BBA - Bioenergetics (v.1659, #2-3)

Keywords: Euromit; Eumitocombat; 6th Framework programme; Mitochondrial medicine; Mitochondrial disease; Oxidative phosphorylation disorders; European; Mitochondrial DNA; Nuclear DNA; Complex I; Complex IV;

Mitochondrial medicine by Salvatore DiMauro (107-114).
After reviewing the history of mitochondrial diseases, I follow a genetic classification to discuss new developments and old conundrums.In the field of mitochondrial DNA (mtDNA) mutations, I argue that we are not yet scraping the bottom of the barrel because: (i) new mtDNA mutations are still being discovered, especially in protein-coding genes; (ii) the pathogenicity of homoplasmic mutations is being revisited; (iii) some genetic dogmas are chipped but not broken; (iv) mtDNA haplotypes are gaining interest in human pathology; (v) pathogenesis is still largely enigmatic.In the field of nuclear DNA (nDNA) mutations, there has been good progress in our understanding of disorders due to faulty intergenomic communication. Of the genes responsible for multiple deletions and depletion of mtDNA, mutations in POLG have been associated with a great variety of clinical phenotypes in humans and to precocious aging in mice. Novel pathogenetic mechanisms include alterations in the lipid milieu of the inner mitochondrial membrane and mutations in genes controlling mitochondrial motility, fission, and fusion.

The epidemiology of mitochondrial disorders—past, present and future by Andrew M. Schaefer; Robert W. Taylor; Douglass M. Turnbull; Patrick F. Chinnery (115-120).
A number of epidemiological studies of mitochondrial disease have been carried out over the last decade, clearly demonstrating that mitochondrial disorders are far more common than was previously accepted. This review summarizes current knowledge of the prevalence of human mitochondrial disorders—data that has important implications for the provision of health care and adequate resources for research into the pathogenesis and treatment of these disorders.
Keywords: Mitochondria; Mitochondrial disease; mtDNA; Epidemiology; Mitochondrial encephalomyopathy;

Biochemical and molecular diagnosis of mitochondrial respiratory chain disorders by David R. Thorburn; Canny Sugiana; Renato Salemi; Denise M. Kirby; Lisa Worgan; Akira Ohtake; Michael T. Ryan (121-128).
Biochemical diagnosis of mitochondrial respiratory chain disorders requires caution to avoid misdiagnosis of secondary enzyme defects, and can be improved by the use of conservative diagnostic criteria. Pathogenic mutations causing mitochondrial disorders have now been identified in more than 30 mitochondrial DNA (mtDNA) genes encoding respiratory chain subunits, ribosomal- and t-RNAs. mtDNA mutations appear to be responsible for most adult patients with mitochondrial disease and approximately a quarter of paediatric patients. A family history suggesting maternal inheritance is the exception rather than the norm for children with mtDNA mutations, many of whom have de novo mutations. Prenatal diagnosis and pre-implantation genetic diagnosis can be offered to some women at risk of transmitting a mtDNA mutation, particularly those at lower recurrence risk. Mutations in more than 30 nuclear genes, including those encoding for respiratory chain subunits and assembly factors, have now been shown to cause mitochondrial disorders, creating difficulties in prioritising which genes should be studied by mutation analysis in individual patients. A number of approaches offer promise to guide the choice of candidate genes, including Blue Native-PAGE immunoblotting and microarray expression analysis.
Keywords: Mitochondrial diseases; Respiratory chain; Mutation; mtDNA; Assembly; Microarray;

Molecular diagnostics of mitochondrial disorders by Agnès Rötig; Sophie Lebon; Elena Zinovieva; Julie Mollet; Emmanuelle Sarzi; Jean-Paul Bonnefont; Arnold Munnich (129-135).
The mitochondrial respiratory chain (RC) results from the expression of both mitochondrial and nuclear genes. The number of disease-causing mutations in nuclear genes is steadily growing and mitochondrial DNA (mtDNA) deletions and mutations account for no more than 15–20% of pediatric patients. Unfortunately, the disease-causing mutations have been identified for only a small number of patients. Thus, elucidating the genetic bases of RC is both essential for genetic diagnosis of patients and for fundamental knowledge of these disorders. The molecular diagnostics of mitochondrial disorders come under both genetic diagnosis and research. Indeed, identification of a new gene in a specific patient allows to perform genetic diagnosis in other families and identification of mutations in already known disease-causing genes allows to constitute a cohort of patients for further functional studies. Thus, elucidating the genetic bases of RC deficiency is an essential task that needs the use of several appropriate strategies. Fine phenotypage of patients and candidate gene screening is the first step for the constitution of a well-characterized cohort of patients. Genetic mapping has to be used in large families. This approach is greatly enhanced in the case of consanguineous families. The consanguinity of the parents should also lead to test genetic markers surrounding the gene loci rather than to directly sequence several candidate genes. However, the main problem is encountered in the cases of sporadic cases for which no genetic approaches can be developed. In these cases, functional complementation by human chromosomes or cDNA is the only presently available strategy.
Keywords: mtDNA; Nuclear genes; Molecular diagnosis;

Clinical and molecular findings in children with complex I deficiency by M. Bugiani; F. Invernizzi; S. Alberio; E. Briem; E. Lamantea; F. Carrara; I. Moroni; L. Farina; M. Spada; M.A. Donati; G. Uziel; M. Zeviani (136-147).
Isolated complex I deficiency, the most frequent OXPHOS disorder in infants and children, is genetically heterogeneous. Mutations have been found in seven mitochondrial DNA (mtDNA) and eight nuclear DNA encoded subunits, respectively, but in most of the cases the genetic basis of the biochemical defect is unknown. We analyzed the entire mtDNA and 11 nuclear encoded complex I subunits in 23 isolated complex I-deficient children, classified into five clinical groups: Leigh syndrome, progressive leukoencephalopathy, neonatal cardiomyopathy, severe infantile lactic acidosis, and a miscellaneous group of unspecified encephalomyopathies. A genetic definition was reached in eight patients (35%). Mutations in mtDNA were found in six out of eight children with Leigh syndrome, indicating a prevalent association between this phenotype and abnormalities in ND genes. In two patients with leukoencephalopathy, homozygous mutations were detected in two different nuclear-encoded complex I genes, including a novel transition in NDUFS1 subunit. In addition to these, a child affected by mitochondrial encephalomyopathy had heterozygous mutations in NDUFA8 and NDUFS2 genes, while another child with neonatal cardiomyopathy had a complex rearrangement in a single NDUFS7 allele. The latter cases suggest the possibility of unconventional patterns of inheritance in complex I defects.
Keywords: Mitochondrial disorder; Children; Complex I deficiency; mtDNA mutation; Nuclear DNA mutation;

The transcription machinery in mammalian mitochondria by Martina Gaspari; Nils-Göran Larsson; Claes M. Gustafsson (148-152).
Initiation of transcription at mitochondrial promoters in mammalian cells requires the simultaneous presence of a monomeric mitochondrial RNA polymerase, mitochondrial transcription factor A, and either transcription factor B1 or B2. We here review recent progress in our understanding of how these basal factors cooperate in the initiation and regulation of mitochondrial transcription. We describe the evolutionary origin of individual transcription factors and discuss how these phylogenetic relationships may facilitate a molecular understanding of the mitochondrial transcription machinery.
Keywords: POLRMT; TFAM; TFB2M; Mitochondrion; Transcription;

Defects in the biosynthesis of mitochondrial heme c and heme a in yeast and mammals by Carlos T. Moraes; Franscisca Diaz; Antoni Barrientos (153-159).
Defects in heme biosynthesis have been associated with a large number of diseases, but mostly recognized in porphyrias, which are neurovisceral or cutaneous disorders caused by the accumulation of biosynthetic intermediates. However, defects in the maturation of heme groups that are part of the oxidative phosphorylation system are now also recognized as important causes of disease. The electron transport chain contains heme groups of the types a, b and c, all of which are directly involved in electron transfer reactions. In this article, we review the effect of mutations in enzymes involved in the maturation of heme a (the prosthetic group of cytochrome c oxidase) and heme c (the prosthetic group of cytochrome c) both in yeast and in humans. COX10 and COX15 are two genes, initially identified in Saccharomyces cerevisiae that have been found to cause infantile cytochrome c oxidase deficiency in humans. They participate in the farnesylation and hydroxylation of heme b, steps that are necessary for the formation of heme a, the prosthetic group required for cytochrome oxidase assembly and activity. Deletion of the cytochrome c heme lyase gene in a single allele has also been associated with a human disease, known as Microphthalmia with Linear Skin defects (MLS) syndrome. The cytochrome c heme lyase is necessary to covalently attach the heme group to the apocytochrome c polypeptide. The production of mouse models recapitulating these diseases is providing novel information on the pathogenesis of clinical syndromes.
Keywords: Heme biosynthesis; Yeast; Mammal;

Molecular genetics of complex I-deficient Chinese hamster cell lines by Immo E. Scheffler; Nagendra Yadava; Prasanth Potluri (160-171).
The work from our laboratory on complex I-deficient Chinese hamster cell mutants is reviewed. Several complementation groups with a complete defect have been identified. Three of these are due to X-linked mutations, and the mutated genes for two have been identified. We describe null mutants in the genes for the subunits MWFE (gene: NDUFA1) and ESSS. They represent small integral membrane proteins localized in the Iα (Iγ) and Iβ subcomplexes, respectively [J. Hirst, J. Carroll, I.M. Fearnley, R.J. Shannon, J.E. Walker. The nuclear encoded subunits of complex I from bovine heart mitochondria. Biochim. Biophys. Acta 1604 (7-10-2003) 135–150.]. Both are absolutely essential for assembly and activity of complex I. Epitope-tagged versions of these proteins can be expressed from a poly-cistronic vector to complement the mutants, or to be co-expressed with the endogenous proteins in other hamster cell lines (mutant or wild type), or human cells. Structure–function analyses can be performed with proteins altered by site-directed mutagenesis. A cell line has been constructed in which the MWFE subunit is conditionally expressed, opening a window on the kinetics of assembly of complex I. Its targeting, import into mitochondria, and orientation in the inner membrane have also been investigated. The two proteins have recently been shown to be the targets for a cAMP-dependent kinase [R. Chen, I.M. Fearnley, S.Y. Peak_Chew, J.E. Walker. The phosphorylation of subunits of complex I from bovine heart mitochondria. J. Biol. Chem. xx (2004) xx–xx.]. The epitope-tagged proteins can be cross-linked with other complex I subunits.
Keywords: Mitochondria; Complex I; Respiration-deficient mammalian cell;

Respiratory chain defects: what do we know for sure about their consequences in vivo? by Jean-Jacques Brière; Dominique Chrétien; Paule Bénit; Pierre Rustin (172-177).
The function and the structure of mitochondria have been the subject of intensive research since the discovery of these organelles. Yet, the investigation of patients with mitochondrial disease reveals that we do not understand a large part of the underlying pathogenic processes. This has disastrous consequences in terms of the therapy possibly proposed to the patients and their family. An attempt is made in this short review to question our present ideas on the potential consequences of mitochondrial dysfunctions and to enlighten new observations which might be valuable in the understanding of the physiopathology of these diseases.
Keywords: Mitochondrial disease; Metabolism; Respiratory chain; ATP; Succinate; Superoxide;

Viral proteins targeting mitochondria: controlling cell death by Patricia Boya; Anne-Laure Pauleau; Delphine Poncet; Rosa-Ana Gonzalez-Polo; Naoufal Zamzami; Guido Kroemer (178-189).
Mitochondrial membrane permeabilization (MMP) is a critical step regulating apoptosis. Viruses have evolved multiple strategies to modulate apoptosis for their own benefit. Thus, many viruses code for proteins that act on mitochondria and control apoptosis of infected cells. Viral proapoptotic proteins translocate to mitochondrial membranes and induce MMP, which is often accompanied by mitochondrial swelling and fragmentation. From a structural point of view, all the viral proapoptotic proteins discovered so far contain amphipathic α-helices that are necessary for the proapoptotic effects and seem to have pore-forming properties, as it has been shown for Vpr from human immunodeficiency virus-1 (HIV-1) and HBx from hepatitis B virus (HBV). In contrast, antiapoptotic viral proteins (e.g., M11L from myxoma virus, F1L from vaccinia virus and BHRF1 from Epstein–Barr virus) contain mitochondrial targeting sequences (MTS) in their C-terminus that are homologous to tail-anchoring domains. These domains are similar to those present in many proteins of the Bcl-2 family and are responsible for inserting the protein in the outer mitochondrial membrane leaving the N-terminus of the protein facing the cytosol. The antiapoptotic proteins K7 and K15 from avian encephalomyelitis virus (AEV) and viral mitochondria inhibitor of apoptosis (vMIA) from cytomegalovirus are capable of binding host-specific apoptosis-modulatory proteins such as Bax, Bcl-2, activated caspase 3, CAML, CIDE-B and HAX. In conclusion, viruses modulate apoptosis at the mitochondrial level by multiple different strategies.
Keywords: Viral protein; Apoptosis; Mitochondrial targeting sequence; Mitochondrial membrane permeabilization; Amphipathic α-helix; C-terminal anchoring domain;

Mitochondrial disease in flies by Howard T. Jacobs; Daniel J.M. Fernández-Ayala; Shweta Manjiry; Esko Kemppainen; Janne M. Toivonen; Kevin M.C. O'Dell (190-196).
The Drosophila mutant technical knockout (tko), affecting the mitochondrial protein synthetic apparatus, exhibits respiratory chain deficiency and a phenotype resembling various features of mitochondrial disease in humans (paralytic seizures, deafness, developmental retardation). We are using this mutant to analyse the cellular and genomic targets of mitochondrial dysfunction, and to identify ways in which the phenotype can be alleviated. Transgenic expression of wild-type tko in different patterns in the mutant background reveals critical times and cell-types for production of components of the mitochondrial disease-like phenotype. Mitochondrial bioenergy deficit during the period of maximal growth, as well as in specific parts of the nervous system, appears to be most deleterious. Inbreeding of tko mutant lines results in a systematic improvement in all phenotypic parameters tested. The resulting sub-lines can be used for genetic mapping and transcriptomic analysis, revealing clues as to the genes and pathways that can modify mitochondrial disease-like phenotypes in a model metazoan.
Keywords: Drosophila; Transcriptomics; Transgene; Protein synthesis; Technical knockout;

While diagnosis and genetic analysis of mitochondrial disorders has made remarkable progress, we still do not understand how given molecular defects are correlated to specific patterns of symptoms and their severity. Towards resolving this dilemma for the largest and therefore most affected respiratory chain enzyme, we have established the yeast Yarrowia lipolytica as a eucaryotic model system to analyse respiratory chain complex I. For in vivo analysis, eYFP protein was attached to the 30-kDa subunit to visualize complex I and mitochondria. Deletions strains for nuclear coded subunits allow the reconstruction of patient alleles by site-directed mutagenesis and plasmid complementation. In most of the pathogenic mutations analysed so far, decreased catalytic activities, elevated K M values, and/or elevated I 50 values for quinone-analogous inhibitors were observed, providing plausible clues on the pathogenic process at the molecular level. Leigh mutations in the 49-kDa and PSST homologous subunits are found in regions that are at the boundaries of the ubiquinone-reducing catalytic core. This supports the proposed structural model and at the same time identifies novel domains critical for catalysis. Thus, Y. lipolytica is a useful lower eucaryotic model that will help to understand how pathogenic mutations in complex I interfere with enzyme function.
Keywords: Yarrowia lipolytica; Mutation; Complex I;

Focused proteomics: towards a high throughput monoclonal antibody-based resolution of proteins for diagnosis of mitochondrial diseases by James Murray; Sky Yonally; Robert Aggeler; Michael F. Marusich; Roderick A. Capaldi (206-211).
The availability of monoclonal antibodies (mAbs) against the proteins of the oxidative phosphorylation chain (OXPHOS) and other mitochondrial components facilitates the analysis and ultimately the diagnosis of mitochondrially related diseases. mAbs against each of the five complexes and pyruvate dehydrogenase (PDH) are the basis of a rapid and simple immunocytochemical approach [Hanson, B.J., Capaldi, R.A., Marusich, M.F. and Sherwood, S.W., J. Histochem. Cytochem. 50 (2002) 1281–1288]. This approach can be used to detect if complexes have altered assembly in mitochondrial disease due to mutations in nuclear encoded genes, such as in Leigh's disease, or in mitochondrially encoded genes, e.g., MELAS. Other mAbs have recently been obtained that can immunocapture each of the five OXPHOS complexes, PDH and the adenine nucleotide translocase (ANT) from very small amounts of tissue such as that obtained from cell culture or needle biopsies from patients. When adapted to a 96-well plate format, these mAbs allow measurement of the specific activity of each of the mitochondrial components individually and analysis of their subunit composition and state of posttranslational modification. The immunocapture protocol should be useful not only in the analysis of genetic mitochondrial diseases but also in evaluating and ultimately diagnosing late-onset mitochondrial disorders including Parkinson's disease, Alzheimer's disease, and late-onset diabetes, which are thought to result from accumulated oxidative damage to mitochondrial proteins such as the OXPHOS chain.
Keywords: OXPHOS; Respiratory chain; Complex; I; II; III; IV; V; ANT; Mitochondria; Monoclonal antibody; Immunoprecipitation;

Shaping the mitochondrial proteome by Toni Gabaldón; Martijn A. Huynen (212-220).
Mitochondria are eukaryotic organelles that originated from a single bacterial endosymbiosis some 2 billion years ago. The transition from the ancestral endosymbiont to the modern mitochondrion has been accompanied by major changes in its protein content, the so-called proteome. These changes included complete loss of some bacterial pathways, amelioration of others and gain of completely new complexes of eukaryotic origin such as the ATP/ADP translocase and most of the mitochondrial protein import machinery. This renewal of proteins has been so extensive that only 14–16% of modern mitochondrial proteome has an origin that can be traced back to the bacterial endosymbiont. The rest consists of proteins of diverse origin that were eventually recruited to function in the organelle. This shaping of the proteome content reflects the transformation of mitochondria into a highly specialized organelle that, besides ATP production, comprises a variety of functions within the eukaryotic metabolism. Here we review recent advances in the fields of comparative genomics and proteomics that are throwing light on the origin and evolution of the mitochondrial proteome.
Keywords: Mitochondria; Mitochondrial evolution; Alpha-proteobacteria; Proteome; Endosymbiosis;

Implications of exercise training in mtDNA defects—use it or lose it? by Tanja Taivassalo; Ronald G. Haller (221-231).
Whether regular exercise is beneficial or should be avoided is a question currently unsettled in patients with heteroplasmic mitochondrial DNA (mtDNA) disorders of skeletal muscle. Deleterious effects of habitual physical inactivity superimposed upon impaired mitochondrial oxidative phosphorylation may contribute to varying degrees of exercise intolerance in these patients. Endurance exercise training is widely known to improve exercise capacity in healthy subjects and various chronic-disease patient populations. Although we have shown that beneficial physiological and biochemical responses to training increase exercise tolerance in patients with mtDNA defects, knowledge of the muscle adaptive response to endurance training within the setting of mitochondrial heteroplasmy remains limited. In order to determine advisability of endurance training as therapy, it remains to be established whether potential endurance training-induced increases in mutant mtDNA levels may be offset by increases in absolute wild-type mtDNA levels, and whether chronic inactivity leads to a selective down-regulation of wild-type mtDNA. Resistance training utilizes a different adaptive exercise approach to induce the transfer of normal mitochondrial templates from satellite cells to mature muscle fibers of patients with sporadic mtDNA disorders. The efficacy and safety of this approach needs to be further established. Our current inability to clearly advise patients to “use it or lose it” underscores the immediate urgency of studying the effects of exercise on skeletal muscle of patients with heteroplasmic mtDNA defects.
Keywords: Mitochondrial myopathy; mtDNA defect; Exercise intolerance; Treatment; Endurance and resistance training; Satellite cell;

Strategies for treating disorders of the mitochondrial genome by Paul M. Smith; Günther F. Ross; Robert W. Taylor; Douglass M. Turnbull; Robert N. Lightowlers (232-239).
Defects of the mitochondrial genome are a significant cause of disease. Patients suffer from a wide variety of clinical presentations, ranging from fatal infantile disease to mild muscle weakness. Most disorders, however, are characterized by inexorable progression. As mutations often cause defects in several components of the complexes that couple oxidative phosphorylation, this terminal state of oxidative metabolism cannot be readily bypassed by dietary means, leading to the search for novel therapies. In this article, we present the theory behind several concepts and report progress. We also discuss some of the recent difficulties encountered in the progress towards an antigenomc approach to treating mtDNA disorders.
Keywords: Antigenomic hypothesis; mtDNA disease; Treatment of mtDNA disease; Alloptic expression; Mithocondrial tRNA import;

Author Index (240-241).

Cumulative Contents (242-243).