RESUMEN
Mitochondrial aconitase is the second enzyme in the tricarboxylic acid (TCA) cycle catalyzing the interconversion of citrate into isocitrate and encoded by the nuclear gene ACO2. A homozygous pathogenic variant in the ACO2 gene was initially described in 2012 resulting in a novel disorder termed "infantile cerebellar retinal degeneration" (ICRD, OMIM#614559). Subsequently, additional studies reported patients with pathogenic ACO2 variants, further expanding the genetic and clinical spectrum of this disorder to include milder and later onset manifestations. Here, we report an international multicenter cohort of 16 patients (of whom 7 are newly diagnosed) with biallelic pathogenic variants in ACO2 gene. Most patients present in early infancy with severe truncal hypotonia, truncal ataxia, variable seizures, evolving microcephaly, and ophthalmological abnormalities of which the most dominant are esotropia and optic atrophy with later development of retinal dystrophy. Most patients remain nonambulatory and do no acquire any language, but a subgroup of patients share a more favorable course. Brain magnetic resonance imaging (MRI) is typically normal within the first months but global atrophy gradually develops affecting predominantly the cerebellum. Ten of our patients were homozygous to the previously reported c.336C>G founder mutation while the other six patients were all compound heterozygotes displaying 10 novel mutations of whom 2 were nonsense predicting a deleterious effect on enzyme function. Structural protein modeling predicted significant impairment in aconitase substrate binding in the additional missense mutations. This study provides the most extensive cohort of patients and further delineates the clinical, radiological, biochemical, and molecular features of ACO2 deficiency.
Asunto(s)
Aconitato Hidratasa/deficiencia , Enfermedades Neurodegenerativas/diagnóstico , Atrofia Óptica/diagnóstico , Distrofias Retinianas/diagnóstico , Aconitato Hidratasa/genética , Adolescente , Ataxia/genética , Cerebelo/patología , Niño , Preescolar , Ciclo del Ácido Cítrico , Exoma/genética , Femenino , Secuenciación de Nucleótidos de Alto Rendimiento , Homocigoto , Humanos , Internacionalidad , Imagen por Resonancia Magnética , Masculino , Microcefalia/genética , Mutación Missense , Enfermedades Neurodegenerativas/genética , Atrofia Óptica/genética , Distrofias Retinianas/genética , Síndrome , Adulto JovenRESUMEN
Mitochondrial respiratory chain dysfunction has been identified in a number of neurodegenerative disorders. Infantile cerebellar-retinal degeneration associated with mutations in the mitochondrial aconitase 2 gene (ACO2) has been recently described as a neurodegenerative disease of autosomal recessive inheritance. To date there is no biomarker for ACO2 deficiency and diagnosis relies on genetic analysis. Here we report global metabolic profiling in eight patients with ACO2 deficiency. Using an LC-MS-based metabolomics platform we have identified several metabolites with affected plasma concentrations including the tricarboxylic acid cycle metabolites cis-aconitate, isocitrate and alpha-ketoglutarate, as well as phosphoenolpyruvate and hydroxybutyrate. Taken together we report a diagnostic metabolic fingerprint for mitochondrial aconitase 2 deficiency.
Asunto(s)
Aconitato Hidratasa/deficiencia , Aconitato Hidratasa/genética , Ácido Aconítico/sangre , Adolescente , Biomarcadores/sangre , Niño , Preescolar , Femenino , Trastornos Heredodegenerativos del Sistema Nervioso/sangre , Trastornos Heredodegenerativos del Sistema Nervioso/diagnóstico , Humanos , Hidroxibutiratos/sangre , Isocitratos/sangre , Ácidos Cetoglutáricos/sangre , Masculino , Metabolómica/métodos , Fosfoenolpiruvato/sangreRESUMEN
Defects in the tricarboxylic acid cycle (TCA) are associated with a spectrum of neurological phenotypes that are often difficult to diagnose and manage. Whole-exome sequencing (WES) led to a rapid expansion of diagnostic capabilities in such disorders and facilitated a better understanding of disease pathogenesis, although functional characterization remains a bottleneck to the interpretation of potential pathological variants. We report a 2-year-old boy of Afro-Caribbean ancestry, who presented with neuromuscular symptoms without significant abnormalities on routine diagnostic evaluation. WES revealed compound heterozygous missense variants of uncertain significance in mitochondrial aconitase (ACO2), which encodes the TCA enzyme ACO2. Pathogenic variants in ACO2 have been described in a handful of families as the cause of infantile cerebellar-retinal degeneration syndrome. Using biochemical and cellular assays in patient fibroblasts, we found that ACO2 expression was quantitatively normal, but ACO2 enzyme activity was <20% of that observed in control cells. We also observed a deficiency in cellular respiration and, for the first time, demonstrate evidence of mitochondrial DNA depletion and altered expression of some TCA components and electron transport chain subunits. The observed cellular defects were completely restored with ACO2 gene rescue. Our findings demonstrate the pathogenicity of two VUS in ACO2, provide novel mechanistic insights to TCA disturbances in ACO2 deficiency, and implicate mitochondrial DNA depletion in the pathogenesis of this recently described disorder.
Asunto(s)
Aconitato Hidratasa/deficiencia , Aconitato Hidratasa/genética , Errores Innatos del Metabolismo/genética , Mutación Missense , Enfermedades Neuromusculares/genética , Preescolar , Ciclo del Ácido Cítrico , ADN Mitocondrial/genética , Exoma , Regulación de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Masculino , Errores Innatos del Metabolismo/etnología , Errores Innatos del Metabolismo/metabolismo , Enfermedades Neuromusculares/etnología , Enfermedades Neuromusculares/metabolismoRESUMEN
Aconitase catalyzes the conversion of citrate to isocitrate in the tricarboxylic acid (TCA) cycle, and its deficiency in humans is associated with an infantile neurodegenerative disorder affecting mainly the cerebellum and retina. Here we investigated the effect of gene knockout and knockdown of the mitochondrial aconitase Acon in Drosophila. Acon-knockout flies were homozygous lethal, indicating that Acon is essential for viability. RNA interference-generated Acon-knockdown flies exhibited a variety of phenotypes, such as reduced locomotor activity, a shortened lifespan, and increased cell death in the developing brain. Metabolomic analysis revealed that acetyl-CoA, citrate/isocitrate, and cis-aconitate were significantly increased, while most metabolites of glycolysis and the TCA cycle were reduced. Reduced triacylglyceride and increased acetyl-CoA suggested that lipids were used as an energy source because of the impaired glycolysis and TCA cycle. The Acon-knockdown model should facilitate further understanding of the pathophysiology of m-aconitase deficiency in humans.
Asunto(s)
Aconitato Hidratasa/deficiencia , Proteínas de Drosophila/deficiencia , Drosophila melanogaster/metabolismo , Acetilcoenzima A/metabolismo , Aconitato Hidratasa/genética , Animales , Secuencia de Bases , Muerte Celular , Ciclo del Ácido Cítrico , Cartilla de ADN/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Metabolismo Energético , Femenino , Técnicas de Silenciamiento del Gen , Técnicas de Inactivación de Genes , Genes de Insecto , Glucólisis , Metabolismo de los Lípidos , Longevidad , Metaboloma , Mitocondrias/metabolismo , Modelos Biológicos , Actividad Motora , Neuronas/citología , Fenotipo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Triglicéridos/metabolismoRESUMEN
Mitochondrial oxidative stress is a contributing factor in the etiology of numerous neuronal disorders. However, the precise mechanism(s) by which mitochondrial reactive oxygen species modify cellular targets to induce neurotoxicity remains unknown. In this study, we determined the role of mitochondrial aconitase (m-aconitase) in neurotoxicity by decreasing its expression. Incubation of the rat dopaminergic cell line, N27, with paraquat (PQ(2+) ) resulted in aconitase inactivation, increased hydrogen peroxide (H(2) O(2) ) and increased ferrous iron (Fe(2+) ) at times preceding cell death. To confirm the role of m-aconitase in dopaminergic cell death, we knocked down m-aconitase expression via RNA interference. Incubation of m-aconitase knockdown N27 cells with PQ(2+) resulted in decreased H(2) O(2) production, Fe(2+) accumulation, and cell death compared with cells expressing basal levels of m-aconitase. To determine the metabolic role of m-aconitase in mediating neuroprotection, we conducted a complete bioenergetic profile. m-Aconitase knockdown N27 cells showed a global decrease in metabolism (glycolysis and oxygen consumption rates) which blocked PQ(2+) -induced H(+) leak and respiratory capacity deficiency. These findings suggest that dopaminergic cells are protected from death by decreasing release of H(2) O(2) and Fe(2+) in addition to decreased cellular metabolism.
Asunto(s)
Aconitato Hidratasa/metabolismo , Dopamina/metabolismo , Peróxido de Hidrógeno/metabolismo , Hierro/metabolismo , Mitocondrias/metabolismo , Aconitato Hidratasa/deficiencia , Aconitato Hidratasa/genética , Análisis de Varianza , Animales , Antimicina A/farmacología , Carbonil Cianuro p-Trifluorometoxifenil Hidrazona/farmacología , Muerte Celular/efectos de los fármacos , Línea Celular Transformada , Fumarato Hidratasa/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Herbicidas/toxicidad , Ionóforos/farmacología , L-Lactato Deshidrogenasa/metabolismo , Mitocondrias/efectos de los fármacos , Consumo de Oxígeno/efectos de los fármacos , Consumo de Oxígeno/fisiología , Paraquat/toxicidad , Protones , ARN Mensajero/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Ratas , Factores de Tiempo , Transfección/métodosRESUMEN
Myopathy with deficiency of succinate dehydrogenase and aconitase is a recessively inherited disorder characterized by childhood-onset early fatigue, dyspnoea and palpitations on trivial exercise. The disease is non-progressive, but life-threatening episodes of widespread weakness, severe metabolic acidosis and rhabdomyolysis may occur. The disease has so far only been identified in northern Sweden. The clinical, histochemical and biochemical phenotype is very homogenous and the patients are homozygous for a deep intronic IVS5 + 382G>C splicing affecting mutation in ISCU, which encodes the differently spliced cytosolic and mitochondrial iron-sulphur cluster assembly protein IscU. Iron-sulphur cluster containing proteins are essential for iron homeostasis and respiratory chain function, with IscU being among the most conserved proteins in evolution. We identified a shared homozygous segment of only 405,000 base pair with the deep intronic mutation in eight patients with a phenotype consistent with the original description of the disease. Two other patients, two brothers, had an identical biochemical and histochemical phenotype which is probably pathognomonic for muscle iron-sulphur cluster deficiency, but they presented with a disease where the clinical phenotype was characterized by early onset of a slowly progressive severe muscle weakness, severe exercise intolerance and cardiomyopathy. The brothers were compound heterozygous for the deep intronic mutation and had a c.149 G>A missense mutation in exon 3 changing a completely conserved glycine residue to a glutamate. The missense mutation was inherited from their mother who was of Finnish descent. The intronic mutation affects mRNA splicing and results in inclusion of pseudoexons in most transcripts in muscle. The pseudoexon inclusion results in a change in the reading frame and appearance of a premature stop codon. In western blot analysis of protein extracts from fibroblasts, there was no pronounced reduction of IscU in any of the patients, but the analysis revealed that the species corresponding to mitochondrial IscU migrates slower than a species present only in whole cells. In protein extracted from isolated skeletal muscle mitochondria the western blot analysis revealed a severe deficiency of IscU in the homozygous patients and appearance of a faint new fraction that could represent a truncated protein. There was only a slight reduction of mitochondrial IscU in the compound heterozygotes, despite their severe phenotype, indicating that the IscU expressed in these patients is non-functional.
Asunto(s)
Proteínas Hierro-Azufre/genética , Miopatías Mitocondriales/genética , Mutación , Aconitato Hidratasa/deficiencia , Adolescente , Adulto , Anciano , Secuencia de Bases , Biopsia , Células Cultivadas , Niño , Preescolar , Femenino , Genotipo , Humanos , Proteínas Hierro-Azufre/deficiencia , Masculino , Mitocondrias Musculares/ultraestructura , Miopatías Mitocondriales/metabolismo , Miopatías Mitocondriales/patología , Proteínas Mitocondriales/deficiencia , Datos de Secuencia Molecular , Músculo Esquelético/patología , Linaje , Fenotipo , Reacción en Cadena de la Polimerasa/métodos , Polimorfismo de Nucleótido Simple , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Adulto JovenRESUMEN
A myopathy with severe exercise intolerance and myoglobinuria has been described in patients from northern Sweden, with associated deficiencies of succinate dehydrogenase and aconitase in skeletal muscle. We identified the gene for the iron-sulfur cluster scaffold protein ISCU as a candidate within a region of shared homozygosity among patients with this disease. We found a single mutation in ISCU that likely strengthens a weak splice acceptor site, with consequent exon retention. A marked reduction of ISCU mRNA and mitochondrial ISCU protein in patient muscle was associated with a decrease in the iron regulatory protein IRP1 and intracellular iron overload in skeletal muscle, consistent with a muscle-specific alteration of iron homeostasis in this disease. ISCU interacts with the Friedreich ataxia gene product frataxin in iron-sulfur cluster biosynthesis. Our results therefore extend the range of known human diseases that are caused by defects in iron-sulfur cluster biogenesis.
Asunto(s)
Tolerancia al Ejercicio/genética , Proteínas Hierro-Azufre/genética , Miopatías Mitocondriales/genética , Sitios de Empalme de ARN/genética , Aconitato Hidratasa/deficiencia , Adulto , Anciano , Secuencia de Aminoácidos , Secuencia de Bases , Análisis Mutacional de ADN , Homocigoto , Humanos , Mitocondrias/enzimología , Miopatías Mitocondriales/enzimología , Datos de Secuencia Molecular , Mutación , Linaje , Polimorfismo de Nucleótido Simple , ARN Mensajero/metabolismo , Succinato Deshidrogenasa/deficiencia , SueciaRESUMEN
The distribution of identical enzymatic activities between different subcellular compartments is a fundamental process of living cells. At present, the Saccharomyces cerevisiae aconitase enzyme has been detected only in mitochondria, where it functions in the tricarboxylic acid (TCA) cycle and is considered a mitochondrial matrix marker. We developed two strategies for physical and functional detection of aconitase in the yeast cytosol: 1) we fused the alpha peptide of the beta-galactosidase enzyme to aconitase and observed alpha complementation in the cytosol; and 2) we created an ACO1-URA3 hybrid gene, which allowed isolation of strains in which the hybrid protein is exclusively targeted to mitochondria. These strains display a specific phenotype consistent with glyoxylate shunt elimination. Together, our data indicate that yeast aconitase isoenzymes distribute between two distinct subcellular compartments and participate in two separate metabolic pathways; the glyoxylate shunt in the cytosol and the TCA cycle in mitochondria. We maintain that such dual distribution phenomena have a wider occurrence than recorded currently, the reason being that in certain cases there is a small fraction of one of the isoenzymes, in one of the locations, making its detection very difficult. We term this phenomenon of highly uneven isoenzyme distribution "eclipsed distribution."
Asunto(s)
Aconitato Hidratasa/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transducción de Señal/fisiología , Acetatos , Aconitato Hidratasa/deficiencia , Aconitato Hidratasa/genética , Secuencia de Aminoácidos , Citosol/enzimología , Etanol , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Proteína 1 Reguladora de Hierro/genética , Proteína 1 Reguladora de Hierro/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Mitocondrias/metabolismo , Datos de Secuencia Molecular , Mutación , Organismos Modificados Genéticamente , Transporte de Proteínas/fisiología , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal/genética , beta-Galactosidasa/metabolismoRESUMEN
The importance of a functional Krebs cycle for energy generation in the procyclic stage of Trypanosoma brucei was investigated under physiological conditions during logarithmic phase growth of a pleomorphic parasite strain. Wild type procyclic cells and mutants with targeted deletion of the gene coding for aconitase were derived by synchronous in vitro differentiation from wild type and mutant (Delta aco::NEO/Delta aco::HYG) bloodstream stage parasites, respectively, where aconitase is not expressed and is dispensable. No differences in intracellular levels of glycolytic and Krebs cycle intermediates were found in procyclic wild type and mutant cells, except for citrate that accumulated up to 90-fold in the mutants, confirming the absence of aconitase activity. Surprisingly, deletion of aconitase did not change differentiation nor the growth rate or the intracellular ATP/ADP ratio in those cells. Metabolic studies using radioactively labeled substrates and NMR analysis demonstrated that glucose and proline were not degraded via the Krebs cycle to CO(2). Instead, glucose was degraded to acetate, succinate, and alanine, whereas proline was degraded to succinate. Importantly, there was absolutely no difference in the metabolic products released by wild type and aconitase knockout parasites, and both were for survival strictly dependent on respiration via the mitochondrial electron transport chain. Hence, although the Krebs cycle enzymes are present, procyclic T. brucei do not use Krebs cycle activity for energy generation, but the mitochondrial respiratory chain is essential for survival and growth. We therefore propose a revised model of the energy metabolism of procyclic T. brucei.
Asunto(s)
Aconitato Hidratasa/metabolismo , Metabolismo Energético/fisiología , Trypanosoma brucei brucei/fisiología , Aconitato Hidratasa/deficiencia , Aconitato Hidratasa/genética , Animales , Ciclo del Ácido Cítrico , Eliminación de Gen , Glucosa/metabolismo , Glutamato Deshidrogenasa/deficiencia , Glutamato Deshidrogenasa/genética , Glutamato Deshidrogenasa/metabolismo , Isocitrato Deshidrogenasa/deficiencia , Isocitrato Deshidrogenasa/genética , Isocitrato Deshidrogenasa/metabolismo , Complejo Cetoglutarato Deshidrogenasa/deficiencia , Complejo Cetoglutarato Deshidrogenasa/genética , Complejo Cetoglutarato Deshidrogenasa/metabolismo , Espectroscopía de Resonancia Magnética , Cianuro de Potasio/farmacología , Transfección , Trypanosoma brucei brucei/efectos de los fármacos , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/crecimiento & desarrolloRESUMEN
Consecutive 60-min microdialysis samples were taken from the tibial anterior muscle in 11 healthy subjects and 4 patients with mitochondrial myopathy before (2-3 samples) and after (3-4 samples, 2 controls and 1 patient excluded) sustained isometric foot dorsiflexions. Before exercise, mean concentrations of lactate, pyruvate, hypoxanthine, urate, aspartate, and glutamate did not significantly differ between controls and patients. After exercise, the controls showed significantly increased concentrations of lactate, pyruvate, and urate, decreased hypoxanthine, and no change in aspartate and glutamate. Similar findings were observed in the patients. Plasma lactate was unchanged. Exercise-induced increase in integrated electromyogram amplitude and rated subjective fatigue were correlated to increased post-exercise lactate concentrations, with no obvious difference between the groups. Microdialysis of skeletal muscle allows the detection and monitoring of biochemical changes in the interstitial space. With the exercise protocol used, however, it was not possible to demonstrate any biochemical difference between healthy controls and patients with mitochondrial myopathy.
Asunto(s)
Miopatías Mitocondriales/metabolismo , Miopatías Mitocondriales/fisiopatología , Fatiga Muscular/fisiología , Aconitato Hidratasa/deficiencia , Adolescente , Adulto , Anciano , Electromiografía , Ejercicio Físico/fisiología , Femenino , Humanos , Contracción Isométrica/fisiología , Ácido Láctico/metabolismo , Masculino , Microdiálisis , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiopatología , Ácido Pirúvico/metabolismo , Succinato Deshidrogenasa/deficienciaAsunto(s)
Aconitato Hidratasa , Proteínas de Unión a Hierro , Mitocondrias/enzimología , Aconitato Hidratasa/deficiencia , Aconitato Hidratasa/genética , Aconitato Hidratasa/fisiología , Ciclo del Ácido Cítrico , Ataxia de Friedreich/etiología , Humanos , Proteínas Hierro-Azufre , Isoenzimas , Mitocondrias/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Succinato Deshidrogenasa/deficiencia , FrataxinaAsunto(s)
Aconitato Hidratasa/deficiencia , Proteínas de Unión a Hierro , Diagnóstico Diferencial , Ataxia de Friedreich/genética , Ataxia de Friedreich/fisiopatología , Humanos , Enfermedades Neuromusculares/etiología , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Pronóstico , Expansión de Repetición de Trinucleótido , FrataxinaAsunto(s)
Errores Innatos del Metabolismo del Piruvato , Piruvatos/metabolismo , Acidosis Láctica/etiología , Aconitato Hidratasa/deficiencia , Metabolismo Energético , Fumarato Hidratasa/deficiencia , Fumarato Hidratasa/genética , Humanos , Complejo Cetoglutarato Deshidrogenasa/deficiencia , Complejo Cetoglutarato Deshidrogenasa/genética , Mitocondrias/metabolismo , Mutación , Enfermedades Neuromusculares/etiología , Piruvato Carboxilasa/genética , Enfermedad por Deficiencia de Piruvato Carboxilasa/genética , Piruvato Deshidrogenasa (Lipoamida)/deficiencia , Piruvato Deshidrogenasa (Lipoamida)/genética , Errores Innatos del Metabolismo del Piruvato/clasificación , Errores Innatos del Metabolismo del Piruvato/genéticaRESUMEN
Friedreich's ataxia (FRDA) is an autosomal recessive disorder with a frequency of 1 in 50 000 live births. In 97% of patients it is caused by the abnormal expansion of a GAA repeat in intron 1 of the FRDA gene on chromosome 9, which encodes a 210 amino acid protein called frataxin. Frataxin is widely expressed and has been localized to mitochondria although its function is unknown. We have investigated mitochondrial function, mitochondrial DNA levels, aconitase activity and iron content in tissues from FRDA patients. There were significant reductions in the activities of complex I, complex II/III and aconitase in FRDA heart. Respiratory chain and aconitase activities were decreased although not significantly in skeletal muscle, but were normal in FRDA cerebellum and dorsal root ganglia, although there was a mild decrease in aconitase activity in the latter. Mitochondrial DNA levels were reduced in FRDA heart and skeletal muscle, although in skeletal muscle this was paralleled by a decline in citrate synthase activity. Increased iron deposition was seen in FRDA heart, liver and spleen in a pattern consistent with a mitochondrial location. The iron accumulation, mitochondrial respiratory chain and aconitase dysfunction and mitochondrial DNA depletion in FRDA heart samples largely paralleled those in the yeast YFH1 knockout model, suggesting that frataxin may be involved in mitochondrial iron regulation or iron sulphur centre synthesis. However, the severe deficiency in aconitase activity also suggests that oxidant stress may induce a self-amplifying cycle of oxidative damage and mitochondrial dysfunction, which may contribute to cellular toxicity.
Asunto(s)
Ataxia de Friedreich/genética , Ataxia de Friedreich/metabolismo , Aconitato Hidratasa/deficiencia , Aconitato Hidratasa/genética , Adolescente , Adulto , ADN Mitocondrial/análisis , Transporte de Electrón/genética , Ataxia de Friedreich/enzimología , Ataxia de Friedreich/patología , Humanos , Hierro/análisis , Persona de Mediana Edad , Coloración y EtiquetadoRESUMEN
Oxidative stress has been implicated in many diseases. The chief source of reactive oxygen species within the cell is the mitochondrion. We have characterized a variety of the biochemical and metabolic effects of inactivation of the mouse gene for the mitochondrial superoxide dismutase (CD1-Sod2(tm1Cje)). The Sod2 mutant mice exhibit a tissue-specific inhibition of the respiratory chain enzymes NADH-dehydrogenase (complex I) and succinate dehydrogenase (complex II), inactivation of the tricarboxylic acid cycle enzyme aconitase, development of a urine organic aciduria in conjunction with a partial defect in 3-hydroxy-3-methylglutaryl-CoA lyase, and accumulation of oxidative DNA damage. These results indicate that the increase in mitochondrial reactive oxygen species can result in biochemical aberrations with features reminiscent of mitochondrial myopathy, Friedreich ataxia, and 3-hydroxy-3-methylglutaryl-CoA lyase deficiency.
Asunto(s)
Mitocondrias Cardíacas/metabolismo , Mitocondrias Musculares/metabolismo , Miopatías Mitocondriales/genética , Fosforilación Oxidativa , Superóxido Dismutasa/deficiencia , Superóxido Dismutasa/genética , Aconitato Hidratasa/deficiencia , Aconitato Hidratasa/metabolismo , Animales , Encéfalo/metabolismo , Ácidos Carboxílicos/metabolismo , Ácidos Carboxílicos/orina , Cruzamientos Genéticos , Daño del ADN , Femenino , Fumarato Hidratasa/metabolismo , Masculino , Ratones , Ratones Mutantes , Mitocondrias/metabolismo , Miopatías Mitocondriales/enzimología , Oxo-Ácido-Liasas/deficiencia , Oxo-Ácido-Liasas/metabolismo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Friedreich ataxia (FRDA) is a common autosomal recessive degenerative disease (1/50,000 live births) characterized by a progressive-gait and limb ataxia with lack of tendon reflexes in the legs, dysarthria and pyramidal weakness of the inferior limbs. Hypertrophic cardiomyopathy is observed in most FRDA patients. The gene associated with the disease has been mapped to chromosome 9q13 (ref. 3) and encodes a 210-amino-acid protein, frataxin. FRDA is caused primarily by a GAA repeat expansion within the first intron of the frataxin gene, which accounts for 98% of mutant alleles. The function of the protein is unknown, but an increased iron content has been reported in hearts of FRDA patients and in mitochondria of yeast strains carrying a deleted frataxin gene counterpart (YFH1), suggesting that frataxin plays a major role in regulating mitochondrial iron transport. Here, we report a deficient activity of the iron-sulphur (Fe-S) cluster-containing subunits of mitochondrial respiratory complexes I, II and III in the endomyocardial biopsy of two unrelated FRDA patients. Aconitase, an iron-sulphur protein involved in iron homeostasis, was found to be deficient as well. Moreover, disruption of the YFH1 gene resulted in multiple Fe-S-dependent enzyme deficiencies in yeast. The deficiency of Fe-S-dependent enzyme activities in both FRDA patients and yeast should be related to mitochondrial iron accumulation, especially as Fe-S proteins are remarkably sensitive to free radicals. Mutated frataxin triggers aconitase and mitochondrial Fe-S respiratory enzyme deficiency in FRDA, which should therefore be regarded as a mitochondrial disorder.
Asunto(s)
Aconitato Hidratasa/deficiencia , Ataxia de Friedreich/metabolismo , Proteínas de Unión a Hierro , Proteínas Hierro-Azufre/deficiencia , Mitocondrias/metabolismo , Ciclo del Ácido Cítrico , Transporte de Electrón , Endocardio/metabolismo , Ataxia de Friedreich/genética , Genes Fúngicos , Humanos , Hierro/metabolismo , Modelos Biológicos , Mutación , Miocardio/metabolismo , Estrés Oxidativo , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , FrataxinaRESUMEN
A hereditary myopathy with lactic acidosis during physical exercise, low physical work capacity, and paroxysmal myoglobinuria (HML), called "Myopathy with deficiency of succinate dehydrogenase and aconitase" (McKusick 255125) has been described in 19 members of nine families who lived in two geographically separate areas in northern Sweden. By using the unique Swedish historical archives, including Catechetical Meeting Records from a number of northern Swedish parishes, it has been possible to trace ancestors of the nine families including all known 19 cases back in time to some key couples, who lived up to 300 years ago (that is seven to ten generations). No common single couple or common links between families in the past was found in these registers as a support for a single or several mutations that had developed far back in time. The mode of inheritance in this family is most likely autosomal recessive. This material will be used for the chromosomal localisation of the gene.
Asunto(s)
Acidosis Láctica/genética , Aconitato Hidratasa/deficiencia , Miopatías Mitocondriales/genética , Succinato Deshidrogenasa/deficiencia , Aconitato Hidratasa/genética , Adolescente , Femenino , Genes Recesivos , Ligamiento Genético/genética , Humanos , Escala de Lod , Masculino , Linaje , Succinato Deshidrogenasa/genética , SueciaRESUMEN
Recently, we described a patient with severe exercise intolerance and episodic myoglobinuria, associated with marked impairment of succinate oxidation and deficient activity of succinate dehydrogenase and aconitase in muscle mitochondria (1). We now report additional enzymatic and immunological characterization of mitochondria. In addition to severe deficiency of complex II, manifested by reduction of succinate dehydrogenase and succinate:coenzyme Q oxidoreductase activities to 12 and 22% of normal, respectively, complex III activity was reduced to 37% and rhodanese to 48% of normal. Furthermore, although complex I activity was not measured, immunoblot analysis of complex I showed deficiency of the 39-, 24-, 13-, and 9-kD peptides with lesser reductions of the 51- and 18-kD peptides. Immunoblots of complex III showed markedly reduced levels of the mature Rieske protein in mitochondria and elevated levels of its precursor in the cytosol, suggesting deficient uptake into mitochondria. Immunoreactive aconitase was also low. These data, together with the previous documentation of low amounts of the 30-kD iron-sulfur protein and the 13.5-kD subunit of complex II, compared to near normal levels of the 70-kD protein suggest a more generalized abnormality of the synthesis, import, processing, or assembly of a group of proteins containing iron-sulfur clusters.
Asunto(s)
Aconitato Hidratasa/deficiencia , Proteínas Hierro-Azufre/metabolismo , Mitocondrias Musculares/enzimología , Miopatías Mitocondriales/enzimología , Complejos Multienzimáticos/metabolismo , NAD(P)H Deshidrogenasa (Quinona)/metabolismo , Oxidorreductasas/metabolismo , Succinato Deshidrogenasa/deficiencia , Succinato Deshidrogenasa/metabolismo , Adulto , Citrato (si)-Sintasa/metabolismo , Complejo II de Transporte de Electrones , Complejo III de Transporte de Electrones/metabolismo , Humanos , Proteínas Hierro-Azufre/análisis , Masculino , Modelos Biológicos , Tiosulfato Azufretransferasa/metabolismoRESUMEN
We evaluated a 22-yr-old Swedish man with lifelong exercise intolerance marked by premature exertional muscle fatigue, dyspnea, and cardiac palpitations with superimposed episodes lasting days to weeks of increased muscle fatigability and weakness associated with painful muscle swelling and pigmenturia. Cycle exercise testing revealed low maximal oxygen uptake (12 ml/min per kg; healthy sedentary men = 39 +/- 5) with exaggerated increases in venous lactate and pyruvate in relation to oxygen uptake (VO2) but low lactate/pyruvate ratios in maximal exercise. The severe oxidative limitation was characterized by impaired muscle oxygen extraction indicated by subnormal systemic arteriovenous oxygen difference (a-v O2 diff) in maximal exercise (patient = 4.0 ml/dl, normal men = 16.7 +/- 2.1) despite normal oxygen carrying capacity and Hgb-O2 P50. In contrast maximal oxygen delivery (cardiac output, Q) was high compared to sedentary healthy men (Qmax, patient = 303 ml/min per kg, normal men 238 +/- 36) and the slope of increase in Q relative to VO2 (i.e., delta Q/delta VO2) from rest to exercise was exaggerated (delta Q/delta VO2, patient = 29, normal men = 4.7 +/- 0.6) indicating uncoupling of the normal approximately 1:1 relationship between oxygen delivery and utilization in dynamic exercise. Studies of isolated skeletal muscle mitochondria in our patient revealed markedly impaired succinate oxidation with normal glutamate oxidation implying a metabolic defect at the level of complex II of the mitochondrial respiratory chain. A defect in Complex II in skeletal muscle was confirmed by the finding of deficiency of succinate dehydrogenase as determined histochemically and biochemically. Immunoblot analysis showed low amounts of the 30-kD (iron-sulfur) and 13.5-kD proteins with near normal levels of the 70-kD protein of complex II. Deficiency of succinate dehydrogenase was associated with decreased levels of mitochondrial aconitase assessed enzymatically and immunologically whereas activities of other tricarboxylic acid cycle enzymes were increased compared to normal subjects. The exercise findings are consistent with the hypothesis that this defect impairs muscle oxidative metabolism by limiting the rate of NADH production by the tricarboxylic acid cycle.