative stress and mitochondrial anomalies. Mitochondria are the prime energy source in most eukaryotic cells, but these highly dynamic organelles are also involved in a multitude of cellular events. Disruption of mitochondrial homeostasis and the subsequent mitochondrial dysfunction plays a key role in the pathophysiology of neurodegenerative diseases. Therefore, maintenance of mitochondrial integrity through different surveillance mechanisms is critical for neuronal survival. In this thesis I have studied in depth some mitochondrial quality control mechanisms in the context of PD and HD, in order to broaden the knowledge about the pathomechanisms leading to cell death. In the first chapter I have studied mitochondrial protein import in in vitro and in vivo models of PD. In vitro, complex I inhibition, a characteristic pathological hallmark in PD, impaired mitochondrial protein import. This was associated with OXPHOS protein downregulation, accumulation of aggregated proteins inside mitochondria and downregulation of mitochondrial chaperones. Therefore, we aimed to reestablish the mitochondrial protein import by overexpressing two key components of the system: translocase of the outer membrane 20 (TOM20) and translocase of the inner membrane 23 (TIM23). Overexpression of TOM20 and TIM23 in vitro restored protein import into mitochondria and ameliorated mitochondrial dysfunction and cell death. Complex I inhibition also impaired mitochondrial protein import and led to dopaminergic neurodegeneration in vivo. Overexpression of TIM23 partially rescued protein import into mitochondria and slightly protected dopaminergic neurons in the SNpc. On the contrary, TOM20 overexpression did not rescue protein import into mitochondria and exacerbated neurodegeneration in both SNpc and striatum. These results highlight mitochondrial protein import dysfunction and the distinct role of two of their components in the pathogenesis of PD and suggest the need for future studies to target other elements in the system. In the second chapter, I have studied the role of huntingtin in mitophagy and how the polyglutamine expansion present in mutant huntingtin can affect its function. For such, I worked with differentiated striatal ST-Q7 (as control) and ST-Q111 (as mutant) cells, expressing full length huntingtin. In these conditions, induced mitophagy was not mediated by Parkin recruitment into depolarized mitochondria. Mutant huntingtin impaired induced mitophagy by altering wildtype huntingtin scaffolding activity at different steps of mitophagy process: (i) ULK1 activation through its release from the mTORC1, (ii) Beclin1-Vps15 complex formation, (iii) interaction of the mitophagy adapters OPTN and NDP52 with huntingtin and (iv) with LC3. As a result, mitochondria from ST-Q111 cells exhibited increased damage and altered mitochondrial respiration. These results uncover impaired mitophagy as a potential pathological mechanism linked with HD. In conclusion, we have discovered new mitochondrial targets for PD and HD emphasizing the important role that mitochondrial quality control plays in neurodegeneration

Le malattie mitocondriali sono disturbi genetici caratterizzati da difetti di fosforilazione ossidativa causati da mutazioni nel DNA mitocondriale, o in geni nucleari i cui prodotti sono legati alla fosforilazione ossidativa o alla biologia mitocondriale. La prima parte del progetto è stata focalizzata sulla generazione e la caratterizzazione di un modello murino di malattia mitocondriale, Ttc19ko. I pazienti con mutazioni in TTC19 sviluppano danni neurologici e deficit di complesso III. Ttc19 è una proteina mitocondriale che sembra essere coinvolta nell’assemblaggio e/o stabilità del complesso III. Abbiamo dimostrato che il modello murino Ttc19ko ha sintomi neurologici, debolezza muscolare e riduzione dell’attività locomotoria spontanea, in analogia con la malattia umana. L’analisi istologica ha rivelato alcune anomalie neurologiche con presenza di accumuli di ubiquitina e GFAP. I topi Ttc19ko hanno una riduzione complessiva del metabolismo energetico, una diminuzione del consumo di O2 e di produzione di CO2. L’attività enzimatica del complesso III è significativamente ridotta nei tessuti e ciò è legato ad un aumento della produzione di ROS. L’analisi BNGE ha mostrato una riduzione della incorporazione della subunità catalitica Rieske nel complesso assemblato. L’immunoprecipitazione di TTC19-Flag in colture cellulari trattate con amminoacidi marcati ha rivelato una maggiore interazione tra Ttc19 e le subunità del pre-complesso III, e una minore interazione con proteine Rieske e Uqcrh, entrambe assemblate tardivamente. Abbiamo inoltre dimostrato che Ttc19 rimane associata al complesso III assemblato. Nell’insieme, questi risultati suggeriscono che Ttc19 è un fattore intrinseco di assemblaggio del complesso III che interagisce con il pre-complesso III facilitando così l'incorporazione della proteina Rieske. La seconda parte del progetto è stata focalizzata sulla messa a punto di una terapia genica su un altro modello murino di malattia mitocondriale, MPV17ko. Mutazioni in MPV17 causano una sindrome epatocerebrale con deplezione del mtDNA, insufficienza epatica a esordio precoce, gravi crisi ipoglicemiche e morte. Il trapianto di fegato e l'alimentazione frequente a base di carboidrati a lento rilascio sono le uniche terapie disponibili, anche se in seguito si sviluppano danni neurologici. Il ruolo fisiologico di MPV17 non è chiaro. Abbiamo dimostrato che MPV17 fa parte di un complesso ad alto peso molecolare a composizione sconosciuta, che è essenziale per il mantenimento del mtDNA nel fegato. In dieta standard, il topo MPV17-/- non mostra quasi alcun sintomo di disfunzione epatica, ma una dieta chetogenica porta questi animali a sviluppare cirrosi e insufficienza epatica grave. Tuttavia, quando l'espressione di MPV17 è ripristinata dalla somministrazione di virus adeno-associato, si assiste ad un ricostituzione del complesso supramolecolare contenente Mpv17, ad un ripristino completo del numero di copie di mtDNA, ed alla prevenzione dell’insufficienza epatica indotta dal dieta chetogenica. Questi risultati aprono nuove prospettive terapeutiche per il trattamento delle sindromi da deplezione del mtDNA indotte da mutazioni nel gene MPV17.
Mitochondrial diseases are genetic disorders characterized by defects in oxidative phosphorylation caused by mutations in mitochondrial DNA, or in nuclear genes whose products are related to oxidative phosphorylation or mitochondrial biology. The first part of the project was focused on the generation and characterization of a mouse model of mitochondrial disease, Ttc19ko. Patients with mutations in TTC19 were characterized by neurological impairments and mitochondrial respiratory complex III deficiency. Ttc19 is a mitochondrial protein that seems to be associated to complex III assembly and/or stability. We showed that Ttc19ko mice have neurological symptoms, muscular weakness and reduction in spontaneous locomotors activity, clearly resembling the human disease. Brain also had neurological abnormalities with presence of ubiquitin and GFAP positive staining. Comprehensive lab animals monitoring system revealed a reduction in O2 consumption, CO2 production and energy expenditure in Ttc19ko mice, indicating an overall reduction of energy metabolism. Complex III activity was significantly reduced in tissues and this was linked to an increased ROS production. BNGE analysis of mitochondrial complex III showed a substantial reduction of the incorporation of the catalytic Rieske iron-sulfur protein into the fully assembled complex. A stable isotope labelling by amino acids in cell culture (SILAC) expressing TTC19-Flag followed by immunoprecipitation and mass spec analysis revealed a higher scored interaction between Ttc19 and the subunits of the pre-complexIII, and a lower scored interaction with Rieske protein and Uqcrh, both of them are late assembled subunits. We also demonstrated that Ttc19 is associated to the fully assembled complex III. Taken together, these results suggests that Ttc19 is an intrinsic assembly factor of complex III that interacts with the pre-complex III thus facilitating the incorporation of the late assembled Rieske protein. The second part of the project was focused on a gene therapy approach on a second mouse model of mitochondrial disease, MPv17ko. Mutations in hMPV17 cause a hepatocerebral form of mtDNA depletion syndrome hallmarked by early-onset liver failure, leading to premature death. Liver transplantation and frequent feeding using slow-release carbohydrates are the only available therapies, although surviving patients develop slowly progressive neuropathy. The physiological role of Mpv17 is still unclear. We showed that Mpv17 is part of a high molecular weight complex of unknown composition, which is essential for mtDNA maintenance in liver. On a standard diet, Mpv17ko mouse shows hardly any symptom of liver dysfunction, but a ketogenic diet leads these animals to liver cirrhosis and failure. However, when expression of human MPV17 is carried out by adeno-associated virus mediated gene replacement, the Mpv17ko mice are able to reconstitute the Mpv17-containing supramolecular complex, restore liver mtDNA copy number and oxidative phosphorylation proficiency and prevent liver failure induced by the KD. These results open new therapeutic perspectives for the treatment of MPV17-related liver-specific MDS.

Abstract The mitochondrial oxidative phosphorylation system (OXPHOS) generates energy but also deleterious reactive oxygen species (ROS). Changes in the cytoskeleton, composed mainly of microfilaments, microtubules and intermediate filaments, have been observed in OXPHOS deficiency. The 3243A>G point mutation in mitochondrial DNA (mtDNA) leads to mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS), which is the most common mitochondrial disease. Interestingly, mitochondrial aberrations have been demonstrated in patients with a mutation in NOTCH3, the genetic cause of cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL). Randomization of vimentin intermediate filament direction and length together with slower population growth was observed in myoblasts with 3243A>G, with no difference in the amount of apoptotic cell death. Upon complex IV inhibition (with or without the microtubule-depolymerizing compound nocodazole) or a lack of mtDNA (ρ0) in osteosarcoma cells the vimentin network collapsed perinuclearly, forming thick bundles, whereas complex I inhibition led to thinner vimentin network bundles. Furthermore, the amount of vimentin was increased in ρ0 cells. Mitochondria accumulated around the nucleus upon complex IV inhibition and in ρ0 cells. Analysis of the total proteome revealed that specific OXPHOS deficiencies led to changes in the expression of cytoskeletal proteins and proteins involved in apoptosis, OXPHOS, glycolysis and oxidative stress response. Muscle histochemical and genetic analysis showed ragged red fibres and cytochrome c oxidase-negative fibres to be associated with 5650G>A in a patient with R133C in NOTCH3 and 5650G>A in MTTA. Immunolabelling of cells with R133C and 5650G>A revealed a sparse tubulin network with asters and less abundant mitochondria by comparison with control cell lines. Comparison of nucleotide diversity between CADASIL pedigrees and controls showed increased mtDNA sequence variation in the CADASIL patients. Also maternal relatives in two CADASIL pedigrees differed from each other in their mtDNA. These findings suggest that defects in OXPHOS lead to selective changes in the vimentin network, which may have a role in the pathophysiology of mitochondrial diseases. They also suggest a relationship between NOTCH3 and mtDNA, and establish the pathogenicity of 5650G>A. The overall results emphasize that a deficiency in the energy converting system together with oxidative stress can lead to cytoskeletal changes.

Complex I (NADH:ubiquinone oxidoreductase), located in the mitochondrial inner membrane, is a major electron entry point to the respiratory chain. It couples the energy released from electron transfer (from NADH to ubiquinone) to the concomitant pumping of protons across the membrane, to generate an electrochemical proton motive force. Mammalian complex I is composed of 45 subunits, 14 of which comprise its simpler bacterial homologues. It is encoded by both the mitochondrial and nuclear genomes, and pathological mutations in both sets of subunits result in severe neuromuscular disorders such as Leigh syndrome. Several structures of mammalian complex I from various organisms have been determined, but the limited resolutions of the structures, which typically refer to poorly characterised enzyme states, has hampered detailed analyses of mechanistic features. The first part of this thesis describes development of a method for purifying complex I from the genetically amenable and medically relevant model organism Mus musculus (mouse), in a pure, stable and active state. The enzyme from mouse heart mitochondria was then comprehensively characterised, to ensure the presence of all the expected subunits and co-factors, and to define its kinetic properties. The second part of this thesis describes structural studies by single particle electron cryomicroscopy (cryo-EM) on the purified mouse enzyme in two distinct states, the 'active' and 'de-active' states. The active state was determined to 3.3 Å resolution, the highest resolution structure of a eukaryotic complex I so far. Subsequently, comparison of the two mouse structures, together with previously determined mammalian and bacterial structures, revealed variations in key structural elements in the membrane domain, which may be crucial for the catalytic mechanism. Moreover, in the high-resolution active mouse complex I structure a nucleotide co-factor was observed bound to the nucleoside kinase subunit NDUFA10. Finally, complex I from the Ndufs4 knockout mouse model, which recapitulates the effects of a human mutation that causes Leigh syndrome, was purified and subjected to kinetic and proteomic analyses. Following cross-linking and preliminary structural studies, it was concluded that the detrimental effects of deleting NDUFS4 are due to lack of stability of the mature complex.

Peripheral arterial disease (PAD) is an atherosclerotic disease in the peripheral arteries, which reduces blood supply to the lower extremities. Intermittent claudication is the symptom that develops early in PAD patients and is accompanied by the haemodynamic finding of a fall in systolic blood pressure at the ankles following exercise. In PAD patients, exercise performance is not well correlated with haemodynamic measurements and other mechanisms have been suggested to account for the impairment. There have been reports about the impaired mitochondrial metabolism (eg, decreased activities of mitochondrial enzymes) and abnormal mitochondrial structure in the skeletal muscle in PAD patients. It is not known, however, whether mitochondrial ATP production is impaired in the skeletal muscle in PAD. Whether the mitochondrial function is impaired in PAD patients, and whether the impaired mitochondrial function in the muscle contributes to the impaired exercise performance in PAD patients is unknown. The object of this work is to explore the mitochondrial function in the skeletal muscle of PAD patients and its relationship to exercise performance. Impaired exercise performance in PAD patients is evaluated using a treadmill walking performance test, which is closely correlated to patients' daily activity performance. Treadmill walking however, in addition to being influenced by local muscle factors, is influenced by central contributions, such as cardiac output and the central nervous system. As walking is limited by intermittent claudication in PAD patients localized in the legs, it would be valuable to develop a local calf muscle performance test to better understand the underlying pathophysiology in PAD. Such a protocol has not been used previously in experiments involving PAD patients. Hence, the research aim for Study 1 was to establish a calf muscle performance test protocol and to investigate its variability. Fourteen healthy control subjects and eight PAD patients undertook the maximal plantar flexion test once a week for five weeks using a Kin-Com Dynamometer. In the traditional assessment, the total impulse and peak impulse are the variables that were measured as representing the calf muscle performance. Both these variables are significantly lower in PAD patients than in controls. Alternatively, by applying simple mathematical models, the muscle function dimensions of endurance, strength and fatigability can be investigated in a single test. Compared with control subjects the PAD patients had lower muscle endurance, lower muscle strength, higher fatigue index, but no difference were found in magnitude or rate-of-fatigue. The variability of the test was different for different estimated parameters of the models, with the highest variability in muscle fatigability (rate of fatigue, CV=75% in controls) and the lowest variability in muscle strength (CV=16% in controls). The variability of the traditional assessment parameters, which included total impulse and peak impulse, was around 13% in controls and 18- 24% in PAD patients for the five tests. Based on these findings the calf muscle performance test can be applied in PAD patients to investigate different muscle function dimensions. While many of the dimensions were impaired in PAD patients compared with controls, the high variability of some of the parameters have to be considered during its application. Having established a local calf muscle performance test, the aim of Study 2 was to explore the relationship between the calf muscle performance and the traditional treadmill walking performance. Seventeen PAD patients and fourteen control subjects were tested using both the calf muscle performance test described earlier and walking performance test. The walking performance was tested using a graded treadmill protocol. The total walking time was significantly lower in PAD than that in control subjects. No variable of calf muscle performance correlated with walking performance in control subjects. However, in PAD patients, a number of calf muscle performance variables correlated with walking performance. The total impulse and the peak impulse in the best legs (higher ABI) tended to correlate with pain-time. In simple mathematical models, the muscle endurance in the worst legs (lower ABI) correlated positively with pain-walking time, and the muscle fatigue-index in the worst legs correlated negatively with total walking time. In conclusion, in PAD patients, some dimensions of calf muscle performance correlated with walking performance. This suggests that some factors of local calf muscle performance might contribute to the impaired walking performance in PAD patients. The research aim for Study 3 was to investigate a number of calf muscle physiological factors, and to ascertain their relationship with calf muscle and walking performance in PAD patients and control subjects. The physiological factors examined include ankle brachial pressure index (ABI), calf muscle weight, calf blood flow, and skeletal muscle mitochondrial ATP production rate (MAPR) in vitro. The calf muscle weight in PAD patients was significantly lower than that in control subjects. In PAD patients, the calf muscle weight was significantly lower in the worst legs than that in the best legs. The ABI was lower in PAD than in controls and significantly lower in PAD worst legs than in PAD best legs. The leg blood flow (measured by venous-occlusion plethysmography) was lower in PAD than that in controls, but there was no significant difference between PAD best legs and PAD worst legs. The MAPR was measured using different substrate combinations. The MAPR (PM, pyruvate + malate), MAPR (PCM, palmitoyl-carnitine + malate) and MAPR (PPKM, pyruvate + palmi_toyl-carnitine + alpha-ketoglutarate + malate) shows the capacity of mitochondria to produce ATP by oxidising glucose or fatty acids or both of these substrates respectively. The MAPR for the three substrate combinations in PAD patients was no different from controls. The relationship between these physiological measurements and exercise performance differed between PAD and controls. In control subjects, the calf muscle weight, ABI, leg blood flow and MAPR were not significantly correlated with walking performance, but correlated with some variables of local calf muscle performance. In PAD patients, the calf muscle weight, ABI and blood flow did not correlate with walking performance. However, the MAPR (PMkg) was positively correlated with total walking time, and MAPR (PPKMg) was positively correlated with pain-free walking time. In calf muscle performance, in the best legs, the body weight was positively correlated with total impulse and peak impulse; the calf muscle weight was positively correlated with contraction number and peak impulse; and the blood flow correlated with peak impulse. In the worst legs, the calf muscle weight and ABI were not significantly correlated with any variables; the leg blood flow was negatively correlated with contraction number; the mitochondrial protein content correlated with total impulse; the MAPR (PM) tended to correlate with peak impulse. These results suggest the importance of all these local muscle physiological factors in calf muscle performance. However, only MAPR was important in the walking performance in PAD patients. The aim of Study 4 was to further explore the relationship between MAPR and exercise performance in PAD patients after exercise training. The effect of 16 weeks of treadmill exercise training on exercise performance and MAPR was evaluated in five PAD patients. In the treadmill walking performance test, total walking time increases ranged from 100 to 150% in these five patients. The pain-free walking time increased in three patients but did not change in the other two. In the calf muscle performance test, the total impulse and contraction number increased in both legs of four patients and decreased in both legs of one patient, but the magnitude of improvement was less than 5% and the peak impulse did not change in a consistent trend. The changes in body weight, calf muscle weight, and ABI in these five patients were less than 5%. However, the increased blood flow measured by venousocclusion plethysmography in both legs ranged from 100 to 150%. The MAPR by oxidising glucose was significantly higher in trained patients than that in untrained patients, which suggested a possible change in mitochondrial function in response to exercise training. Such change in mitochondrial function may have a potential role in contributing to calf muscle performance and walking performance after exercise training. In summary, for the first time, a local calf muscle performance test has been established to allow better understanding of calf muscle pathophysiology in PAD patients. Using this test, it has been shown that calf muscle performance is significantly impaired in PAD patients compared with control subjects. The impairment is characterised by lower muscle endurance, lower muscle strength and higher fatigability. The impaired local calf muscle performance might contribute to the impaired overall walking performance in PAD patients. The MAPR, especially 5 through oxidising glucose, contributed to walking performance. In this pilot exercise training study, a 20 weeks exercise training program failed to improve the calf muscle performance and walking performance in PAD patients. The higher MAPR in oxidising glucose in trained PAD patients again suggested the importance of muscle glucose oxidation as a contributing factor in the exercise performance in PAD patients.

Mitochondria are central to cellular metabolism; therefore, changes in nutrient availability are expected to impact the organelle’s function. To assess the interdependence of mitochondria and nutrients, two models were investigated: rapidly proliferating cells, and fibroblasts derived from patients with mitochondrial disorders. In Human Embryonic Kidney (HEK) cells, amino acid withdrawal increases mitochondrial function (Johnson et al., 2014). Investigation of two unrelated cell lines, A549 and HeLa cells, revealed perturbed regulation of cytosolic translation, and reduced mitochondrial function during amino acid starvation. This was associated with reduced survival compared to HEK cells, demonstrating that these cells differ in their nutrient-processing pathways in response to amino acid deprivation. Assessment of mitochondrial protein translocases for a role in retrograde signalling revealed that Tim17A, a component of the TIM23 translocase, is rapidly downregulated during amino acid withdrawal, in all cell lines investigated. Among the amino acids, cysteine metabolism has previously been suggested to be important for the spontaneous recovery from certain mitochondrial translation disorders (Boczonadi et al., 2013; Zeharia et al., 2009). Investigation into the regulation of the transsulphuration pathway (TSP), which produces cysteine as well as catabolising it to hydrogen sulphide (H2S), revealed interindividual differences in TSP expression in fibroblasts with a mutation in mitochondrial tRNA modifying enzymes. Strikingly, one patient surviving to adulthood in a normally fatal infantile disease showed upregulation of H2S-detoxifying enzyme, SQR, and its catabolite, thiosulphate. Furthermore, the TSP and SQR were induced using a pharmacological model of mitochondrial dysfunction, highlighting the response of this pathway to mitochondrial disease. Together, these data demonstrate the central role of the mitochondria in nutrient metabolism and stress response, and the possibility to adapt metabolic pathways to ameliorate the pathological consequences of mitochondrial defects.

Mitochondrial diseases are a clinically heterogeneous group of inherited disorders associated with defects in the oxidative phosphorylation system, with an estimated incidence in between 1:5,000 and 1:10,000 live births. Mitochondrial respiratory chain function depends on the coordinated expression of both mitochondrial and nuclear genomes. Thus, also mutations affecting nuclear-encoded mitochondrial proteins are responsible for mitochondrial disease onset. During the last decades, an increasing number of novel nuclear disease genes have been identified. Among those genes, human MPV17 and APOPT1 have already been linked to mitochondrial diseases but their role in mitochondrial physiology and disease remains still puzzling. Mutations in the human MPV17 nuclear gene, encoding a small hydrophobic mitochondrial inner membrane protein, are a prominent cause of a pediatric hepatocerebral form of mitochondrial DNA depletion syndrome. APOPT1 mutations are responsible for an infantile or childhood mitochondrial encephalopathy hallmarked by profound deficiencies in both COX activity and amount. In order to dissect out the role of these two genes, in this PhD project we focused our attention on the functional and molecular characterization of Drosophila melanogaster orthologs of MPV17 and APOPT1. We found that dMpv17 down-regulation in flies causes a profound mitochondrial DNA depletion in the fat bodies (a Drosophila organ analogous to human liver). Depletion is also detected, albeit moderate, in dMpv17 KD cells. Our results reveal that dMpv17 can form a channel when inserted in an artificial planar lipid bilayer. Moreover, we also show that the Drosophila protein could interact with dMic19, a component of the MICOS complex, as well as dMrp4, that could play a role in dMpv17 gating regulation. The analysis of mitochondrial morphology in dMpv17 down-regulated cells together with the interaction with Mic19 suggest a possible role for dMpv17 in the maintenance of the structural and functional stability of the inner mitochondrial membrane. Further, we confirmed that Drosophila is a reliable model for studying human mitochondrial disease also in the case of APOPT1. Indeed, our preliminary data show that dApopt1 down-regulation in flies causes motor impairment and COX deficiency, characteristic features of the human disease. Not only COX activity but also coxI transcript is decreased in dApopt1 down-regulated flies. Finally, we show that H2O2 treatment and, in turn, oxidative stress induce an increase in dApopt1 transcript. Finally, our data shed new light on the possible role of dMpv17 and dApopt1 in physiological and pathological conditions.
Le malattie mitocondriali sono un gruppo ampio e eterogeneo di disordini ereditari causati da difetti del metabolismo energetico mitocondriale attribuibili a un malfunzionamento della catena respiratoria mitocondriale. La loro incidenza è stata stimata tra 1:1500 e 1:10000 nati vivi. Queste sindromi sono il risultato di un gran numero di mutazioni rilevabili sia nel genoma nucleare sia in quello mitocondriale. Negli ultimi decenni, il numero di geni scoperti essere responsabili dell’insorgenza di malattie mitocondriali è enormemente aumentato. Mutazioni nella proteina MPV17, localizzata nella membrana mitocondriale interna, sono state associate ad una particolare forma di sindrome da deplezione di DNA mitocondriale che colpisce primariamente il fegato e il sistema nervoso in età pediatrica. Invece, mutazioni in APOPT1 sono state identificate in pazienti caratterizzati da sintomi neurologici di vari entità associati a perdita della parola e della capacità motoria e accompagnati da un significativo deficit di citocromo C ossidasi a livello muscolare. Dal momento che la funzione di queste due proteine risulta essere ancora sconosciuta, abbiamo cercato di determinare il loro ruolo a livello mitocondriale e nello sviluppo di queste malattie studiando i geni ortologhi in Drosophila, dMpv17 e dApopt1. Abbiamo dimostrato che la down-regolazione dell’espressione di dMpv17 in vivo comporta una diminuzione significativa nel numero di copie di DNA mitocondriale nei fat bodies, un analogo funzionale del fegato dei mammiferi. Inoltre, alcuni risultati ottenuti molto recentemente suggeriscono che dMPV17 sarebbe in grado di formare un canale in un planar lipid bilayer. Infine, abbiamo identificato come suoi possibili interattori un componente del complesso MICOS e dMrp4, una proteina facente parte della famiglia dei trasportatori ABC che potrebbe regolare la sua attività di canale. L’interazione con il complesso MICOS e i risultati della microscopia elettronica sulla morfologia dei mitocondri nelle cellule silenziate per dMpv17, che hanno evidenziato una diminuzione nel numero e nella lunghezza delle creste, fanno ipotizzare un possibile ruolo nel mantenimento della struttura e, quindi, della funzionalità della membrana mitocondriale interna. Studiando l’ortologo di APOPT1, dApopt1, abbiamo osservato che la sua down-regolazione in vivo comporta un marcato difetto locomotorio accompagnato da una significativa riduzione dell’attività della citocromo C ossidasi, sintomi descritti anche nella patologia umana. Infine, poiché la proteina umana sembra avere un ruolo nella risposta allo stress ossidativo, abbiamo dimostrato che l’espressione di dApopt1 è indotta dal trattamento con H2O2. I risultati relativi alla caratterizzazione di dMpv17 e quelli preliminari riguardanti dApopt1 contribuiscono a fare luce sul ruolo di queste proteine sia a livello fisiologico che patologico, e confermano la validità di Drosophila come organismo modello per lo studio delle malattie genetiche umane

Human mitochondrial diseases known as mitochondrial encephalomyopathies affecting the oxidative phosphorylation (OXPHOS) system can result from a large number of different mutations, both in the nuclear genome or in the maternally inherited mitochondrial DNA (mtDNA). The manifestations of mitochondrial diseases are extremely diverse, including numerous symptoms of variable severity, affecting tissues highly dependent on mitochondrial energy, such as brain, muscle, heart, and the sensorineural epithelia. Moreover these disorders can appear early in life, in children or in young adults (Wallace et al. 1999). We used Drosophila melanogaster as a model organism to study a number of different mitochondrial disorders. The main reasons why the fruit fly is considered a good model are several. It is small, easy to grow, it has a short life cycle and generation time, it has a large number of progeny and finally it has a small completed sequenced genome, (Adams et al. 2000), with a large number of orthologs to human genes. In particular we employed Drosophila to characterize the molecular function of three different genes whose dysfunctions result in encephalomyopathies affecting the functionality of the mitochondrial respiratory chain at different levels. However, in Drosophila, mutations in these genes cause a common larval lethal phenotype. Furthermore another gene characterized in the present work is involved in the maintenance of mitochondrial DNA (mtDNA). Structural alterations of the mitochondrial genome or decrease of its copy number have profound consequences on mitochondrial function and cause severe human diseases which ultimately, also lead to death (Spinazzola and Zeviani 2005). These genes, and the pathologies deriving from their impairments, were studied in Drosophila with different approaches which follow a common general scheme. Firstly we emloyed an approach which would allow ubiquitous or tissue-specific gene knock down, produced by exploiting the yeast UAS/GAL4 binary system. Specific dsRNA allows the targeted silencing of the interesting gene which can be spatially and temporally modulated (Brand and Perrimon 1993). When possible, we adopted procedures to obtain a gene knock out model. Generation of knock out lines is now a well established method which involves the recombination between transgenic lines bearing insertional elements present within the fruit fly genome. It is rather straightforward to obtain genomic deletions through the combination of FRT bearing transgenes flanking the region of interest, in the presence of FLP recombinase (Parks et al. 2004). Furthermore, when possible, confirmation of the genetic specificity of the observed defects in the Drosophila models was obtained by implementing appropriate gene rescue strategies.
Le malattie di mitocondriali umane, note anche come encefalomiopatie mitocondriali, vanno ad agire sulla funzionalità della fosforilazione ossidativa (OXPHOS) e sono il risultato di un gran numero di mutazioni, che possono localizzarsi sia a livello del genoma nucleare sia a livello di quello mitocondriale (mtDNA a eredità materna). Le malattie mitocondriali producono sintomi estremamente diversi, di gravità variabile, che vanno a colpire tessuti ad elevata richiesta energetica, quali cervello, muscolo, cuore, ed epiteli sensorio-neurali. Inoltre questi disturbi possono apparire precocemente, in giovane età, nei bambini o in età adulta (Wallace et al. 1999). In particolare si è utilizzato Drosophila melanogaster come un organismo modello per studiare varie malattie mitocondriali. Le principali ragioni che fanno del moscerino della frutta un ottimo organismo modello sono molteplici. Innanzitutto ha dimensioni ridotte, è facile da crescere, possiede ciclo di vita e tempo di generazione assai brevi, produce un buon numero di progenie ed è caratterizzato da un genoma piccolo completamente sequenziato, (Adams et al. 2000), con la presenza di gran numero di geni ortologhi a quelli umani. In particolare, grazie all’utilizzo di Drosophila, sono stati analizzati, a livello molecolare, tre diversi geni, le cui disfunzioni danno luogo ad encefalomiopatie, che vanno a colpire la funzionalità della catena respiratoria mitocondriale a diversi livelli. Nel complesso mutazioni in questi geni causano un fenotipo letale. Inoltre, è stato caratterizzato anche un altro gene, coinvolto nella stabilità e nel mantenimento del genoma mitocondriale (mtDNA). Alterazioni strutturali dell’ mtDNA o decremento del numero di copie dello stesso, hanno profonde conseguenze sulla funzionalità mitocondriale e determinano l’insorgenza di gravi malattie umane, per la maggioranza letali (Spinazzola e Zeviani 2005). Questi geni, e le patologie che derivano da un loro malfunzionamento, sono stati studiati in Drosophila con approcci diversi che tuttavia seguono un schema generale comune. In primo luogo, la versatilità del nostro organismo modello rende possibile il silenziamento post-trascrizionale del gene in questione, sfruttando il sistema binario di lievito UAS-GAL4 (Brand e Perrimon 1993). In tal modo è possibile ottenere l’abbattimento del gene, abbattimento che può essere modulato sia temporalmente che spazialmente. Inoltre, qualora le condizioni lo permettano, è di grande utilità la generazione di un modello knock out (KO) del gene. Il KO in drosophila è tuttora un metodo di facile generazione, che prevede un meccanismo di ricombinazione tra linee di drosophila contenenti elementi inserzionali localizzati alle estremità del gene da eliminare. In dettaglio il meccanismo descritto da (Parks et al. 2004) sfrutta la ricombinazione specifica tra siti FRT, localizzati all’interno dell’elemento trasponibile, in presenza di flippasi (FLP) che catalizza l’excisione del gene d’interesse. Una volta eseguito il silenziamento post-trascrizionale o il KO del gene d’interesse, al fine di validare i modelli ottenuti, il passo successivo è quello che prevede il recupero del fenotipo osservato. Esperimenti di “rescue” prevedono la realizzazione di un costrutto contente il gene d’interesse sotto il controllo di uno specifico promotore, in grado di attivarne l’espressione in un contesto “loss of gene”. Se l'espressione del gene sintetico risulta in grado di recuperare il fenotipo KO/KD, la caratterizzazione genica è completata. Nello specifico, in questo lavoro è stato condotto lo studio di quattro diversi geni seguendo lo stesso schema comune, mirato a descrivere la funzione molecolare di questi geni coinvolti in devastanti sindromi mitocondriali.

Les maladies mitochondriales sont des pathologies fréquentes du métabolisme caractérisées par une forte hétérogénéité clinique et génétique, notamment par la dépendance à 2 génomes, nucléaire (ADNn) et mitochondrial (ADNmt), et le concept d’hétéroplasmie (HT). L’objectif de ce travail de thèse a été de développer une stratégie d’analyse de l’ADNmt par séquençage haut-débit (NGS), puis de l’appliquer à l’étude des maladies mitochondriales et des pathologies liées au vieillissement : glaucome à angle ouvert (GPAO) et vieillissement ovarien précoce. Après validation des performances de notre stratégie NGS pour la détection et la quantification des variations de l’ADNmt, nous avons confirmé l’intérêt de l’analyse systématique de la totalité de l’ADNmt avec l’identification de nouveaux variants et l’utilisation de cellules uroépithéliales pour le diagnostic des maladies mitochondriales. Cependant, ces progrès génèrent de nouveaux défis dont l’interprétation des faibles HT et la priorisation des variants de signification inconnue. Pour les pathologies liées au vieillissement, nous avons mis en évidence le possible rôle protecteur des haplogroupes T et H chez les femmes, respectivement dans la survenue et la sévérité du GPAO, suggérant une modulation de l’influence de l’ADNmt par le genre et donc l’importance de la stratification par sexe dans les études d’association. En revanche, nous n’observons pas d’accumulation d’anomalies de l’ADNmt dans le vieillissement ovarien précoce. En perspective, nous rapportons l’identification d’une mutation de l’ADNn dans un phénotype atypique, rappelant la complexité de l’étude des pathologies mitochondriales, du fait de ce double génome
Mitochondrial diseases are common metabolic disorders characterized by strong clinical and genetic heterogeneity, in particular due to the dependence on 2 genomes, nuclear (nDNA) and mitochondrial DNA (mtDNA), and the concept of mitochondrial heteroplasmy. The purpose of this work was to develop a strategy for the analysis of the mtDNA through next-generation sequencing (NGS), and then to apply it to the study of mitochondrial diseases and those related to aging: primary open-angle glaucoma (POAG) and ovarian aging. After validating the performances of our NGS strategy for the detection and quantification of mtDNA variations, we confirmed the power of systematic analysis of the whole mitochondrial genome with the use of uroepithelial cells for mitochondrial diseases diagnosis and the identification of novel mtDNA variants. However, these advances generate new challenges such as the interpretation of low percentages of mtDNA mutations or the prediction of the pathogenicity of new variants. For aging-related diseases, we have identified the possible protective role of the mitochondrial haplogroups T and H in women, respectively in the occurrence and severity of POAG, suggesting that mtDNA influence is drivenby gender, and thus the importance of gender stratification for association studies. By contrast, we did not observe any accumulation of mtDNA abnormalities in early ovarian aging. In perspective, we report the identification of a nDNA mutation in an atypical phenotype, highlighting the complexity of mitochondrial diseases diagnosis, due to this double genome

Human mitochondrial RNase P protein 1 (MRPP1) and 2 (MRPP2) form a methyltransferase complex with N1-methylation activity towards purine-9 on mitochondrial tRNA. A third component, mitochondrial RNase P protein 3 (MRPP3), adds RNase P 5'-end ribonuclease processing activity to the complex. MRPP2 alone is a dehydrogenase reductase (SDR) involved in many reactions e.g. steroid metabolism and isoleucine β-oxidation. Inherited MRPP2 missense mutations cause the rare neurological HSD10 disease, where, due to the multifunctional nature of MRPP2, no clear genotype-phenotype relationship has been established. In this study, investigations are performed on the molecular properties of the individual proteins, as well as interactions within the methyltransferase/RNase P MRPP1-MRPP2-MRPP3 complex, to shed light on disease-causing mutations. At the molecular level, the N-terminal region of MRPP1, with no known structural fold, is responsible for dimerisation, whilst the C-terminal region contains a methyltransferase (MT) domain. The structure of the MRPP1 MT-domain to 1.96 Å resolution reveals the binding of the methyl donor S-adenosyl-methionine (SAM). Assembly of the MRPP1-MRPP2 methyltransferase complex requires intact proteins, and formation of the MRPP1-MRPP2-MRPP3 RNase P complex further requires the pre-tRNA substrate. Low-resolution small angle X-ray scattering (SAXS) models of the complexes, with and without tRNA substrate, provide clues into the protein-tRNA interaction. Reported patient mutations in MRPP2 alters the biochemical properties of the protein with each effect correlating with the structural location of the mutation. From the biochemical data, a correlation exists between phenotype and MRPP2 protein abundance. A novel missense mutation, causing a mild phenotype, show devastating effects on all functions involving MRPP2, thus no clear correlation exists between disease severity and residual activity. A patient mutation in MRPP3, leading to the rare disorder Perrault syndrome, targets the active site and leads to a decrease in product formation. The mutation does not affect MRPP1-MRPP2-MRPP3 complex assembly, hence agreeing well with it being a catalytic mutant. Together, these structural and biochemical data provide a better understanding of the molecular facets affected by HSD10 disease patient mutations, to further explore the diverse disease severity observed in patients. Long term, this will hopefully lead to novel treatments designed for patients suffering from HSD10 disease.

Changes in mitochondrial dynamics, including alterations in the fusion/fission balance, have repeatedly been observed in ageing and neurodegenerative disease, including ALS. We investigated the effect of ageing or disease on mitochondrial network complexity in primary dermal fibroblasts, derived from ALS patients or controls. Increased network complexity during ageing was observed in the control cohort, though motility was unaffected. Conversely, network complexity decreased in the sporadic ALS patient cohort upon ageing. We speculate that this alteration in mitochondrial dynamics may contribute to the significant detrimental effect age has on disease prognosis in ALS. In mutant TARDBP (mTARDBP) patient fibroblasts, a trend towards increased network complexity compared to age-matched controls was observed. Moreover, despite no discernible differences in ATP levels in the mTARDBP fibroblasts compared to control samples, microarray analysis hinted at changes in two metabolic pathways, glycolysis and the TCA cycle, both known to influence mitochondrial dynamics. Subtle changes in the interaction of the ER and mitochondria were also observed in the mTDP-43 fibroblasts. Furthermore, expression of two autophagy genes, MAP1S and Atg12, was significantly altered in the mTARDBP fibroblasts. However, LC3-II western blotting in either mTDP-43 or mC9ORF72 fibroblasts did not reveal any significant differences, suggesting that changes in autophagy is not the primary cellular process responsible for the mitochondrial morphology alterations. HSP is characterised by defects in axonal transport, including mitochondrial transport. A mouse model of SPG4-mediated HSP showed axonal swellings in cortical neurons, correlating with impaired axonal transport. We showed that Tro19622 (a microtubule-interacting drug) did not ameliorate the number of axonal swellings in the mutant neurons, though alterations in microtubule integrity were apparent. Conversely, HDAC6 inhibitor, Tubastatin A, reduced the number of axonal swellings in SPG4-mutant neurons to wild type levels, suggesting that microtubule acetylation state could be an interesting therapeutic target for future investigation in HSP.

Par définition, les maladies mitochondriales résultent d’un défaut dans le processus des oxydations phosphorylantes (OXPHOS). Celui-ci permet aux cellules de se fournir en ATP, soit la principale source d’énergie qu’elles peuvent utiliser. Dans ce processus, quatre complexes (I-IV) insérés dans la membrane mitochondriale interne transfèrent à l’oxygène moléculaire les équivalents réducteurs libérés par l’oxydation de carbohydrates et d’acides gras. Cette activité génère une force proton motrice utilisée pour la synthèse d’ATP à partir d’ADP et de phosphate inorganique par le complexe V ou ATP synthase.Des maladies dont NARP (Neuropathy Ataxia Retinitis Pigmentosa) et MILS (Maternally Inherited Leigh Syndrome) ont été associées à des mutations de la sous-unité a de l’ATP synthase. Son gène (ATP6) est dans le génome mitochondrial. Celui-ci est présent jusqu’à plusieurs milliers de copies par cellule. Les mutations du gène ATP6 coexistent souvent avec des copies sauvages du génome mitochondrial dans les cellules et tissus des patients, ce qui rend leur étude difficile. La levure Saccharomyces cerevisiae dont on peut modifier à loisir le génome mitochondrial permet de s’affranchir de cette hétérogénéité génétique (appelée hétéroplasmie). De plus, grâce à sa bonne capacité fermentaire, elle est capable de survivre à l’inactivation du système OXPHOS.J’ai au cours de ma thèse exploité ces caractéristiques pour mieux définir les conséquences sur l’ATP synthase de cinq mutations du gène ATP6 identifiées chez des patients : m.8993T>G, m.9191T>C, m.8969G>A, m.8909T>C, et m.9166T>C. Le pouvoir pathogène des trois premières a été établi. Les deux dernières sont des nouveaux variants de l’ADN mitochondrial. Via l’identification de suppresseurs intragéniques, et à la lumière de structures à haute résolution de l’ATP synthase décrites récemment, j’ai pu définir les bases moléculaires des mécanismes pathogènes induits par les mutations m.8993T>G, m.9191T>C, et m.8969G>A. Le variant m.8909T>C a été identifié en combinaison avec une mutation pathogène bien connue dans un ARN de transfert (m.3243A>G). Nous avons trouvé qu’un équivalent de cette nouvelle mutation a en levure des effets délétères sur l’assemblage ou la stabilité de la sous-unité a comparables à ceux induits par des mutations du gène ATP6 (m.8993T>C, m.9176T>C) dont le pouvoir pathogène a été établi, et qu’elle a donc potentiellement la capacité d’affecter seule la santé humaine. Mes études en levure sont cohérentes avec des études ayant conclu récemment à la pathogénicité du variant m.9166T>C et permettent de mieux comprendre comment il impacte l’ATP synthase.J’ai identifié un mécanisme de suppression actif sur des modèles levure de mutations pathogènes de la sous-unité a. Il implique le transporteur des oxodicarboxylates (Odc1) localisé dans la membrane mitochondriale interne. J’ai trouvé que la surexpression d’Odc1 permet une plus grande activité du cycle de Krebs (ou TCA). Ce cycle intervient dans l’oxydation de substrats organiques dont les équivalents réducteurs sont ensuite transférés à l’oxygène par la chaîne respiratoire. Il tourne à bas régime dans les mutants de l’ATP synthase dont l’activité canal à protons est altérée. La suppression-Odc1 dépendante entraîne un découplage partiel de la membrane interne, de sorte que le cycle TCA est stimulé malgré le défaut en ATP synthase. Cet effet permet une plus grande production d’ATP via la phosphorylation d’ADP couplée directement à une des réactions du cycle de Krebs. Ces résultats ouvrent des perspectives intéressantes pour le traitement des maladies associées à des altérations de l’ATP synthase, et possiblement d’autres désordres métaboliques. Cette étude apporte de plus un éclairage nouveau sur le contrôle de la biogenèse du complexe IV par l’ATP synthase
By definition, mitochondrial diseases result from a defect in the process of oxidative phosphorylation (OXPHOS). This is responsible for the production of ATP, the main source of cellular energy. In this process, four multiprotein complexes (I-IV) inserted into the inner mitochondrial membrane transfer to molecular oxygen the reducing equivalents released by the oxidation of carbohydrates and fatty acids. This activity generates a proton motive force used for the synthesis of ATP from ADP and inorganic phosphate by the Complex V or ATP synthase.Diseases including NARP (Neuropathy Ataxia Retinitis Pigmentosa) and MILS (Maternally Inherited Leigh Syndrome) have been associated with mutations in the subunit a of ATP synthase. Its gene (ATP6) is in the mitochondrial genome. This genome is present in up to several thousand copies per cell. Mutations in the ATP6 gene often coexist with wild-type copies of the mitochondrial genome in patients' cells and tissues (heteroplasmy), which makes their study difficult. The yeast Saccharomyces cerevisiae, whose mitochondrial genome can be modified at will, makes it possible to overcome this genetic heterogeneity owing to its incapacity to stably maintaining heteroplasmy. In addition, thanks to its good fermentation capacity, this organism is able to survive mutations that inactivate the OXPHOS system.During my thesis, I exploited these characteristics to better define the consequences on ATP synthase of five ATP6 gene mutations identified in patients: m.8969G>A, m.9191T>C, m.8993T>G, m.8909T>C, and m.9166T>C. The pathogenicity of the first three has been established. The last two are new mitochondrial DNA variants. Through the identification of intragenic suppressors, and in the light of high-resolution structures of ATP synthase described recently, I was able to define the molecular bases of the pathogenic mechanisms induced by the m.8993T>G, m.9191T>C and m.8969G>A mutations. The m.8909T>C variant was identified in combination with a well-known pathogenic mutation in tRNALeu (m.3243A>G). We have found that an equivalent of this new mutation in yeast has deleterious effects on the assembly/stability of the subunit a comparable to those induced by mutations of the ATP6 gene (m.8993T>C, m.9176T>C) with a well-established pathogenicity, and therefore has the potential to affect human health on its own. My studies in yeast are consistent with studies that recently concluded on the pathogenicity of the m.9166T>C variant and allow to better understand how it impacts ATP synthase.I have identified an active suppressor mechanism in yeast models of pathogenic subunit a mutations. It involves the oxodicarboxylate transporter (Odc1) located in the inner mitochondrial membrane. I have found that artificially overexpressing Odc1 allows for greater Krebs cycle (or TCA) activity. This cycle is involved in the oxidation of organic substrates whose reducing equivalents are then transferred to oxygen by the respiratory chain. It runs low in ATP synthase mutants with impaired proton channel activity. The Odc1-dependent suppressor activity results from a partial uncoupling of the inner membrane so that the TCA cycle is stimulated despite the presence of defect in ATP synthase. This effect allows a greater production of ATP via ADP phosphorylation coupled with one of the reactions of the Krebs cycle. These results open interesting perspectives for the treatment of diseases associated with alterations in ATP synthase, and possibly other metabolic disorders. This study also sheds new light on the control of complex IV biogenesis by ATP synthase

Mitochondrial respiratory chain deficiency and increased oxidative stress have been closely associated with major age-associated neurodegenerative diseases. I hypothesized that mitochondrial oxidative phosphorylation defects or elevated oxidative stress, which could arise in a stochastic manner during our normal aging process, might modulate the formation of protein aggregates or production of misfolded proteins, contributing to the initiation of these diseases. To test this hypothesis, we (i) have developed and characterized mouse and cellular models of Alzheimer's and Huntington's diseases expressing aggregate-prone pathogenic proteins, beta-amyloid and mutant huntingtin (Chapters 1 and 2), (ii) have developed mouse models that exhibit neuron-specific defects in mitochondrial oxidative phosphorylation (Chapters 2 and 3), and (iii) have evaluated the alterations in the amount of aggregate loads upon genetic and pharmacological manipulations of mitochondrial oxidative phosphorylation activities (Chapters 1 and 2). The evaluation of the impacts of mitochondrial defects on the amount of huntingtin aggregates has revealed that a defect in complex III promotes the accumulation of huntingtin aggregates via the impairment of proteasome activity (Chapter 1). On the other hand, ablation of complex IV activity in a subset of postmitotic neurons revealed that complex IV deficiency does not promote either oxidative stress or the deposition of amyloid plaques in a mouse model of Alzheimer's disease, questioning the mitochondrial origin of Alzheimer's disease (Chapter 2). However, as shown previously, the tight correlation between oxidative stress and accumulation of amyloid plaques was found. Chapter 3 involved the generation of an improved mouse model, in which mitochondrial defects can be induced in a subset of forebrain neurons (cortex, hippocampus, and striatum) in a doxycycline-dependent manner. This system relies on the regulated expression of a mitochondria-targeted restriction enzyme, PstI, which digests mitochondrial DNA and thereby impairs the activity of oxidative phosphorylation. In conclusion, our studies highlighted the disease-specific complex pathways that may modulate the accumulation of misfolded proteins during aging. Future studies employing the newly-developed mouse model may reveal a contribution of age-associated global defects of oxidative phosphorylation to oxidative stress and neurodegenerative diseases.

La F1FO-ATP synthase mitochondriale produit la majorité de l’énergie cellulaire chezles eucaryotes aérobes sous forme d’ATP par le processus des oxydations phosphorylantes.Chez la plupart des espèces, cette enzyme possède une origine génétique double, nucléaire etmitochondriale. Dans la première partie de ce travail, je décris la construction de modèles delevure de mutations du gène mitochondrial ATP6 de l’ATP synthase découvertes chez despatients atteints de maladies neurologiques (9185T>C and 9191T>C) ou dans des tumeurs(8716A>G, 8914C>A, 8932C>T, 8953A>G and 9131T>C). Le gène ATP6 code une sousunitéessentielle (a/6) du domaine FO de l’ATP synthase. J’ai trouvé que la mutation 9185T>Cn’affecte pas l’assemblage de l’ATP synthase, mais conduit à une diminution de la vitesse desynthèse d’ATP d’environ 30%. La mutation 9191T>C empêche presque entièrementl’incorporation de la sous-unité a/6 dans l’ATP synthase. Les cinq mutations identifiées dansles tumeurs ont un effet modeste à nul, indiquant que ces mutations ne favorisent pas latumorigenèse en affectant le processus énergétique mitochondrial, comme évoquéprécédemment. J’ai ensuite étudié la régulation de la synthèse des sous-unités a/6 et 9 dans lesmitochondries de levures. La sous-unité 9 est présente sous la forme d’un anneau de 10 copiesqui interagit avec la sous-unité 6. Durant la catalyse, la rotation de cet anneau provoque deschangements conformationnels favorisant la synthèse d’ATP dans le secteur F1 de l’ATPsynthase. Je montre que la synthèse de ces protéines est couplée à leur assemblage, demanière à ce qu’elles soient produites dans une stoechiométrie adéquate et pour éviterl’accumulation d’intermédiaires d’ATP synthase potentiellement délétères
Mitochondrial F1FO-ATP synthase produces most of the cellular energy in aerobiceukaryotes under the form of ATP in the process of oxidative phosphorylation. This enzymehas in most species a double genetic origin, nuclear and mitochondrial. In the first part of thiswork, I describe the construction of yeast models of ATP synthase mutations in themitochondrial ATP6 gene, that have been found in patients presenting with neurologicaldisorders (9185T>C and 9191T>C) and in tumors (8716A>G, 8914C>A, 8932C>T,8953A>G and 9131T>C). The ATP6 gene encodes an essential subunit (called a/6) of theATP synthase proton-translocating domain (FO). The 9185T>C mutation had no effect on theassembly of ATP synthase, but reduces the rate of ATP synthesis by 30%. The 9191T>Cmutation almost completely prevented incorporation of the subunit a/6 into the ATP synthase.The five mutations found in tumors had modest, if at all, effect, indicating that thesemutations probably do not favor tumorigenesis, as was hypothesized. In the second part of mythesis, I studied the regulation of synthesis of subunits a/6 and 9 in yeast mitochondria. Thesubunit 9 is present in 10 copies forming a ring that interacts with subunit 6. Protonmovements through the FO induce the rotation of the subunit 9-ring, which results inconformational changes that promote ATP synthesis in the catalytic sector (F1) of ATPsynthase. I discovered mechanisms that enable the coupling of the synthesis of these proteinsto their assembly, as a means to ensure the production of subunits 6 and 9 in the rightstoichiometry and to avoid accumulation of potentially harmful assembly intermediates of theATP synthase

Mitochondria are highly dynamic organelles and their function is crucial for the maintenance of cellular homeostasis. Alterations in mitochondrial homeostasis has emerged as a hallmark of several diseases, like cancer and genetic disorders. Mitochondrial fitness and its energetic state can impact cell viability and proliferation, and its modulation via pharmacological tools can be used to treat several mitochondria-related diseases. The potassium channel Kv1.3 is a voltage-gated potassium channel emerged as a novel oncological target. It is overexpressed in several tissues compared to normal ones, enhance cell proliferation, cell migration and metastasis. Direct inhibition of Kv1.3 using its known membrane-permeant inhibitor PAP-1 alters mitochondrial function and leads to reactive oxygen species (ROS)-mediated death. We generated two classes of PAP-1 derivatives in order to improve its effect. The first class comprises the mitochondria-targeted compounds PAPTP and PCARBTP. These molecules have a positively charged triphenyl-phosphonium group linked to the PAP-1 structure, so they can reach mitochondria in a more efficient way and can be used at lower concentrations compared to their precursor. We have demonstrated that these drugs selectively kill cancer cells in vitro, ex vivo and in vivo. The mechanism of selectivity involves both the overexpression of Kv1.3 and the high basal ROS level, present mainly in cancer cells. On the other hand, low, sub-lethal concentrations of PAPTP and PCARBTP cause a mild increase in mitochondrial ROS production, favoring cell survival and proliferation through the modulation of mitochondrial homeostasis. The second class of PAP-1 derivatives comprises the more soluble compounds PEGME and PTGME. Recapitulating the molecular mechanism of the mitochondria-targeted compounds, these more soluble derivatives act in vitro, ex vivo and in vivo. Moreover, PEGME and PTGME inhibited respiratory chain complex I (CI). To clarify this aspect, we revealed for the first time that mitoKv1.3 likely localizes in proximity of CI in the IMM, at the level of the supercomplexes. To detect what else the new mitochondria-targeted derivatives of PAP-1 make inside cancer cells at sub-lethal concentrations, we investigated the effects of mitoKv1.3 inhibition on Wnt signaling. We demonstrated that the reduction of mitochondrial ATP due to the use of mitochondria-affecting drugs or to genetic dysfunction due to complex III (CIII) deficiency, decreases calcium uptake into the endoplasmic reticulum (ER), leading to ER stress and to impaired Wnt signaling. Importantly, both the recovery of ATP level or the inhibition of ER stress restored Wnt activity downregulated by mitochondria-affected compounds. This research revealed for the first time an unexpected mechanism related to the control of Wnt signaling by mitochondrial ATP, opening a new possibility to use compounds affecting mitochondrial homeostasis to fight tumors, or to use mitochondrial ATP-increasing drugs to ameliorate patients’ symptoms. In the context of genetic dysfunction, we focused also on the use of the bacterial redox cycler to ameliorate CIII related disease. This redox cycler can mimic CIII activity, by accepting electrons from ubiquinol and reducing cytochrome c in vitro. We observed that sub-lethal doses of the drug recovers mitochondrial function both by increasing mitochondrial ATP production and by promoting mitohormesis, in vitro in CIII deficient cells from patients with CIII disease. Importantly, this redox cycler can increase also in vivo ATP production, and rescue respiration rate and locomotor ability in flies and zebrafish models of CIII deficiency. These results strongly suggest that the application of this drug in the sublethal concentration range might be a promising therapeutic tool against complex III diseases.

Thesis (Ph. D.)--University of Oregon, 2004.
Typescript. Includes vita and abstract. Includes bibliographical references (leaves 113-119). Also available for download via the World Wide Web; free to University of Oregon users.

Mitochondrial dysfunction occurs in patients with mitochondrial disease, in neurodegenerative conditions and as part of the ageing process. It affects predominantly tissues with high metabolic requirements such as central nervous system and skeletal muscle. In patients with mitochondrial disease, both mitochondrial and nuclear genetic defects commonly cause a biochemical defect in muscle. However, due to the multi-copy nature of mitochondrial DNA, muscle displays a mosaic pattern of deficiency when the mitochondrial genome is affected. This particular pattern makes these defects challenging to quantify. Current standard methods to diagnose and investigate mitochondrial disease in affected tissues present several limitations. Biochemical studies are only suitable for cases with a high proportion of cells with mitochondrial respiratory chain deficiency. Moreover, histochemical assays only provide qualitative assessment of complex II and IV activities and are not capable of evaluating other complexes, such as complex I - the commonest affected respiratory complex in mitochondrial pathology. This project aimed therefore at developing a novel assay to accurately quantify mitochondrial dysfunction in human skeletal muscle. Once optimised, this assay was further used to explore: the mechanisms underlying mitochondrial pathology, its potential in helping the current diagnostic setting, as well as its potential to assess the effectiveness of therapeutic approaches aimed at treating mitochondrial dysfunction. This work described the development and validation of a novel quadruple immunofluorescent technique. This assay quantifies accurately key subunits of respiratory complexes I and IV together with mitochondrial mass, using a single 10μm section. The additional labelling of a cell membrane marker allows semi-automatic and computer-based sampling of large numbers of individual muscle fibres. Using this technique, this study characterised a variety of mitochondrial and nuclear genetic defects and demonstrated that specific genotypes exhibit distinct biochemical signatures in muscle. In patients with suspected mitochondrial disease, this assay provided clues on the possible genetic causes. Furthermore, this novel assay evaluated the effect of an endurance exercise program in patients with mitochondrial myopathy and was able to detect subtle changes in respiratory complexes levels.

Thesis (MScMedSc)--Stellenbosch University, 2013.
ENGLISH ABSTRACT: INTRODUCTION: The mechanism behind obesity-related cardiomyopathies is at present not completely known, however, cardiac insulin resistance has been implicated as one of the main arbitrators of obesity-related cardiovascular disease. A few studies have associated perturbations in the insulin-mediated PI3K/PKB/Akt pathway in mediating this insulin resistance. Moreover, this pathway has been shown to regulate myocardial apoptosis, which in turn has been implicated in a number of cardiovascular diseases. Currently, few studies have compared the early onset and advanced effects of obesity on the heart. AIMS: To compare the early and advanced stages of obesity in terms of myocardial (i) PI3K/PKB/Akt signalling, (ii) apoptotic signalling and (iii) mitochondrial integrity. Furthermore, we aim to assess the cardiac mitochondrial (i) PI3K/PKB/Akt signalling, (ii) apoptotic signalling and (iii) integrity during the advanced stages of obesity. METHODS: Male Wistar rats were randomly assigned to either a control or diet-induced obesity (DIO) group. Controls were fed a standard rat chow diet and the DIO group fed a high caloric diet (standard rat chow supplemented with sucrose and condensed milk). The diets were implemented for either 8 or 20 weeks and thereafter, the body weight, intra-peritoneal fat mass, and fasting blood glucose and insulin levels (including intra-peritoneal glucose tolerance tests (IPGTTs)) were determined. Freeze-clamped hearts from both groups were subjected to cytosolic western blot analysis for PI3K p85 subunit, PKB/Akt, GSK-3α/β, Bad, Bax and Bcl-2. A fraction of each heart was also subjected to WB analysis of the mitochondrial electron transport chain (ETC) complexes (I-V). Thereafter, the above mentioned proteins were also probed for in mitochondria isolated from the 20 weeks group after administering insulin and exposing the hearts to ischemia. Oxidative phosphorylation (OXPHOS) capacity analysis was then conducted on mitochondria isolated from 20 weeks DIO and control groups and thereafter a citrate synthase (CS) activity assay was performed on these mitochondria. RESULTS: After the 8 and 20 weeks diet, the DIOs had significantly increased intra-peritoneal fat mass, fasting plasma glucose and insulin levels, compared to their controls. Cytosolic WB analysis: The tp85, pp85 and pPKB/Akt levels were significantly higher in the DIOs in comparison to the controls after 8 weeks of diet. Furthermore, pBad and Bax expression were significantly elevated in these animals. After 20 weeks of diet, the DIOs had significantly decreased pp85, tPKB/Akt and pPKB/Akt levels. The tBad was significantly elevated, while the Bad phosphorylated over total expression (P/T) ratio was significantly decreased, in these animals. CS activity assay: CS activity was significantly decreased in the DIOs, versus the controls, at 20 weeks. Mitochondrial ETC WB analysis: The subunit expression in complexes I-III and V did not differ significantly after 8 weeks however, the expression was significantly lower in complexes I and II after 20 weeks. Interestingly, the complexes III and V expression was significantly elevated. Mitochondrial OXPHOS analysis: The ADP/O ratio with (1) glutamate or (2) palmitoyl-L- carnitine as substrate, showed a significant decrease in the DIOs at 20 weeks. Mitochondrial WB analysis: The pp85 subunit was significantly elevated in the control and DIO groups, exposed to insulin and ischemia, in comparison to the untreated controls. The Bcl-2 levels were significantly decreased in the insulin and ischemia DIOs, when matched against the untreated DIOs. The tBad expression did not differ significantly between the insulin and untreated controls, while the tBad was significantly augmented in the ischemia controls versus untreated controls. All significant differences were taken as p<0.05. CONCLUSION: The results indicate that the initial stage of diet-induced obesity is associated with cardioprotection as there is augmented PI3K/PKB/Akt pathway signalling and a decrease in apoptotic markers. In contrast, during the advanced stages of obesity a decreased activity in PI3K/PKB/Akt pathway is associated with myocardial apoptosis and decreased mitochondrial function and integrity.
AFRIKAANSE OPSOMMING: INLEIDING: Die meganisme verantwoordelik vir vetsug-verwante kardiomiopatieë is huidiglik nie bekend nie maar kardiale insulienweerstandigheid word geïmpliseer as een van die hoof bemiddelaars van vetsug-verwante hartsiektes. Verskeie studies het versteurings in die insulien-gemediëerde PI3K/PKB/Akt pad geassosieer met die bevordering van hierdie insulienweerstandigheid. Daarbenewens is dit getoon dat hierdie pad betrokke is in die regulering van miokardiale apoptose, wat op sy beurt geïmpliseer is in 'n aantal kardiovaskulêre siektes. Daar is tans min studies beskikbaar wat die vroeë en laat gevolge van obesiteit op die hart vergelyk. DOELWITTE: Om die vroeë en gevorderde stadiums van vetsug te vergelyk in terme van miokardiale (i) PI3K/PKB/Akt seintransduksie, (ii) apoptotiese seintransduksie en (iii) mitokondriale integriteit. Verder, het die studie ten doel om die kardiale mitokondriale (i) PI3K/PKB/Akt en (ii) apoptotiese seintransduksie en (iii) integriteit in die gevorderde stadiums van vetsug te bepaal. METODES: Manlike Wistar rotte is ewekansig toegewys aan óf 'n kontrole of dieet-geïnduseerde vetsug (DIO) groep. Kontroles is met 'n normale rotkos dieet en die DIO groep met 'n hoë kalorie dieet (normale rotkos aangevul met sukrose en kondensmelk) gevoed. Die dieet is vir 8 of 20 weke volgehou en daarna was die liggaamsgewig, intra-peritoneale vet massa, en vastende bloed glukose en insulien vlakke (insluitende intra-peritoneale glukose toleransie toets (IPGTT`s)) bepaal. Gevriesklampte harte van beide groepe is onderwerp aan sitosoliese WB-analise vir die PI3K p85 subeenheid, PKB / Akt, GSK-3α/β, Bad, Bax en Bcl-2. `n Fraksie van hierdie harte is ook onderwerp aan westerse klad analise (WK-analise) van die mitokondriale elektron vervoer ketting (EVK) komplekse (I-V). Daarna is bogenoemde proteïene ondersoek in mitokondrieë geïsoleer uit die 20 weke groep ná die toediening van insulien en die blootstelling van die harte aan iskemie. Die oksigraaf mitokondriale oksidatiewe fosforilering (OXPHOS) kapasiteit analise is dan op mitokondrieë van 20 weke DIO en kontrole groepe uitgevoer en daarna is 'n sitraatsintase (SS) aktiwiteitstoets gedoen. RESULTATE: Na die 8 en 20 weke dieet, het die intra-peritoneale vet massa, vastende plasma glukose en insulien vlakke in die DIOs aansienlik toegeneem, in vergelyking met hul kontroles. Sitosoliese WK-analise: Die tp85, pp85 en pPKB/Akt vlakke was beduidend hoër in die DIOs in vergelyking met die kontroles, na 8 weke van die dieet. Verder is die pBad en Bax vlakke beduidend verhoog in hierdie diere. Na 20 weke van die dieet, het die pp85, tPKB/Akt en pPKB/Akt vlakke beduidend afgeneem in die DIOs, in vergelyking met die kontroles. Die tBad was beduidend verhoog, terwyl die Bad verhouding van gefosforileerde oor die totale proteïen uitdrukking (P/T)-verhouding) beduidend verminder het in hierdie diere. SS aktiwiteitstoets: SS aktiwiteit is beduidend verminder in die DIOs, teenoor die kontroles, op 20 weke. Mitokondriale EVK WK-analise: Die subeenheid uitdrukking in komplekse I-III en V was nie beduidend verskillend na 8 weke nie. Na 20 weke egter, was die uitdrukking aansienlik laer in komplekse I en II. Interessant genoeg, is die uitdrukking aansienlik verhoog in komplekse III en V. Mitokondriale OXPHOS analise: Die ADP/O verhouding met (1) glutamaat of (2) palmitiel-L-karnitien as substraat, het beduidend afgeneem in die DIOs teen 20 weke. Mitokondriale WK-analise: Die pp85 subeenheid was beduidend verhoog in die kontrole en DIO groepe, blootgestel aan insulien en iskemie, in vergelyking met die onbehandelde kontroles. Die Bcl-2 vlakke was beduidend verminder in die insulien en isgemie DIOs, in vergelyking met onbehandelde DIOs. Die tBad uitdrukking het nie beduidend verskil tussen die insulien en onbehandelde kontroles nie, terwyl die tBad beduidend verhoog was in die isgemie kontroles versus onbehandelde kontroles. Alle beduidende verskille is geneem as p<0.05. GEVOLGTREKKING: Die resultate dui daarop dat die eerste fase van dieet-geïnduseerde obesiteit geassosieer is met kardiale beskerming want `n toename in PI3K/PKB/Akt seintransduksie en 'n afname in apoptotiese merkers is waargeneem. In teenstelling, in die gevorderde stadium van vetsug is daar 'n afname in aktiwiteit in die PI3K/PKB/Akt pad wat verband hou met verhoogde miokardiale apoptose en verminderde mitokondriale funksie en integriteit.

The scope of my thesis was the identification of genes responsible for: 1) an adult-onset neurological syndrome, with leukodystrophy and motor-neuron disease, in two half-siblings; 2) an infantile hypertrophic cardiomyopathy with lactic acidosis and mitochondrial respiratory chain defects. Since all genetic screenings performed on the basis of clinical manifestations did not provide a diagnosis for these patients, we carried out whole exome sequencing. My work contributed to the publications of three papers. In the second chapter of this thesis, there is the article concerning the identification and characterization of the first GFAP-ε mutation,causing an adult form of Alexander disease in two affected siblings. The male presented a severe motor-neuron disease whereas his sister showed a mild movement disorder with cognitive impairment. In addition to the GFAP-ε mutation, we found a variant in HDAC6 on chromosome X, present only in the male patient; HDAC6 is a candidate MND susceptibility gene and the identified missense variant is probably responsible for his different phenotype. Cellular models were used to experimentally prove the altered functionality of mutant GFAP-ε and HDAC6. The third chapter contains the paper reporting the first mutations in MTO1, responsible for a mitochondrial disorder associated with hypertrophic cardiomyopathy, lactic acidosis and mitochondrial respiratory chain defects. Then chapter four consists of the article where we described and characterized news MTO1 mutations found in other patients. In both articles characterization of the identified mutations and validation of their deleterious effects were assessed in patients’ specimens (fibroblasts) and in yeast Saccharomyces cerevisiae.

Thesis (MTech (Medical Technology))--Cape Technikon, 1996.
Haematological defects observed in clonal deletions of mtDNA and the inhibition of mitochondrial function by benzene and chloramphenicol, suggest a role for mtDNA in the pathogenesis of radiation - induced preleukaemia (MDS). The fact that leukaemia cells contain abnormal mitochondria and abnormally structured mtDNA, makes it reasonable to assume mtDNA mutations could be central to the pathogenesis of both MDS and leukaemia. It was decided to examine MDS patients for the presence of mtDNA length mutations (dimers and cocantameres). Such topological forms have already been reported in the literature in association with human leukaemia. These steric considerations suggest that mtDNA dimers are probably non-functional due to supercoiling. Thus, it was felt that a progressive accumulation of non-functional dimers in the haematopoietic compartment could account for many of the clinical features associated with MDS. Transmission electron microscopy was used to examine haematopoietic mtDNA in the bone marrow of six patients with MDS. Abnormal mtDNA dimer formation was found in all instances. The proportional number of these dimers were found to roughly correlate with the Myeloid/ Erythroid cell ratio in the bone marrow, and it appeared likely that the dimers were generated in the myeloid compartment during early MDS.

Inherited metabolic diseases are genetic disorders caused by the alteration of a specific metabolic reaction. We focused on the study of the genetic bases of two main types of metabolic disorders, mitochondrial diseases and urea cycle defects. In this work, we report the characterization of novel genes that are potentially involved in mitochondrial respiratory chain defects: we identified three novel COX-assembly genes required for Cytochrome c Oxidase (COX) biogenesis and two novel genes involved in coenzyme Q biosynthesis.These genes represent new candidates to be screened in patients with isolated COX defect or primary coenzyme Q deficiency, without mutations in other known genes. We then studied a family affected by coenzyme Q deficiency and found a homozygous mutation in the COQ2 gene, this is the first report of a mutation in a ubiquinone biosynthetic gene. Moreover, we developed a C.elegans model of COX defect in order to study the pathogenesis of this disease. Finally, we performed a mutational screening in a cohort of patients affected by argininosuccininc aciduria and developed a functional complementation assay using S.cerevisiae strain to prove the pathogenesis of the novel mutations; we also established some genotype-phenotype correlations.

Les maladies mitochondriales regroupent un ensemble de pathologies liées à un déficit de la chaînerespiratoire mitochondriale. Au laboratoire, nous focalisons notre intérêt sur les mitochondriopathies liées à undéfaut de stabilité de l’ADN mitochondrial (ADNmt), qui se traduit par des délétions multiples et/ou unedéplétion (diminution du nombre de copies). Ces pathologies sont caractérisées par une importantehétérogénéité clinique et génétique et sont secondaires à des mutations dans des gènes nucléaires codantpour des protéines impliquées dans le maintien de l’ADNmt. De nos jours, la recherche des gènesresponsables d’instabilité de l’ADNmt s’avère négative chez plus de 70% des malades, d’où un grand intérêtpour améliorer les techniques d’identification des mutations et la recherche de nouveaux gènes impliquésdans ces pathologies
Non communiqué

Існує безліч нозологічних одиниць спадкових хвороб, які повністю ще не ідентифіковані. Вони маскуються під відомі хвороби, їх лікують, як відомі хвороби, але ефект не настає. Такими захворюваннями донедавна були мітохондріальні хвороби. У 1981 році в лабораторії Медичного дослідницького центру у Кембриджі науковою групою Сенгера Ф. була розшифрована нуклеотидна структура ДНК мітохондрій (мтДНК) людини, пізніше названою хромосомою 25 або М-хромосомою. При цитуванні документа, використовуйте посилання http://essuir.sumdu.edu.ua/handle/123456789/5893

Thesis (MTech(Medical Technology))--Cape Technikon, Cape Town, 1996
Haematol.ogical defects observed in cl.onal deletions of mtDNA and the inhibition of mitochondrial function by benzene and chloramphenicol., suggest a role for mtDNA in the pathogenesis of radiation - induced preleukaemia (MDS). The fact that leukaemia cell.s contain abnormal mitochondria and abnormally structured mtDNA, makes it reasonable to assume mtDNA mutations could be central to the pathogenesis of both MDS and l.eukaemia. It was decided to examine MDS patients for the presence of mtDNA length mutations (dimers and cocantameres). Such topol.ogical forms have already been reported in the literature in association with human leukaemia. These steric considerations suggest that mtDNA dimers are probably non-functional due to supercoil.ing. Thus, it was felt that a progressive accumulation of non-functional dimers in the haematopoietic compartment could account for many of the cl.inical. features associated with MDS. Transmission electron microscopy was used to examine haematopoietic mtDNA in the bone marrow of six patients with MDS. Abnormal mtDNA dimer formation was found in al.l instances. The proportional. number of these dimers were found to roughly correlate with the Myeloid/ Erythroid cell. ratio in the bone marrow, and it appeared likely that the dimers were generated in the myeloid compartment during early MDS. Control.l.ed radiation studies were performed on 20 wistar rats in an attempt to elucidate the approximate time when abnormal mtDNA dimer formation occurred, fol.l.owing fractionated gamma or gamma-neutron irradiation. Gamma-irradiated rats demonstrated abnormal mtDNA dimer formation at the time hypoplastic marrow recovery was first observed.

BACKGROUND: Mitochondrial complex I deficiency often leads to a devastating neurodegenerative disorder of childhood. In most cases, the underlying genetic defect is unknown. Recessive nuclear gene mutations, rather than mitochondrial DNA mutations, account for the majority of cases. AIM: Our aim was to identify the genetic basis of complex I deficiency in 34 patients with isolated complex I deficiency, by studying six of the 39 nuclear encoded complex I subunit genes (NDUFV1, NDUFS1, NDUFS2, NDUFS4, NDUFS7 and NDUFS8). These genes have been conserved throughout evolution and carry out essential aspects of complex I function. METHODS: RNA was extracted from patient fibroblasts and cDNA made by reverse transcription. Overlapping amplicons that together spanned the entire coding area of each gene were amplified by PCR. The genes were screened for mutations using denaturing High Performance Liquid Chromatography (dHPLC). Patient samples with abnormal dHPLC profiles underwent direct DNA sequencing. RESULTS: Novel mutations were identified in six of 34 (18%) patients with isolated complex I deficiency. Five patients had two mutations identified and one patient had a single mutation in NDUFS4 identified. All patients with mutations had a progressive encephalopathy and five out of six had Leigh syndrome or Leigh like syndrome. Mutations were found in three nuclear encoded subunit genes, NDUFV1, NDUFS2 and NDUFS4. Three novel NDUFV1 mutations were identified (R386H, K111E and P252R). The R386H mutation was found in two apparently unrelated patients. Four novel NDUFS2 mutations were identified (R221X, M292T, R333Q and IVS9+4A<G). The novel NDUFS4 mutation c.221delC was found in two patients - one in homozygous form and the other heterozygous. Specific genotype and phenotype correlations were not identified. CONCLUSIONS: Nuclear encoded complex I subunit gene mutations are an important contributor to the aetiology of isolated complex I deficiency in childhood. Screening of these genes is an essential part of the investigation of complex I deficiency.

Huntington's disease (HD) is an autosomal-dominant inherited neurodegenerative disorder, characterized by progressive behavioral, motor and cognitive deficits (HDCRG, 1993; Ross & Margolis, 2001). The predominant neuropathological hallmark of HD is the selective loss of medium spiny neurons within the striatum that extends to other brain regions with the progression of the disease (Martin & Gusella, 1986). Although mutant huntingtin (mHtt) represents a key factor in the pathogenesis of the disease, the molecular mechanisms underlying the preferential vulnerability of the striatum to mHtt toxicity remain unclear. Compelling evidence suggest that mitochondrial defects may play a crucial role in the disease, however, it is still debated whether mitochondrial dysfunction represents just an epiphenomenon of the cellular degeneration or it has an actual pathogenic role. In this Thesis, the general purpose was to identify possible mitochondrial impaired mechanisms and to investigate their role in the selective striatal vulnerability of HD, in order to provide important insights for the development of new therapeutic strategies. The first aim was to clarify whether mitochondrial injuries perpetrate the dopaminergic neurotoxicity in HD striatal degeneration. Since Cdk5 has been proposed as a critical regulator of mitochondrial fission (Meuer et al., 2007) and as a deleterious player in the striatal vulnerability upon dopamine signalling (Paoletti et al., 2008), we hypothesize a new detrimental role for Cdk5 in HD pathology by mediating dopaminergic neurotoxicity through deregulation of mitochondrial dynamic processes. On the other hand, several studies have proposed mitochondrial Ca mishandling as a component of Ca controversial and a conclusive cause remains uncertain. We propose that the propensity of mitochondria to undergo fragmentation in HD could result in the disruption of ER-mitochondria contacts, which are essential for the proper buffering of Ca whether defects in ER-mitochondria associated membranes could be responsible for the alteration of mitochondrial Ca handling in HD. dyshomeostasis in HD. However, the results appear 2+ 2+ by mitochondria. Therefore, the second aim of this study was to investigate.
La enfermedad de Huntington (EH) es un trastorno neurodegenerativo de herencia autosómica dominante, causado por la expansión del trinucleótido CAG en el gen IT15 que codifica para la proteína huntingtina (htt) (HDCRG, 1993). Los pacientes con EH desarrollan alteraciones neurológicas tales como trastornos psiquiátricos, motores y cognitivos (Ross & Margolis, 2001). El sello neuropatológico más característico de este trastorno es la atrofia del cuerpo estriado que se extiende a otras regiones del cerebro con la progresión de la enfermedad (Martin & Gusella, 1986). Aunque la huntingtina mutada (mHtt) representa un factor clave en la patogénesis de la enfermedad, los mecanismos implicados en la selectiva degeneración estriatal todavía se desconocen. Estudios previos de nuestro grupo de investigación han demostrado que el aumento de la vulnerabilidad de las células estriatales a la mHtt tras activación dopaminérgica implica la actividad aberrante de Cdk5 (Paoletti et al., 2008). Por otra parte, estudios recientes han involucrado también Cdk5 en la regulación de la fisión mitocondrial (Meuer et al., 2007). En este escenario hemos planteado la hipótesis de que la desregulación de Cdk5 inducida por la mHtt podría contribuir a la patología estriatal en la EH. Por ello el primer objetivo de esta Tesis ha sido definir si el aumento de la actividad de Cdk5 inducida por la mHtt incrementa la vulnerabilidad estriatal a insultos excitotóxicos mediante la alteración de la dinámica mitocondrial. Por otra parte, estudios recientes identifican una defectuosa captación del calcio mitocondrial como otro mecanismo responsable de la patogénesis y progresión de la EH (Giacomello et al., 2011). Un excesiva fragmentación mitocondrial podría alterar la dispersión de los orgánulos en el espacio intracelular alterando su capacidad de captación del Ca endoplásmico (RE). Por lo tanto, el segundo objetivo de esta Tesis fue investigar el papel de los sitios de contacto entre mitocondria y RE en la alteración de la señalización del Ca y la interacción con otras membranas, tales como las del retículo 2+ que caracteriza la EH.

Thesis (MSc)--Stellenbosch University, 2014.
ENGLISH ABSTRACT: Diuraphis noxia (Kurdjumov, Hemiptera, Aphididae) commonly known as the Russian wheat aphid (RWA), is a small phloem-feeding pest of wheat (Triticum aestivum L). Virulent D. noxia biotypes that are able to feed on previously resistant wheat cultivars continue to develop and therefor the identification of factors contributing to virulence is vital. Since energy metabolism plays a key role in the survival of organisms, genes and processes involved in the production and regulation of energy may be key contributors to virulence: such as mitochondria and the NAD+/NADH that reflects the health and metabolic activities of a cell. The involvement of carotenoids in the generation of energy through a photosynthesis-like process may be an important factor, as well as its contribution to aphid immunity through mediation of oxidative stress. The complete mitochondrial genome of global Diuraphis noxia populations was characterised using Next Generation sequencing, and was found to be 15 721bp in size and consisting of 38 genes typically found within most insects. Single nucleotide polymorphism (SNP) analyses of the genomes of nine populations revealed 125 SNPs in the protein coding genes with the majority of the SNPs occurring in the ND genes, and the least in the ND4L gene. Low SNP variant frequency was found for the atp6 and atp8 genes, which differed from other reports in the Hemiptera. Variable ND5 expression levels were observed among the biotypes, although no correlation was apparent between ND5 expression and the virulence associated with each biotype. Whereas atp6 transcription was higher in the highly virulent biotype (SAM) under normal and stressful conditions in comparison to the least virulent biotype (SA1). A significantly higher NAD+/NADH ratio was also observed for the SAM biotype under stressful conditions in comparison to the lesser virulent biotypes. UPLC-MS analysis did not reveal any lycopene or β-carotene due to low compound concentrations in the extracted samples but various hydrophobic compounds were present in different concentrations among the biotypes. The carotene desaturase expression profile revealed that SA1 had the lowest relative expression of the gene involved in carotenoid products, while SAM had the highest, under normal and stressful conditions. The results indicate that sequence conservation in mitochondrial genes are associated with key energy processes to maintain a state of homeostasis under variable conditions and that the generation of energy is a contributing factor to the virulence development of D. noxia. The results also show that carotenoids may possibly contribute to fitness of D. noxia through reactive oxygen species scavenging or the production of additional energy, but further investigation is needed for confirmation.
AFRIKAANSE OPSOMMING: Diuraphis noxia (Kurdjumov, Hemiptera, Aphididae) algemeen bekend as die Russiese koringluis (RWA), is ‘n klein floëem-voedende pes van koring (Triticum aestivum L). Virulente D. noxia biotipes wat instaat is om op voorheen bestande koring kultivars te voed gaan ontwikkel voortdurend, en daarom is die identifisering van faktore wat kan bydrae tot virulensie so belangrik. Omdat energie-metabolisme ‘n sleutelrol in die oorlewing van organismes speel, kan gene en prosesse wat by die produksie en regulering van energie betrokke is belangrike bydraers tot virulensie lewer: soos onder andere mitokondria en die NAD+/NADH-verhouding wat die gesondheid en metaboliese aktiwiteit van ‘n sel reflekteer. Die betrokkenheid van karotenoïede in die produksie van energie deur 'n fotosintese-verwante proses kan 'n belangrike faktor bydraend tot luis fiksheid wees, asook die bydra daarvan tot plantluis-immuniteit deur bemiddeling van oksidatiewe stres. Die volledige mitochondriale genoom van globale Diuraphis noxia populasies is met behulp van volgende generasie DNA volgordebepaling gekarrakteriseer, en daar is bevind dat dit 15 721 bp in grootte is en uit 38 gene bestaan wat tipies binne insekte voorkom. Enkelnukleotied- polimorfisme (SNP) ontleding van die genome van nege populasies het onthul dat daar 125 SNPs in die proteïen-koderende gene voorkom, met die meerderheid van die SNPs in die ND-gene, en die minste in die ND4L-geen. Lae SNP-frekwensies is gevind vir die atp6- en atp8- gene, wat verskil van verslae oor ander Hemiptera. Veranderlike ND5-uitdrukkingsvlakke onder die biotipes is waargeneem, alhoewel geen korrelasie duidelik was tussen ND5-uitdrukking en die virulensie geassosieer met elke biotipe nie. Die transkripsie van atp6 was hoër in die hoogs virulente biotipe (SAM) onder normale en stresvolle toestande in vergelyking met die minste virulente biotipe (SA1). ‘n Aansienlike hoër NAD+/NADH-verhouding is ook waargeneem vir die SAM-biotipe onder spanningsvolle omstandighede in vergelyking met die minder virulente biotipes. UPLC-MS-analise het geen likopeen of β-karoteen geïdentifiseer nie as gevolg van lae verbinding konsentrasies in die onttrekte monsters, maar verskeie hidrofobiese verbindings was in verskillende konsentrasies tussen die biotipes teenwoordig. Die karoteen desaturase-uitdrukkingsprofiel het aangetoon dat SA1 die laagste relatiewe uitdrukking van gene betrokke by karotenoïed produksie het, terwyl SAM die hoogste relatiewe uitdrukking onder normale en spanningsvolle omstandighede het. Die resultate van die studie dui daarop dat die volgorde bewaring in mitochondriale gene verband hou met die sleutel energie prosesse om 'n toestand van homeostase onder wisselende omstandighede te handhaaf en dat die produksie van energie 'n bydraende faktor tot die ontwikkeling van virulensie in D. noxia is. Die resultate toon ook aan dat karotenoïede moontlik kan bydra tot fiksheid van D. noxia deur reaktiewe suurstofspesies te aas of deur die produksie van addisionele energie, maar verdere ondersoeke word benodig ter bevestiging.

Le malattie mitocondriali (MD) sono un gruppo clinicamente eterogeneo di patologie dovute al difetto di funzione dei mitocondri, in particolare della catena respiratoria mitocondriale (RC) e della fosforilazione ossidativa (OXPHOS). Le funzioni mitocondriali sono sotto il controllo di due diversi genomi: DNA mitocondriale (mtDNA) e genoma nucleare (nDNA). Le forme infantili sono più spesso associate a mutazioni nel nDNA; negli ultimi anni tecnologie di sequenziamento di nuova generazione (Next Generation Sequencing-NGS) hanno permesso di identificare nuovi geni malattia; in collaborazione con altri centri abbiamo contribuito alla definizione del fenotipo associato ai nuovi geni malattia identificati. L’applicazione di tale tecnica è stata estesa anche allo studio del mtDNA: anche se sono state descritte più di 100 mutazioni e delezioni nel mtDNA in associazione a uno spettro estremamente eterogeneo di presentazioni cliniche, solo alcune di esse sono associate a sindromi cliniche ben definite nell'infanzia. Abbiamo effettuato una valutazione sistematica dei dati clinici, strumentali, metabolici e biochimici di una vasta coorte di pazienti affetti dal MD più comune nell'infanzia: la sindrome di Leigh. Abbiamo analizzato in questa popolazione la correlazione genotipo-fenotipo nei casi associati al gene nDNA e mtDNA al fine di identificare indizi diagnostici per la sindrome di Leigh correlata al mtDNA. In casi geneticamente irrisolti e vari fenotipi (sindrome di Leigh, leucodistrofia ...), abbiamo eseguito lo screening del mtDNA utilizzando tecnologie di sequenziamento di nuova generazione (NGS) al fine di valutare, con elevata precisione, mutazioni puntiformi e delezioni singole o multiple di grandi dimensioni, entrambe in omoplasma o stato eteroplasmico. Abbiamo identificato mutazioni sia nuove che note associate a fenotipo inatteso (es. MTCO3). I nostri dati definiscono e ampliano meglio lo spettro fenotipico del mtDNA-MD nell'infanzia.
Mitochondrial diseases (MD) are a clinically heterogeneous group of disorders that arise as a result of dysfunction of the mitochondrial respiratory chain (RC) and oxidative phosphorylation (OXPHOS). Mitochondrial functions are under the control of two different genomes: mitochondrial DNA (mtDNA) and nuclear genome (nDNA). Childhood phenotypes are often associated with nDNA mutations; in recent years, new-generation sequencing technologies (Next Generation Sequencing-NGS) have identified novel causative genes; in collaboration with other centers we contributed to the definition of phenotype associated with the new identified disease genes. The application of this technique has also been extended to the study of mtDNA: even if more than 100 mutations and deletions in mtDNA have been described in association with an extremely heterogeneous spectrum of clinical presentations, only a few of them are associated with well-defined clinical syndromes in childhood. We performed a systematic evaluation of clinical, instrumental, metabolic and biochemical data of a large cohort of patients affected by the most common MD in childhood: Leigh syndrome. We analyzed in this population, genotype-phenotype correlation in nDNA and mtDNA gene associated cases in order to identify diagnostic clues for mtDNA related Leigh syndrome. In genetically unresolved cases and various phenotypes (Leigh syndrome, leukodystropy..), we performed mtDNA screening using next-generation sequencing (NGS) technologies in order to assess, with high accuracy, point mutations and single or multiple large deletions, both in homoplasmic or heteroplasmic state. We identified both novel and known mutations associated to unexpected phenotype (i.e. CO3 gene). Our data better define and expand the phenotypic spectrum of mtDNA-MD in childhood.

Le système nerveux consiste en plusieurs types cellulaires quels interagissent avec eux-mêmes pour conduire des potentiels d’action du soma au travers l’axone vers la synapse. Dans les nerfs périphériques, les cellules de Schwann interagissent avec les neurones par enrouler autour l’axone et créer une gaine de myéline. Cette gaine de myéline permit une conduction rapide des potentiels d’action de nœud de Ranvier vers nœud de Ranvier, quels sont petits régions non-myélinisés de l’axone. En plus, les cellules de Schwann transportent du lactate vers le neurone et le neurone utilise ce lactate pour la production d’énergie sous la forme d’ATP. Cette production est nécessaire, parce que beaucoup de processus dans des cellules, comme la conduction des potentiels d’action, ont besoin d’ATP. Trois mécanismes sont impliqués à la production d’ATP : la glycolyse dans le cytosol et le cycle de Krebs et la chaîne de transport d'électrons dans les mitochondries. Par contre, la production d’ATP par des mitochondries résulte en la production du dérivés réactifs de l'oxygène (DRO), quels provoquent du stress oxydative. DRO peuvent être présent en plusieurs formes et ces formes différentes ont des traits spécifiques, mais tous les formes peuvent endommager la cellule. Par exemple, l’ion superoxyde est très réactif donc ils agissent extrêmement vite avec les molécules dans son environnement. Par contre, peroxyde d'hydrogène est un type de DRO quel est moins réactif, mais peut diffuser à plus longue distances et peut oxyder des cibles plus distales. Heureusement, les cellules sont équipées avec un système antioxydant compétant en consistent une groupe d’enzymes antioxydant, quels réduisent du DRO à l’eau. Si les mitochondries dysfonctionnent ou si l’équilibre entre DRO et antioxydants devient en disbalance, des neuropathies pourraient se développer, comme sclérose latérale amyotrophique (SLA), sclérose en plaques (SEP), maladie d'Alzheimer et maladie de Parkinson. Au SNP, des neuropathies périphériques comme la maladie Charcot-Marie-Tooth (CMT) peuvent se développer en raison d’excès du DRODans cette thèse, je présente un modèle élaboré pour la production d’ATP et DRO par les mitochondries in vivo. Je montrerai comment les cellules de Schwann utilisent l’effet Warburg, la transition de métabolisme de phosphorylation oxydative à glycolyse, pour la production de lactate, quel est transporté vers le neurone pour la production d’énergie. Je montrerai aussi que sans l’effet Warburg dans des cellules de Schwann, le métabolisme neuronal est détérioré menant à la production d’ATP diminué, déficits neuronales et des problèmes moteurs. Suite à l’activité nerveuse, la production d’ATP par des mitochondries augmente, mais aussi la production de DRO. Pourtant les deux productions ne sont pas en parallèle. En plus, je montre que la physiologie mitochondriale est affectée par des neuropathies. Aux souris avec défectueux mitofusin2, un modèle pour CMT2A, le contact entre le réticulum endoplasmique et les mitochondries est diminué, à côté d’un changement de morphologie et fonction mitochondriale. En plus, démyélinisation résulte dans des déficits de la production ATP et DRO, montrent une dissociation pathologique entre la production d’ATP et DRO.Pour obtenir ces résultats, des techniques d'imagerie avancées étaient utilisé pour imager les nerfs périphériques des souris transgéniques. Ces transgènes sont introduit dans les souris par injection des vecteurs virales, quels induisent l’expression des sondes fluorescents dans les cellules neuronales. Ces sondes fluorescentes étaient détectées par microscopie multiphotonique. En plus du modèle de la production d’ATP et DRO dans des nerfs périphériques, je présente un protocole pour introduire des vecteurs viraux dans le nerf sciatique
The nervous system consists of several cell types that interact with each other in order to conduct action potentials from the neuronal soma through axons to the synapse. In peripheral nerves, Schwann cells interact with neurons by wrapping around the axon and creating a myelin sheath. This myelin sheath allows for fast conduction of action potentials from node of Ranvier to node of Ranvier, which are small unmyelinated areas of the axon. In addition, Schwann cells transfer lactate to the neuron, which the axonal mitochondria use to produce energy in the form of ATP. This is necessary, because many cellular processes, such as the conduction of action potentials use ATP. The production of ATP involves three mechanisms: anaerobic glycolysis in the cytosol and the Kreb’s cycle and electron transport chain within mitochondria. However, the production of ATP by mitochondria also results in the production of reactive oxygen species (ROS), which cause cell damage. ROS can be present in several different forms and these different forms have specific properties. For example, superoxide anions are highly reactive and subsequently react rapidly with the molecules in their environment. Hydrogen peroxide on the other hand is less reactive but hence can diffuse over longer distances and react with their targets more distally. Fortunately, the cell contains a competent antioxidant system, which can reduce ROS to water. When mitochondria malfunction or when the equilibrium between ROS and antioxidants becomes in disbalance, neuropathies can develop, such as amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS), Alzheimer’s disease or Parkinson’s disease. In the PNS, peripheral neuropathies can develop such as Charcot-Marie-Tooth disease as a result from an excess of ROS.In this thesis, I will provide an elaborate model for ATP and ROS production by axonal mitochondria in vivo. I will show how Schwann cells use the Warburg effect, the shift in metabolism from oxidative phosphorylation to anaerobic glycolysis, to produce lactate, which is then transported to the neuron for energy production. I also demonstrate that without the Warburg effect in Schwann cells neuronal metabolism would be impaired, leading to impaired ATP production, neuronal deficits and motor problems. Following action potential firing, not only ATP is produced by mitochondria, but also ROS, although with independent dynamics. In addition, I show that mitochondrial physiology is affected by several neuropathologies. In mitofusin2 deficient mice, a model for CMT2A, contact between the endoplasmic reticulum and mitochondria is impaired next to affected mitochondrial morphology and function. Also demyelination causes deficits in mitochondrial ATP and ROS production, showing a pathologic decoupling between ATP and ROS.To obtain these results, advanced imaging techniques were used to image peripheral nerves of transgenic mice. These transgenes were introduced in mice via injection of viral vectors which induce expression of fluorescent probes in neuronal cells. These fluorescent probes were detected via multiphoton microscopy. Next to the model for ATP and ROS production in peripheral nerves, I provide a protocol for introducing viral vectors into mouse sciatic nerves

Les défauts de génome mitochondrial provoquent des maladies neuromusculaires, pour lequel aucun traitement efficace n'a été mis au point. La plupart des mutations mitochondriales sont hétéroplasmique, ce qui signifie que l'ADN mitochondrial (ADNmt) de type sauvage et muté coexistent dans la même cellule, et le changement de proportion entre deux types d'ADNmt pourrait rétablir les fonctions mitochondriales. Le but du projet était le développement du système pour cibler l'ARN thérapeutique dans les cellules humaines vivantes. Au cours de ma thèse j'ai synthétisé une série de nouveaux ARN anti-réplicatifs contenant modifications chimiques pour augmenter leur stabilité dans la cellule, et mis au point la nouvelle méthode de synthèse chimique des molécules d'ARN contenant cholestérol fixé par l'intermédiaire d'un pont biodégradable. Ces ARN étaient capable de pénétrer dans les cellules humains, d'être adressées dans les mitochondries et de diminuer la proportion d' ADNmt muté
Defects in mitochondrial genome cause neuromuscular diseases, for which no efficient therapy has been developed. Since most mitochondrial mutations are heteroplasmic, wild type and mutated mitochondrial DNA (mtDNA) coexist in the same cell, and the shift in proportion between two mtDNA types could restore mitochondrial functions. The aim of the project was development of carrier-free system for targeting the therapeutic mitochondrially importable RNA into living human cells. During my PhD study, I have synthesized a set of new anti-replicative RNAs containing various chemical modifications, aiming to increase their stability in the cell, and developed a new method for the chemical synthesis of RNA molecules containing cholesterol attached through a biodegradable bridge. Cholesterol containing antireplicative RNAs were characterised by efficient cellular uptake, partial colocalisation with mitochondria and ability to decrease the proportion of mutant mtDNA

Mitochondria is no longer viewed as merely a powerhouse of the cell. It is now apparentthat mitochondria play a central role in signaling, maintaining cellular homeostasis and cell fate.Mitochondrial dysfunction has been linked to many human diseases caused by cellular metabolicderegulation, such as obesity, diabetes, neurodegenerative disease, cardiovascular disease andcancer. Eukaryotic organisms have evolved an efficient way in sensing, communicating andresponding to cellular stress and regulating mitochondrial activity correspondingly through acomplex network of intercommunicating protein kinases and their downstream effectors. Thisdissertation focuses on the interplay of two of the master metabolic regulators in the cell: AMPKand PASK, and characterization of the functions of their downstream substrates: OSBP andMED13. AMPK is an energy sensing kinase that maintains energy homeostasis in the cell,whereas PASK is a nutrient sensing kinase that regulates glucose partitioning and respiration inthe cell. Both kinases play important roles in mitochondrial function and regulation, anddeficiency in either kinase has been found to associate with various human pathologies. Furthercharacterization of the cross-talk and molecular mechanisms of both kinases in controllingmitochondrial health and function may aid in the identification of new targets for treatingmetabolic diseases.

[EN] Post-transcriptional modification of the wobble uridine (U34) of a tRNA set is an evolutionary conserved process, produced by homologous proteins from the MnmA/MTU1, MnmE/GTPBP3 and MnmG/MTO1 families. Mutations in the human genes MTU1 and GTPBP3 or MTO1 produce acute infantile liver failure, and hypertrophic cardiomyopathy and lactic acidosis, respectively, which usually cause lethality in the first months of life. It is assumed that the primary cause of these diseases is the lack of the modifications introduced by the MTU1 protein in position 2 (tiol) and GTPBP3 and MTO1 proteins (taurinomethylation) in position 5 at U34 in a subgroup of mt-tRNAs. Nevertheless, the molecular mechanisms underlying these diseases (and other diseases associated with such modifications) are not clear. The reason why the typical defects of oxidative phosphorylation (due to impaired mitochondrial translation) produce such wide range of phenotypes is still unknown. Our hypothesis sustains that the mitochondria-nucleus retrograde signaling pathways triggered by the hypomodification at position 2 and 5 of U34 are different, and that each nuclear response is modulated by the genetic and epigenetic programs of cells and organisms. In this work, we have used the nematode Caenorhabditis elegans as a model organism to study the effects of inactivating the homologue proteins to MTU1, GTPBP3 and MTO1, which we have named as MTTU-1, MTCU-1 and MTCU-2, respectively. We have proved that these nuclear encoded proteins are located in mitochondria and are involved in U34 modification of mt-tRNAs. The mtcu-1 and mtcu-2 mutants show a reduction in fertility, while the mttu-1 mutant shows a reduction in fertility and a lengthening of the reproductive cycle (both phenotypes are thermosensitive). The phenotypes exhibited by the mttu-1, mtcu-1 and mtcu-2 mutants support our hypothesis, in which the mttu-1 single mutation, on the one hand, and the mtcu-1 and mtcu-2 single mutations, on the other hand, trigger different retrograde signaling pathways which produce specific nucear expression. Thus, a nuclear dependent phenotypic trait (as transcription or mt-tRNAs stability) and the expression of nuclear genes as ucp-4, hsp-6, hsp-60 and other genes involved in mitochondrial metabolism show a differential pattern in both group of mutants. hsp-6 and hsp-60 genes (UPRmt markers) are downregulated in mttu-1 single mutant, which could be related to fertility and reproductive cycle thermosensitivity. The three single mutants exhibit reduced expression of glycolysis and ß-oxidation genes (usually more drastic in the mttu-1 mutant), an induction of a glutaminolysis marker, and an induction of the ucp-4 gene, which encodes a transporter of the succinate to the mitochondria. Due to all three single mutants display a mild OXPHOS dysfunction, we propose that the observed changes in the expression of genes involved in the mitochondrial metabolism reveal a TCA cycle reprogramming aimed to compensate the reduction of acetil-CoA (coming from glycolysis and fatty acid oxidation) though the activation of anaplerotic pathways characterized by the succinate import to mitochondria by UCP-4 and the incorporation of 2-oxoglurate from glutaminolysis. We also analyze the effects of the simultanous suppression of modifications at positions 2 and 5 of U34 in C. elegans. The double mutant mtcu-2;mttu-1 displayed a severe OXPHOS dysfunction and a 5-fold higher AMP/ATP ratio, which was associated with embryonic lethality, developmental arrest in primary larval stages, penetrant sterility in adults and extended lifespan. This lifespan extension is modulated by signaling pathways which depend on AMPK (specifically on AAK-1 catalitic subunit) and steroid hormones, through DAF-9 and DAF-12 proteins. This work shows the important gene reprogramming related to mitochondrial metabolism in response to U34 hypomodification of mt-tRNAs, and shows new connexions between signaling pathways that extend lifespan.
[ES] La modificación post-transcripcional de la uridina de tambaleo (U34) de ciertos tRNAs es un proceso conservado evolutivamente, realizado por proteínas homólogas de las familias MnmA/MTU1, MnmE/GTPBP3 y MnmG/MTO1, y biológicamente relevante. De hecho, mutaciones en los genes humanos MTU1 y GTPBP3 o MTO1 causan fallo hepático infantil agudo y cardiomiopatía hipertrófica infantil, respectivamente, que producen letalidad durante los primeros meses de vida. Se asume que la causa primaria de estas enfermedades es la ausencia de las modificaciones introducidas por la proteína MTU1 en la posición 2 (tiol) y las proteínas GTPBP3 y MTO1 (taurinometil) en la posición 5 de la U34 en un grupo de mt-tRNAs. Se desconocen los mecanismos subyacentes y las razones por las que el déficit de OXPHOS resultante en todos los casos (atribuido a alteraciones de la traducción mitocondrial de proteínas) produce fenotipos tan diversos. Nuestra hipótesis es que la señalización retrógrada mitocondria-núcleo promovida por la hipomodificación de los mt-tRNAs en 2 ó 5 de la U34 es diferente y la respuesta nuclear viene modulada por el programa genético y epigenético de células y organismos. Hemos utilizado el nematodo C. elegans como modelo para estudiar los efectos producidos por la inactivación de las proteínas homólogas de MTU1, GTPBP3 y MTO1 a las que hemos denominado MTTU-1, MTCU-1 y MTCU-2. Hemos comprobado que estas proteínas, codificadas por el núcleo, son de localización mitocondrial y están implicadas en la modificación de la U34 de los mt-tRNAs. Los mutantes mtcu-1 y mtcu-2 presentan una reducción en su fertilidad y, en el caso del mutante simple mttu-1, fenotipos asociados a termosensibilidad. Los fenotipos exhibidos por los mutantes mttu-1, mtcu-1 y mtcu-2 sustentan la hipótesis de que la mutación mttu-1, y las mutaciones mtcu-1 y mtcu-2 promueven señales retrógradas diferentes que producen patrones de expresión nuclear específicos. Así, un rasgo fenotípico dependiente de genes nucleares (como lo es la transcripción y/o estabilidad de los mt-tRNAs) y la expresión de genes nucleares como ucp-4, hsp-6, hsp-60 y otros implicados en el metabolismo mitocondrial muestran un patrón diferente en los dos grupos de mutantes. Los genes hsp-6 y hsp-60 (marcadores de la UPRmt) están regulados a la baja en el mutante mttu-1. Los tres mutantes simples exhiben una reducción en la expresión de genes de la glicólisis y de la ß-oxidación de los ácidos grasos, una inducción en un marcador de glutaminolisis y una inducción en el gen ucp-4 (mayor en mttu-1) implicado en el transporte de succinato a la mitocondria. Dado que los tres mutantes simples presentan una disfunción OXPHOS relativamente suave, proponemos que los cambios de expresión en genes que modulan el metabolismo mitocondrial revelan una reprogramación del ciclo del TCA que compensa la disminución en el aporte de acetil-CoA procedente de glicólisis y oxidación de ácidos grasos con la activación de rutas anapleróticas del ciclo del TCA (importe de succinato a la mitocondria por UCP-4 y aporte de ¿-cetoglutarato procedente de la glutaminolisis). También analizamos los efectos de la anulación simultánea de las modificaciones en las posiciones 2 y 5 de la U34. El doble mutante mttu-1;mtcu-2 presenta una disfunción OXPHOS severa, con una ratio AMP/ATP 5 veces superior al control, que resulta en letalidad embrionaria, detención del desarrollo en estadios larvarios tempranos y esterilidad completa en los adultos que presentan, por otra parte, una longevidad unas dos veces superior a la cepa control. Este incremento de la longevidad está modulado por rutas de señalización que dependen de la subunidad catalítica AAK-1 (AMPK), y de hormonas esteroideas (proteínas DAF-9 y DAF-12). El trabajo muestra la importante reprogramación de genes relacionados con el metabolismo mitocondrial en respuesta a la hipomodificación de la U34 de los mt-tRNAs y
[CAT] La modificació post-transcripcional de la uridina de balanceig (U34) de certs tRNAs és un procés conservat evolutivament realitzat per proteïnes homòlogues a les de les famílies MnmA/MTU1, MnmE/GTPBP3 i MnmG/MTO1 i biològicament relevant. De fet, mutacions en els gens humans MTU1 i GTPBP3 o MTO1 causen fallada hepàtica infantil aguda i cardiomiopatia hipertròfica infantil amb acidosis làctica, respectivament, que produïxen letalitat durant els primers mesos de vida. S'assumix que la causa primària d'aquestes malalties és l'absència de les modificacions introduïdes per la proteïna MTU1 a la posició 2 (tiol) i per les proteïnes GTPBP3 i MTO1 (taurinometil) a la posició 5 de la U34 en un grup de mt-tRNAs. Es desconeixen els mecanismes subjacents en estes malalties i les raons per les quals el dèficit de la OXPHOS resultant en tots els casos (atribuït a alteracions de la traducció mitocondrial de proteïnes) produïx fenotips tan diversos. La nostra hipòtesi és que la senyalització retrògrada mitocondria-nucli promoguda per la hipomodificació dels mt-tRNAs en 2 o 5 de la U34 és diferent i la resposta nuclear en cada cas es dependent del programa genètic i epigenètic de cèl¿lules i organismes. Hem utilitzat el nematode C. elegans com a organisme model per a estudiar els efectes produïts per la inactivació de les proteïnes homòlogues de MTU1, GTPBP3 i MTO1 a les que hem denominat MTTU-1, MTCU-1 i MTCU-2. Hem comprovat que aquestes proteïnes, codificades pel nucli, són de localització mitocondrial i estan implicades en la modificació de la U34 dels mt-tRNAs. Els mutants mtcu-1 i mtcu-2 presenten una reducció en la seua fertilitat i, en el cas del mutant mttu-1, fenotipus associats a termosensibilitat. Els fenotipus exhibits pels mutants mttu-1, mtcu-1 i mtcu-2 sustenten la hipòtesi que la mutació mttu-1, i les mutacions mtcu-1 i mtcu-2 promouen senyals retrògrads diferents que produïxen patrons d'expressió nuclears específics. Així, un tret fenotípic dependent de gens nuclears (com ho és la transcripció i/o l'estabilitat dels mt-tRNAs) i l'expressió de gens nuclears com ucp-4, hsp-6, hsp-60 i altres implicats en el metabolisme mitocondrial mostren un patró diferent en els dos grups de mutants. Els gens hsp-6 i hsp-60 (marcadors de la UPRmt) estan regulats a la baixa en el mutant mttu-1. Els tres mutants simples exhibixen una reducció en l'expressió de gens de la glicòlisi i de la ß-oxidació dels àcids grassos, una inducció en un marcador de glutaminolisi i una inducció en el gen ucp-4 (major en el mutant mttu-1) implicat en el transport de succinat a la mitocondria. Atés que els tres mutants simples presenten una disfunció OXPHOS relativament suau, proposem que els canvis d'expressió en gens que modulen el metabolisme mitocondrial revelen una reprogramació del cicle del TCA que compensa la disminució en l'aportació d'acetil-CoA procedent de la glicòlisi i de l'oxidació d'àcids grassos amb l'activació de rutes anaplerótiques del cicle del TCA (importació de succinat a la mitocondria per UCP-4 i aportació de ¿-cetoglutarat de la glutaminolisi). També s'analitzen els efectes de l'anul¿lació simultània de les modificacions en 2 i 5 de la U34. El doble mutant mttu-1;mtcu-2 presenta una disfunció OXPHOS severa, amb una ràtio AMP/ATP 5 vegades superior al control, que resulta en letalitat embrionària, detenció del desenvolupament en estadis larvaris primerencs, esterilitat completa en els adults i una longevitat unes 2 vegades superior al control. Aquest increment de la longevitat està modulat per rutes de senyalització que depenen de la subunitat catalítica AAK-1 (AMPK), i d'hormones esteroidees (a través de les proteïnes DAF-9 i DAF-12). En resum, aquest treball mostra per primera vegada a nivell d'un animal model la important reprogramació de gens relacionats amb el metabolisme mitocondrial en resposta a la hipomodificació de la U34 dels mt-tRNAs i
Navarro González, MDC. (2016). Caenorhabditis elegans as a research tool to study mitochondrial diseases associated with defects in tRNA modification [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/61978
TESIS

Sporadic inclusion body myositis (sIBM) is the most common myopathy in elderly. This disease causes muscle wasting with both distal and proximal affectation. Quadriceps and finger flexors are muscle typically affected. At pathological level, three different features are present in muscle biopsies: inflammation, mitochondrial abnormalities and degeneration. The presence of T-cell infiltrate, ragged-red fibers and rimmed vacuoles are some features linked to these pathological processes. Protein misfolding and aggregation lead to the formation of the mentioned rimmed vacuoles, composed by many different misfolded proteins: β-amyloid, caveolin, phosphor-Tau among others. The accumulation of β-amyloid is also a hallmark of Alzheimer’s disease (AD), which presents some parallelisms with sIBM. In this work, we aimed to evaluate the mitochondrial state in muscle from sIBM patients but also in peripheral blood mononuclear cells (PBMC) to describe the molecular abnormalities that mitochondria could present in this disease. In addition, we aimed to measure plasmatic molecules related to inflammation, mitochondria and degeneration, in search for plasmatic biomarkers in sIBM patients but also in dermatomyositis and polymyositis, both diseases from the inflammatory myopathy group, like sIBM. Regarding mitochondrial analysis, muscle biopsies from 23 sIBM patients were analyzed, as well as 18 controls free of muscle disease. In addition, PBMC from 14 sIBM patients and from 20 controls were assessed as well. MtDNA levels and also mitochondrial respiratory chain complex IV (COX) activity were significantly decreased in muscle from sIBM patients compared to controls. Interestingly, when analyzing PBMC, dysfunction in COX activity was also found. In this tissue, a deregulation in mitochondrial protein synthesis was also found. As 57% of the sIBM patients presented mtDNA deletions, we aimed to evaluate if the presence of mtDNA deletions correlate with impaired mitochondrial parameters. sIBM patients with mtDNA deletions presented the lowest amount of mtDNA, and those patients without deletions showed values more similar to controls. A similar pattern was found when correlating the presence of MFN-2, a protein involved in mitochondrial dynamics. Again, patients with mtDNA deletions presented the lowest amount of this protein, and patients without deletions showed an intermediate values between patients with deletions and controls. Regarding the analysis of plasmatic molecules related to sIBM pathological features, inflammatory, mitochondrial and amyloidogenic molecules were analyzed in plasma samples from 21 sIBM, 20 controls and also in 14 plasma samples from dermatomyositis (DM) and polymyositis (PM) patients, which constitute an inflammatory myopathy different from sIBM group. Inflammatory (IL-6 and TNF-α) and mitochondrial-related (circulating mtDNA, FGF-21 and CoQ) molecules did not show significant differences between groups. However, amyloidogenic molecules (BACE-1, PS-1 and sAPPβ) were increased in sIBM patients respect to controls but also respect to the DM and PM group confirming its implication in sIBM pathogenesis. Sensitivity and specificity test showed that BACE-1 would be the most suitable biomarker for sIBM diagnosis. This thesis describes at molecular level the mitochondrial implication in the disease, and also reinforces the amyloidogenic component in sIBM. In addition, it proposes a plasmatic and non-invasive biomarker that could help in the sIBM diagnosis, especially in discriminating between other inflammatory myopathies, like polymyositis.
La miositis per cossos d’inclusió en la seva forma esporàdica (MCI) és la miopatia més comú en individus de més de 50 anys tot i ser una malaltia rara. Cursa amb atròfia muscular progressiva distal i proximal i actualment no es coneix cura. A nivell histopatològic presenta un component inflamatori, un component mitocondrial i un component degeneratiu. Degut al seu component degeneratiu i a la similitud de les proteïnes que formen aquests cossos d’inclusió, s’ha establert un possible paral·lelisme amb la malaltia d’Alzheimer. Els objectius d’aquesta tesi doctoral són explorar a nivell molecular les alteracions mitocondriales en la MCI en múscul, però també en cèl·lules mononuclears de sang perifèrica (CMSP), ja que és un teixit menys invasiu. A més, com a segon objectiu principal pretén d’estudiar mol·lècules relacionades amb la inflamació, amb el mitocondri i amb la degeneració en plasma d’aquests pacients per tal de demostrar la seva implicación amb la etiopatogènia i a més per establir nous marcadors menys invasius que permetin diagnosticar la malaltia i diferenciarla d’altres malalties similars com la dermatomiositis i la polimiositis. Fent referència a l’estudi mitocondrial, tant la quantitat de DNA mitocondrial com l’activitat del complex IV de la cadena mitocondrial (COX) es van trobar disminuïts en músculs dels pacients amb MCI. D’altra banda, amb l’estudi de les CMSP, també vam trobar disminuïda l’activitat de la COX, i a més una desregulación de la síntesis de proteïnes mitocondrials. Donat que un 57% dels pacients va presentar delecions múltiples al DNA mitocondrial, la presència d’aquestes delecions correlacionava amb una menor quantitat de DNA mitocondrial i a més amb un decrement de proteïna MFN-2, implicada en la dinàmica mitocondrial. Amb l’estudi de les molècul·les plasmàtiques, es van analitzar en plasma de pacients amb MCI, en controls però també en pacients amb dermatomiositis i polimiositis mol·lècules relacionades amb la inflamació (IL-6 i TNF-α), amb el mitocondri (DNA mitocondrial circulant, FGF-21 i enzim CoQ) i amb la amiloidogènesi (BACE-1, PS-1 i sAPPβ). Les mol.lècules amiloidogèniques es trobaven incrementades en els pacients amb MCI respecte controls i altres miopatíes inflamatòries, demostrant la seva impliació en la etiopatogènia i obtenint un cert valor diagnòstic. Amb aquesta tesi, s’ha demostrat la implicació mitocondrial en la etiopatogènia de la MCI, i s’han trobat alteracions en plasma de mol·lècules amiloidogèniques que, a més, tenen potencial diagnòstic per diferenciar aquesta malaltia d’altres miopatíes inflamatòries com la polimiositis.

Las enfermedades mitocondriales pueden ser englobadas dentro de las enfermedades raras de la edad pediátrica. Esta tesis recoge aspectos clínicos, bioquímicos y genéticos de 241 pacientes con enfermedades mitocondriales diagnosticados en el hospital "Necker-Enfants Malades, Paris", en el período comprendido entre los años 1977 al 2002.. Este amplio espectro de información incluye características generales como son la descripción de las formas de presentación clínicas y procedimientos diagnósticos, pero también recoge aspectos poco conocidos como son los estudios clínicos de seguimiento a largo plazo de estos pacientes, déficits enzimáticos muy inusuales y nuevos hallazgos clínicos, bioquímicos y moleculares no reportados previamente.
En cuanto a las manifestaciones clínicas, a parte de la forma de presentación clásica con afectación multiorgánica e hiperlactacidemia severa en los casos de debut precoz, se exponen algunos signos guía que pueden ser indicativos de algún déficit en concreto. En particular, los movimientos anormales asociado a un síndrome rígido acinético infantil nos pueden apuntar hacia un déficit de piruvato carboxilasa, mientras que hipoglucemias recurrentes aisladas son orientativas de un déficit de complejo III de la cadena respiratoria mitocondrial.
Los estudios bioquímicos se demuestran de mayor ayuda en trastornos del metabolismo del piruvato y del ciclo de Krebs que en déficits de la cadena respiratoria. En concreto, una hiperlactacidemia asociada a una relación lactato/piruvato normal asociado a una actividad normal de la enzima piruvato deshidrogenasa, orientan hacia un déficit en el transportador mitocondrial del piruvato.
Un punto en ocasiones conflictivo en el estudio de la cadena respiratoria mitocondrial, es el tipo de tejido a analizar. Lo más habitual es realizar una biopsia muscular Por el contrario se realizan escasos estudios en tejido hepático si los resultados en músculo han sido normales y no hay signos clínicos ó biológicos de afectación hepática. Los resultados de este estudio muestran que el análisis de la cadena respiratoria en tejido hepático pueden ser de gran ayuda en el diagnóstico, especialmente cuando el paciente presenta manifestaciones neurológicas predominantes.
Por otro lado, existen escasos estudios de seguimiento clínico a largo plazo en el campo de los errores congénitos del metabolismo, en cambio, éstos son cruciales para tener una idea precisa del futuro de estos niños así como la posibilidad de establecer factores pronósticos. En este sentido, los neonatos con diversos déficits de la cadena respiratoria mitocondrial tienen una elevada mortalidad y una muy pobre calidad de vida aunque se recogen algunas formas "reversibles" y "benignas".
Los datos relativos a mutaciones de genes nucleares recogidos en este trabajo son escasos ya que exponer las características genéticas ó establecer posibles relaciones entre el genotipo y el fenotipo de estas enfermedades no estaban dentro de los objetivos de este trabajo. A pesar de ello se reporta un nuevo gen nuclear responsable de un cuadro clínico de hipoglicemias de repetición asociado con un déficit del complejo III.
Mitochondrial disorders are rare causes of disease in childhood. This thesis reports clinical, biochemical and genetic aspects of 241 paediatric patients with mitochondrial disorders diagnosed in the "Hôpital Necker-Enfants Malades, Paris" from 1977 to 2002. This wide spectrum of information includes general characteristics such as clinical presentation and diagnostic approach, poorly documented features such as follow-up studies or uncommon enzymatic deficiencies, and new particular findings, not reported before.
Concerning clinical presentation, other than the well known multiorganic involvement associated with severe hyperlactacidemia in young children, some guideline signs that may indicate a particular defect are exposed. Movement disorders associated with rigid akinetic syndrome in pyruvate carboxylase deficiency or recurrent isolated hypoglycaemias in complex III deficiency are examples of these rare but very orientative signs.
Biochemical tests appear to be of great help in deficiencies that do not concern the respiratory chain. Consequently, some "redox cycle", amino acids or organic acids profiles are more likely to correspond to defects in pyruvate or Krebs cycle metabolism. In particular, hyperlactacidemia with normal lactate/pyruvate ratio and normal pyruvate dehydrogenase activity would point towards impaired mitochondrial pyruvate carrier.
Other diagnostic dilemma in MRC is the tissue of choice. It is unusual to perform a liver biopsy if the results of the activities in muscle have been normal but there is no apparent hepatic disease. The results of this study show that the analyse of the hepatic tissue could be very helpful, especially in patients with predominant neurological disease.
Follow-up studies in inborn errors of metabolism are very infrequent. However, they are crutial to have a global and accurate idea about the future of these children as well as to establish possible prognostic factors. In this respect, newborns with respiratory chain deficiencies have a very high mortality and a poor quality of life, although some "reversible" and "benign" forms could be found.
Little data about nuclear genes is here reported. Genetic characteristics or trying to establish phenotype-genotype correlations, are not among the aims of this study. Nevertheless a new nuclear gene related to isolated repetitive hypoglycaemias and complex III deficiency is also reported.

Abstract Various genetic aetiologies — including mitochondrial diseases, chromosomal disorders, and other monogenic diseases — are involved in status epilepticus (SE), a common neurologic emergency occurring in children and adults that exhibits high rates of morbidity and mortality. The exact frequency of mitochondrial SE is currently undefined. Furthermore, patients with pathogenic variants of POLG1 encoding mitochondrial DNA polymerase gamma have an increased risk of acute liver failure (ALF) induced by the common antiepileptic drug, valproic acid (VPA), which is problematic due to these patients also often experiencing drug-resistant seizures. Overall, the role of liver transplantation (LT) in VPA-ALF due to mitochondrial disease has been controversial. In the present work, large retrospective cohort studies were conducted for two main purposes: (1) to determine the genetic aetiologies of SE among Finnish paediatric and adult patients by specifically focusing on the common mitochondrial genetic defects associated with an increased risk of SE and (2) to examine whether common POLG1 p.Q1236H and p.E1143G variants are connected to liver or pancreatic toxicity upon exposure to VPA monotherapy. This thesis also describes the characteristics of VPA-ALF associated with the pathogenic POLG1 variant p.W748S and the prognosis of LT in a retrospective case series. Mitochondrial diseases explained 4.5% of SE cases in the study cohort. Patients with mitochondrial SE suffered from refractory SE significantly more often than patients with other forms of genetic or non-genetic SE. Additionally, mortality rates were higher in patients with mitochondrial or chromosomal disorders compared with the other groups, reflecting the severity of the underlying condition and the higher frequency of refractory SE. POLG1 variants p.Q1236H and p.E1143G could not be identified as risk factors for VHT or pancreatic toxicity, implying that VPA treatment might be suitable for patients harbouring these variants when other pathogenic variants are absent. Finally, the homozygous status of the pathogenic POLG1 variant p.W748S and older age of the patient during the presentation of VPA-ALF seem to be associated with higher survival rates following LT, which should be considered in the management of VPA-ALF
Tiivistelmä Useita perinnöllisiä syitä, kuten mitokondriotauteja, kromosomihäiriöitä ja muita geenimuutoksia on tunnistettu status epilepticuksen (SE) eli pitkittyneen epileptisen kohtauksen taustalla. SE on yleinen neurologinen hätätilanne, johon liittyy merkittävää oheissairastavuutta ja kuolleisuutta sekä lapsilla että aikuisilla. Mitokondriotauteihin liittyvän SE:n tarkkaa esiintyvyyttä ei tiedetä. Potilailla, joilla on patogeenisia variantteja mitokondrioiden DNA-polymeraasia koodaavassa tuman POLG1-geenissä, on todettu kohonnut riski yleisesti käytetyn epilepsialääkkeen valproaatin (VPA) aiheuttaman akuutin maksavaurion kehittymiselle. Tämä tekee lääkehoidon valinnasta ongelmallista, koska näillä potilailla on usein epilepsialääkkeille resistenttejä kohtauksia. Maksansiirron merkitys akuutin maksavaurion hoidossa mitokondriotauteja sairastavilla potilailla on ollut kiistanalainen. Tutkimuksen tavoitteena oli selvittää SE:n perinnöllisiä syitä suomalaisilla lapsi- ja aikuispotilailla retrospektiivisesti kerätyssä laajassa potilasaineistossa. Tutkimuksessa keskityttiin yleisimpiin mitokondriaalisiin perinnöllisiin muutoksiin, joiden on aiemmin todettu liittyvän SE:n lisääntyneeseen riskiin. Tutkimuksen toisena päätavoitteena oli selvittää väestössä yleisten POLG1-geenin muutosten eli varianttien p.Q1236H ja p.E1143G yhteyttä maksatoksisuuteen tai haimatoksisuuteen VPA-monoterapian aikana. Lisäksi tutkittiin VPA:n aiheuttaman maksavaurion kliinisiä erityispiirteitä patogeeniseen POLG1-varianttiin p.W748S liittyen sekä mutaatiostatuksen vaikutusta maksansiirron jälkeiseen ennusteeseen. Mitokondriotaudit selittivät 4,5 % SE-tapauksista tämän väitöskirjatyön potilasaineistossa ja näillä potilailla SE pitkittyi hoitoresistentiksi tai erittäin resistentiksi merkitsevästi muita potilasryhmiä useammin. Kuolleisuus oli suurin potilailla, joilla todettiin mitokondriotauti tai kromosomihäiriö, liittyen todennäköisimmin vakavaan taustasairauteen ja hoitoresistentin SE:n suurempaan esiintyvyyteen. Tutkittuja POLG1-variantteja p.Q1236H ja p.E1143G ei voitu tunnistaa maksa- tai haimatoksisuuden riskitekijöiksi, mikä tarkoittaa, että VPA-hoito voisi sopia näille potilaille, mikäli muita patogeenisiä variantteja ei todeta. Patogeenisen POLG1-variantin p.W748S homotsygoottisuus ja nuoruusikä tai varhainen aikuisikä maksavaurion ajankohtana ovat maksansiirron ennustetta parantavia tekijöitä, mikä tulisi ottaa huomioon hoitopäätöksiä tehtäessä