Search

Mitochondrial carriers (MCs) are a superfamily of nuclear-encoded proteins that are mostly localized in the inner mitochondrial membrane and transport numerous metabolites, nucleotides, cofactors and inorganic anions. Their unique sequence features, i.e., a tripartite structure, six transmembrane α-helices and a three-fold repeated signature motif, allow MCs to be easily recognized. This review describes how the functions of MCs from Saccharomyces cerevisiae, Homo sapiens and Arabidopsis thaliana (listed in the first table) were discovered after the genome sequence of S. cerevisiae was determined in 1996. In the genomic era, more than 50 previously unknown MCs from these organisms have been identified and characterized biochemically using a method consisting of gene expression, purification of the recombinant proteins, their reconstitution into liposomes and transport assays (EPRA). Information derived from studies with intact mitochondria, genetic and metabolic evidence, sequence similarity, phylogenetic analysis and complementation of knockout phenotypes have guided the choice of substrates that were tested in the transport assays. In addition, the diseases associated to defects of human MCs have been briefly reviewed. This article is part of a Special Issue entitled: Mitochondrial Channels edited by Pierre Sonveaux, Pierre Maechler and Jean-Claude Martinou., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Background: The Coq protein complex assembled from several Coq proteins is critical for coenzyme Q6 (CoQ6) biosynthesis in yeast. Secondary CoQ10 deficiency is associated with mitochondrial DNA (mtDNA) mutations in patients. We previously demonstrated that carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) suppressed CoQ10 levels and COQ5 protein maturation in human 143B cells., Methods: This study explored the putative COQ protein complex in human cells through two-dimensional blue native-polyacrylamide gel electrophoresis and Western blotting to investigate its status in 143B cells after FCCP treatment and in cybrids harboring the mtDNA mutation that caused myoclonic epilepsy with ragged-red fibers (MERRF) syndrome. Ubiquinol-10 and ubiquinone-10 levels were detected by high-performance liquid chromatography. Mitochondrial energy status, mRNA levels of various PDSS and COQ genes, and protein levels of COQ5 and COQ9 in cybrids were examined., Results: A high-molecular-weight protein complex containing COQ5, but not COQ9, in the mitochondria was identified and its level was suppressed by FCCP and in cybrids with MERRF mutation. That was associated with decreased mitochondrial membrane potential and mitochondrial ATP production. Total CoQ10 levels were decreased under both conditions, but the ubiquinol-10:ubiquinone-10 ratio was increased in mutant cybrids. The expression of COQ5 was increased but COQ5 protein maturation was suppressed in the mutant cybrids., Conclusions: A novel COQ5-containing protein complex was discovered in human cells. Its destabilization was associated with reduced CoQ10 levels and mitochondrial energy deficiency in human cells treated with FCCP or exhibiting MERRF mutation., General Significance: The findings elucidate a possible mechanism for mitochondrial dysfunction-induced CoQ10 deficiency in human cells., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Coenzyme Q (CoQ, or ubiquinone) is a remarkable lipid that plays an essential role in mitochondria as an electron shuttle between complexes I and II of the respiratory chain, and complex III. It is also a cofactor of other dehydrogenases, a modulator of the permeability transition pore and an essential antioxidant. CoQ is synthesized in mitochondria by a set of at least 12 proteins that form a multiprotein complex. The exact composition of this complex is still unclear. Most of the genes involved in CoQ biosynthesis (COQ genes) have been studied in yeast and have mammalian orthologues. Some of them encode enzymes involved in the modification of the quinone ring of CoQ, but for others the precise function is unknown. Two genes appear to have a regulatory role: COQ8 (and its human counterparts ADCK3 and ADCK4) encodes a putative kinase, while PTC7 encodes a phosphatase required for the activation of Coq7. Mutations in human COQ genes cause primary CoQ(10) deficiency, a clinically heterogeneous mitochondrial disorder with onset from birth to the seventh decade, and with clinical manifestation ranging from fatal multisystem disorders, to isolated encephalopathy or nephropathy. The pathogenesis of CoQ(10) deficiency involves deficient ATP production and excessive ROS formation, but possibly other aspects of CoQ(10) function are implicated. CoQ(10) deficiency is unique among mitochondrial disorders since an effective treatment is available. Many patients respond to oral CoQ(10) supplementation. Nevertheless, treatment is still problematic because of the low bioavailability of the compound, and novel pharmacological approaches are currently being investigated. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2-6, 2016', edited by Prof. Paolo Bernardi., (Copyright © 2016. Published by Elsevier B.V.)

One of the important elements of operation of cytochrome bc1 (mitochondrial respiratory complex III) is a large scale movement of the head domain of iron-sulfur protein (ISP-HD), which connects the quinol oxidation site (Qo) located within the cytochrome b, with the outermost heme c(1) of cytochrome c(1). Several mitochondrial disease-related mutations in cytochrome b are located at the cytochrome b-ISP-HD interface, thus their molecular effects can be associated with altered motion of ISP-HD. Using purple bacterial model, we recently showed that one of such mutations - G167P shifts the equilibrium position of ISP-HD towards positions remote from the Qo site as compared to the native enzyme [Borek et al., J. Biol. Chem. 290 (2015) 23781-23792]. This resulted in the enhanced propensity of the mutant to generate reactive oxygen species (ROS) which was explained on the basis of the model evoking "semireverse" electron transfer from heme bL to quinone. Here we examine another mutation from that group - G332D (G290D in human), finding that it also shifts the equilibrium position of ISP-HD in the same direction, however displays less of the enhancement in ROS production. We provide spectroscopic indication that G332D might affect the electrostatics of interaction between cytochrome b and ISP-HD. This effect, in light of the measured enzymatic activities and electron transfer rates, appears to be less severe than structural distortion caused by proline in G167P mutant. Comparative analysis of the effects of G332D and G167P confirms a general prediction that mutations located at the cytochrome b-ISP-HD interface influence the motion of ISP-HD and indicates that "pushing" ISP-HD away from the Qo site is the most likely outcome of this influence. It can also be predicted that an increase in ROS production associated with the "pushing" effect is quite sensitive to overall severity of this change with more active mutants being generally more protected against elevated ROS. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2-6, 2016', edited by Prof. Paolo Bernardi., (Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.)

Next Generation Sequencing (NGS) technologies are revolutionizing the diagnostic screening for rare disease entities, including primary mitochondrial disorders, particularly those caused by nuclear gene defects. NGS approaches are able to identify the causative gene defects in small families and even single individuals, unsuitable for investigation by traditional linkage analysis. These technologies are contributing to fill the gap between mitochondrial disease cases defined on the basis of clinical, neuroimaging and biochemical readouts, which still outnumber by approximately 50% the cases for which a molecular-genetic diagnosis is attained. We have been using a combined, two-step strategy, based on targeted genes panel as a first NGS screening, followed by whole exome sequencing (WES) in still unsolved cases, to analyze a large cohort of subjects, that failed to show mutations in mtDNA and in ad hoc sets of specific nuclear genes, sequenced by the Sanger's method. Not only this approach has allowed us to reach molecular diagnosis in a significant fraction (20%) of these difficult cases, but it has also revealed unexpected and conceptually new findings. These include the possibility of marked variable penetrance of recessive mutations, the identification of large-scale DNA rearrangements, which explain spuriously heterozygous cases, and the association of mutations in known genes with unusual, previously unreported clinical phenotypes. Importantly, WES on selected cases has unraveled the presence of pathogenic mutations in genes encoding non-mitochondrial proteins (e.g. the transcription factor E4F1), an observation that further expands the intricate genetics of mitochondrial disease and suggests a new area of investigation in mitochondrial medicine. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2-6, 2016', edited by Prof. Paolo Bernardi., (Copyright © 2016. Published by Elsevier B.V.)

This review resumes the association between mitochondrial function and diseases, especially neurodegenerative diseases. Additionally, it summarizes the major role of IGF-1 as a mitochondrial protector, as studied in several experimental models (cirrhosis, aging …). The contribution of mitochondrial dysfunction to impairments in insulin metabolic signaling is also suggested by gene array analysis showing that reductions in gene expression, that regulates mitochondrial ATP production, are associated with insulin resistance and type 2 diabetes mellitus. Moreover, reductions in oxidative capacity of mitochondrial electron transport chain are manifested in obese, insulin-resistant and diabetic patients. Genetic and environmental factors, oxidative stress, and alterations in mitochondrial biogenesis can adversely affect mitochondrial function, leading to insulin resistance and several pathological conditions, such as type 2 diabetes. Finally, it remains essential to know the exact mechanisms involved in mitochondrial generation and metabolism, mitophagy, apoptosis, and oxidative stress to establish new targets in order to develop potentially effective therapies. One of the newest targets to recover mitochondrial dysfunction could be the administration of IGF-1 at low doses. In the last years, it has been observed that IGF-1 therapy has several beneficial effects: restores physiological IGF-1 levels; improves insulin resistance and lipid metabolism; exerts mitochondrial protection; and has hepatoprotective, neuroprotective, antioxidant and antifibrogenic effects. In consequence, treatment of mitochondrial dysfunctions with low doses of IGF-1 could be a powerful and useful effective therapy to restore normal mitochondrial functions., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Mitochondria are fundamental organelles with a complex internal architecture that fulfill important diverse functions including iron-sulfur cluster assembly and cell respiration. Intense work for more than 30 years has identified the key protein import components and the pathways involved in protein targeting and assembly. More recently, oxidative folding has been discovered as one important mechanism for mitochondrial proteostasis whilst several human disorders have been linked to this pathway. We describe the molecular components of this pathway in view of their putative redox regulation and we summarize available evidence on the connections of these pathways to human disorders., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Background: Nuclear reprogramming with pluripotency factors enables somatic cells to gain the properties of embryonic stem cells. Mitochondrial resetting and metabolic reprogramming are suggested to be key early events in the induction of human skin fibroblasts to induced pluripotent stem cells (iPSCs)., Scope of Review: We review recent advances in the study of the molecular basis for mitochondrial resetting and metabolic reprogramming in the regulation of the formation of iPSCs. In particular, the recent progress in using iPSCs for mitochondrial disease modeling was discussed., Major Conclusions: iPSCs rely on glycolysis rather than oxidative phosphorylation as a major supply of energy. Mitochondrial resetting and metabolic reprogramming thus play crucial roles in the process of generation of iPSCs from somatic cells., General Significance: Neurons, myocytes, and cardiomyocytes are cells containing abundant mitochondria in the human body, which can be differentiated from iPSCs or trans-differentiated from fibroblasts. Generating these cells from iPSCs derived from skin fibroblasts of patients with mitochondrial diseases or by trans-differentiation with cell-specific transcription factors will provide valuable insights into the role of mitochondrial DNA heteroplasmy in mitochondrial disease modeling and serves as a novel platform for screening of drugs to treat patients with mitochondrial diseases., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Mitochondrial disorders are an important group of genetic conditions characterized by impaired oxidative phosphorylation. Mitochondrial disorders come with an impressive variability of symptoms, organ involvement, and clinical course, which considerably impact the quality of life and quite often shorten the lifespan expectancy. Although the last 20 years have witnessed an exponential increase in understanding the genetic and biochemical mechanisms leading to disease, this has not resulted in the development of effective therapeutic approaches, amenable of improving clinical course and outcome of these conditions to any significant extent. Therapeutic options for mitochondrial diseases still remain focused on supportive interventions aimed at relieving complications. However, new therapeutic strategies have recently been emerging, some of which have shown potential efficacy at the pre-clinical level. This review will present the state of the art on experimental therapy for mitochondrial disorders., (Crown Copyright © 2015. Published by Elsevier B.V. All rights reserved.)

Oxidative phosphorylation system (OXPHOS) deficiencies are rare diseases but constitute the most frequent inborn errors of metabolism. We analyzed the autophagy route in 11 skin fibroblast cultures derived from patients with well characterized and distinct OXPHOS defects. Mitochondrial membrane potential determination revealed a tendency to decrease in 5 patients' cells but reached statistical significance only in 2 of them. The remaining cells showed either no change or a slight increase in this parameter. Colocalization analysis of mitochondria and autophagosomes failed to show evidence of increased selective elimination of mitochondria but revealed more intense autophagosome staining in patients' fibroblasts compared with controls. Despite the absence of increased mitophagy, Parkin recruitment to mitochondria was detected in both controls' and patients' cells and was slightly higher in cells harboring complex I defects. Western blot analysis of the autophagosome marker LC3B, confirmed significantly higher levels of the protein bound to autophagosomes, LC3B-II, in patients' cells, suggesting an increased bulk autophagy in OXPHOS defective fibroblasts. Inhibition of lysosomal proteases caused significant accumulation of LC3B-II in control cells, whereas in patients' cells this phenomenon was less pronounced. Electron microscopy studies showed higher content of late autophagic vacuoles and lysosomes in OXPHOS defective cells, accompanied by higher levels of the lysosomal marker LAMP-1. Our findings suggest that in OXPHOS deficient fibroblasts autophagic flux could be partially hampered leading to an accumulation of autophagic vacuoles and lysosomes., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Background: The neuropathology of mitochondrial disease is well characterised. However, pathophysiological mechanisms at the level of biochemistry and cell biology are less clear. Progress in this area has been hampered by the limited accessibility of neurologically relevant material for analysis., Scope of Review: Here we discuss the recent development of a variety of model systems that have greatly extended our capacity to understand the biochemical features associated with mitochondrial neuropathology. These include animal and cell based models, with mutations in both nuclear and mitochondrial DNA encoded genes, which aim to recapitulate the neuropathology and cellular biochemistry of mitochondrial diseases., Major Conclusions: Analysis of neurological tissue and cells from these models suggests that although there is no unifying mode of pathogenesis, dysfunction of the oxidative phosphorylation (OXPHOS) system is often central. This can be associated with altered reactive oxygen species (ROS) generation, disruption of the mitochondrial membrane potential (ΔΨm) and inadequate ATP synthesis. Thus, other cellular processes such as calcium (Ca(2+)) homeostasis, cellular signaling and mitochondrial morphology could be altered, ultimately compromising viability of neuronal cells., General Significance: Mechanisms of neuronal dysfunction in mitochondrial disease are only just beginning to be characterised, are system dependent and complex, and not merely driven by energy deficiency. The diversity of pathogenic mechanisms emphasises the need for characterisation in a wide range of models, as different therapeutic strategies are likely to be needed for different diseases. This article is part of a Special Issue entitled Frontiers of Mitochondrial Research., (© 2013.)

Background: Mitochondrial DNA (mtDNA) mutations are an important cause of mitochondrial diseases, for which there is no effective treatment due to complex pathophysiology. It has been suggested that mitochondrial dysfunction-elicited reactive oxygen species (ROS) plays a vital role in the pathogenesis of mitochondrial diseases, and the expression levels of several clusters of genes are altered in response to the elevated oxidative stress. Recently, we reported that glycolysis in affected cells with mitochondrial dysfunction is upregulated by AMP-activated protein kinase (AMPK), and such an adaptive response of metabolic reprogramming plays an important role in the pathophysiology of mitochondrial diseases., Scope of Review: We summarize recent findings regarding the role of AMPK-mediated signaling pathways that are involved in: (1) metabolic reprogramming, (2) alteration of cellular redox status and antioxidant enzyme expression, (3) mitochondrial biogenesis, and (4) autophagy, a master regulator of mitochondrial quality control in skin fibroblasts from patients with mitochondrial diseases., Major Conclusion: Induction of adaptive responses via AMPK-PFK2, AMPK-FOXO3a, AMPK-PGC-1α, and AMPK-mTOR signaling pathways, respectively is modulated for the survival of human cells under oxidative stress induced by mitochondrial dysfunction. We suggest that AMPK may be a potential target for the development of therapeutic agents for the treatment of mitochondrial diseases., General Significance: Elucidation of the adaptive mechanism involved in AMPK activation cascades would lead us to gain a deeper insight into the crosstalk between mitochondria and the nucleus in affected tissue cells from patients with mitochondrial diseases. This article is part of a Special Issue entitled Frontiers of Mitochondrial Research., (© 2013.)

Background: Most neurodegenerative diseases are associated with mitochondrial dysfunction. In humans, mutations in mitochondrial genes result in a range of phenotypic outcomes which do not correlate well with the underlying genetic cause. Other neurodegenerative diseases are caused by mutations that affect the function and trafficking of lysosomes, endosomes and autophagosomes. Many of the complexities of these human diseases can be avoided by studying them in the simple eukaryotic model Dictyostelium discoideum., Scope of Review: This review describes research using Dictyostelium to study cytopathological pathways underlying a variety of neurodegenerative diseases including mitochondrial, lysosomal and vesicle trafficking disorders., Major Conclusions: Generalised mitochondrial respiratory deficiencies in Dictyostelium produce a consistent pattern of defective phenotypes that are caused by chronic activation of a cellular energy sensor AMPK (AMP-activated protein kinase) and not ATP deficiency per se. Surprisingly, when individual subunits of Complex I are knocked out, both AMPK-dependent and AMPK-independent, subunit-specific phenotypes are observed. Many nonmitochondrial proteins associated with neurological disorders have homologues in Dictyostelium and are associated with the function and trafficking of lysosomes and endosomes. Conversely, some genes associated with neurodegenerative disorders do not have homologues in Dictyostelium and this provides a unique avenue for studying these mutated proteins in the absence of endogeneous protein., General Significance: Using the Dictyostelium model we have gained insights into the sublethal cytopathological pathways whose dysregulation contributes to phenotypic outcomes in neurodegenerative disease. This work is beginning to distinguish correlation, cause and effect in the complex network of cross talk between the various organelles involved. This article is part of a Special Issue entitled Frontiers of Mitochondrial Research., (© 2013.)

In Parkinson's disease mitochondrial dysfunction can lead to a deficient ATP supply to microtubule protein motors leading to mitochondrial axonal transport disruption. Compromised axonal transport will then lead to a disorganized distribution of mitochondria and other organelles in the cell, as well as, the accumulation of aggregated proteins like alpha-synuclein. Moreover, axonal transport disruption can trigger synaptic accumulation of autophagosomes packed with damaged mitochondria and protein aggregates promoting synaptic failure. We previously observed that neuronal-like cells with an inherent mitochondrial impairment derived from PD patients contain a disorganized microtubule network, as well as, alpha-synuclein oligomer accumulation. In this work we provide new evidence that an agent that promotes microtubule network assembly, NAP (davunetide), improves microtubule-dependent traffic, restores the autophagic flux and potentiates autophagosome-lysosome fusion leading to autophagic vacuole clearance in Parkinson's disease cells. Moreover, NAP is capable of efficiently reducing alpha-synuclein oligomer content and its sequestration by the mitochondria. Most interestingly, NAP decreases mitochondrial ubiquitination levels, as well as, increases mitochondrial membrane potential indicating a rescue in mitochondrial function. Overall, we demonstrate that by improving microtubule-mediated traffic, we can avoid mitochondrial-induced damage and thus recover cell homeostasis. These results prove that NAP may be a promising therapeutic lead candidate for neurodegenerative diseases that involve axonal transport failure and mitochondrial impairment as hallmarks, like Parkinson's disease and related disorders., (© 2013.)

Multiple respiratory chain deficiencies represent a common cause of mitochondrial diseases and are associated with a wide range of clinical symptoms. We report a subject, born to consanguineous parents, with growth retardation and neurological deterioration. Multiple respiratory chain deficiency was found in muscle and fibroblasts of the subject as well as abnormal assembly of complexes I and IV. A microsatellite genotyping of the family members detected only one region of homozygosity on chromosome 17q24.2-q25.3 in which we focused our attention to genes involved in mitochondrial translation. We sequenced MRPL12, encoding the mitochondrial ribosomal protein L12 and identified a c.542C>T transition in exon 5 changing a highly conserved alanine into a valine (p.Ala181Val). This mutation resulted in a decreased steady-state level of MRPL12 protein, with altered integration into the large ribosomal subunit. Moreover, an overall mitochondrial translation defect was observed in the subject's fibroblasts with a significant reduction of synthesis of COXI, COXII and COXIII subunits. Modeling of MRPL12 shows Ala181 positioned in a helix potentially involved in an interface of interaction suggesting that the p.Ala181Val change might be predicted to alter interactions with the elongation factors. These results contrast with the eubacterial orthologues of human MRPL12, where L7/L12 proteins do not appear to have a selective effect on translation. Therefore, analysis of the mutated version found in the subject presented here suggests that the mammalian protein does not function in an entirely analogous manner to the eubacterial L7/L12 equivalent., (Copyright © 2013 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Mitochondrial dysfunction contributes to degenerative neurological disorders, consequently there is a need for mitochondria-targeted therapies that are effective within the brain. One approach to deliver pharmacophores is by conjugation to the lipophilic triphenylphosphonium (TPP) cation that accumulates in mitochondria driven by the membrane potential. While this approach has delivered TPP-conjugated compounds to the brain, the amounts taken up are lower than by other organs., Methods: To discover why uptake of hydrophobic TPP compounds by the brain is relatively poor, we assessed the role of the P-glycoprotein (Mdr1a/b) and breast cancer resistance protein (Bcrp) ATP binding cassette (ABC) transporters, which drive the efflux of lipophilic compounds from the brain thereby restricting the uptake of lipophilic drugs. We used a triple transgenic mouse model lacking two isoforms of P-glycoprotein (Mdr1a/1b) and the Bcrp., Results: There was a significant increase in the uptake into the brain of two hydrophobic TPP compounds, MitoQ and MitoF, in the triple transgenics following intra venous (IV) administration compared to control mice. Greater amounts of the hydrophobic TPP compounds were also retained in the liver of transgenic mice compared to controls. The uptake into the heart, white fat, muscle and kidneys was comparable between the transgenic mice and controls., Conclusion: Efflux of hydrophobic TPP compounds by ABC transporters contributes to their lowered uptake into the brain and liver., General Significance: These findings suggest that strategies to bypass ABC transporters in the BBB will enhance delivery of mitochondria-targeted antioxidants, probes and pharmacophores to the brain., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Oxidative phosphorylation disorders are often associated with increased oxidative stress and antioxidant therapy is frequently given as treatment. However, the role of oxidative stress in oxidative phosphorylation disorders or patients is far from clear and consequently the preventive or therapeutic effect of antioxidants is highly anecdotic. Therefore, we performed a systematic study of a panel of oxidative stress parameters (reactive oxygen species levels, damage and defense) in fibroblasts of twelve well-characterized oxidative phosphorylation patients with a defect in the POLG1 gene, in the mitochondrial DNA-encoded tRNA-Leu gene (m.3243A>G or m.3302A>G) and in one of the mitochondrial DNA-encoded NADH dehydrogenase complex I (CI) subunits. All except two cell lines (one POLG1 and one tRNA-Leu) showed increased reactive oxygen species levels compared with controls, but only four (two CI and two tRNA-Leu) cell lines provided evidence for increased oxidative protein damage. The absence of a correlation between reactive oxygen species levels and oxidative protein damage implies differences in damage prevention or correction. This was investigated by gene expression studies, which showed adaptive and compensating changes involving antioxidants and the unfolded protein response, especially in the POLG1 group. This study indicated that patients display individual responses and that detailed analysis of fibroblasts enables the identification of patients that potentially benefit from antioxidant therapy. Furthermore, the fibroblast model can also be used to search for and test novel, more specific antioxidants or explore ways to stimulate compensatory mechanisms., (© 2012 Elsevier B.V. All rights reserved.)

Respiratory complexes are believed to play a role in the function of the mitochondrial permeability transition pore (PTP), whose dysregulation affects the process of cell death and is involved in a variety of diseases, including cancer and degenerative disorders. We investigated here the PTP in cells devoid of mitochondrial DNA (ρ(0) cells), which lack respiration and constitute a model for the analysis of mitochondrial involvement in several pathological conditions. We observed that mitochondria of ρ(0) cells maintain a membrane potential and that this is readily dissipated after displacement of hexokinase (HK) II from the mitochondrial surface by treatment with either the drug clotrimazole or with a cell-permeant HK II peptide, or by placing ρ(0) cells in a medium without serum and glucose. The PTP inhibitor cyclosporin A (CsA) could decrease the mitochondrial depolarization induced by either HK II displacement or by nutrient depletion. We also found that a fraction of the kinases ERK1/2 and GSK3α/β is located in the mitochondrial matrix of ρ(0) cells, and that glucose and serum deprivation caused concomitant ERK1/2 inhibition and GSK3α/β activation with the ensuing phosphorylation of cyclophilin D, the mitochondrial target of CsA. GSK3α/β inhibition with indirubin-3'-oxime decreased PTP-induced cell death in ρ(0) cells following nutrient ablation. These findings indicate that ρ(0) cells are equipped with a functioning PTP, whose regulatory mechanisms are similar to those observed in cancer cells, and suggest that escape from PTP opening is a survival factor in this model of mitochondrial diseases. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012)., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Defective complex I (CI) is the most common type of oxidative phosphorylation disease, with an incidence of 1 in 5000 live births. Here, whole genome expression profiling of fibroblasts from CI deficient patients was performed to gain insight into the cell pathological mechanism. Our results suggest that patient fibroblasts responded to oxidative stress by Nrf2-mediated induction of the glutathione antioxidant system and Gadd45-mediated activation of the DNA damage response pathway. Furthermore, the observed reduced expression of selenoproteins, might explain the disturbed calcium homeostasis previously described for the patient fibroblasts and might be linked to endoplasmic reticulum stress. These results suggest that both glutathione and selenium metabolism are potentially therapeutic targets in CI deficiency., (© 2011 Elsevier B.V. All rights reserved.)

Complex I (NADH:ubiquinone oxidoreductase) is the largest multimeric enzyme complex of the mitochondrial respiratory chain, which is responsible for electron transport and the generation of a proton gradient across the mitochondrial inner membrane to drive ATP production. Eukaryotic complex I consists of 14 conserved subunits, which are homologous to the bacterial subunits, and more than 26 accessory subunits. In mammals, complex I consists of 45 subunits, which must be assembled correctly to form the properly functioning mature complex. Complex I dysfunction is the most common oxidative phosphorylation (OXPHOS) disorder in humans and defects in the complex I assembly process are often observed. This assembly process has been difficult to characterize because of its large size, the lack of a high resolution structure for complex I, and its dual control by nuclear and mitochondrial DNA. However, in recent years, some of the atomic structure of the complex has been resolved and new insights into complex I assembly have been generated. Furthermore, a number of proteins have been identified as assembly factors for complex I biogenesis and many patients carrying mutations in genes associated with complex I deficiency and mitochondrial diseases have been discovered. Here, we review the current knowledge of the eukaryotic complex I assembly process and new insights from the identification of novel assembly factors. This article is part of a Special Issue entitled: Biogenesis/Assembly of Respiratory Enzyme Complexes., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Isolated complex I deficiency is a frequent cause of respiratory chain defects in childhood. In this study, we report our systematic approach with blue native PAGE (BN-PAGE) to study mitochondrial respiratory chain assembly in skin fibroblasts from patients with Leigh syndrome and CI deficiency. We describe five new NDUFS4 patients with a similar and constant abnormal BN-PAGE profile and present a meta-analysis of the literature. All NDUFS4 mutations that have been tested with BN-PAGE result in a constant and similar abnormal assembly profile with a complete loss of the fully assembled complex I usually due to a truncated protein and the loss of its canonical cAMP dependent protein kinase phosphorylation consensus site. We also report the association of abnormal brain MRI images with this characteristic BN-PAGE profile as the hallmarks of NDUFS4 mutations and the first founder NDUFS4 mutations in the North-African population., (© 2012 Elsevier B.V. All rights reserved.)

Background: Although causative mutations have been identified for numerous mitochondrial disorders, few disease-modifying treatments are available. Two examples of treatable mitochondrial disorders are coenzyme Q(10) (CoQ(10) or ubiquinone) deficiency and mitochondrial neurogastrointestinal encephalomyopathy (MNGIE)., Scope of Review: Here, we describe clinical and molecular features of CoQ(10) deficiencies and MNGIE and explain how understanding their pathomechanisms have led to rationale therapies. Primary CoQ(10) deficiencies, due to mutations in genes required for ubiquinone biosynthesis, and secondary deficiencies, caused by genetic defects not directly related to CoQ(10) biosynthesis, often improve with CoQ(10) supplementation. In vitro and in vivo studies of CoQ(10) deficiencies have revealed biochemical alterations that may account for phenotypic differences among patients and variable responses to therapy. In contrast to the heterogeneous CoQ(10) deficiencies, MNGIE is a single autosomal recessive disease due to mutations in the TYMP gene encoding thymidine phosphorylase (TP). In MNGIE, loss of TP activity causes toxic accumulations of the nucleosides thymidine and deoxyuridine that are incorporated by the mitochondrial pyrimidine salvage pathway and cause deoxynucleoside triphosphate pool imbalances, which, in turn cause mtDNA instability. Allogeneic hematopoetic stem cell transplantation to restore TP activity and eliminate toxic metabolites is a promising therapy for MNGIE., Major Conclusions: CoQ(10) deficiencies and MNGIE demonstrate the feasibility of treating specific mitochondrial disorders through replacement of deficient metabolites or via elimination of excessive toxic molecules., General Significance: Studies of CoQ(10) deficiencies and MNGIE illustrate how understanding the pathogenic mechanisms of mitochondrial diseases can lead to meaningful therapies. This article is part of a Special Issue entitled: Biochemistry of Mitochondria, Life and Intervention 2010., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Background: Mitochondrial DNA depletion syndromes are a group of heterogeneous autosomal recessive disorders associated with a severe reduction in mitochondrial DNA in the affected tissues. Sodium pyruvate has been reported to have a therapeutic effect in mitochondrial diseases., Methods: We analyzed the effects of 0.5g/kg of sodium pyruvate administered through a nasogastric tube in a one-year-old patient with myopathic mitochondrial DNA depletion syndrome. To evaluate the improvement, we used the Newcastle Paediatric Mitochondrial Disease Scale (NPMDS) and manual muscle testing. As the improvement of motor functions in this severely disabled infant could not be comprehensively detected by NPMDS, we also observed the infant's ability to perform several tasks such as pouting, winking, and number of times she could tap a toy xylophone with a stick. Blood lactate and pyruvate levels were also monitored., Results: After one month's treatment, the NPMDS score in section IV, the domain for the quality of life, improved from 17 to13. The infant became capable of raising her forearm, lower leg and wrist against gravity. The maximum number of times she could repeat each task increased and the movements became brisker and stronger. No significant change of the blood lactate level or lactate-to-pyruvate ratio, both of which were mildly increased at the initiation of the therapy, was observed despite the clinical improvement., Conclusion: Sodium pyruvate administered at 0.5g/kg improved the muscle strength and the NPMDS score of an infant with myopathic mitochondrial DNA depletion syndrome., General Significance: Sodium pyruvate may be effective for ameliorating the clinical manifestations of mitochondrial diseases. This article is part of a Special Issue entitled: Biochemistry of Mitochondria., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Malignant brain tumors are a significant health problem in children and adults. Conventional therapeutic approaches have been largely unsuccessful in providing long-term management. As primarily a metabolic disease, malignant brain cancer can be managed through changes in metabolic environment. In contrast to normal neurons and glia, which readily transition to ketone bodies (β-hydroxybutyrate) for energy under reduced glucose, malignant brain tumors are strongly dependent on glycolysis for energy. The transition from glucose to ketone bodies as a major energy source is an evolutionary conserved adaptation to food deprivation that permits the survival of normal cells during extreme shifts in nutritional environment. Only those cells with a flexible genome and normal mitochondria can effectively transition from one energy state to another. Mutations restrict genomic and metabolic flexibility thus making tumor cells more vulnerable to energy stress than normal cells. We propose an alternative approach to brain cancer management that exploits the metabolic flexibility of normal cells at the expense of the genetically defective and metabolically challenged tumor cells. This approach to brain cancer management is supported from recent studies in mice and humans treated with calorie restriction and the ketogenic diet. Issues of implementation and use protocols are presented for the metabolic management of brain cancer., (Copyright © 2010. Published by Elsevier B.V.)

Background: Mitochondrial dysfunction contributes to a range of pathologies, consequently there is a need to monitor mitochondrial function and to intervene pharmacologically to prevent mitochondrial damage. One approach to this is to deliver antioxidants, probes and pharmacophores to mitochondria by conjugation to the lipophilic triphenylphosphonium (TPP) cation that is taken up selectively by mitochondria driven by the membrane potential., Conclusions: Oral administration of TPP-conjugated antioxidants protects against mitochondrial damage in vivo. However, there is also a need to deliver molecules rapidly to mitochondria to respond quickly to pathologies and for the real-time assessment of mitochondrial function., Methods: To see if this was possible we investigated how rapidly TPP cations were taken up by mitochondria in vivo following intravenous (iv) administration., Results: AlkylTPP cations were accumulated selectively by mitochondria within mice within 5 min of iv injection. The extent of uptake was enhanced 10-30-fold relative to simple alkylTPP cations by attaching functional groups to the TPP cation via long, hydrophobic alkyl chains. Conclusions: Mitochondria-targeted antioxidants, probes and pharmacophores can be delivered into mitochondria within minutes of iv administration., General Significance: These findings greatly extend the utility of mitochondria-targeted lipophilic cations as therapies and probes., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Autism Spectrum Disorders encompass severe developmental disorders characterized by variable degrees of impairment in language, communication and social skills, as well as by repetitive and stereotypic patterns of behaviour. Substantial percentages of autistic patients display peripheral markers of mitochondrial energy metabolism dysfunction, such as (a) elevated lactate, pyruvate, and alanine levels in blood, urine and/or cerebrospinal fluid, (b) serum carnitine deficiency, and/or (c) enhanced oxidative stress. These biochemical abnormalities are accompanied by highly heterogeneous clinical presentations, which generally (but by no means always) encompass neurological and systemic symptoms relatively unusual in idiopathic autistic disorder. In some patients, these abnormalities have been successfully explained by the presence of specific mutations or rearrangements in their mitochondrial or nuclear DNA. However, in the majority of cases, abnormal energy metabolism cannot be immediately linked to specific genetic or genomic defects. Recent evidence from post-mortem studies of autistic brains points toward abnormalities in mitochondrial function as possible downstream consequences of dysreactive immunity and altered calcium (Ca(2+)) signalling., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Mutations in human SCO2 gene, encoding the mitochondrial inner membrane Sco2 protein, have been found to be responsible for fatal infantile cardioencephalomyopathy and cytochrome c oxidase (COX) deficiency. One potentially fruitful therapeutic approach for this mitochondrial disorder should be considered the production of human recombinant full length L-Sco2 protein and its deliberate transduction into the mitochondria. Recombinant L-Sco2 protein, fused with TAT, a Protein Transduction Domain (PTD), was produced in bacteria and purified from inclusion bodies (IBs). Following solubilisation with l-arginine, this fusion L-Sco2 protein was transduced in cultured mammalian cells of different origin (U-87 MG, T24, K-562, and patient's primary fibroblasts) and assessed for stability, transduction into mitochondria, processing and impact on recovery of COX activity. Our results indicate that: a) l-Arg solution was effective in solubilising recombinant fusion L-Sco2 protein, derived from IBs; b) fusion L-Sco2 protein was delivered successfully via a time- and concentration-dependent process into the mitochondria of human U-87 MG and T24 cells; c) fusion L-Sco2 protein was also transduced in human K-562 cells, transiently depleted of SCO2 transcripts and thus COX deficient; transduction of this fusion protein led to partial recovery of COX activity in such cells; d) [(35)S]Methionine-labelled fusion L-Sco2 protein, produced in a cell free transcription/translation system and incubated with intact isolated mitochondria derived from K-562 cells, was efficiently processed to yield the corresponding mature Sco2 protein, thus justifying the potential of the transduced fusion L-Sco2 protein to successfully activate COX holoenzyme; and finally, e) recombinant fusion L-Sco2 protein was successfully transduced into the mitochondria of primary fibroblasts derived from SCO2/COX deficient patient and facilitated recovery of COX activity. These findings provide the rationale of delivering recombinant proteins via PTD technology as a model for therapeutic approach of mitochondrial disorders., (Copyright 2010 Elsevier B.V. All rights reserved.)

Cyclophilins are a family of peptidyl-prolyl cis-trans isomerases whose enzymatic activity can be inhibited by cyclosporin A. Sixteen cyclophilins have been identified in humans, and cyclophilin D is a unique isoform that is imported into the mitochondrial matrix. Here we shall (i) review the best characterized functions of cyclophilin D in mitochondria, i.e. regulation of the permeability transition pore, an inner membrane channel that plays an important role in the execution of cell death; (ii) highlight new regulatory interactions that are emerging in the literature, including the modulation of the mitochondrial F1FO ATP synthase through an interaction with the lateral stalk of the enzyme complex; and (iii) discuss diseases where cyclophilin D plays a pathogenetic role that makes it a suitable target for pharmacologic intervention., (Copyright © 2009 Elsevier B.V. All rights reserved.)

p66Shc, the growth factor adaptor protein, can have a substantial impact on mitochondrial metabolism through regulation of cellular response to oxidative stress. We investigated relationships between the extent of p66Shc phosphorylation at Ser36, mitochondrial dysfunctions and an antioxidant defense reactions in fibroblasts derived from five patients with various mitochondrial disorders (two with mitochondrial DNA mutations and three with methylglutaconic aciduria and genetic defects localized, most probably, in nuclear genes). We found that in all these fibroblasts, the extent of p66Shc phosphorylation at Ser36 was significantly increased. This correlated with a substantially decreased level of mitochondrial superoxide dismutase (SOD2) in these cells. This suggest that SOD2 is under control of the Ser36 phosphorylation status of p66Shc protein. As a consequence, an intracellular oxidative stress and accumulation of damages caused by oxygen free radicals are observed in the cells., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Background: Complex I (CI) deficiency is the most frequent cause of OXPHOS disorders. Recent studies have shown increases in reactive oxygen species (ROS) production and mitochondrial network disturbances in patients' fibroblasts harbouring mutations in CI subunits., Objectives: The present work evaluates the impact of mutations in the NDUFA1 and NDUFV1 genes of CI on mitochondrial bioenergetics and dynamics, in fibroblasts from patients suffering isolated CI deficiency., Results: Decreased oxygen consumption rate and slow growth rate were found in patients with severe CI deficiency. Mitochondrial diameter was slightly increased in patients' cells cultured in galactose or treated with 2'-deoxyglucose without evidence of mitochondrial fragmentation. Expression levels of the main proteins involved in mitochondrial dynamics, OPA1, MFN2, and DRP1, were slightly augmented in all patients' cells lines. The study of mitochondrial dynamics showed delayed recovery of the mitochondrial network after treatment with the uncoupler carbonyl cyanide m-chlorophenyl hydrazone (cccp) in patients with severe CI deficiency. Intracellular ROS levels were not increased neither in glucose nor galactose medium in patients' fibroblasts., Conclusion: Our main finding was that severe CI deficiency in patients harbouring mutations in the NDUFA1 and NDUFV1 genes is linked to a delayed mitochondrial network recovery after cccp treatment. However, the CI deficiency is neither associated with massive mitochondrial fragmentation nor with increased ROS levels. The different genetic backgrounds of patients with OXPHOS disorders would explain, at least partially, differences in the pathophysiological manifestations of CI deficiency., (Copyright 2010 Elsevier B.V. All rights reserved.)

The term lipotoxicity elicits visions of steatotic liver, fat laden skeletal muscles and engorged lipid droplets that spawn a number of potentially harmful intermediates that can wreak havoc on signal transduction and organ function. Prominent among these so-called lipotoxic mediators are signaling molecules such as long chain acyl-CoAs, ceramides and diacyglycerols; each of which is thought to engage serine kinases that disrupt the insulin signaling cascade, thereby causing insulin resistance. Defects in skeletal muscle fat oxidation have been implicated as a driving factor contributing to systemic lipid imbalance, whereas exercise-induced enhancement of oxidative potential is considered protective. The past decade of diabetes research has focused heavily on the foregoing scenario, and indeed the model is grounded in strong experimental evidence, albeit largely correlative. This review centers on mechanisms that connect lipid surplus to insulin resistance in skeletal muscle, as well as those that underlie the antilipotoxic actions of exercise. Emphasis is placed on recent studies that challenge accepted paradigms., (Copyright (c) 2009. Published by Elsevier B.V.)

Mitochondrial dysfunction in skeletal muscle has been suggested to underlie the development of insulin resistance and type 2 diabetes mellitus. Reduced mitochondrial capacity will contribute to the accumulation of lipid intermediates, desensitizing insulin signaling and leading to insulin resistance. Why mitochondrial function is reduced in the (pre-)diabetic state is, however, so far unknown. Although it is tempting to suggest that skeletal muscle insulin resistance may result from an inherited or acquired reduction in mitochondrial function in the pre-diabetic state, it cannot be excluded that mitochondrial dysfunction may in fact be the consequence of the insulin-resistant/diabetic state. Lipotoxicity, the deleterious effects of accumulating fatty acids in skeletal muscle cells, may lie at the basis of mitochondrial dysfunction: next to producing energy, mitochondria are also the major source of reactive oxygen species (ROS). Fatty acids accumulating in the vicinity of mitochondria are vulnerable to ROS-induced lipid peroxidation. Subsequently, these lipid peroxides could have lipotoxic effects on mtDNA, RNA and proteins of the mitochondrial machinery, leading to mitochondrial dysfunction. Indeed, increased lipid peroxidation has been reported in insulin resistant skeletal muscle and the mitochondrial uncoupling protein-3, which has been suggested to prevent lipid-induced mitochondrial damage, is reduced in subjects with an impaired glucose tolerance and in type 2 diabetic patients. These findings support the hypothesis that fat accumulation in skeletal muscle may precede the reduction in mitochondrial function that is observed in type 2 diabetes mellitus., (Copyright (c) 2009 Elsevier B.V. All rights reserved.)

Multiple sclerosis (MS) is a relatively common and etiologically unknown disease with no cure. It is the leading cause of neurological disability in young adults, affecting over two million people worldwide. Traditionally, MS has been considered a chronic, inflammatory disorder of the central white matter in which ensuing demyelination results in physical disability. Recently, MS has become increasingly viewed as a neurodegenerative disorder in which axonal injury, neuronal loss, and atrophy of the central nervous system leads to permanent neurological and clinical disability. In this article, we discuss the latest developments on MS research, including etiology, pathology, genetic association, EAE animal models, mechanisms of neuronal injury and axonal transport, and therapeutics. In this article, we also focus on the mechanisms of mitochondrial dysfunction that are involved in MS, including mitochondrial DNA defects, and mitochondrial structural/functional changes.

Taking advantage of a series of questions raised by an association of patients with mitochondrial disease, this review, after a brief overview of basic concepts of mitochondrial bioenergetics and genetics, discusses the pros and cons of a number of practical options in the field of mitochondrial therapy. This makes it clear that, in contrast to the spectacular progress in our understanding of the biochemical and molecular bases of the mitochondrial diseases defined restrictively as disorders due to defects in the mitochondrial respiratory chain, we are still extremely limited in our ability to treat these conditions. We finally discussed the emerging genetic-based strategies that show some promise, even if much work remains to be done.

Mitochondria are an essential organelle, not only to the human cell, but to all eukaryotic life. This essentiality is reflected in the large number of mutations in genes encoding mitochondrial proteins that lead to disease. Aside from their relevance to disease, mitochondria are, given their endosymbiotic origin, very interesting from an evolutionary point of view. Here, in the year that marks the bicentenary of Darwin's birth and the 150th anniversary of the publication of "On the origin of species" we review approaches that implicitly or explicitly use evolutionary analyses to find new genes involved in mitochondrial disease and to predict their function and involvement in pathways. We show how the phenotypic spectrum of mitochondrial disease is linked to the evolutionary origin of mitochondrial proteins, how combinations of evolutionary data and genomics data have been used to predict the mitochondrial proteome and functional links between the mitochondrial proteins and how the evolution of the mitochondrial proteome has been used to predict new mitochondrial disease genes. For the latter we review and reanalyze the eukaryotic evolution of the NADH:ubiquinone oxidoreductase (complex I) and the proteins involved in its assembly.

Proper cell functioning requires precise coordination between mitochondrial ATP production and local energy demand. Ionic calcium (Ca(2+)) plays a central role in this coupling because it activates mitochondrial oxidative phosphorylation (OXPHOS) during hormonal and electrical cell stimulation. To determine how mitochondrial dysfunction affects cytosolic and mitochondrial Ca(2+)/ATP handling, we performed life-cell quantification of these parameters in fibroblast cell lines derived from healthy subjects and patients with isolated deficiency of the first OXPHOS complex (CI). In resting patient cells, CI deficiency was associated with a normal mitochondrial ([ATP](m)) and cytosolic ([ATP](c)) ATP concentration, a normal cytosolic Ca(2+) concentration ([Ca(2+)](c)), but a reduced Ca(2+) content of the endoplasmic reticulum (ER). Furthermore, cellular NAD(P)H levels were increased, mitochondrial membrane potential was slightly depolarized, reactive oxygen species (ROS) levels were elevated and mitochondrial shape was altered. Upon stimulation with bradykinin (Bk), the peak increases in [Ca(2+)](c), mitochondrial Ca(2+) concentration ([Ca(2+)](m)), [ATP](c) and [ATP](m) were reduced in patient cells. In agreement with these results, ATP-dependent Ca(2+) removal from the cytosol was slower. Here, we review the interconnection between cytosolic, endoplasmic reticular and mitochondrial Ca(2+) and ATP handling, and summarize our findings in patient fibroblasts in an integrative model.

Complex III deficiencies are among the least common respiratory-chain abnormalities identified to date in humans. Nevertheless, their unexplained tissue specificity and broad clinical spectrum make them a valuable model for investigating respiratory-chain diseases. In this review, we briefly discuss the properties of complex III and the assay conditions relevant to the screening of high-risk patients. We then review the most recent advances in the field, which include the characterization of several disease genes and of the corresponding clinical presentations. Finally, we discuss genetic and biochemical aspects that may help to understand complex III-associated diseases.

In mammals, the majority of cellular ATP is produced by the mitochondrial F1F(O)-ATP synthase through an elaborate catalytic mechanism. While most subunits of this enzymatic complex are encoded by the nuclear genome, a few essential components are encoded in the mitochondrial genome. The biogenesis of this multi-subunit enzyme is a sophisticated multi-step process that is regulated on levels of transcription, translation and assembly. Defects that result in diminished abundance or functional impairment of the F1F(O)-ATP synthase can cause a variety of severe neuromuscular disorders. Underlying mutations have been identified in both the nuclear and the mitochondrial DNA. The pathogenic mechanisms are only partially understood. Currently, the therapeutic options are extremely limited. Alternative methods of treatment have however been proposed, but still encounter several technical difficulties. The application of novel scientific approaches promises to deepen our understanding of the molecular mechanisms of the ATP synthase, unravel novel therapeutic pathways and improve the unfortunate situation of the patients suffering from such diseases.

Assembly of the oxidative phosphorylation (OXPHOS) system in the mitochondrial inner membrane is an intricate process in which many factors must interact. The OXPHOS system is composed of four respiratory chain complexes, which are responsible for electron transport and generation of the proton gradient in the mitochondrial intermembrane space, and of the ATP synthase that uses this proton gradient to produce ATP. Mitochondrial human disorders are caused by dysfunction of the OXPHOS system, and many of them are associated with altered assembly of one or more components of the OXPHOS system. The study of assembly defects in patients has been useful in unraveling and/or gaining a complete understanding of the processes by which these large multimeric complexes are formed. We review here current knowledge of the biogenesis of OXPHOS complexes based on investigation of the corresponding disorders.

Defects in the oxidative phosphorylation system (OXPHOS) are responsible for a group of extremely heterogeneous and pleiotropic pathologies commonly known as mitochondrial diseases. Although many mutations have been found to be responsible for OXPHOS defects, their pathogenetic mechanisms are still poorly understood. An important contribution to investigate the in vivo function of several mitochondrial proteins and their role in mitochondrial dysfunction, has been provided by mouse models. Thanks to their genetic and physiologic similarity to humans, mouse models represent a powerful tool to investigate the impact of pathological mutations on metabolic pathways. In this review we discuss the main mouse models of mitochondrial disease developed, focusing on the ones that directly affect the OXPHOS system.

Isolated complex I deficiency is the most common cause of respiratory chain dysfunction. Defects in human complex I result in energy generation disorders and they are also implicated in neurodegenerative disease and altered apoptotic signaling. Complex I dysfunction often occurs as a result of its impaired assembly. The assembly process of complex I is poorly understood, complicated by the fact that in mammals, it is composed of 45 different subunits and is regulated by both nuclear and mitochondrial genomes. However, in recent years we have gained new insights into complex I biogenesis and a number of assembly factors involved in this process have also been identified. In most cases, these factors have been discovered through their gene mutations that lead to specific complex I defects and result in mitochondrial disease. Here we review how complex I is assembled and the factors required to mediate this process.

Cardiolipin (CL), the signature lipid of mitochondria, plays a critical role in mitochondrial function and biogenesis. The availability of yeast mutants blocked in CL synthesis has facilitated studies of the biological role of this lipid. Perturbation of CL synthesis leads to growth defects not only during respiratory growth but also under conditions in which respiration is not essential. CL was shown to play a role in mitochondrial protein import, cell wall biogenesis, aging and apoptosis, ceramide synthesis, and translation of electron transport chain components. The genetic disorder Barth syndrome (BTHS) is caused by mutations in the tafazzin gene resulting in decreased total CL levels, accumulation of monolysocardiolipin (MLCL), and decreased unsaturated fatty acyl species of CL. The variation in clinical presentation of BTHS indicates that other physiological factors play a significant role in modifying the phenotype resulting from tafazzin deficiency. Elucidating the functions of CL is expected to shed light on the role of this important lipid in BTHS and other disorders of mitochondrial dysfunction.

A strikingly large number of mitochondrial DNA (mtDNA) mutations have been found to be the cause of respiratory chain and oxidative phosphorylation defects. These mitochondrial disorders were the first to be investigated after the small mtDNA had been sequenced in the 80s. Only recently numerous diseases resulting from mutations in nuclear genes encoding mitochondrial proteins have been characterized. Among these, nine are caused by defects of mitochondrial carriers, a family of nuclear-coded proteins that shuttle a variety of metabolites across the mitochondrial membrane. Mutations of mitochondrial carrier genes involved in mitochondrial functions other than oxidative phosphorylation are responsible for carnitine/acylcarnitine carrier deficiency, HHH syndrome, aspartate/glutamate isoform 2 deficiency, Amish microcephaly, and neonatal myoclonic epilepsy; these disorders are characterized by specific metabolic dysfunctions, depending on the physiological role of the affected carrier in intermediary metabolism. Defects of mitochondrial carriers that supply mitochondria with the substrates of oxidative phosphorylation, inorganic phosphate and ADP, are responsible for diseases characterized by defective energy production. Herein, all the mitochondrial carrier-associated diseases known to date are reviewed for the first time. Particular emphasis is given to the molecular basis and pathogenetic mechanism of these inherited disorders.

Mitochondrial F(1)F(0)-ATPase was studied in lymphocytes from patients with neuropathy, ataxia, and retinitis pigmentosa (NARP), caused by a mutation at leu-156 in the ATPase 6 subunit. The mutation giving the milder phenotype (Leu156Pro) suffered a 30% reduction in proton flux, and a similar loss in ATP synthetic activity. The more severe mutation (Leu156Arg) also suffered a 30% reduction in proton flux, but ATP synthesis was virtually abolished. Oligomycin sensitivity of the proton translocation through F(0) was enhanced by both mutations. We conclude that in the Leu156Pro mutation, rotation of the c-ring is slowed but coupling of ATP synthesis to proton flux is maintained, whereas in the Leu156Arg mutation, proton flux appears to be uncoupled. Modelling indicated that, in the Leu156Arg mutation, transmembrane helix III of ATPase 6 is unable to span the membrane, terminating in an intramembrane helix II-helix III loop. We propose that the integrity of transmembrane helix III is essential for the mechanical function of ATPase 6 as a stator element in the ATP synthase, but that it is not relevant for oligomycin inhibition.

Defects of the oxidative ATP production pathway lead to an amazing variety of disease phenotypes, ranging from childhood encephalomyopathies to hereditary tumor formation. A key enzyme of tricarboxylic cycle, fumarate hydratase (FH), is involved in encephalopathies, but also in leiomyoma formation, and occasionally also in various types of cancer. MELAS (mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes) and NARP (neuropathy ataxia retinitis pigmentosa) are progressive neurological disorders, caused by mitochondrial DNA mutations and respiratory chain (RC) deficiency. These diseases lead to disability and premature death, but not to tumorigenesis. We studied the cellular consequences of FH and RC deficiencies, aiming to identify general responses to energy metabolism defect and those specific for FH-deficiency, suggestively connected to tumorigenesis. Unlike in RC deficiency, the FH-deficient diploid human fibroblasts showed no signs of oxidative stress, but had a reduced redox state with high glutathione levels. The cytoplasmic FH isoform, previously described, but with an unknown function, was completely lacking in all FH-deficient lines. Fumarate was increased in two of our FH-lines, but accumulation of HIF-1alpha was not detected. Glycolysis was induced in both MELAS and in FH-deficiency. Accumulation of fumarate in primary fibroblasts did not activate a hypoxia response, suggesting that hypoxia activation due to fumarate accumulation may be a tissue-specific response. The lack of cytoplasmic form of FH and the reduced redox environment were typical for all FH-mutant lines, and their role in FH-related tumorigenesis requires further attention.

Mitochondrial dysfunction and oxidative stress play a central role in the pathophysiology of nonalcoholic fatty liver disease (NAFLD). This study aimed to elucidate the mechanism(s) responsible for mitochondrial dysfunction in nonalcoholic fatty liver. Fatty liver was induced in rats with a choline-deficient (CD) diet for 30 days. We examined the effect of CD diet on various parameters related to mitochondrial function such as complex I activity, oxygen consumption, reactive oxygen species (ROS) generation and cardiolipin content and oxidation. The activity of complex I was reduced by 35% in mitochondria isolated from CD livers compared with the controls. These changes in complex I activity were associated with parallel changes in state 3 respiration. Hydrogen peroxide (H(2)O(2)) generation was significantly increased in mitochondria isolated from CD livers. The mitochondrial content of cardiolipin, a phospholipid required for optimal activity of complex I, decreased by 38% as function of CD diet, while there was a significantly increase in the level of peroxidized cardiolipin. The lower complex I activity in mitochondria from CD livers could be completely restored to the level of control livers by exogenously added cardiolipin. This effect of cardiolipin could not be replaced by other phospholipids nor by peroxidized cardiolipin. It is concluded that CD diet causes mitochondrial complex I dysfunction which can be attributed to ROS-induced cardiolipin oxidation. These findings provide new insights into the alterations underlying mitochondrial dysfunction in NAFLD.

The NADH:ubiquinone oxidoreductase or complex I of the mitochondrial respiratory chain is an intricate enzyme with a vital role in energy metabolism. Mutations affecting complex I can affect at least three processes; they can impair the oxidation of NADH, reduce the enzyme's ability to pump protons for the generation of a mitochondrial membrane potential and increase the production of damaging reactive oxygen species. We have previously developed a nematode model of complex I-associated mitochondrial dysfunction that features hallmark characteristics of mitochondrial disease, such as lactic acidosis and decreased respiration. We have expressed the Saccharomyces cerevisiae NDI1 gene, which encodes a single subunit NADH dehydrogenase, in a strain of Caenorhabditis elegans with an impaired complex I. Expression of Ndi1p produces marked improvements in animal fitness and reproduction, increases respiration rates and restores mitochondrial membrane potential to wild type levels. Ndi1p functionally integrates into the nematode respiratory chain and mitigates the deleterious effects of a complex I deficit. However, we have also shown that Ndi1p cannot substitute for the absence of complex I. Nevertheless, the yeast Ndi1p should be considered as a candidate for gene therapy in human diseases involving complex I.

Isolated complex I deficiency is the most common enzymatic defect of the oxidative phosphorylation (OXPHOS) system, causing a wide range of clinical phenotypes. We reported before that the rates at which reactive oxygen species (ROS)-sensitive dyes are converted into their fluorescent oxidation products are markedly increased in cultured skin fibroblasts of patients with nuclear-inherited isolated complex I deficiency. Using video-imaging microscopy we show here that these cells also display a marked increase in NAD(P)H autofluorescence. Linear regression analysis revealed a negative correlation with the residual complex I activity and a positive correlation with the oxidation rates of the ROS-sensitive dyes 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein and hydroethidine for a cohort of 10 patient cell lines. On the other hand, video-imaging microscopy of cells expressing reduction-oxidation sensitive GFP1 in either the mitochondrial matrix or cytosol showed the absence of any detectable change in thiol redox state. In agreement with this result, neither the glutathione nor the glutathione disulfide content differed significantly between patient and healthy fibroblasts. Finally, video-rate confocal microscopy of cells loaded with C11-BODIPY(581/591) demonstrated that the extent of lipid peroxidation, which is regarded as a measure of oxidative damage, was not altered in patient fibroblasts. Our results indicate that fibroblasts of patients with isolated complex I deficiency maintain their thiol redox state despite marked increases in ROS production.