Abstract Neurological features are common in mitochondrial diseases because tissues depending upon oxidative phosphorylation bear the brunt of the pathogenesis. The 3243A>G mutation in the MTTL1 gene in mitochondrial DNA is regarded as the most frequent mitchondrial point mutation and classically presents with mitochondrial encephalopathy, lactic acidosis and stroke-like episodes (MELAS). Myopathy and peripheral neuropathy have been documented in patients with mitochondrial diseases, but not properly characterised in patients with the 3243A>G mutation. We have previously determined the prevalence of patients with this mutation in a defined population in northern Finland. The clinical spectrum and molecular aspects of myopathy and peripheral neuropathy are analysed here in a population-based cohort of patients with 3243A>G. Fifty patients were examined neurologically in order to define the frequency of myopathy and its histological, ultrastructural and clinical features. The frequency and phenotypic variability of peripheral neuropathy were determined in 32 patients and muscle computed tomography findings recorded in 24 patients. Finally, variations in mutation heteroplasmy were analysed in 10 patients using single muscle fibre PCR analysis. The frequency of peripheral neuropathy was 22% (95% confidence interval (CI), 9–40%) and that of clinical myopathy 50% (95% CI, 36–64%). Moderate limb weakness was the most common myopathic feature, but mild weakness and external ophthalmoplegia were also present. CT scans revealed myopathic changes in 54% of the patients (95% CI, 33–76%), most frequently in the pelvic muscles. The incidence of myopathy was highest in the fifth decade of life, and higher age and male gender increased the risk of neuropathy. Muscle histology was abnormal in 72% of the cases examined (95% CI, 55–86%). The presence of intramitochondrial crystals and COX-negative fibres and variations in the size and shape of mitochondria were more common in the muscle of myopathic patients. Single muscle fibre analysis pointed to a correlation between the mutation load in ragged red fibres and in adjacent histologically normal fibres, and the proportion of 3243A>G in histologically normal muscle fibres showed a pattern compatible with random genetic drift. The results indicate that myopathy and peripheral neuropathy are common in patients with the 3243A>G and that myopathy is highly variable in presentation. Segregation of 3243A>G in individual muscle fibres showed a complex process with random and non-random elements.

Abstract The 3243A>G mutation in mitochondrial DNA (mtDNA), the most common cause of the syndrome of mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes, is also associated with many other phenotypes such as hearing loss, diabetes mellitus, epilepsy, cognitive decline, myopathy and cardiomyopathy. The prevalence of the mutation has been shown to be 16.3/100 000 adults in Northern Finland. The present study was performed to estimate the frequency and progression of cardiac abnormalities and to examine causes of death in patients with 3243A>G. Left ventricular hypertrophy (LVH) was found in echocardiography in 56% of patients with 3243A>G and in 15% of age and sex-matched controls. The median thickness of the diastolic interventricular septum or posterior wall was 14 mm in the patients with LVH. The prevalence of LVH determined by echocardiography increased from 40% to 56% in 25 patients with 3243A>G during three years of follow-up, this trend being especially marked among the diabetic patients. The ultra-low-frequency (ULF) and very-low-frequency (VLF) components of the spectral analysis of heart rate variability (HRV) were lower among the patients with 3243A>G than in matched controls (p = 0.02 in ULF and p = 0.04 in VLF), and the short-term fractal scaling exponent in detrended fluctuation analysis of HRV was lower in the patients with 3243A>G (1.16 ± 0.18 vs. 1.28 ± 0.13) (p < 0.01). Survival analysis of a birth cohort from pedigrees with 3243A>G revealed excess mortality before the age of 50 years. Neurological and cardiovascular diseases accounted for 32% of all the underlying causes of death in families with 3243A>G. Death was sudden and unexpected in 31% of cases in which 3243A>G was considered to be involved in the cause of death. The results show that cardiac abnormalities are frequent and progressive in patients with the 3243A>G mtDNA mutation and that cardiac autonomic regulation is disturbed. Patients with the 3243A>G mutation and their first degree maternal relatives died younger than was presupposed by their life expectancy at birth or at 15 years. The most common causes of death were neuropsychiatric and cardiovascular diseases.

Abstract Human mitochondria contain a circular genome called mitochondrial DNA (mtDNA). It encodes subunits of the respiratory chain enzymes involved in energy conservation in oxidative phosphorylation and the necessary RNA needed for their expression. Errors in these genes have been shown to cause diseases, called mitochondrial diseases, which mainly affect tissues with high energy-demand, such as brain, heart, and skeletal muscle, or to lead to the production of harmful by-products in the form of reactive oxygen species (ROS) during cellular respiration. ROS damage lipids, proteins, and DNA, especially mtDNA. Accumulation of mtDNA mutations has also been associated with aging. Mitochondrial complex I is located in the inner mitochondrial membrane and catalyzes NADH-ubiquinone oxidoreduction coupled to the translocation of four protons from the inside of the mitochondrion to the intermembranous space. Bacteria contain a homologous but simpler enzyme, NDH-1, with the same catalytic mechanism and which is therefore considered the catalytical core of mitochondrial complex I. Seven of the conserved membranous subunits in complex I are encoded in the mtDNA and are targets for mutations causing mitochondrial diseases, like MELAS syndrome or Leber hereditary optic neuropathy (LHON). We used Paracoccus denitrificans and Escherichia coli NDH-1 enzymes to reveal the role of selected conserved charged residues and MELAS or LHON amino acid substitutions in enzyme catalysis. The growth phenotypes and NDH-1-dependent activities in mutant bacterial membranes were characterized, in addition to the sensitivity to selected complex I inhibitors. In order to enable ROS production measurements in the bacterial model of human mitochondrial diseases, we evaluated the reliability of two superoxide detecting probes, lucigenin and coelenterazine. Elimination of the acidic residue in ND1 (position E228) previously found to cause MELAS, was found detrimental for NDH-1 assembly and activity. Also, elimination of the acidic residue at position E36 in ND4L resulted in an inactive enzyme. ND1-E216A, ND4L-E72Q and -E36Q/I39D/A69D/E72Q substitutions decreased NDH-1 activity somewhat (normal activity in the last mutant), but displayed a negative growth phenotype under NDH-1 dependent conditions, suggestive of impaired energy conservation in these mutants. ND1-Y229, whose substitution causes MELAS, charged residues in loop five of ND1, and ND1-E157, whose substitution causes LHON, were also found important for the enzyme activity. Coelenterazine was found a reliable probe for quantitative superoxide production measurement in mitochondrial or bacterial membranes, and its sensitivity is not affected by the reduction level of the respiratory chain. Therefore, coelenterazine is suitable for quantitative superoxide production measurements.

Chaque mitochondrie contient son propre génome en de multiples copies d’ADN. Les mutations de l'ADN mitochondriales (ADNmt) sont responsables de sévères dysfonctions de la chaîne respiratoire. Le ratio entre la proportion de copies sauvages et mutantes, qualifiée d'hétéroplasmie, détermine la sévérité de la pathologie. Une des mutations les plus répandues de l'ADNmt est la mutation m.3243A>G, affectant l'ARN de transfert de la leucine. Ce variant est à l'origine du syndrome mitochondrial MELAS. Il n’existe à l’heure actuelle aucun traitement curatif pour ce syndrome. Nous avons développé une série de cybrides neuronaux porteurs de la mutation m.3243A>G a différents taux d’hétéroplasmie. Nous avons mis en évidence que de fort taux de mutations sont responsables de sévères dysfonctions de la chaîne respiratoire, d’un défaut d’assemblage précoce du complexe I ainsi qu’une réduction du renouvellement mitochondrial. Différentes stratégies métaboliques ont été employées pour compenser ces déficits. L’exposition des cellules a une restriction glucidique ou à la diète cétogène associant réduction glucidique et ajout de corps cétoniques, améliore significativement les fonctions mitochondriales après 4 semaines. Ces effets passent notamment par une restauration de l’assemblage et de l’activité du complexe I médiée ces interventions métaboliques. Par ailleurs, l’administration de la diète cétogène à un patient atteint du syndrome MELAS a déjà montré des résultats encourageants. De telles approches pourraient alors, constituées des stratégies thérapeutiques futures dans le traitement du syndrome MELAS et des maladies mitochondriales. / Each mitochondrion contains its own genome in multiple copies. Mitochondrial DNA (mtDNA) mutations are responsible for respiratory chain defects. The ratio of mutant to normal mtDNA, a condition known as heteroplasmy, may determine the disease severity. The m.3243A>G mutation, which affects the leucine tRNA, is one of the most common mtDNA mutation. This variant is responsible for the MELAS syndrome, a neurodegenerative disease, characterized by pseudostrokes. Unfortunately there are no curative treatments for MELAS syndrome. We have developed series of cybrid neuronal cells lines carrying the m.3243A>G mutation with different mutant loads, within the same nuclear background. High mutation load is associated to severe respiratory chain dysfunction, an early complex I assembly defect and a mitochondrial turn-over deficit. Different strategies were used to compensate the defects in the mutant cells. Cell exposure to low glucose or ketogenic diet, combining glucose reduction and the addition of ketone bodies, greatly improves mitochondrial functions after 4 weeks. Those effects are linked to a significant increase of complex I assembly and activity mediated by those metabolic interventions. In addition, a MELAS patient treated with ketogenic diet showed significant clinical improvement. Thus, metabolic approaches may constitute promising therapeutic strategies against MELAS syndrome and mitochondrial disorders.

Abstract NADH-ubiquinone oxidoreductase (complex I) is one of the largest enzymes in mammals. Seven (ND1-ND6 and ND4L) of its 45 subunits are encoded in mitochondrial DNA, mutations of which are usually behind mitochondrial diseases such as Leber hereditary optic neuropathy (LHON) and MELAS-syndrome. The rest of the genes are located in the nucleus. Bacterial homologs of complex I (NDH-1) consist of only 13–14 subunits, comprising the catalytic core of the enzyme. These complexes are simpler but perform a similar function. Escherichia coli NDH-1 was employed here to generate amino acid replacements at conserved sites in NuoH, NuoJ and NuoK, counterparts of ND1, ND6 and ND4L, to elucidate their role in complex I. Consequences of homologous amino acid substitutions brought about by ND1-affecting LHON/MELAS-overlap syndrome-associated m.3376G>A and m.3865A>G mutations and the ND6-affecting m.14498T>C substitution associated with LHON were also studied to validate their pathogenicity. Effects of the site-directed mutations were evaluated on the basis of enzyme activity, inhibitor sensitivity and growth phenotype. Highly conserved glutamate-residues 36 and 72 within transmembrane helices of NuoK in positions similar to proton translocating transmembrane proteins were found essential for electron transfer to ubiquinone and growth on medium necessitating normal proton transfer by NDH-1. NuoH and NuoJ replacements at sites corresponding to targets of m.3376G>A and m.14498T>C decreased ubiquinone reductase activity and altered the ubiquinone binding site, while the counterpart of m.3865A>G was without a major effect. Other NuoH and NuoJ mutations studied also affected the interactions of ubiquinone and inhibitors with NDH-1. The results corroborate the pathogenicity of the m.14498T>C and m.3376G>A mutations and demonstrate that the overlap syndrome-associated modification affects complex I in a pattern which appears to combine the effects of separate mutations responsible for LHON and MELAS. Change in ubiquinone binding affinity is a likely pathomechanism of all LHON-associated mutations. Effects of the NuoH, NuoJ and NuoK subunit substitutions also indicate that ND1 and ND6 subunits contribute to the ubiquinone-interacting site of complex I and the site is located in the vicinity of the membrane surface, while ND4L is likely involved in proton pumping activity of the enzyme. / Tiivistelmä 45 alayksiköstä muodostuva NADH-ubikinoni oksidoreduktaasi (kompleksi I) on nisäkkäiden suurimpia entsyymejä. Sen mitokondriaalisessa DNA:ssa koodattujen alayksiköiden ND1-ND6 ja ND4L geeneihin liittyvät mutaatiot ovat yleisiä mitokondriosairauksien, kuten Leberin perinnöllisen näköhermoatrofian (LHON) ja MELAS-oireyhtymän, syitä. Bakteerien vastaava entsyymi (NDH-1) koostuu vain 13–14 alayksiköstä. Tästä huolimatta sen katalysoima reaktio on samankaltainen kuin kompleksi I:n. NDH-1:n katsotaankin edustavan entsyymin katalyyttistä ydintä. Tässä työssä tutkittiin ND1, ND6 ja ND4L alayksiköiden tehtävää kompleksi I:ssä niiden Escherichia coli bakteerissa olevien vastineiden (NuoH, NuoJ ja NuoK) kohdennetun mutageneesin avulla. Samaa lähestymistapaa käytettiin LHON/MELAS-oireyhtymässä todettujen ND1 alayksikön mutaatioiden, m.3376G>A ja m.3865A>G, ja LHON:ssa havaitun ND6:n m.14498T>C mutaation aiheuttamien aminohappomuutosten seurauksien selvittämiseen. Tehtyjen mutaatioiden vaikutuksia arvioitiin entsyymiaktiivisuus-mittauksin ja kasvukokein. NuoK:n solukalvon läpäisevissä rakenteissa olevien kahden glutamaatti-aminohappotähteen sijainti muistuttaa protoneita kalvon läpi kuljettavissa proteiineissa todettua. NuoK:n glutamaattien havaittiinkin olevan tärkeitä elektronien ja protonien kuljetukselle kompleksi I:ssä. m.3376G>A ja m.14498T>C mutaatioiden aiheuttamien aminohappomuutosten vastineet NDH-1:ssä alensivat NDH-1:n elektroninsiirtoaktiivisuutta ja heikensivät ubikinonin sitoutumista, kun taas m.3865A>G mutaatiolla ei ollut vaikutusta. Muut NuoH ja NuoJ alayksiköihin tehdyt aminohappovaihdokset johtivat huonontuneeseen ubikinonin ja kompleksi I:n inhibiittoreiden sitoutumiseen. Saadut tulokset vahvistavat m.3376G>A ja m.14498T>C mutaatioiden patogeenisyyden. Ne myös osoittavat, että LHON/MELAS-oireyhtymään liitetyn mutaation biokemiallisissa vaikutuksissa yhdistyvät sekä LHON:ssa että MELAS-oireyhtymässä todettujen mutaatioiden seuraukset. Esitetyt tulokset tukevat näkemystä siitä, että ubikinonin ja kompleksi I:n välisessä vuorovaikutuksessa tapahtuva muutos on kaikille LHON:aan liitetyille mutaatioille yhteinen vaikutusmekanismi. NuoH:n, NuoJ:n ja NuoK:n kohdennetusta mutageneesista saatujen tulosten perusteella ND1 ja ND6 alayksiköt ovat osa ubikinonin sitoutumispaikkaa entsyymikompleksissa, kun taas ND4L osallistuu protoninkuljetukseen.

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Molecular biology has entered an era of systematic and automated experimentation. High-throughput techniques have moved biology from small-scale experiments focused on specific genes and proteins to genome and proteome-wide screens. One result of this endeavor is the compilation of complex networks of interacting proteins. Molecular biologists hope to understand life's complex molecular machines by studying these networks. This thesis addresses tree open problems centered upon their analysis and quality assessment. First, we introduce power graph analysis as a novel approach to the representation and visualization of biological networks. Power graphs are a graph theoretic approach to lossless and compact representation of complex networks. It groups edges into cliques and bicliques, and nodes into a neighborhood hierarchy. We demonstrate power graph analysis on five examples, and show its advantages over traditional network representations. Moreover, we evaluate the algorithm performance on a benchmark, test the robustness of the algorithm to noise, and measure its empirical time complexity at O (e1.71)- sub-quadratic in the number of edges e. Second, we tackle the difficult and controversial problem of data quality in protein interaction networks. We propose a novel measure for accuracy and completeness of genome-wide protein interaction networks based on network compressibility. We validate this new measure by i) verifying the detrimental effect of false positives and false negatives, ii) showing that gold standard networks are highly compressible, iii) showing that authors' choice of confidence thresholds is consistent with high network compressibility, iv) presenting evidence that compressibility is correlated with co-expression, co-localization and shared function, v) showing that complete and accurate networks of complex systems in other domains exhibit similar levels of compressibility than current high quality interactomes. Third, we apply power graph analysis to networks derived from text-mining as well to gene expression microarray data. In particular, we present i) the network-based analysis of genome-wide expression profiles of the neuroectodermal conversion of mesenchymal stem cells. ii) the analysis of regulatory modules in a rare mitochondrial cytopathy: emph{Mitochondrial Encephalomyopathy, Lactic acidosis, and Stroke-like episodes} (MELAS), and iii) we investigate the biochemical causes behind the enhanced biocompatibility of tantalum compared with titanium.

Molecular biology has entered an era of systematic and automated experimentation. High-throughput techniques have moved biology from small-scale experiments focused on specific genes and proteins to genome and proteome-wide screens. One result of this endeavor is the compilation of complex networks of interacting proteins. Molecular biologists hope to understand life's complex molecular machines by studying these networks. This thesis addresses tree open problems centered upon their analysis and quality assessment. First, we introduce power graph analysis as a novel approach to the representation and visualization of biological networks. Power graphs are a graph theoretic approach to lossless and compact representation of complex networks. It groups edges into cliques and bicliques, and nodes into a neighborhood hierarchy. We demonstrate power graph analysis on five examples, and show its advantages over traditional network representations. Moreover, we evaluate the algorithm performance on a benchmark, test the robustness of the algorithm to noise, and measure its empirical time complexity at O (e1.71)- sub-quadratic in the number of edges e. Second, we tackle the difficult and controversial problem of data quality in protein interaction networks. We propose a novel measure for accuracy and completeness of genome-wide protein interaction networks based on network compressibility. We validate this new measure by i) verifying the detrimental effect of false positives and false negatives, ii) showing that gold standard networks are highly compressible, iii) showing that authors' choice of confidence thresholds is consistent with high network compressibility, iv) presenting evidence that compressibility is correlated with co-expression, co-localization and shared function, v) showing that complete and accurate networks of complex systems in other domains exhibit similar levels of compressibility than current high quality interactomes. Third, we apply power graph analysis to networks derived from text-mining as well to gene expression microarray data. In particular, we present i) the network-based analysis of genome-wide expression profiles of the neuroectodermal conversion of mesenchymal stem cells. ii) the analysis of regulatory modules in a rare mitochondrial cytopathy: emph{Mitochondrial Encephalomyopathy, Lactic acidosis, and Stroke-like episodes} (MELAS), and iii) we investigate the biochemical causes behind the enhanced biocompatibility of tantalum compared with titanium.