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ABSTRACT: Abstract Aims: Cell regulation by signaling reactive oxygen species (sROS) is often incorrectly studied through extracellular oxidant addition. Here, we used the membrane-permeable antioxidant Trolox to examine the role of sROS in mitochondrial morphology, oxidative phosphorylation (OXPHOS), and cytosolic calcium (Ca(2+)) handling in healthy human skin fibroblasts. Results and Innovation: Trolox treatment reduced the levels of 5-(and-6)-chloromethyl-2',7'-dichlorodihydro-fluorescein (CM-H(2)DCF) oxidizing ROS, lowered cellular lipid peroxidation, and induced a less oxidized mitochondrial thiol redox state. This was paralleled by increased glutathione- and mitofusin-dependent mitochondrial filamentation, increased expression of fully assembled mitochondrial complex I, elevated activity of citrate synthase and OXPHOS enzymes, and a higher cellular O(2) consumption. In contrast, Trolox did not alter hydroethidium oxidation, cytosolic thiol redox state, mitochondrial NAD(P)H levels, or mitochondrial membrane potential. Whole genome expression profiling revealed that Trolox did not trigger significant changes in gene expression, suggesting that Trolox acts downstream of this process. Cytosolic Ca(2+) transients, induced by the hormone bradykinin, were of a higher amplitude and decayed faster in Trolox-treated cells. These effects were dose-dependently antagonized by hydrogen peroxide. Conclusions: Our findings suggest that Trolox-sensitive sROS are upstream regulators of mitochondrial mitofusin levels, morphology, and function in healthy human skin fibroblasts. This information not only facilitates the interpretation of antioxidant effects in cell models (of oxidative-stress), but also contributes to a better understanding of ROS-related human pathologies, including mitochondrial disorders. Antioxid. Redox Signal. 17, 1657-1669.
Antioxidants & Redox Signaling 05/2012; 17(12):1657-69. · 8.20 Impact Factor
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ABSTRACT: Mitochondrial complex I (CI) is a multi-subunit enzyme that forms the major entry point of nicotinamide adenine dinucleotide (NADH) electrons into the respiratory chain. Mutations in the NDUFS4 gene, encoding an accessory subunit of this complex, cause a Leigh-like phenotype in humans. To study the nature and penetrance of the CI defect in different tissues, we investigated the role of NDUFS4 in mice with fatal mitochondrial encephalomyopathy, caused by a systemic inactivation of the Ndufs4 gene. We report that the absence of NDUFS4 in different mouse tissues results in decreased activity and stability of CI. This CI instability leads to an increased disconnection of electron influx of the NADH dehydrogenase module from the holo-complex. However, the formation of respiratory supercomplexes still allows formation of active CI in these Ndufs4 knock-out mice. These results reveal the importance of these supramolecular interactions not only for stabilization but also for the assembly of CI, which becomes especially relevant in pathological conditions.
Human Molecular Genetics 09/2011; 21(1):115-20. · 7.64 Impact Factor
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ABSTRACT: Whilst the pathophysiology and genetics of mitochondrial disease are slowly being unraveled, currently no effective remedy for mitochondrial disorders is available. One particular strategy in mitochondrial medicine presently under study is metabolic manipulation. This approach is aimed at counteracting the deranged cell biological homeostasis caused by mitochondrial dysfunction, using dietary modifications or small molecule therapy. Cell biological alterations caused by mitochondrial dysfunction include increased reactive oxygen species production, enhanced lipid peroxidation and altered cellular calcium homeostasis. This review covers the five principles of metabolic manipulation: (1) prevention of oxidative damage by reactive oxygen species, (2) amelioration of lipid peroxidation, (3) correction of altered membrane potential, (4) restoration of calcium homeostasis, and (5) transcription regulation interference. We hypothesize that a combination of compounds targeting different metabolic pathways will abolish cellular disturbance arising as a consequences of mitochondrial dysfunction, and thereby improve or stabilize clinical features. However, only a handful of compounds have reached efficacy testing in mammals, and it remains unknown to what extent metabolic manipulation will affect the whole organism. Until a potent remedy is found, patients will remain dependent on supportive, not curative, interventions.
Journal of Inherited Metabolic Disease 04/2011; 34(2):315-25. · 3.58 Impact Factor
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Journal of Inherited Metabolic Disease 03/2011; 34(2):247-8. · 3.58 Impact Factor
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Mateusz Kolanczyk,
Markus Pech,
Tomasz Zemojtel,
Hiroshi Yamamoto,
Ivan Mikula,
Maria-Antonietta Calvaruso,
Mariël van den Brand,
Ricarda Richter,
Bjoern Fischer,
Anita Ritz, [......], Jan Smeitink,
Uwe Kornak,
Danny Chan,
Martin Vingron,
Pavel Martasek,
Robert N Lightowlers,
Leo Nijtmans,
Markus Schuelke,
Knud H Nierhaus,
Stefan Mundlos
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ABSTRACT: Nitric oxide associated-1 (NOA1) is an evolutionarily conserved guanosine triphosphate (GTP) binding protein that localizes predominantly to mitochondria in mammalian cells. On the basis of bioinformatic analysis, we predicted its possible involvement in ribosomal biogenesis, although this had not been supported by any experimental evidence. Here we determine NOA1 function through generation of knockout mice and in vitro assays. NOA1-deficient mice exhibit midgestation lethality associated with a severe developmental defect of the embryo and trophoblast. Primary embryonic fibroblasts isolated from NOA1 knockout embryos show deficient mitochondrial protein synthesis and a global defect of oxidative phosphorylation (OXPHOS). Additionally, Noa1⁻/⁻ cells are impaired in staurosporine-induced apoptosis. The analysis of mitochondrial ribosomal subunits from Noa1⁻/⁻ cells by sucrose gradient centrifugation and Western blotting showed anomalous sedimentation, consistent with a defect in mitochondrial ribosome assembly. Furthermore, in vitro experiments revealed that intrinsic NOA1 GTPase activity was stimulated by bacterial ribosomal constituents. Taken together, our data show that NOA1 is required for mitochondrial protein synthesis, likely due to its yet unidentified role in mitoribosomal biogenesis. Thus, NOA1 is required for such basal mitochondrial functions as adenosine triphosphate (ATP) synthesis and apoptosis.
Molecular biology of the cell 01/2011; 22(1):1-11. · 5.98 Impact Factor
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Martijn Kranendijk,
Eduard A Struys,
Emile van Schaftingen,
K Michael Gibson,
Warsha A Kanhai,
Marjo S van der Knaap,
Jeanne Amiel,
Neil R Buist,
Anibh M Das,
Johannis B de Klerk, [......],
Elisabeth Holme,
Edwin P Kirk,
Stanley H Korman,
Eva Morava,
Andrew Morris, Jan Smeitink,
Rám N Sukhai,
Hilary Vallance,
Cornelis Jakobs,
Gajja S Salomons
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ABSTRACT: Heterozygous somatic mutations in the genes encoding isocitrate dehydrogenase-1 and -2 (IDH1 and IDH2) were recently discovered in human neoplastic disorders. These mutations disable the enzymes' normal ability to convert isocitrate to 2-ketoglutarate (2-KG) and confer on the enzymes a new function: the ability to convert 2-KG to d-2-hydroxyglutarate (D-2-HG). We have detected heterozygous germline mutations in IDH2 that alter enzyme residue Arg(140) in 15 unrelated patients with d-2-hydroxyglutaric aciduria (D-2-HGA), a rare neurometabolic disorder characterized by supraphysiological levels of D-2-HG. These findings provide additional impetus for investigating the role of D-2-HG in the pathophysiology of metabolic disease and cancer.
Science 10/2010; 330(6002):336. · 31.20 Impact Factor
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Jessica Nouws,
Leo Nijtmans,
Sander M Houten,
Mariël van den Brand,
Martijn Huynen,
Hanka Venselaar,
Saskia Hoefs,
Jolein Gloerich,
Jonathan Kronick,
Timothy Hutchin,
Peter Willems,
Richard Rodenburg,
Ronald Wanders,
Lambert van den Heuvel, Jan Smeitink,
Rutger O Vogel
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ABSTRACT: Acyl-CoA dehydrogenase 9 (ACAD9) is a recently identified member of the acyl-CoA dehydrogenase family. It closely resembles very long-chain acyl-CoA dehydrogenase (VLCAD), involved in mitochondrial beta oxidation of long-chain fatty acids. Contrary to its previously proposed involvement in fatty acid oxidation, we describe a role for ACAD9 in oxidative phosphorylation. ACAD9 binds complex I assembly factors NDUFAF1 and Ecsit and is specifically required for the assembly of complex I. Furthermore, ACAD9 mutations result in complex I deficiency and not in disturbed long-chain fatty acid oxidation. This strongly contrasts with its evolutionary ancestor VLCAD, which we show is not required for complex I assembly and clearly plays a role in fatty acid oxidation. Our results demonstrate that two closely related metabolic enzymes have diverged at the root of the vertebrate lineage to function in two separate mitochondrial metabolic pathways and have clinical implications for the diagnosis of complex I deficiency.
Cell metabolism 09/2010; 12(3):283-94. · 17.35 Impact Factor
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Jan Smeitink
New England Journal of Medicine 03/2010; 362(12):1144-5. · 53.30 Impact Factor
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ABSTRACT: Mitochondrial disorders are a heterogeneous group of often multisystemic and early fatal diseases, which are amongst the most common inherited human diseases. These disorders are caused by defects in the oxidative phosphorylation (OXPHOS) system, which comprises five multisubunit enzyme complexes encoded by both the nuclear and the mitochondrial genomes. Due to the multitude of proteins and intricacy of the processes required for a properly functioning OXPHOS system, identifying the genetic defect that underlies an OXPHOS deficiency is not an easy task, especially in the case of combined OXPHOS defects. In the present communication we give an extensive overview of the proteins and processes (in)directly involved in mitochondrial translation and the biogenesis of the OXPHOS system and their roles in combined OXPHOS deficiencies. This knowledge is important for further research into the genetic causes, with the ultimate goal to effectively prevent and cure these complex and often devastating disorders.
Journal of Biomedicine and Biotechnology 01/2010; 2010:737385. · 2.44 Impact Factor
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Paulien Smits,
Sandy Mattijssen,
Eva Morava,
Mariël van den Brand,
Frans van den Brandt,
Frits Wijburg,
Ger Pruijn, Jan Smeitink,
Leo Nijtmans,
Richard Rodenburg,
Lambert van den Heuvel
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ABSTRACT: Combined oxidative phosphorylation (OXPHOS) system deficiencies are a group of mitochondrial disorders that are associated with a range of clinical phenotypes and genetic defects. They occur in approximately 30% of all OXPHOS disorders and around 4% are combined complex I, III and IV deficiencies. In this study we present two mutations in the mitochondrial tRNA(Trp) (MT-TW) and tRNA(Arg) (MT-TR) genes, m.5556G>A and m.10450A>G, respectively, which were detected in two unrelated patients showing combined OXPHOS complex I, III and IV deficiencies and progressive multisystemic diseases. Both mitochondrial tRNA mutations were almost homoplasmic in fibroblasts and muscle tissue of the two patients and not present in controls. Patient fibroblasts showed a general mitochondrial translation defect. The mutations resulted in lowered steady-state levels and altered conformations of the tRNAs. Cybrid cell lines showed similar tRNA defects and impairment of OXPHOS complex assembly as patient fibroblasts. Our results show that these tRNA(Trp) and tRNA(Arg) mutations cause the combined OXPHOS deficiencies in the patients, adding to the still expanding group of pathogenic mitochondrial tRNA mutations.
European journal of human genetics: EJHG 10/2009; 18(3):324-9. · 3.56 Impact Factor
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Riekelt H Houtkooper,
Richard J Rodenburg,
Charlotte Thiels,
Henk van Lenthe,
Femke Stet,
Bwee Tien Poll-The,
Janet E Stone,
Colin G Steward,
Ronald J Wanders, Jan Smeitink,
Willem Kulik,
Frédéric M Vaz
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ABSTRACT: Barth syndrome (BTHS) is an X-linked recessive disorder caused by mutations in the tafazzin (or TAZ) gene and is clinically characterized by (cardio)myopathy, neutropenia, and growth abnormalities. Biochemical abnormalities include decreased levels of the mitochondrial phospholipid cardiolipin, increased levels of monolysocardiolipin, and a lower degree of unsaturation of the (monolyso)cardiolipin acyl chains. Diagnostic testing for BTHS is routinely performed by TAZ gene sequencing, and recently a BTHS screening method in bloodspots has been developed, but both methods have important limitations. Because a validated confirmatory method is not yet available, we set up and validated a high-performance liquid chromatography-mass spectrometry (HPLC-MS) method for BTHS in cultured fibroblasts, lymphocytes, and skeletal muscle based on cardiolipin, monolysocardiolipin, and the monolysocardiolipin/cardiolipin ratio. In addition, we performed retrospective analysis of 121 muscle samples of patients with myopathy of which mitochondrial origin was presumed, and we identified one patient with cardiolipin abnormalities similar to BTHS patients. Molecular analysis revealed a bona fide mutation in the TAZ gene. We conclude that (monolyso)cardiolipin analysis by HPLC-MS not only is a powerful tool to diagnose patients with clinical signs and symptoms of BTHS but also should be used in patients suffering from mitochondrial myopathies with unknown etiology.
Analytical Biochemistry 05/2009; 387(2):230-7. · 3.00 Impact Factor
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ABSTRACT: Malfunction of mitochondrial complex I caused by nuclear gene mutations causes early-onset neurodegenerative diseases. Previous work using cultured fibroblasts of complex-I-deficient patients revealed elevated levels of reactive oxygen species (ROS) and reductions in both total Ca(2+) content of the endoplasmic reticulum (ER(Ca)) and bradykinin(Bk)-induced increases in cytosolic and mitochondrial free Ca(2+) ([Ca(2+)](C); [Ca(2+)](M)) and ATP ([ATP](C); [ATP](M)) concentration. Here, we determined the mitochondrial membrane potential (Delta psi) in patient skin fibroblasts and show significant correlations with cellular ROS levels and ER(Ca), i.e., the less negative Delta psi, the higher these levels and the lower ER(Ca). Treatment with 6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox) normalized Delta psi and Bk-induced increases in [Ca(2+)](M) and [ATP](M). These effects were accompanied by an increase in ER(Ca) and Bk-induced increase in [Ca(2+)](C). Together, these results provide evidence for an integral role of increased ROS levels in complex I deficiency and point to the potential therapeutic value of antioxidant treatment.
Journal of Molecular Medicine 04/2009; 87(5):515-22. · 4.67 Impact Factor
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Pediatric Anesthesia 02/2009; 19(1):62-3. · 2.10 Impact Factor
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Despina Contopoulos-Ioannidis,
Athanasios Evangeliou,
Henk ter Laak,
Bert de Vries,
Rolph Pfundt,
Hans Scheffer, Jan Smeitink,
Meropi Tzoufi,
Alexandros Makis,
Evangelos Marinos,
Richard Hess,
David Adams,
Marjan Huizing,
Eva Morava
American Journal of Medical Genetics Part A 12/2008; 146A(23):3100-3. · 2.39 Impact Factor
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ABSTRACT: Defects in mitochondrial oxidative phosphorylation (OXPHOS) are a frequent cause of severe inherited metabolic disorders and also contribute to aging. The OXPHOS system constitutes five multi-subunit complexes embedded in the mitochondrial inner membrane. Correct function of this system requires proper assembly of the approximately 80 proteins in the complexes, as well as numerous assembly factors. Blue native electrophoresis has become a crucial tool to investigate OXPHOS-related defects in mitochondrial disease patients. In addition, OXPHOS-assembly profiles can be obtained by two dimensional blue native/SDS gel electrophoresis, which provides additional information for identifying disease-causing mutations and insight in the role of specific proteins in the biogenesis of the OXPHOS system. Here we provide a practical guide on how to set-up the basic technique to study OXPHOS defects in patient-derived cells and tissues.
Methods 11/2008; 46(4):281-7. · 4.01 Impact Factor
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ABSTRACT: Besides characteristic neurological and musculoskeletal symptoms, children with mitochondrial dysfunction often present with feeding problems and failure to thrive. Substrate depletion for the respiratory chain has an effect on energy expenditure. Secondary mitochondrial dysfunction has been reported in severe chronic malnutrition. We evaluated the nutritional state, the growth parameters, and the metabolic condition in 172 children undergoing muscle biopsy for a suspected disorder of the oxidative phosphorylation system (OXPHOS). We performed dietary evaluation and initiated nutritional intervention when needed prior to the biopsy. Mitochondrial dysfunction was confirmed by detection of enzyme-complex deficiencies and/or by mutations in 83 children, in 14 patients no biochemical abnormalities were found. In the whole study group, as well as in the subgroup with enzyme-complex deficiency and/or mutation, a significant correlation was found between the mitochondrial production of adenosine triphosphate (ATP) and the age related BMI. Nutritional state and growth should be considered by interpreting the results of ATP-production in fresh muscle biopsy. Due to a positive correlation between the age appropriate BMI and the ATP-production, we strongly advise optimizing the nutritional state preceding the muscle biopsy in children with a suspected OXPHOS-disorder. Dietary intervention remains although challenging due to frequent gastrointestinal problems and eating disorders.
Pediatric Research 10/2008; · 2.70 Impact Factor
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Giuseppe Gasparre,
Eric Hervouet,
Elodie de Laplanche,
Jocelyne Demont,
Lucia Fiammetta Pennisi,
Marc Colombel,
Florence Mège-Lechevallier,
Jean-Yves Scoazec,
Elena Bonora,
Roel Smeets, Jan Smeitink,
Vladimir Lazar,
James Lespinasse,
Sophie Giraud,
Catherine Godinot,
Giovanni Romeo,
Hélène Simonnet
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ABSTRACT: Mutations in mitochondrial DNA (mtDNA) are frequent in cancers but it is not yet clearly established whether they are modifier events involved in cancer progression or whether they are a consequence of tumorigenesis. Here we show a benign tumor type in which mtDNA mutations that lead to complex I (CI) enzyme deficiency are found in all tumors and are the only genetic alteration detected. Actually renal oncocytomas are homogeneous tumors characterized by dense accumulation of mitochondria and we had found that they are deficient in electron transport chain complex I (CI, NADH-ubiquinone oxidoreductase). In this work total sequencing of mtDNA showed that 9/9 tumors harbored point mutations in mtDNA, seven in CI genes, one in complex III, and one in the control region. 7/8 mutations were somatic. All tumors were somatically deficient for CI. The clonal amplification of mutated mtDNA in 8/9 tumors demonstrates that these alterations are selected and therefore favor or trigger growth. No nuclear DNA rearrangement was detected beside mtDNA defects. We hypothesize that functional deficiency of the oxidative phosphorylation CI could create a loop of amplification of mitochondria during cell division, impair substrates oxidation and increase intermediary metabolites availability.
Human Molecular Genetics 05/2008; 17(7):986-95. · 7.64 Impact Factor
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Marcel M Verbeek,
Gerry C H Steenbergen-Spanjers,
Michèl A A P Willemsen,
Frans A Hol, Jan Smeitink,
Jürgen Seeger,
Padraic Grattan-Smith,
Monique M Ryan,
Georg F Hoffmann,
Maria A Donati,
Nenad Blau,
Ronald A Wevers
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ABSTRACT: Tyrosine hydroxylase (TH) deficiency (OMIM 191290) is one cause of early-onset dopa-responsive dystonia. We describe seven cases from five unrelated families with dopa-responsive dystonia and low homovanillic acid in cerebrospinal fluid who were suspected to suffer from TH deficiency. Analysis of part of the TH promotor showed five homozygous and two heterozygous mutations in the highly conserved cyclic adenosine monophosphate response element. Our data suggest that, if no mutations are found in the coding regions of the gene in patients strongly suspected of TH deficiency, the search for pathogenic mutations should be extended to regulatory promotor elements.
Annals of Neurology 11/2007; 62(4):422-6. · 11.09 Impact Factor
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ABSTRACT: The characteristic clinical presentation, especially the appearance of muscle symptoms, is quite unique in children carrying the mtA8344G mutation. The diagnosis of MERRF syndrome is seldom made in the pediatric age. Fatigue is a common finding in children of pubertal age. Fatigue in combination with recurrent resting muscle pain occurs frequently in the initial phase of various hereditary muscle disorders and in several autoimmune, endocrine and metabolic syndromes. In the absence of obvious biochemical/metabolic abnormalities and in the lack of neurological symptoms the complaints are frequently labelled as fibromyalgia or chronic fatigue syndrome. In patients with behavioural or psychiatric abnormalities one might even start to question the organic etiology of the complaints. We describe a family carrying the classic MTTK mutation with a variable degree of heteroplasmy, presenting in childhood as isolated recurrent muscle pain as the first symptom of the disease.
European Journal of Paediatric Neurology 08/2007; 11(4):243-6. · 2.12 Impact Factor
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ABSTRACT: In this chapter the biochemical diagnosis of OXPHOS disorders is presented. The laboratory investigations in suspected patients
are started with the examination of body fluids. The most important metabolite to be measured is lactate, that is frequendy
found to be elevated in blood, urine and cerebrospinal fluid of patients with OXPHOS disorders. The next step in the diagnostic
procedure consists of the examination of tissues. The biochemical diagnostic investigations are preferably performed in muscle
tissue because in most patients the defect is expressed in muscle. Biopsy material is preferred above autopsy material. Biochemical
examination of a fresh muscle sample is to be preferred because mitochondria are intact in fresh muscle thus allowing measurement
of the overall oxidative capacity of the mitochondria. In a frozen muscle sample only enzyme activities of the OXPHOS complexes
can be measured. In the latter case patients with a disturbance in the oxidative phosphorylation not localized in one of the
OXPHOS complexes remain undiagnosed. Practical guidelines for the biochemical examinations of muscle are provided. In certain
circumstances it is necessary to examine also fibroblasts. This is an absolute prerequisite in case prenatal diagnosis is
requested. The interpre-tation of the biochemical investigations is discussed with special emphasis on the observed residual
enzyme activities.
03/2007: pages 79-94;
