Nonshivering thermogenesis capacity associated to mitochondrial DNA haplotypes and gender in the greater white-toothed shrew, Crocidura russula.

A selection gradient was recently suggested as one possible cause for a clinal distribution of mitochondrial DNA (mtDNA) haplotypes along an altitudinal transect in the greater white-toothed shrew, Crocidura russula (Ehinger et al. 2002). One mtDNA haplotype (H1) rare in lowland, became widespread when approaching the altitudinal margin of the distribution. As H1 differs from the main lowland haplotype by several nonsynonymous mutations (including on ATP6), and as mitochondria play a crucial role in metabolism and thermogenesis, distribution patterns might stem from differences in the thermogenic capacity of different mtDNA haplotypes. In order to test this hypothesis, we measured the nonshivering thermogenesis (NST) associated with different mtDNA haplotypes. Sixty-two shrews, half of which had the H1 haplotype, were acclimated in November at semioutdoor conditions and measured for NST throughout winter. Our results showed the crucial role of NST for winter survival in C. russula. The individuals that survived winter displayed a higher significant increase in NST during acclimation, associated with a significant gain in body mass, presumably from brown fat accumulation. The NST capacity (ratio of NST to basal metabolic rate) was exceptionally high for such a small species. NST was significantly affected by a gender x haplotype interaction after winter-acclimation: females bearing the H1 haplotype displayed a better thermogenesis at the onset of the breeding season, while the reverse was true for males. Altogether, our results suggest a sexually antagonistic cyto-nuclear selection on thermogenesis.

Lack of the mitochondrial protein acylglycerol kinase causes Sengers syndrome.

Exome sequencing of an individual with congenital cataracts, hypertrophic cardiomyopathy, skeletal myopathy, and lactic acidosis, all typical symptoms of Sengers syndrome, discovered two nonsense mutations in the gene encoding mitochondrial acylglycerol kinase (AGK). Mutation screening of AGK in further individuals with congenital cataracts and cardiomyopathy identified numerous loss-of-function mutations in an additional eight families, confirming the causal nature of AGK deficiency in Sengers syndrome. The loss of AGK led to a decrease of the adenine nucleotide translocator in the inner mitochondrial membrane in muscle, consistent with a role of AGK in driving the assembly of the translocator as a result of its effects on phospholipid metabolism in mitochondria.

New Findings in a Global Approach to Dissect the Whole Phenotype of PLA2G6 Gene Mutations.

Mutations in PLA2G6 gene have variable phenotypic outcome including infantile neuroaxonal dystrophy, atypical neuroaxonal dystrophy, idiopathic neurodegeneration with brain iron accumulation and Karak syndrome. The cause of this phenotypic variation is so far unknown which impairs both genetic diagnosis and appropriate family counseling. We report detailed clinical, electrophysiological, neuroimaging, histologic, biochemical and genetic characterization of 11 patients, from 6 consanguineous families, who were followed for a period of up to 17 years. Cerebellar atrophy was constant and the earliest feature of the disease preceding brain iron accumulation, leading to the provisional diagnosis of a recessive progressive ataxia in these patients. Ultrastructural characterization of patients' muscle biopsies revealed focal accumulation of granular and membranous material possibly resulting from defective membrane homeostasis caused by disrupted PLA2G6 function. Enzyme studies in one of these muscle biopsies provided evidence for a relatively low mitochondrial content, which is compatible with the structural mitochondrial alterations seen by electron microscopy. Genetic characterization of 11 patients led to the identification of six underlying PLA2G6 gene mutations, five of which are novel. Importantly, by combining clinical and genetic data we have observed that while the phenotype of neurodegeneration associated with PLA2G6 mutations is variable in this cohort of patients belonging to the same ethnic background, it is partially influenced by the genotype, considering the age at onset and the functional disability criteria. Molecular testing for PLA2G6 mutations is, therefore, indicated in childhood-onset ataxia syndromes, if neuroimaging shows cerebellar atrophy with or without evidence of iron accumulation.

Chromosomal versus mitochondrial DNA evolution: tracking the evolutionary history of the southwestern european populations of the Sorex araneus group (Mammalia Insectivora)

The shrews of the Sorer araneus group have undergone a spectacular chromosome evolution. The karyotype of Sorer granarius is generally considered ancestral to those of Sorer coronatus and S. araneus. However, a sequence of 777 base pairs of the cytochrome b gene of the mitochondrial DNA (mtDNA) produces a quite different picture: S. granarius is closely related to the populations of S. araneus from the Pyrenees and from the northwestern Alps, whereas S. coronatus and S. araneus from Italy and the southern Alps represent two well-separated lineages. It is suggested that mtDNA and chromosomal evolution are in this case largely independant processes. Whereas mtDNA haplotypes are closely linked to the geographical history of the populations, chromosomal mutations were probably transmitted from one population to another. Available data suggest that the impressive chromosome polymorphism of this group is quite a recent phenomenon.

A nuclear defect in the 4p16 region predisposes to multiple mitochondrial DNA deletions in families with Wolfram syndrome.

Wolfram syndrome is a progressive neurodegenerative disorder transmitted in an autosomal recessive mode. We report two Wolfram syndrome families harboring multiple deletions of mitochondrial DNA. The deletions reached percentages as high as 85-90% in affected tissues such as the central nervous system of one patient, while in other tissues from the same patient and from other members of the family, the percentages of deleted mitochondrial DNA genomes were only 1-10%. Recently, a Wolfram syndrome gene has been linked to markers on 4p16. In both families linkage between the disease locus and 4p16 markers gave a maximum multipoint lod score of 3.79 at theta = 0 (Pi<0.03) with respect to D4S431. In these families, the syndrome was caused by mutations in this nucleus-encoded gene which deleteriously interacts with the mitochondrial genome. This is the first evidence of the implication of both genomes in a recessive disease.

A nuclear defect in the 4p16 region predisposes to multiple mitochondrial DNA deletions in families with Wolfram syndrome.

Wolfram syndrome is a progressive neurodegenerative disorder transmitted in an autosomal recessive mode. We report two Wolfram syndrome families harboring multiple deletions of mitochondrial DNA. The deletions reached percentages as high as 85-90% in affected tissues such as the central nervous system of one patient, while in other tissues from the same patient and from other members of the family, the percentages of deleted mitochondrial DNA genomes were only 1-10%. Recently, a Wolfram syndrome gene has been linked to markers on 4p16. In both families linkage between the disease locus and 4p16 markers gave a maximum multipoint lod score of 3.79 at theta = 0 (Pi<0.03) with respect to D4S431. In these families, the syndrome was caused by mutations in this nucleus-encoded gene which deleteriously interacts with the mitochondrial genome. This is the first evidence of the implication of both genomes in a recessive disease.

No longitudinal mitochondrial DNA sequence changes in HIV-infected individuals with and without lipoatrophy.

The potential for mitochondrial (mt) DNA mutation accumulation during antiretroviral therapy (ART), and preferential accumulation in patients with lipoatrophy compared with control participants, remains controversial. We sequenced the entire mitochondrial genome, both before ART and after ART exposure, in 29 human immunodeficiency virus (HIV)-infected Swiss HIV Cohort Study participants initiating a first-line thymidine analogue-containing ART regimen. No accumulation of mtDNA mutations or deletions was detected in 13 participants who developed lipoatrophy or in 16 control participants after significant and comparable ART exposure (median duration, 3.3 and 3.7 years, respectively). In HIV-infected persons, the development of lipoatrophy is unlikely to be associated with accumulation of mtDNA mutations detectable in peripheral blood.

Mitochondrial DNA damage and animal longevity: insights from comparative studies

Chemical reactions in living cells are under strict enzyme control and conform to a tightly regulated metabolic program. However, uncontrolled and potentially deleterious endogenous reactions occur, even under physiological conditions. Aging, in this chemical context, could be viewed as an entropic process, the result of chemical side reactions that chronically and cumulatively degrade the function of biological systems. Mitochondria are a main source of reactive oxygen species (ROS) and chemical sidereactions in healthy aerobic tissues and are the only known extranuclear cellular organelles in animal cells that contain their own DNA (mtDNA). ROS can modify mtDNA directly at the sugar-phosphate backbone or at the bases, producing many different oxidatively modified purines and pyrimidines, as well as single and double strand breaks and DNA mutations. In this scenario, natural selection tends to decrease the mitochondrial ROS generation, the oxidative damage to mtDNA, and the mitochondrial mutation rate in long-lived species, in agreement with the mitochondrial oxidative stress theory of aging.

Lack of GDAP1 induces neuronal calcium and mitochondrial defects in a knockout mouse model of charcot-marie-tooth neuropathy.

Mutations in GDAP1, which encodes protein located in the mitochondrial outer membrane, cause axonal recessive (AR-CMT2), axonal dominant (CMT2K) and demyelinating recessive (CMT4A) forms of Charcot-Marie-Tooth (CMT) neuropathy. Loss of function recessive mutations in GDAP1 are associated with decreased mitochondrial fission activity, while dominant mutations result in impairment of mitochondrial fusion with increased production of reactive oxygen species and susceptibility to apoptotic stimuli. GDAP1 silencing in vitro reduces Ca2+ inflow through store-operated Ca2+ entry (SOCE) upon mobilization of endoplasmic reticulum (ER) Ca2+, likely in association with an abnormal distribution of the mitochondrial network. To investigate the functional consequences of lack of GDAP1 in vivo, we generated a Gdap1 knockout mouse. The affected animals presented abnormal motor behavior starting at the age of 3 months. Electrophysiological and biochemical studies confirmed the axonal nature of the neuropathy whereas histopathological studies over time showed progressive loss of motor neurons (MNs) in the anterior horn of the spinal cord and defects in neuromuscular junctions. Analyses of cultured embryonic MNs and adult dorsal root ganglia neurons from affected animals demonstrated large and defective mitochondria, changes in the ER cisternae, reduced acetylation of cytoskeletal α-tubulin and increased autophagy vesicles. Importantly, MNs showed reduced cytosolic calcium and SOCE response. The development and characterization of the GDAP1 neuropathy mice model thus revealed that some of the pathophysiological changes present in axonal recessive form of the GDAP1-related CMT might be the consequence of changes in the mitochondrial network biology and mitochondria-endoplasmic reticulum interaction leading to abnormalities in calcium homeostasis.

Analysis of the mitochondrial DNA of Schizophrenia patients by next generation sequencing

Mitochondrial DNA (mtDNA), a maternally inherited 16.6-Kb molecule crucial for energy production, is implicated in numerous human traits and disorders. It has been hypothesized that the presence of mutations in the mtDNA may contribute to the complex genetic basis of schizophreniadisease, due to the evidence of maternal inheritance and the presence of schizophrenia symptoms in patients affected of a mitochondrial disorder related to a mtDNA mutation. The present project aims to study the association of variants of mitochondrial DNA (mtDNA), and an increased risk of schizophrenia in a cohort of patients and controls from the same population. The entire mtDNA of 55 schizophrenia patients with an apparent maternal transmission of the disease and 38 controls was sequenced by Next Generation Sequencing (Ion Torrent PGM, Life Technologies) and compared to the reference sequence. The current method for establishing mtDNA haplotypes is Sanger sequencing, which is laborious, timeconsuming, and expensive. With the emergence of Next Generation Sequencing technologies, this sequencing process can be much more quickly and cost-efficiently. We have identified 14 variants that have not been previously reported. Two of them were missense variants: MTATP6 p.V113M and MTND5 p.F334L ,and also three variants encoding rRNA and one variant encoding tRNA. Not significant differences have been found in the number of variants between the two groups. We found that the sequence alignment algorithm employed to align NGS reads played a significant role in the analysis of the data and the resulting mtDNA haplotypes. Further development of the bioinformatics analysis and annotation step would be desirable to facilitate the application of NGS in mtDNA analysis.

Mutations in the heat-shock protein A9 (HSPA9) gene cause the EVEN-PLUS syndrome of congenital malformations and skeletal dysplasia.

We and others have reported mutations in LONP1, a gene coding for a mitochondrial chaperone and protease, as the cause of the human CODAS (cerebral, ocular, dental, auricular and skeletal) syndrome (MIM 600373). Here, we delineate a similar but distinct condition that shares the epiphyseal, vertebral and ocular changes of CODAS but also included severe microtia, nasal hypoplasia, and other malformations, and for which we propose the name of EVEN-PLUS syndrome for epiphyseal, vertebral, ear, nose, plus associated findings. In three individuals from two families, no mutation in LONP1 was found; instead, we found biallelic mutations in HSPA9, the gene that codes for mHSP70/mortalin, another highly conserved mitochondrial chaperone protein essential in mitochondrial protein import, folding, and degradation. The functional relationship between LONP1 and HSPA9 in mitochondrial protein chaperoning and the overlapping phenotypes of CODAS and EVEN-PLUS delineate a family of "mitochondrial chaperonopathies" and point to an unexplored role of mitochondrial chaperones in human embryonic morphogenesis.

Mitochondrial disease in Southwestern Finland. Population-based molecular genetic and clinical studies

Mitochondria are present in all eukaryotic cells. They enable these cells utilize oxygen in the production of adenosine triphosphate in the oxidative phosphorylation system, the mitochondrial respiratory chain. The concept ‘mitochondrial disease’ conventionally refers to disorders of the respiratory chain that lead to oxidative phosphorylation defect. Mitochondrial disease in humans can present at any age, and practically in any organ system. Mitochondrial disease can be inherited in maternal, autosomal dominant, autosomal recessive, or X-chromosomal fashion. One of the most common molecular etiologies of mitochondrial disease in population is the m.3243A>G mutation in the MT-TL1 gene, encoding mitochondrial tRNALeu(UUR). Clinical evaluation of patients with m.3243A>G has revealed various typical clinical features, such as stroke-like episodes, diabetes mellitus and sensorineural hearing loss. The prevalence and clinical characteristics of mitochondrial disease in population are not well known. This thesis consists of a series of studies, in which the prevalence and characteristics of mitochondrial disease in the adult population of Southwestern Finland were assessed. Mitochondrial haplogroup Uk was associated with increased risk of occipital ischemic stroke among young women. Large-scale mitochondrial DNA deletions and mutations of the POLG1 gene were the most common molecular etiologies of progressive external ophthalmoplegia. Around 1% of diabetes mellitus emerging between the ages 18 – 45 years was associated with the m.3243A>G mutation. Moreover, among these young diabetic patients, mitochondrial haplogroup U was associated with maternal family history of diabetes. These studies demonstrate the usefulness of carefully planned molecular epidemiological investigations in the study of mitochondrial disorders.

Proteomic analysis of cytosolic proteins associated with petite mutations in Candida glabrata

The incidence of superficial or deep-seated infections due to Candida glabrata has increased markedly, probably because of the low intrinsic susceptibility of this microorganism to azole antifungals and its relatively high propensity to acquire azole resistance. To determine changes in the C. glabrata proteome associated with petite mutations, cytosolic extracts from an azole-resistant petite mutant of C. glabrata induced by exposure to ethidium bromide, and from its azole-susceptible parent isolate were compared by two-dimensional polyacrylamide gel electrophoresis. Proteins of interest were identified by peptide mass fingerprinting or sequence tagging using a matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometer. Tryptic peptides from a total of 160 Coomassie-positive spots were analyzed for each strain. Sixty-five different proteins were identified in the cytosolic extracts of the parent strain and 58 in the petite mutant. Among the proteins identified, 10 were higher in the mutant strain, whereas 23 were lower compared to the parent strain. The results revealed a significant decrease in the enzymes associated with the metabolic rate of mutant cells such as aconitase, transaldolase, and pyruvate kinase, and changes in the levels of specific heat shock proteins. Moreover, transketolase, aconitase and catalase activity measurements decreased significantly in the ethidium bromide-induced petite mutant. These data may be useful for designing experiments to obtain a better understanding of the nuclear response to impairment of mitochondrial function associated with this mutation in C. glabrata.

Significance of DNA base excision repair in the maintenance of mitochondrial function in Saccharomyces cerevisiae /

Mitochondria have an important role in cell metabolism, being the major site of ATP production via oxidative phosphorylation (OXPHOS). Accumulation of mtDNA mutations have been linked to the development of respiratory dysfunction, apoptosis, and aging. Base excision repair (BER) is the major and the only certain repair pathway existing in mitochondria that is in responsible for removing and repairing various base modifications as well as abasic sites (AP sites). In this research, Saccharomyces cerevisiae (S. cerevisiae) BER gene knockout strains, including 3 single DNA glycosylase gene knockout strains and Ap endonuclease (Apn 1 p) knockout strain were used to examine the importance of this DNA repair pathway to the maintenance of respiratory function. Here, I show that individual DNA glycosylases are nonessential in maintenance of normal function in yeast mitochondria, corroborating with previous research in mammalian experimental models. The yeast strain lacking Apn 1 p activity exhibits respiratory deficits, including inefficient and significantly low intracellular ATP level, which maybe due to partial uncoupling of OXPHOS. Growth of this yeast strain on respiratory medium is inhibited, but no evidence was found for increased ROS level in Apn 1 p mitochondria. This strain also shows an increased cell size, and this observation combined with an uncoupled OXPHOS may indicate a premature aging in the Apnlp knockout strain, but more evidence is needed to support this hypothesis. However, the BER is necessary for maintenance of mitochondrial function in respiring S.cerevisiae.

Heteroplasmy in mammal mitochondrial deoxyribonucleic acid

La nature a développé diverses stratégies afin d’assurer le commencement de la vie dans des conditions d’homoplasmie, c’est-à-dire des conditions telles que les cellules sont dotées du même ADN mitochondrial. Toutefois, des nouveaux haplotypes de l’acide désoxyribonucléique mitochondrial (ADNmt) peuvent apparaitre et croître de plusieurs façons tout au long de la durée d’une vie menant à l’hétéroplasmie. Par exemple, l’hétéroplasmie de l’ADNmt peut être créée artificiellement par des technologies reproductives assistées, ainsi que naturellement par le processus de vieillissement. De ce fait, la thèse de ce doctorat fut divisée en deux principaux objectifs. Le premier étant celui d’analyser les changements survenus dans l’hétéroplasmie de l’ADNmt produit par le transfert nucléaire des cellules somatiques (SCNT) lors du développement de l’embryon jusqu’au fœtus et aux tissus adultes de bovins clonés. En ce qui concerne le second objectif, il s’agit d’analyser les changements survenus dans l’hétéroplasmie de l’ADNmt causés par le vieillissement dans une cellule somatique adulte et dans des tissus germinaux durant l’ovogénèse, ainsi qu’au début de l’embryogenèse et dans la procédure de culture in vitro sur des souris. Dans la première série d’expériences sur des bovins, des fibroblastes fœtaux transportant une mutation d’ADNmt (insertion de 66 pb) furent fusionnés avec des ovocytes receveurs transportant l’ADNmt du type sauvage. La présence d’ADNmt venant de la cellule donneuse a été analysée à différents stades de développement, soit sur des embryons âgés de 17 jours (n=17), des fœtus âgés de 40 jours (n=3), des fœtus âgés de 60 jours (n=3), un fœtus âgé de 240 jours et 3 clones post-nataux âgés de 18 à 24 mois. Chaque individu s’est avéré être hétéroplasmique et 99 % (103/104) des échantillons de tissus analysés étaient également hétéroplasmiques. Cependant, l’ovaire venant du fœtus de 240 jours fut le seul à être homoplasmique pour l’ADNmt de l’ovocyte receveur. Dans la plupart des échantillons analysés (95,2 %, soit 99/104) la moyenne d’hétéroplasmie était de 1,46 %. Par contre, un fœtus âgé de 40 jours a présenté un niveau élevé d’hétéroplasmie (20,9 %), indiquant ainsi que des évènements rares d’augmentation de l’ADNmt des cellules donneuses peuvent survenir. Étant donné que la majorité des clones SCNT montrait de l’hétéroplasmie de l’ADNmt à des proportions comparables à celles des cellules donneuses au moment de la reconstruction de l’embryon, on a pu conclure que l’hétéroplasmie produite par des techniques de transfert nucléaire utilisant des cellules somatiques est due à une ségrégation neutre de l’ADNmt. Dans la seconde série d’expériences sur des souris, des femelles de différents âges, c.à.d. jeunes (0 – 8 mois), moyennes (8 – 16 mois) et vieilles (16 – 24 mois), ont été synchronisées (gonadotrophines) et sacrifiées dans le but d’obtenir des ovocytes au stade de vésicule germinal, et des ovocytes au stade métaphase-II produits in vivo et in vitro. De plus, des embryons in vivo et in vitro au stade de deux-cellules et des embryons au stade de blastocystes ont été obtenus de femelles jeunes. Différents tissus somatiques, venant de femelles des trois stades d’âge ont été obtenus : cerveau, foie, muscle et du cumulus ovocytaire. De plus, l’effet du vieillissement a été mesuré selon la fertilité de la femelle. En effet, les effets sur l’hétéroplasmie du vieillissement, du stade de développement et de la culture in vitro ont été mesurés dans des ovocytes et dans des embryons. Les effets du vieillissement sur les mitochondries ont été mesurés par rapport au nombre total de copies de l’ADNmt, au pourcentage des délétions communes et sur l’expression de trois gènes : Ndufs4, Mt-nd2 and Mt-nd4. Il a été possible d’observer que la fertilité des femelles dans la colonie de souris diminuait avec l’âge. En fait, le vieillissement affectait l’ADNmt dans les tissus somatiques, cependant il n’avait pas d’effet sur le cumulus, les ovocytes et les embryons. Le nombre de délétions de l’ADNmt augmentait pendant la reprise de la méiose et celui-ci diminuait au début du développement embryonnaire. La culture in vitro n’affectait pas la quantité d’ADNmt dans la plupart des tissus germinaux. Puisque nous n’avons pas trouvé d’effet de l’âge dans la majorité des paramètres mitochondriaux analysés dans les ovocytes et les embryons, il est suggéré que la délétion commune de l’ADNmt dans les tissus germinaux est davantage reliée au statut cellulaire de la production d’énergie qu’au processus de vieillissement. Deux sources différentes de mutations de l’ADNmt produites dans les ovocytes normaux ou reconstitués ont produit différents résultats d’hétéroplasmie au début de l’embryogénèse. Chez les bovins, l’hétéroplasmie artificielle impliquant une petite insertion (66 pb) dans la région non codante (D-loop) de l’ADNmt a été vraisemblablement non nocive pour l’embryon, tolérant la persistance de l’ADNmt étranger pendant les différents stades du développement des clones. Chez les souris, l’hétéroplasmie naturelle produite par une grande délétion (4974 pb délétion commune) dans la région codante de l’ADNmt a été vraisemblablement nocive pour l’embryon et par conséquent éliminée pour assurer l’homoplasmie au début du développement embryonnaire.