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1.
Muscle Nerve ; 57(2): 193-199, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-28833236

RESUMO

INTRODUCTION: The aim of this study was to determine the safety and therapeutic potential of L-carnitine and valproic acid (VPA) in infants with spinal muscular atrophy (SMA). METHODS: Our investigation was an open-label phase 2 multicenter trial of L-carnitine and VPA in infants with SMA type I with retrospective comparison to an untreated, matched cohort. Primary outcomes were: safety and adverse events; secondary outcomes were survival, time to death/>16 hours/day of ventilator support; motor outcomes; and maximum ulnar compound motor action potential amplitude. RESULTS: A total of 245 AEs were observed in 35 of the 37 treated subjects (95%). Respiratory events accounted for 49% of all adverse events, resulting in 14 deaths. Survival was not significantly different between treated and untreated cohorts. DISCUSSION: This trial provides evidence that, in infants with SMA type I, L-carnitine/VPA is ineffective at altering survival. The substantial proportion of infants reaching end-points within 6 months of enrollment underscores the urgent need for pre-symptomatic treatment in SMA type I. Muscle Nerve 57: 193-199, 2018.


Assuntos
Carnitina/uso terapêutico , GABAérgicos/uso terapêutico , Atrofias Musculares Espinais da Infância/tratamento farmacológico , Ácido Valproico/uso terapêutico , Complexo Vitamínico B/uso terapêutico , Potenciais de Ação/efeitos dos fármacos , Carnitina/efeitos adversos , Estudos de Coortes , Quimioterapia Combinada , Feminino , GABAérgicos/efeitos adversos , Humanos , Lactente , Masculino , Resultados Negativos , Respiração Artificial , Estudos Retrospectivos , Atrofias Musculares Espinais da Infância/fisiopatologia , Análise de Sobrevida , Resultado do Tratamento , Ácido Valproico/efeitos adversos , Complexo Vitamínico B/efeitos adversos
2.
Int J Mol Sci ; 18(6)2017 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-28556799

RESUMO

Mitochondria play a pivotal role in energy metabolism, but whether insulin signaling per se could regulate mitochondrial function has not been identified yet. To investigate whether mitochondrial function is regulated by insulin signaling, we analyzed muscle and liver of insulin receptor (IR)+/--insulin receptor substrate-1 (IRS-1)+/- double heterozygous (IR-IRS1dh) mice, a well described model for insulin resistance. IR-IRS1dh mice were studied at the age of 6 and 12 months and glucose metabolism was determined by glucose and insulin tolerance tests. Mitochondrial enzyme activities, oxygen consumption, and membrane potential were assessed using spectrophotometric, respirometric, and proton motive force analysis, respectively. IR-IRS1dh mice showed elevated serum insulin levels. Hepatic mitochondrial oxygen consumption was reduced in IR-IRS1dh animals at 12 months of age. Furthermore, 6-month-old IR-IRS1dh mice demonstrated enhanced mitochondrial respiration in skeletal muscle, but a tendency of impaired glucose tolerance. On the other hand, 12-month-old IR-IRS1dh mice showed improved glucose tolerance, but normal muscle mitochondrial function. Our data revealed that deficiency in IR/IRS-1 resulted in normal or even elevated skeletal muscle, but impaired hepatic mitochondrial function, suggesting a direct cross-talk between insulin signaling and mitochondria in the liver.


Assuntos
Proteínas Substratos do Receptor de Insulina/metabolismo , Insulina/metabolismo , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Receptor de Insulina/metabolismo , Animais , Glicemia/metabolismo , Glucose/metabolismo , Proteínas Substratos do Receptor de Insulina/genética , Fígado/metabolismo , Camundongos , Músculo Esquelético/metabolismo , Receptor de Insulina/genética , Transdução de Sinais/genética , Transdução de Sinais/fisiologia
3.
Mol Cell Pediatr ; 3(1): 22, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27317552

RESUMO

BACKGROUND: Neonatal lactic acidosis can be associated to severe inborn errors of metabolism. Rapid identification of the underlying disorder may improve the clinical management through reliable counseling of the parents and adaptation of the treatment. METHODS: We present the case of a term newborn with persistent hypoglycemia on postnatal day 1, who developed severe lactic acidosis, aggravating under intravenous glucose administration. Routine metabolic investigations revealed elevated pyruvate and lactate levels in urine, and magnetic resonance spectroscopy showed a lactic acid peak and decreased N-acetylaspartate levels. Mitochondrial disorders, e.g., pyruvate dehydrogenase (PDH) deficiency, were the major differential diagnoses. However, both hypoglycemia and the elevated lactate to pyruvate ratio in serum (=55.2) were not typical for PDH deficiency. We used "Mendeliome sequencing", a next-generation sequencing approach targeting all genes which have been previously linked to single-gene disorders, to obtain the correct diagnosis. RESULTS: On day 27 of life, we identified a homozygous stop mutation in the PDHX gene, causing pyruvate dehydrogenase E3-binding protein deficiency. After starting the ketogenic diet, the infant recovered and is showing delayed but progressive development. CONCLUSIONS: Mendeliome sequencing was successfully used to disentangle the underlying cause of severe neonatal lactic acidosis. Indeed, it is one of several targeted sequencing approaches that allow rapid and reliable counseling of the parents, adaptation of the clinical management, and renunciation of unnecessary, potentially invasive and often costly diagnostic measures.

4.
J Inherit Metab Dis ; 39(1): 115-24, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26025547

RESUMO

BACKGROUND: Severe methylenetetrahydrofolate reductase (MTHFR) deficiency is a rare inborn defect disturbing the remethylation of homocysteine to methionine (<200 reported cases). This retrospective study evaluates clinical, biochemical genetic and in vitro enzymatic data in a cohort of 33 patients. METHODS: Clinical, biochemical and treatment data was obtained from physicians by using a questionnaire. MTHFR activity was measured in primary fibroblasts; genomic DNA was extracted from cultured fibroblasts. RESULTS: Thirty-three patients (mean age at follow-up 11.4 years; four deceased; median age at first presentation 5 weeks; 17 females) were included. Patients with very low (<1.5%) mean control values of enzyme activity (n = 14) presented earlier and with a pattern of feeding problems, encephalopathy, muscular hypotonia, neurocognitive impairment, apnoea, hydrocephalus, microcephaly and epilepsy. Patients with higher (>1.7-34.8%) residual enzyme activity had mainly psychiatric symptoms, mental retardation, myelopathy, ataxia and spasticity. Treatment with various combinations of betaine, methionine, folate and cobalamin improved the biochemical and clinical phenotype. During the disease course, patients with very low enzyme activity showed a progression of feeding problems, neurological symptoms, mental retardation, and psychiatric disease while in patients with higher residual enzyme activity, myelopathy, ataxia and spasticity increased. All other symptoms remained stable or improved in both groups upon treatment as did brain imaging in some cases. No clear genotype-phenotype correlation was obvious. DISCUSSION: MTHFR deficiency is a severe disease primarily affecting the central nervous system. Age at presentation and clinical pattern are correlated with residual enzyme activity. Treatment alleviates biochemical abnormalities and clinical symptoms partially.


Assuntos
Homocistinúria/enzimologia , Homocistinúria/genética , Metilenotetra-Hidrofolato Redutase (NADPH2)/deficiência , Metilenotetra-Hidrofolato Redutase (NADPH2)/genética , Espasticidade Muscular/enzimologia , Espasticidade Muscular/genética , Ataxia/genética , Betaína/uso terapêutico , Criança , Feminino , Ácido Fólico/uso terapêutico , Estudos de Associação Genética/métodos , Homocistinúria/tratamento farmacológico , Humanos , Deficiência Intelectual/genética , Masculino , Metionina/uso terapêutico , Espasticidade Muscular/tratamento farmacológico , Mutação/genética , Fenótipo , Transtornos Psicóticos/tratamento farmacológico , Transtornos Psicóticos/enzimologia , Transtornos Psicóticos/genética , Estudos Retrospectivos , Doenças da Medula Espinal/genética , Vitamina B 12/uso terapêutico
5.
Cell Metab ; 21(5): 667-77, 2015 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-25955204

RESUMO

Aging is a progressive decline of body function, during which many tissues accumulate few cells with high levels of deleted mitochondrial DNA (mtDNA), leading to a defect of mitochondrial functions. Whether this mosaic mitochondrial deficiency contributes to organ dysfunction is unknown. To investigate this, we generated mice with an accelerated accumulation of mtDNA deletions in the myocardium, by expressing a dominant-negative mutant mitochondrial helicase. These animals accumulated few randomly distributed cardiomyocytes with compromised mitochondrial function, which led to spontaneous ventricular premature contractions and AV blocks at 18 months. These symptoms were not caused by a general mitochondrial dysfunction in the entire myocardium, and were not observed in mice at 12 months with significantly lower numbers of dysfunctional cells. Therefore, our results suggest that the disposition to arrhythmia typically found in the aged human heart might be due to the random accumulation of mtDNA deletions and the subsequent mosaic respiratory chain deficiency.


Assuntos
Envelhecimento , Arritmias Cardíacas/etiologia , DNA Mitocondrial/genética , Mitocôndrias/genética , Doenças Mitocondriais/complicações , Doenças Mitocondriais/genética , Oxigênio/metabolismo , Animais , Arritmias Cardíacas/genética , Arritmias Cardíacas/metabolismo , Arritmias Cardíacas/patologia , Respiração Celular , Complexo IV da Cadeia de Transporte de Elétrons/genética , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Deleção de Genes , Camundongos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Doenças Mitocondriais/metabolismo , Doenças Mitocondriais/patologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia
6.
J Hepatol ; 60(4): 816-23, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24291365

RESUMO

BACKGROUND & AIMS: To determine if diabetic and insulin-resistant states cause mitochondrial dysfunction in liver or if there is long term adaptation of mitochondrial function to these states, mice were (i) fed with a high-fat diet to induce obesity and T2D (HFD), (ii) had a genetic defect in insulin signaling causing whole body insulin resistance, but not full blown T2D (IR/IRS-1(+/-) mice), or (iii) were analyzed after treatment with streptozocin (STZ) to induce a T1D-like state. METHODS: Hepatic lipid levels were measured by thin layer chromatography. Mitochondrial respiratory chain (RC) levels and function were determined by Western blot, spectrophotometric, oxygen consumption and proton motive force analysis. Gene expression was analyzed by real-time PCR and microarray. RESULTS: HFD caused insulin resistance and hepatic lipid accumulation, but RC was largely unchanged. Livers from insulin resistant IR/IRS-1(+/-) mice had normal lipid contents and a normal RC, but mitochondria were less well coupled. Livers from severely hyperglycemic and hypoinsulinemic STZ mice had massively depleted lipid levels, but RC abundance was unchanged. However, liver mitochondria isolated from these animals showed increased abundance and activity of the RC, which was better coupled. CONCLUSIONS: Insulin resistance, induced either by obesity or genetic manipulation and steatosis do not cause mitochondrial dysfunction in mouse liver. Also, mitochondrial dysfunction is not a prerequisite for liver steatosis. However, severe insulin deficiency and high blood glucose levels lead to an enhanced performance and better coupling of the RC. This may represent an adaptation to fuel overload and the high energy-requirement of an unsuppressed gluconeogenesis.


Assuntos
Adaptação Fisiológica , Diabetes Mellitus Tipo 2/fisiopatologia , Resistência à Insulina/fisiologia , Mitocôndrias Hepáticas/fisiologia , Animais , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/fisiopatologia , Diabetes Mellitus Tipo 2/etiologia , Dieta Hiperlipídica/efeitos adversos , Fígado Gorduroso/etiologia , Fígado Gorduroso/fisiopatologia , Expressão Gênica , Proteínas Substratos do Receptor de Insulina/deficiência , Proteínas Substratos do Receptor de Insulina/genética , Proteínas Substratos do Receptor de Insulina/metabolismo , Canais Iônicos/metabolismo , Fígado/metabolismo , Fígado/fisiopatologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Translocases Mitocondriais de ADP e ATP/metabolismo , Proteínas Mitocondriais/metabolismo , Obesidade/etiologia , Obesidade/fisiopatologia , Fosforilação Oxidativa , Força Próton-Motriz , Receptor de Insulina/deficiência , Receptor de Insulina/genética , Receptor de Insulina/metabolismo , Transdução de Sinais , Proteína Desacopladora 2
7.
Nucleic Acids Res ; 41(21): 9848-57, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23982517

RESUMO

Mitochondrial topoisomerase I is a genetically distinct mitochondria-dedicated enzyme with a crucial but so far unknown role in the homeostasis of mitochondrial DNA metabolism. Here, we present data suggesting a negative regulatory function in mitochondrial transcription or transcript stability. Deficiency or depletion of mitochondrial topoisomerase I increased mitochondrial transcripts, whereas overexpression lowered mitochondrial transcripts, depleted respiratory complexes I, III and IV, decreased cell respiration and raised superoxide levels. Acute depletion of mitochondrial topoisomerase I triggered neither a nuclear mito-biogenic stress response nor compensatory topoisomerase IIß upregulation, suggesting the concomitant increase in mitochondrial transcripts was due to release of a local inhibitory effect. Mitochondrial topoisomerase I was co-immunoprecipitated with mitochondrial RNA polymerase. It selectively accumulated and rapidly exchanged at a subset of nucleoids distinguished by the presence of newly synthesized RNA and/or mitochondrial RNA polymerase. The inactive Y559F-mutant behaved similarly without affecting mitochondrial transcripts. In conclusion, mitochondrial topoisomerase I dampens mitochondrial transcription and thereby alters respiratory capacity. The mechanism involves selective association of the active enzyme with transcriptionally active nucleoids and a direct interaction with mitochondrial RNA polymerase. The inhibitory role of topoisomerase I in mitochondrial transcription is strikingly different from the stimulatory role of topoisomerase I in nuclear transcription.


Assuntos
DNA Topoisomerases Tipo I/metabolismo , Regulação da Expressão Gênica , Mitocôndrias/enzimologia , Mitocôndrias/genética , Transcrição Genética , Animais , Linhagem Celular Tumoral , Células Cultivadas , Humanos , Camundongos , Mitocôndrias/metabolismo , RNA/metabolismo , RNA Mitocondrial
8.
Am J Hum Genet ; 93(1): 181-90, 2013 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-23830518

RESUMO

Myopathies are a clinically and etiologically heterogeneous group of disorders that can range from limb girdle muscular dystrophy (LGMD) to syndromic forms with associated features including intellectual disability. Here, we report the identification of mutations in transport protein particle complex 11 (TRAPPC11) in three individuals of a consanguineous Syrian family presenting with LGMD and in five individuals of Hutterite descent presenting with myopathy, infantile hyperkinetic movements, ataxia, and intellectual disability. By using a combination of whole-exome or genome sequencing with homozygosity mapping, we identified the homozygous c.2938G>A (p.Gly980Arg) missense mutation within the gryzun domain of TRAPPC11 in the Syrian LGMD family and the homozygous c.1287+5G>A splice-site mutation resulting in a 58 amino acid in-frame deletion (p.Ala372_Ser429del) in the foie gras domain of TRAPPC11 in the Hutterite families. TRAPPC11 encodes a component of the multiprotein TRAPP complex involved in membrane trafficking. We demonstrate that both mutations impair the binding ability of TRAPPC11 to other TRAPP complex components and disrupt the Golgi apparatus architecture. Marker trafficking experiments for the p.Ala372_Ser429del deletion indicated normal ER-to-Golgi trafficking but dramatically delayed exit from the Golgi to the cell surface. Moreover, we observed alterations of the lysosomal membrane glycoproteins lysosome-associated membrane protein 1 (LAMP1) and LAMP2 as a consequence of TRAPPC11 dysfunction supporting a defect in the transport of secretory proteins as the underlying pathomechanism.


Assuntos
Deficiência Intelectual/genética , Transtornos dos Movimentos/genética , Doenças Musculares/genética , Distrofia Muscular do Cíngulo dos Membros/genética , Deleção de Sequência , Proteínas de Transporte Vesicular/metabolismo , Adolescente , Adulto , Ataxia/genética , Mapeamento Cromossômico , Consanguinidade , Creatina Quinase/sangue , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/patologia , Exoma , Feminino , Complexo de Golgi/genética , Complexo de Golgi/metabolismo , Complexo de Golgi/patologia , Homozigoto , Humanos , Proteína 2 de Membrana Associada ao Lisossomo , Glicoproteínas de Membrana Associadas ao Lisossomo/genética , Glicoproteínas de Membrana Associadas ao Lisossomo/metabolismo , Lisossomos/metabolismo , Masculino , Transtornos dos Movimentos/patologia , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Doenças Musculares/patologia , Distrofia Muscular do Cíngulo dos Membros/patologia , Linhagem , Ligação Proteica , Transporte Proteico , Sítios de Splice de RNA , Síria , Proteínas de Transporte Vesicular/genética , Adulto Jovem
9.
Mitochondrion ; 11(6): 954-63, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21945727

RESUMO

We report a fragmented mitochondrial network and swollen and irregularly shaped mitochondria with partial to complete loss of the cristae in fibroblasts of a patient with a novel TMEM70 gene deletion, which could be completely restored by complementation of the TMEM70 genetic defect. Comparative genomics analysis predicted the topology of TMEM70 in the inner mitochondrial membrane, which could be confirmed by immunogold labeling experiments, and showed that the TMEM70 gene is not restricted to higher multi-cellular eukaryotes. This study demonstrates that the role of complex V in mitochondrial cristae morphology applies to human mitochondrial disease pathology.


Assuntos
Adenosina Trifosfatases/deficiência , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Mitocôndrias/ultraestrutura , Proteínas Mitocondriais/genética , Deleção de Sequência , Proteínas de Transporte , Células Cultivadas , Fibroblastos/ultraestrutura , Teste de Complementação Genética , Humanos , Recém-Nascido , Masculino , ATPases Mitocondriais Próton-Translocadoras
10.
J Inherit Metab Dis ; 34(1): 197-201, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21153446

RESUMO

Combined respiratory chain deficiency accounts for about 30% of mitochondrial respiratory chain deficiencies and is frequently associated with mtDNA depletion, deletions or point mutations. However combined respiratory chain deficiency may also be caused by mutations in nuclear genes affecting mitochondrial translation. Here we describe a 2-year-old girl, who developed an acute, isolated, severe liver failure with mitochondrial pathology and decreased respiratory chain enzyme activities both in liver and skeletal muscle at 4 months of age. Her liver function improved significantly within a month, liver function tests returned to normal. Liver cirrhosis remained without any further complications so far. Pathogenic compound heterozygous mutations were identified in the TRMU gene. This condition is one of the few mitochondrial disorders with a life-threatening onset showing recovery later in life, therefore a prompt diagnosis and treatment of these patients has great importance in clinical practice. We suggest that TRMU deficiency should be considered in infants with acute liver disease.


Assuntos
Cirrose Hepática/diagnóstico , Falência Hepática Aguda/diagnóstico , Doenças Mitocondriais/diagnóstico , Proteínas Mitocondriais/genética , tRNA Metiltransferases/genética , Sequência de Bases , Pré-Escolar , Feminino , Humanos , Cirrose Hepática/etiologia , Cirrose Hepática/genética , Falência Hepática Aguda/complicações , Falência Hepática Aguda/genética , Doenças Mitocondriais/complicações , Doenças Mitocondriais/genética , Dados de Sequência Molecular , Mutação/fisiologia
11.
Eur J Cell Biol ; 90(4): 342-55, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21131095

RESUMO

Ras-related GTPases of the Miro family have been implicated in mitochondrial homeostasis and microtubule-dependent transport. They consist of two GTP-binding domains separated by calcium-binding motifs and of a C-terminal transmembrane domain that targets the protein to the outer mitochondrial membrane. We disrupted the single Miro-encoding gene in Dictyostelium discoideum and observed a substantial growth defect that we attribute to a decreased mitochondrial mass and cellular ATP content. However, mutant cells even showed an increased rate of oxygen consumption, while glucose consumption, mitochondrial transmembrane potential and production of reactive oxygen species were unaltered. Processes characteristic of the multicellular stage of the D. discoideum life cycle were also unaltered. Although mitochondria occasionally use microtubules for transport in D. discoideum, their size and distribution were not visibly affected. We found Miro in all branches of the eukaryotic tree with the exception of a few protist lineages (mainly those lacking typical mitochondria). Trypanosomatids and ciliates possess structurally unique homologs lacking the N-terminal or the C-terminal GTPase domain, respectively. We propose that in D. discoideum, as in yeasts and plants, Miro plays roles in mitochondrial homeostasis, but the ability to build a complex that regulates its association to kinesin for microtubule-dependent transport probably arose in metazoans.


Assuntos
Dictyostelium/enzimologia , Mitocôndrias/enzimologia , Proteínas rho de Ligação ao GTP/metabolismo , Sequência de Aminoácidos , Evolução Biológica , Transporte Biológico/genética , Cálcio/metabolismo , Dictyostelium/genética , Glucose/metabolismo , Cinesina/genética , Cinesina/metabolismo , Microtúbulos/genética , Microtúbulos/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Dados de Sequência Molecular , Consumo de Oxigênio , Filogenia , Proteínas rho de Ligação ao GTP/classificação , Proteínas rho de Ligação ao GTP/genética
12.
Mol Genet Metab ; 100(3): 251-6, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20382551

RESUMO

Mitochondrial complex I deficiency is the most frequently encountered defect of the oxidative phosphorylation system. To identify the genetic cause of the complex I deficiency, we screened the gene encoding the NDUFS1 subunit. We report 3 patients with low residual complex I activity expressed in cultured fibroblasts, which displayed novel mutations in the NDUFS1 gene. One mutation introduces a premature stop codon, 3 mutations cause a substitution of amino acids and another mutation a deletion of one amino acid. The fibroblasts of the patients display a decreased amount and activity of complex I. In addition, a disturbed assembly pattern was observed. These results suggest that NDUFS1 is a prime candidate to screen for disease-causing mutations in patients with a very low residual complex I activity in cultured fibroblasts.


Assuntos
Complexo I de Transporte de Elétrons/deficiência , Complexo I de Transporte de Elétrons/genética , Doenças Mitocondriais/enzimologia , Doenças Mitocondriais/genética , Mutação , NADH Desidrogenase/genética , Sequência de Aminoácidos , Substituição de Aminoácidos , Sequência de Bases , Encéfalo/patologia , Células Cultivadas , Criança , Pré-Escolar , Códon sem Sentido , Análise Mutacional de DNA , Feminino , Fibroblastos/enzimologia , Humanos , Lactente , Imagem por Ressonância Magnética , Masculino , Doenças Mitocondriais/patologia , Deleção de Sequência , Homologia de Sequência de Aminoácidos
13.
Genes Dev ; 22(4): 476-88, 2008 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-18281461

RESUMO

Prohibitins comprise an evolutionarily conserved and ubiquitously expressed family of membrane proteins with poorly described functions. Large assemblies of PHB1 and PHB2 subunits are localized in the inner membrane of mitochondria, but various roles in other cellular compartments have also been proposed for both proteins. Here, we used conditional gene targeting of murine Phb2 to define cellular activities of prohibitins. Our experiments restrict the function of prohibitins to mitochondria and identify the processing of the dynamin-like GTPase OPA1, an essential component of the mitochondrial fusion machinery, as the central cellular process controlled by prohibitins. Deletion of Phb2 leads to the selective loss of long isoforms of OPA1. This results in an aberrant cristae morphogenesis and an impaired cellular proliferation and resistance toward apoptosis. Expression of a long OPA1 isoform in PHB2-deficient cells suppresses these defects, identifying impaired OPA1 processing as the primary cellular defect in the absence of prohibitins. Our results therefore assign an essential function for the formation of mitochondrial cristae to prohibitins and suggest a coupling of cell proliferation to mitochondrial morphogenesis.


Assuntos
Apoptose , Proliferação de Células , GTP Fosfo-Hidrolases/metabolismo , Mitocôndrias/metabolismo , Proteínas Repressoras/fisiologia , Animais , Feminino , GTP Fosfo-Hidrolases/genética , Integrases/metabolismo , Masculino , Fusão de Membrana/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia de Fluorescência , Mitocôndrias/ultraestrutura , Morfogênese , Mutagênese Sítio-Dirigida , Isoformas de Proteínas
14.
Exp Cell Res ; 313(14): 3076-89, 2007 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-17509565

RESUMO

Energy-producing pathways, adenine nucleotide levels, oxidative stress response and Ca(2+) homeostasis were investigated in cybrid cells incorporating two pathogenic mitochondrial DNA point mutations, 3243A>G and 3302A>G in tRNA(Leu(UUR)), as well as Rho(0) cells and compared to their parental 143B osteosarcoma cell line. All cells suffering from a severe respiratory chain deficiency were able to proliferate as fast as controls. The major defect in oxidative phosphorylation was efficiently compensated by a rise in anaerobic glycolysis, so that the total ATP production rate was preserved. This enhancement of glycolysis was enabled by a considerable decrease of cellular total adenine nucleotide pools and a concomitant shift in the AMP+ADP/ATP ratios, while the energy charge potential was still in the normal range. Further important consequences were an increased production of superoxide which, however, was neither escorted by major changes in the antioxidative defence systems nor was it leading to substantial oxidative damage. Most interestingly, the lowered mitochondrial membrane potential led to a disturbed intramitochondrial calcium homeostasis, which most likely is a major pathomechanism in mitochondrial diseases.


Assuntos
Trifosfato de Adenosina/metabolismo , Cálcio/metabolismo , Transporte de Elétrons/fisiologia , Glicólise/fisiologia , Mitocôndrias/metabolismo , Aminoácidos/metabolismo , Antioxidantes/metabolismo , Linhagem Celular , Homeostase , Humanos , Concentração de Íons de Hidrogênio , Ácido Láctico/metabolismo , Potenciais da Membrana/fisiologia , Oxirredução , Estresse Oxidativo , Fenótipo , Espécies Reativas de Oxigênio/metabolismo
15.
J Invest Dermatol ; 127(5): 1084-93, 2007 May.
Artigo em Inglês | MEDLINE | ID: mdl-17185981

RESUMO

The energy metabolism of the epidermis has been the subject of controversy; thus we characterized the mitochondrial phenotype of human primary keratinocytes and fibroblasts, in cell culture and in human skin sections. We found that keratinocytes respire as much as fibroblasts, however, maximal activities of the respiratory chain (RC) complexes were 2- to 5-fold lower, whereas expression levels of RC proteins were similar. Maximal activities of aconitase and isocitrate dehydrogenase, two mitochondrial enzymes especially vulnerable to superoxide, were lower than in fibroblasts. Indeed, superoxide anion levels were much higher in keratinocytes, and keratinocytes displayed higher lipid peroxidation levels and a lower reduced glutathione/oxidized glutathione ratio, indicating enhanced oxidative stress. Although superoxide dismutase activity and especially expression of the mitochondrial superoxide dismutase, Mn-SOD, were drastically lower in keratinocytes, explaining the high superoxide levels, glutathione peroxidase activity and protein were almost undetectable in fibroblasts. Catalase activity and hydrogen peroxide levels were similar. In summary, we could show that keratinocytes actively use the mitochondrial RC not only for adenosine 5' triphosphate synthesis but also for the accumulation of superoxide anions, even at the expense of mitochondrial functional capacity, indicating that superoxide-driven mitochondrial impairment might be a prerequisite for keratinocyte differentiation.


Assuntos
Queratinócitos/metabolismo , Mitocôndrias/fisiologia , Superóxido Dismutase/fisiologia , Superóxidos/metabolismo , Aconitato Hidratase/metabolismo , Diferenciação Celular/fisiologia , Células Cultivadas , Criança , Transporte de Elétrons/fisiologia , Metabolismo Energético/fisiologia , Células Epidérmicas , Epiderme/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Isocitrato Desidrogenase/metabolismo , Queratinócitos/citologia , Peroxidação de Lipídeos/fisiologia , Masculino , Estresse Oxidativo/fisiologia
16.
Nucleic Acids Res ; 34(22): 6404-15, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-17130166

RESUMO

The gene encoding mt-tRNA(Leu(UUR)), MT-TL1, is a hotspot for pathogenic mtDNA mutations. Amongst the first to be described was the 3302A>G transition which resulted in a substantial accumulation in patient muscle of RNA19, an unprocessed RNA intermediate including mt-16S rRNA, mt-tRNA(Leu(UUR)) and MTND1. We have now been able to further assess the molecular aetiology associated with 3302A>G in transmitochondrial cybrids. Increased steady-state levels of RNA19 was confirmed, although not to the levels previously reported in muscle. This data was consistent with an increase in RNA19 stability. The mutation resulted in decreased mt-tRNA(Leu(UUR)) levels, but its stability was unchanged, consistent with a defect in RNA19 processing responsible for low tRNA levels. A partial defect in aminoacylation was also identified, potentially caused by an alteration in tRNA structure. These deficiencies lead to a severe defect in respiration in the transmitochondrial cybrids, consistent with the profound mitochondrial disorder originally associated with this mutation.


Assuntos
Genes Mitocondriais , Miopatias Mitocondriais/genética , Mutação Puntual , RNA de Transferência de Leucina/genética , Proliferação de Células , Células Clonais , Transporte de Elétrons , Genótipo , Humanos , Mitocôndrias/metabolismo , Miopatias Mitocondriais/metabolismo , Músculo Esquelético/metabolismo , RNA/química , RNA/genética , RNA/metabolismo , Precursores de RNA/metabolismo , Estabilidade de RNA , RNA Mitocondrial , RNA de Transferência de Leucina/química , RNA de Transferência de Leucina/metabolismo , Aminoacilação de RNA de Transferência
17.
Hum Mol Genet ; 14(24): 3857-64, 2005 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-16278235

RESUMO

We have disrupted expression of the mitochondrial Friedreich ataxia protein frataxin specifically in murine hepatocytes to generate mice with impaired mitochondrial function and decreased oxidative phosphorylation. These animals have a reduced life span and develop multiple hepatic tumors. Livers also show increased oxidative stress, impaired respiration and reduced ATP levels paralleled by reduced activity of iron-sulfur cluster (Fe/S) containing proteins (ISP), which all leads to increased hepatocyte turnover by promoting both apoptosis and proliferation. Accordingly, phosphorylation of the stress-inducible p38 MAP kinase was found to be specifically impaired following disruption of frataxin. Taken together, these findings indicate that frataxin may act as a mitochondrial tumor suppressor protein in mammals.


Assuntos
Proteínas de Ligação ao Ferro/genética , Neoplasias Hepáticas/genética , Fígado/fisiologia , Longevidade/genética , Mitocôndrias/metabolismo , Animais , Apoptose/genética , Proliferação de Células , Hepatócitos/metabolismo , Hepatócitos/patologia , Proteínas de Ligação ao Ferro/metabolismo , Proteínas com Ferro-Enxofre/metabolismo , Fígado/patologia , Neoplasias Hepáticas/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/genética , Estresse Oxidativo , Fosforilação , Espécies Reativas de Oxigênio/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
18.
Nucleic Acids Res ; 33(17): 5647-58, 2005.
Artigo em Inglês | MEDLINE | ID: mdl-16199753

RESUMO

We have studied the consequences of two homoplasmic, pathogenic point mutations (T7512C and G7497A) in the tRNA(Ser(UCN)) gene of mitochondrial (mt) DNA using osteosarcoma cybrids. We identified a severe reduction of tRNA(Ser(UCN)) to levels below 10% of controls for both mutations, resulting in a 40% reduction in mitochondrial protein synthesis rate and in a respiratory chain deficiency resembling that in the patients muscle. Aminoacylation was apparently unaffected. On non-denaturating northern blots we detected an altered electrophoretic mobility for G7497A containing tRNA molecules suggesting a structural impact of this mutation, which was confirmed by structural probing. By comparing in vitro transcribed molecules with native RNA in such gels, we also identified tRNA(Ser(UCN)) being present in two isoforms in vivo, probably corresponding to the nascent, unmodified transcripts co-migrating with the in vitro transcripts and a second, faster moving isoform corresponding to the mature tRNA. In cybrids containing either mutations the unmodified isoforms were severely reduced. We hypothesize that both mutations lead to an impairment of post-transcriptional modification processes, ultimately leading to a preponderance of degradation by nucleases over maturation by modifying enzymes, resulting in severely reduced tRNA(Ser(UCN)) steady state levels. We infer that an increased degradation rate, caused by disturbance of tRNA maturation and, in the case of the G7497A mutant, alteration of tRNA structure, is a new pathogenic mechanism of mt tRNA point mutations.


Assuntos
DNA Mitocondrial/genética , Doenças Mitocondriais/genética , Mutação Puntual , Processamento Pós-Transcricional do RNA , RNA de Transferência de Serina/metabolismo , RNA/metabolismo , Aminoacilação , Sequência de Bases , Linhagem Celular , Criança , Pré-Escolar , Complexo I de Transporte de Elétrons/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Humanos , Masculino , Doenças Mitocondriais/metabolismo , Proteínas Mitocondriais/biossíntese , Dados de Sequência Molecular , RNA/química , RNA/genética , Precursores de RNA/metabolismo , Estabilidade de RNA , RNA Mitocondrial , RNA de Transferência de Serina/química , RNA de Transferência de Serina/genética
19.
J Clin Neurophysiol ; 22(1): 43-8, 2005.
Artigo em Inglês | MEDLINE | ID: mdl-15689712

RESUMO

The diagnostic accuracy in pediatric neurology has been considerably improved by new methods such as magnetic resonance imaging and molecular genetic analysis. However, standard diagnostic techniques continue to play an important role. The authors analyzed the diagnostic value of electromyography (EMG) and nerve conduction studies (NCS) in a retrospective study of 498 pediatric patients. The overall consistency between EMG results and the final clinical diagnosis in all children examined was 98%. In myogenic diseases, the concordance between EMG and clinical findings was lower (80%), because some patients with congenital myopathies showed normal EMG findings in this group. Peripheral neurogenic diseases were in all but one of the cases diagnosed correctly (99.5%). No decrease in diagnostic reliability was found in the younger age group. EMG and NCS examinations have to be adapted to the needs of children by an experienced examiner, but continue to be valuable diagnostic methods in pediatric neurology.


Assuntos
Eletromiografia/métodos , Doenças do Sistema Nervoso/diagnóstico , Condução Nervosa/fisiologia , Adolescente , Adulto , Fatores Etários , Criança , Pré-Escolar , Estudos de Coortes , Diagnóstico Diferencial , Feminino , Humanos , Lactente , Masculino , Doenças do Sistema Nervoso/classificação , Doenças do Sistema Nervoso/fisiopatologia , Exame Físico , Valor Preditivo dos Testes , Estudos Retrospectivos , Sensibilidade e Especificidade , Processamento de Sinais Assistido por Computador
20.
Cardiovasc Res ; 64(2): 198-207, 2004 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-15485678

RESUMO

During hypertrophy, proliferation of mitochondria does not keep pace with the increasing energy demand of the heart. This probably contributes importantly to cardiac failure, together with other phenotypic changes occurring during the growth process. The problem may be even aggravated if defects of mitochondrial function itself and not external factors cause the hypertrophic process. Here we review the basic mechanisms controlling mitochondrial biogenesis, especially the pathways coordinating expression of nuclear encoded mitochondrial genes and the small mitochondrial genome, and how these mechanisms may be connected to the cardiomyocyte differentiation program during development as well as under physiological and pathological circumstances.


Assuntos
Cardiomegalia/patologia , Cardiomegalia/fisiopatologia , Mitocôndrias Cardíacas/patologia , Cardiomegalia/metabolismo , Replicação do DNA , DNA Mitocondrial/metabolismo , Metabolismo Energético , Regulação da Expressão Gênica , Humanos , Mitocôndrias Cardíacas/metabolismo
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