A NOVEL MUTATION IN BCS1L IN A PATIENT WITH AN ISOLATED MITOCHONDRIAL COMPLEX III DEFICIENCY

Background: Isolated complex III deficiencies are caused by mutations in the mitochondrial CytB gene, in the BCS1L gene coding for a CIII assembly factor and in the UQCRQ gene that codes for the ubiquinone binding protein of complex III. Objective: Description of clinical features, mitochondrial function and molecular genetic analysis in a patient with an isolated complex III deficiency. Patient: A 17 year old boy, born to consanguineous parents who presented with hypoglycemia, glycosuria, deafness, growth retardation, Fanconi Syndrome and severe lactic acidosis in the neonatal period. Methods: Activities and assembly of OXPHOS complexes were investigated spectrophotometrically and by BN-PAGE. mt-DNAwas screened for deletions. Cytochrome b (CytB) and the BCS1L gene were sequenced. Results: Isolated complex III deficiency was detected in the patient's skeletal muscle. Using BN-PAGE blotting a complex III of lower molecular weight was detected. Staining the 2D reveals a missing subunit. No mutation was detected in the mitochondrial CytB gene. Sequence analysis of BCS1L revealed a novel homozygous point mutation p.M48V. Conclusion: The patients decreased complex III activity is most likely caused by incomplete assembly of complex III due to the homozygous p. M48V mutation in the BCS1L gene.

Repercussion of a deficiency in mitochondrial ß-oxidation on the carbon flux of short-chain fatty acids to the peroxisomal ß-oxidation cycle in Aspergillus nidulans.

The fungus Aspergillus nidulans contains both a mitochondrial and peroxisomal ß-oxidation pathway. This work was aimed at studying the influence of mutations in the foxA gene, encoding a peroxisomal multifunctional protein, or in the scdA/echA genes, encoding a mitochondrial short-chain dehydrogenase and an enoyl-CoA hydratase, respectively, on the carbon flux to the peroxisomal ß-oxidation pathway. A. nidulans transformed with a peroxisomal polyhydroxyalkanoate (PHA) synthase produced PHA from the polymerization of 3-hydroxyacyl-CoA intermediates derived from the peroxisomal ß-oxidation of external fatty acids. PHA produced from erucic acid or heptadecanoic acid contained a broad spectrum of monomers, ranging from 5 to 14 carbons, revealing that the peroxisomal ß-oxidation cycle can handle both long and short-chain intermediates. While the ∆foxA mutant grown on erucic acid or oleic acid synthesized 10-fold less PHA compared to wild type, the same mutant grown on octanoic acid or heptanoic acid produced 3- to 6-fold more PHA. Thus, while FoxA has an important contribution to the degradation of long-chain fatty acids, the flux of short-chain fatty acids to peroxisomal ß-oxidation is actually enhanced in its absence. While no change in PHA was observed in the ∆scdA∆echA mutant grown on erucic acid or oleic acid compared to wild type, there was a 2- to 4-fold increased synthesis of PHA in ∆scdA∆echA cells grown in octanoic acid or heptanoic acid. These results reveal that a compensatory mechanism exists in A. nidulans that increases the flux of short-chain fatty acids towards the peroxisomal ß-oxidation cycle when the mitochondrial ß-oxidation pathway is defective.

The mitochondrial targeting function of randomly generated peptide sequences correlates with predicted helical amphiphilicity.

A pool of oligonucleotides encoding a start methionine and nine random amino acids was inserted at the 5'-end of the gene for the yeast cytochrome oxidase subunit IV lacking its own mitochondrial targeting sequence. Approximately one-quarter of the randomly generated sequences targeted subunit IV to its correct intramitochondrial location in vivo. Sequence analysis of 89 randomly generated sequences showed that their efficiencies as mitochondrial targeting signals correlated with the potential to fold into an amphiphilic alpha-helix. Functional targeting sequences were enriched in arginine and isoleucine residues but contained few aspartate, glutamate, and proline residues. Nonfunctional sequences predicted to have significant helical amphiphilicity often had at least one acidic or multiple helix-breaking residues that would be expected to interfere with targeting functioning. These results support the hypothesis that the signal for targeting a protein into the mitochondrial matrix is usually a positively charged amphiphilic helix.

Molecular phylogeny and evolution of Sorex shrews (Soricidae: insectivora) inferred from mitochondrial DNA sequence data.

Shrews of the genus Sorex are characterized by a Holarctic distribution, and relationships among extant taxa have never been fully resolved. Phylogenies have been proposed based on morphological, karyological, and biochemical comparisons, but these analyses often produced controversial and contradictory results. Phylogenetic analyses of partial mitochondrial cytochrome b gene sequences (1011 bp) were used to examine the relationships among 27 Sorex species. The molecular data suggest that Sorex comprises two major monophyletic lineages, one restricted mostly to the New World and one with a primarily Palearctic distribution. Furthermore, several sister-species relationships are revealed by the analysis. Based on the split between the Soricinae and Crocidurinae subfamilies, we used a 95% confidence interval for both the calibration of a molecular clock and the subsequent calculation of major diversification events within the genus Sorex. Our analysis does not support an unambiguous acceleration of the molecular clock in shrews, the estimated rate being similar to other estimates of mammalian mitochondrial clocks. In addition, the data presented here indicate that estimates from the fossil record greatly underestimate divergence dates among Sorex taxa.

Forensic identification of urine samples: a comparison between nuclear and mitochondrial DNA markers.

Urine samples from 20 male volunteers of European Caucasian origin were stored at 4 degrees C over a 4-month period in order to compare the identification potential of nuclear DNA (nDNA) and mitochondrial DNA (mtDNA) markers. The amount of nDNA recovered from urines dramatically declined over time. Consequently, nDNA likelihood ratios (LRs) greater than 1,000 were obtained for 100, 70 and 55% of the urines analysed after 6, 60 and 120 days, respectively. For the mtDNA, HVI and HVII sequences were obtained for all samples tested, whatever the period considered. Nevertheless, the highest mtDNA LR of 435 was relatively low compared to its nDNA equivalent. Indeed, LRs obtained with only three nDNA loci could easily exceed this value and are quite easier to obtain. Overall, the joint use of nDNA and mtDNA markers enabled the 20 urine samples to be identified, even after the 4-month period.

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.

Multilayered genetic and omics dissection of mitochondrial activity in a mouse reference population

The manner by which genotype and environment affect complex phenotypes is one of the fundamental questions in biology. In this study, we quantified the transcriptome--a subset of the metabolome--and, using targeted proteomics, quantified a subset of the liver proteome from 40 strains of the BXD mouse genetic reference population on two diverse diets. We discovered dozens of transcript, protein, and metabolite QTLs, several of which linked to metabolic phenotypes. Most prominently, Dhtkd1 was identified as a primary regulator of 2-aminoadipate, explaining variance in fasted glucose and diabetes status in both mice and humans. These integrated molecular profiles also allowed further characterization of complex pathways, particularly the mitochondrial unfolded protein response (UPR(mt)). UPR(mt) shows strikingly variant responses at the transcript and protein level that are remarkably conserved among C. elegans, mice, and humans. Overall, these examples demonstrate the value of an integrated multilayered omics approach to characterize complex metabolic phenotypes.

The biogeography of mitochondrial and nuclear discordance in animals.

Combining nuclear (nuDNA) and mitochondrial DNA (mtDNA) markers has improved the power of molecular data to test phylogenetic and phylogeographic hypotheses and has highlighted the limitations of studies using only mtDNA markers. In fact, in the past decade, many conflicting geographic patterns between mitochondrial and nuclear genetic markers have been identified (i.e. mito-nuclear discordance). Our goals in this synthesis are to: (i) review known cases of mito-nuclear discordance in animal systems, (ii) to summarize the biogeographic patterns in each instance and (iii) to identify common drivers of discordance in various groups. In total, we identified 126 cases in animal systems with strong evidence of discordance between the biogeographic patterns obtained from mitochondrial DNA and those observed in the nuclear genome. In most cases, these patterns are attributed to adaptive introgression of mtDNA, demographic disparities and sex-biased asymmetries, with some studies also implicating hybrid zone movement, human introductions and Wolbachia infection in insects. We also discuss situations where divergent mtDNA clades seem to have arisen in the absence of geographic isolation. For those cases where foreign mtDNA haplotypes are found deep within the range of a second taxon, data suggest that those mtDNA haplotypes are more likely to be at a high frequency and are commonly driven by sex-biased asymmetries and/or adaptive introgression. In addition, we discuss the problems with inferring the processes causing discordance from biogeographic patterns that are common in many studies. In many cases, authors presented more than one explanation for discordant patterns in a given system, which indicates that likely more data are required. Ideally, to resolve this issue, we see important future work shifting focus from documenting the prevalence of mito-nuclear discordance towards testing hypotheses regarding the drivers of discordance. Indeed, there is great potential for certain cases of mitochondrial introgression to become important natural systems within which to test the effect of different mitochondrial genotypes on whole-animal phenotypes.

Mitochondrial DNA (mt DNA) A3243G mutation associated with an annular perimacular retinal atrophy

Résumé:Background:La mutation 3243 de 1'ADN mitochondrial est associee avec le syndrome l\/HDD (surdite, diabète transmis par la mère) et le syndrome MELAS (Myopathie, Encéphalopathie, acidose Lactique et attaques cérébrales). Elle est aussi associe à des troubles cardiaques, digestifs, endo- et exocrines. Nous rapportons deux cas de maculopathie associée à cette mutation.Histoire et symptomes: pCas l: il s'agit d'une femme de 60 ans soufrant d'un diabète et d'une surdité sans plainte visuelle lors de la présentation. Son acuité visuelle était de 10/ l0 des deux yeux.Cas 2: il s'agit d'une femme de 54 ans souffrant d'une surdité et d'un diabète qui se plaint d'une baisse de vision principalement de l'oeil gauche. Son acuité visuelle était de 6/10 pour l'oeil droit et de 0.5/l0 pour l'oeil gauche.Les deux patientes présentaient une atrophie choriorétinienne aréolaire centrale. La patiente 1 a été suivie durant plus de 15 ans. Une évolution lente et progressive de la maculopathie a été observée. Lors de la dernière visite, l'acuité visuelle était de 6/ l0 dans les deux yeux. Elle présentait un handicap marqué des suites du scotome annulaire.Thérapie et pronostic:AucunConclusion:Les deux patientes présentaient une atrophie rétinienne annulaire périmaculaire. Les patients atteints d'une mutation 3243 de l'ADN mitochondrial devraient bénéficier d'un examen du fond d'oeil à la recherche d'une maculopahtie, même s'ils sont asymptomatiques.Inversement, la découverte d'une telle maculopathie géographique devrait suggérer la possibilité d'une mutation au locus 3243 de l'ADN mitochondrial, surtout en présence d'un diabète et/ou d'une surdité.

Mitochondrial uncoupling as a regulator of life-history trajectories in birds: an experimental study in the zebra finch.

Mitochondria have a fundamental role in the transduction of energy from food into ATP. The coupling between food oxidation and ATP production is never perfect, but may nevertheless be of evolutionary significance. The 'uncoupling to survive' hypothesis suggests that 'mild' mitochondrial uncoupling evolved as a protective mechanism against the excessive production of damaging reactive oxygen species (ROS). Because resource allocation and ROS production are thought to shape animal life histories, alternative life-history trajectories might be driven by individual variation in the degree of mitochondrial uncoupling. We tested this hypothesis in a small bird species, the zebra finch (Taeniopygia guttata), by treating adults with the artificial mitochondrial uncoupler 2,4-dinitrophenol (DNP) over a 32-month period. In agreement with our expectations, the uncoupling treatment increased metabolic rate. However, we found no evidence that treated birds enjoyed lower oxidative stress levels or greater survival rates, in contrast to previous results in other taxa. In vitro experiments revealed lower sensitivity of ROS production to DNP in mitochondria isolated from skeletal muscles of zebra finch than mouse. In addition, we found significant reductions in the number of eggs laid and in the inflammatory immune response in treated birds. Altogether, our data suggest that the 'uncoupling to survive' hypothesis may not be applicable for zebra finches, presumably because of lower effects of mitochondrial uncoupling on mitochondrial ROS production in birds than in mammals. Nevertheless, mitochondrial uncoupling appeared to be a potential life-history regulator of traits such as fecundity and immunity at adulthood, even with food supplied ad libitum.

Mfn2 downregulation in excitotoxicity causes mitochondrial dysfunction and delayed neuronal death.

Mitochondrial fusion and fission is a dynamic process critical for the maintenance of mitochondrial function and cell viability. During excitotoxicity neuronal mitochondria are fragmented, but the mechanism underlying this process is poorly understood. Here, we show that Mfn2 is the only member of the mitochondrial fusion/fission machinery whose expression is reduced in in vitro and in vivo models of excitotoxicity. Whereas in cortical primary cultures, Drp1 recruitment to mitochondria plays a primordial role in mitochondrial fragmentation in an early phase that can be reversed once the insult has ceased, Mfn2 downregulation intervenes in a delayed mitochondrial fragmentation phase that progresses even when the insult has ceased. Downregulation of Mfn2 causes mitochondrial dysfunction, altered calcium homeostasis, and enhanced Bax translocation to mitochondria, resulting in delayed neuronal death. We found that transcription factor MEF2 regulates basal Mfn2 expression in neurons and that excitotoxicity-dependent degradation of MEF2 causes Mfn2 downregulation. Thus, Mfn2 reduction is a late event in excitotoxicity and its targeting may help to reduce excitotoxic damage and increase the currently short therapeutic window in stroke.

Control of mitochondrial pH by uncoupling protein 4 in astrocytes promotes neuronal survival.

Brain activity is energetically costly and requires a steady and highly regulated flow of energy equivalents between neural cells. It is believed that a substantial share of cerebral glucose, the major source of energy of the brain, will preferentially be metabolized in astrocytes via aerobic glycolysis. The aim of this study was to evaluate whether uncoupling proteins (UCPs), located in the inner membrane of mitochondria, play a role in setting up the metabolic response pattern of astrocytes. UCPs are believed to mediate the transmembrane transfer of protons, resulting in the uncoupling of oxidative phosphorylation from ATP production. UCPs are therefore potentially important regulators of energy fluxes. The main UCP isoforms expressed in the brain are UCP2, UCP4, and UCP5. We examined in particular the role of UCP4 in neuron-astrocyte metabolic coupling and measured a range of functional metabolic parameters including mitochondrial electrical potential and pH, reactive oxygen species production, NAD/NADH ratio, ATP/ADP ratio, CO2 and lactate production, and oxygen consumption rate. In brief, we found that UCP4 regulates the intramitochondrial pH of astrocytes, which acidifies as a consequence of glutamate uptake, with the main consequence of reducing efficiency of mitochondrial ATP production. The diminished ATP production is effectively compensated by enhancement of glycolysis. This nonoxidative production of energy is not associated with deleterious H2O2 production. We show that astrocytes expressing more UCP4 produced more lactate, which is used as an energy source by neurons, and had the ability to enhance neuronal survival.

Mitochondrial DNA (mtDNA) A 3243G mutation associated with an annular perimacular retinal atrophy

BACKGROUND: A point mutation at the locus 3243 of the mitonchondrial DNA (mtDNA) is associated with either the MIDD syndrome (maternally inherited diabetes, deafness), the MELAS syndrome (myopathy, encephalitis, lactic acidosis, stroke) or cardiac, digestive, endocrine or exocrine dysfunctions. We report a peculiar maculopathy in two patients with an mtDNA 3243 mutation. HISTORY AND SIGNS: Case 1: A visually asymptomatic 40-year-old woman was examined for screening of diabetic retinopathy. Visual acuity was 10 / 10 in both eyes. Case 2: A 54-year-old woman with deafness and diabetes complained of visual loss. Visual acuity was 6 / 10 for the right eye and 0.5 / 10 for the left eye. Both patients exhibited a chorioretinal areolar atrophy. Case 1 was followed over 15 years and exhibited a slow progression of the maculopathy with moderate loss of visual acuity to 6 / 10 in both eyes, but marked handicap from the annular scotoma. THERAPY AND OUTCOME: None. CONCLUSION: Both patients presented a perimacular annular retinal atrophy. Patients harbouring mtDNA 3243 mutation should be examined for the presence of a maculopathy, even if they are asymptomatic. Conversely, the finding of such a geographic maculopathy should suggest the possibility of a point mutation at the locus 3243 of the mitochondrial DNA, especially in the presences of diabetes mellitus and/or deafness