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1.
J Inherit Metab Dis ; 2019 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-31652339

RESUMO

Assessing long-term mortality and identifying predictors of death in adults with mitochondrial diseases. We retrospectively included adult patients with genetically proven mitochondrial diseases referred to our centre between January 2000 and June 2016, and collected information relative to their genetic testing, clinical assessments, and vital status. We performed single and multiple variable analyses in search of predictors of total mortality, and calculated hazard ratios (HR) and 95% confidence intervals (CI). We included 267 patients (women 59%; median age 43.3 [31.3-54.2] years), including 111 with mitochondrial DNA (mtDNA) single large-scale deletions, 65 with m.3243A>G, 24 with m.8344A>G, 32 with other mtDNA point mutations, and 36 patients with nuclear genes mutations. Over a median follow-up of 8.9 years (0.3 to 18.7), 61 patients (22.8%) died, at a median age of 50.7 (37.9-51.9) years. Primary cause of death was cardiovascular disease in 16 patients (26.2%), respiratory in 11 (18.0%), and gastrointestinal in 5 (8.1%). By multiple variable analysis, diabetes (HR 2.75; 95% CI 1.46-5.18), intraventricular cardiac conduction defects (HR 3.38; 95% CI 1.71-6.76) and focal brain involvement (HR 2.39; 95% CI 1.25-4.57) were independent predictors of death. Adult patients with mitochondrial diseases present high morbidity that can be independently predicted by the presence of diabetes, intraventricular cardiac conduction defects, and focal brain involvement.

2.
PLoS One ; 14(8): e0221886, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31461494

RESUMO

BACKGROUND: ATP synthase, the mitochondrial complex V, plays a major role in bioenergetics and its defects lead to severe diseases. Lack of a consensual protocol for the assay of complex V activity probably explains the under-representation of complex V defect among mitochondrial diseases. The aim of this work was to elaborate a fast, simple and reliable method to check the maximal complex V capacity in samples relevant to clinical diagnosis. METHODS: Using homogenates from four different murine organs, we tested the use of dodecylmaltoside, stability of the activity, linearity with protein amount, sensitivity to oligomycin and to exogenous inhibitory factor 1 (IF1), influence of freezing, and impact of mitochondrial purification. RESULTS: We obtained organ-dependent, reproducible and stable complex V specific activities, similar with fresh and frozen organs. Similar inhibition by oligomycin and exogenous IF1 demonstrated tight coupling between F1 and F0 domains. The Michaelis constant for MgATP had close values for all organs, in the 150-220 µM range. Complex V catalytic turnover rate, as measured in preparations solubilized in detergent using immunotitration and activity measurements, was more than three times higher in extracts from brain or muscle than in extracts from heart or liver. This tissue specificity suggested post-translational modifications. Concomitant measurement of respiratory activities showed only slightly different complex II/complex V ratio in the four organs. In contrast, complex I/complex V ratio differed in brain as compared to the three other organs because of a high complex I activity in brain. Mitochondria purification preserved these ratios, except for brain where selective degradation of complex I occurred. Therefore, mitochondrial purification could introduce a biased enzymatic evaluation. CONCLUSION: Altogether, this work demonstrates that a reliable assay of complex V activity is perfectly possible with very small samples from frozen biopsies, which was confirmed using control and deficient human muscles.

3.
Int J Oncol ; 54(6): 2149-2156, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30942448

RESUMO

Mitotane (also termed o,p'­DDD) is the most effective therapy for advanced adrenocortical carcinoma (ACC). Mitotane­induced dyslipidemia is treated with statins. Mitotane and statins are known to exert anti­proliferative effects in vitro; however, the effects of statins have never been directly evaluated in patients with ACC and ACC cells, at least to the best of our knowledge. Thus, in this study, we aimed to examine the effects of the rosuvastatin on ACC cells. It has been shown that the combined use of mitotane and statins significantly increases the tumor control rate in patients with ACC; however, it would be of interest to elucidate the molecular mechanisms involved in this potentiation. In this study, we examined the effects of mitotane, rosuvastatin and their combination in NCI­H295R human ACC cells using proliferation assays, gene expression analyses and free intracellular cholesterol measurements. The results revealed that mitotane dose­dependently reduced cell viability, induced apoptosis and increased intracellular free cholesterol levels, considered as one of the key features of mitotane action, while rosuvastatin alone reduced cell viability and increased apoptosis at high concentrations. We also demonstrated that rosuvastatin potentiated the effects of mitotane by reducing cell viability, inducing apoptosis, increasing intracellular free cholesterol levels, and by decreasing the expression of 3­hydroxy­3­methylglutaryl­CoA reductase (HMGCR) and ATP binding cassette subfamily a member 1 (ABCA1), genes involved in cholesterol metabolism, and inhibiting steroidogenesis. Collectively, potentiating the effects of mitotane with the use of rosuvastatin may provide novel therapeutic strategies for ACC, given that the combination of these drugs, pending clinical validation, may lead to the better management of ACC.


Assuntos
Neoplasias do Córtex Suprarrenal/tratamento farmacológico , Carcinoma Adrenocortical/tratamento farmacológico , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Mitotano/farmacologia , Rosuvastatina Cálcica/farmacologia , Neoplasias do Córtex Suprarrenal/patologia , Carcinoma Adrenocortical/patologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sinergismo Farmacológico , Feminino , Humanos , Mitotano/uso terapêutico , Rosuvastatina Cálcica/uso terapêutico
5.
FASEB J ; 33(6): 7168-7179, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30848931

RESUMO

Polymerase γ catalytic subunit (POLG) gene encodes the enzyme responsible for mitochondrial DNA (mtDNA) synthesis. Mutations affecting POLG are the most prevalent cause of mitochondrial disease because of defective mtDNA replication and lead to a wide spectrum of clinical phenotypes characterized by mtDNA deletions or depletion. Enhancing mitochondrial deoxyribonucleoside triphosphate (dNTP) synthesis effectively rescues mtDNA depletion in different models of defective mtDNA maintenance due to dNTP insufficiency. In this study, we studied mtDNA copy number recovery rates following ethidium bromide-forced depletion in quiescent fibroblasts from patients harboring mutations in different domains of POLG. Whereas control cells spontaneously recovered initial mtDNA levels, POLG-deficient cells experienced a more severe depletion and could not repopulate mtDNA. However, activation of deoxyribonucleoside (dN) salvage by supplementation with dNs plus erythro-9-(2-hydroxy-3-nonyl) adenine (inhibitor of deoxyadenosine degradation) led to increased mitochondrial dNTP pools and promoted mtDNA repopulation in all tested POLG-mutant cells independently of their specific genetic defect. The treatment did not compromise POLG fidelity because no increase in multiple deletions or point mutations was detected. Our study suggests that physiologic dNTP concentration limits the mtDNA replication rate. We thus propose that increasing mitochondrial dNTP availability could be of therapeutic interest for POLG deficiency and other conditions in which mtDNA maintenance is challenged.-Blázquez-Bermejo, C., Carreño-Gago, L., Molina-Granada, D., Aguirre, J., Ramón, J., Torres-Torronteras, J., Cabrera-Pérez, R., Martín, M. Á., Domínguez-González, C., de la Cruz, X., Lombès, A., García-Arumí, E., Martí, R., Cámara, Y. Increased dNTP pools rescue mtDNA depletion in human POLG-deficient fibroblasts.

6.
Life Sci Alliance ; 2(1)2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30683687

RESUMO

Mitochondria have a compartmentalized gene expression system dedicated to the synthesis of membrane proteins essential for oxidative phosphorylation. Responsive quality control mechanisms are needed to ensure that aberrant protein synthesis does not disrupt mitochondrial function. Pathogenic mutations that impede the function of the mitochondrial matrix quality control protease complex composed of AFG3L2 and paraplegin cause a multifaceted clinical syndrome. At the cell and molecular level, defects to this quality control complex are defined by impairment to mitochondrial form and function. Here, we establish the etiology of these phenotypes. We show how disruptions to the quality control of mitochondrial protein synthesis trigger a sequential stress response characterized first by OMA1 activation followed by loss of mitochondrial ribosomes and by remodelling of mitochondrial inner membrane ultrastructure. Inhibiting mitochondrial protein synthesis with chloramphenicol completely blocks this stress response. Together, our data establish a mechanism linking major cell biological phenotypes of AFG3L2 pathogenesis and show how modulation of mitochondrial protein synthesis can exert a beneficial effect on organelle homeostasis.

8.
Orphanet J Rare Dis ; 13(1): 120, 2018 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-30025539

RESUMO

BACKGROUND: Mitochondrial acyl-CoA dehydrogenase family member 9 (ACAD9) is essential for the assembly of mitochondrial respiratory chain complex I. Disease causing biallelic variants in ACAD9 have been reported in individuals presenting with lactic acidosis and cardiomyopathy. RESULTS: We describe the genetic, clinical and biochemical findings in a cohort of 70 patients, of whom 29 previously unpublished. We found 34 known and 18 previously unreported variants in ACAD9. No patients harbored biallelic loss of function mutations, indicating that this combination is unlikely to be compatible with life. Causal pathogenic variants were distributed throughout the entire gene, and there was no obvious genotype-phenotype correlation. Most of the patients presented in the first year of life. For this subgroup the survival was poor (50% not surviving the first 2 years) comparing to patients with a later presentation (more than 90% surviving 10 years). The most common clinical findings were cardiomyopathy (85%), muscular weakness (75%) and exercise intolerance (72%). Interestingly, severe intellectual deficits were only reported in one patient and severe developmental delays in four patients. More than 70% of the patients were able to perform the same activities of daily living when compared to peers. CONCLUSIONS: Our data show that riboflavin treatment improves complex I activity in the majority of patient-derived fibroblasts tested. This effect was also reported for most of the treated patients and is mirrored in the survival data. In the patient group with disease-onset below 1 year of age, we observed a statistically-significant better survival for patients treated with riboflavin.

9.
J Pharmacol Exp Ther ; 365(3): 711-726, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29669730

RESUMO

Although mitochondriotoxicity plays a major role in drug-induced hepatotoxicity, alteration of mitochondrial DNA (mtDNA) homeostasis has been described only with a few drugs. Because it requires long drug exposure, this mechanism of toxicity cannot be detected with investigations performed in isolated liver mitochondria or cultured cells exposed to drugs for several hours or a few days. Thus, a first aim of this study was to determine whether a 2-week treatment with nine hepatotoxic drugs could affect mtDNA homeostasis in HepaRG cells. Previous investigations with these drugs showed rapid toxicity on oxidative phosphorylation but did not address the possibility of delayed toxicity secondary to mtDNA homeostasis impairment. The maximal concentration used for each drug induced about 10% cytotoxicity. Two other drugs, zalcitabine and linezolid, were used as positive controls for their respective effects on mtDNA replication and translation. Another goal was to determine whether drug-induced mitochondriotoxicity could be modulated by lipid overload mimicking nonalcoholic fatty liver. Among the nine drugs, imipramine and ritonavir induced mitochondrial effects suggesting alteration of mtDNA translation. Ritonavir toxicity was stronger in nonsteatotic cells. None of the nine drugs decreased mtDNA levels. However, increased mtDNA was observed with five drugs, especially in nonsteatotic cells. The mtDNA levels could not be correlated with the expression of key factors involved in mitochondrial biogenesis, such as peroxisome proliferator-activated receptor-γ coactivator 1α (PGC1α), PGC1ß, and AMP-activated protein kinase α-subunit. Hence, drug-induced impairment of mtDNA translation might not be rare, and increased mtDNA levels could be a frequent adaptive response to slight energy shortage. Nevertheless, this adaptation could be impaired by lipid overload.

10.
Int J Mol Sci ; 19(3)2018 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-29518970

RESUMO

Mammalian respiratory complex I (CI) biogenesis requires both nuclear and mitochondria-encoded proteins and is mostly organized in respiratory supercomplexes. Among the CI proteins encoded by the mitochondrial DNA, NADH-ubiquinone oxidoreductase chain 1 (ND1) is a core subunit, evolutionary conserved from bacteria to mammals. Recently, ND1 has been recognized as a pivotal subunit in maintaining the structural and functional interaction among the hydrophilic and hydrophobic CI arms. A critical role of human ND1 both in CI biogenesis and in the dynamic organization of supercomplexes has been depicted, although the proof of concept is still missing and the critical amount of ND1 protein necessary for a proper assembly of both CI and supercomplexes is not defined. By exploiting a unique model in which human ND1 is allotopically re-expressed in cells lacking the endogenous protein, we demonstrated that the lack of this protein induces a stall in the multi-step process of CI biogenesis, as well as the alteration of supramolecular organization of respiratory complexes. We also defined a mutation threshold for the m.3571insC truncative mutation in mitochondrially encoded NADH:ubiquinone oxidoreductase core subunit 1 (MT-ND1), below which CI and its supramolecular organization is recovered, strengthening the notion that a certain amount of human ND1 is required for CI and supercomplexes biogenesis.


Assuntos
Alelos , Complexo I de Transporte de Elétrons/química , Complexo I de Transporte de Elétrons/genética , Mutação , NADH Desidrogenase/química , NADH Desidrogenase/genética , Respiração Celular , DNA Mitocondrial/genética , Complexo I de Transporte de Elétrons/metabolismo , Mitocôndrias/genética , Mitocôndrias/metabolismo , NADH Desidrogenase/metabolismo , Consumo de Oxigênio , Ligação Proteica , Relação Estrutura-Atividade
11.
Mol Cell ; 69(4): 594-609.e8, 2018 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-29452639

RESUMO

Accumulating evidence indicates that the MDM2 oncoprotein promotes tumorigenesis beyond its canonical negative effects on the p53 tumor suppressor, but these p53-independent functions remain poorly understood. Here, we show that a fraction of endogenous MDM2 is actively imported in mitochondria to control respiration and mitochondrial dynamics independently of p53. Mitochondrial MDM2 represses the transcription of NADH-dehydrogenase 6 (MT-ND6) in vitro and in vivo, impinging on respiratory complex I activity and enhancing mitochondrial ROS production. Recruitment of MDM2 to mitochondria increases during oxidative stress and hypoxia. Accordingly, mice lacking MDM2 in skeletal muscles exhibit higher MT-ND6 levels, enhanced complex I activity, and increased muscular endurance in mild hypoxic conditions. Furthermore, increased mitochondrial MDM2 levels enhance the migratory and invasive properties of cancer cells. Collectively, these data uncover a previously unsuspected function of the MDM2 oncoprotein in mitochondria that play critical roles in skeletal muscle physiology and may contribute to tumor progression.

12.
J Clin Invest ; 128(4): 1671-1687, 2018 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-29447131

RESUMO

Synthetic lethality is an efficient mechanism-based approach to selectively target DNA repair defects. Excision repair cross-complementation group 1 (ERCC1) deficiency is frequently found in non-small-cell lung cancer (NSCLC), making this DNA repair protein an attractive target for exploiting synthetic lethal approaches in the disease. Using unbiased proteomic and metabolic high-throughput profiling on a unique in-house-generated isogenic model of ERCC1 deficiency, we found marked metabolic rewiring of ERCC1-deficient populations, including decreased levels of the metabolite NAD+ and reduced expression of the rate-limiting NAD+ biosynthetic enzyme nicotinamide phosphoribosyltransferase (NAMPT). We also found reduced NAMPT expression in NSCLC samples with low levels of ERCC1. These metabolic alterations were a primary effect of ERCC1 deficiency, and caused selective exquisite sensitivity to small-molecule NAMPT inhibitors, both in vitro - ERCC1-deficient cells being approximately 1,000 times more sensitive than ERCC1-WT cells - and in vivo. Using transmission electronic microscopy and functional metabolic studies, we found that ERCC1-deficient cells harbor mitochondrial defects. We propose a model where NAD+ acts as a regulator of ERCC1-deficient NSCLC cell fitness. These findings open therapeutic opportunities that exploit a yet-undescribed nuclear-mitochondrial synthetic lethal relationship in NSCLC models, and highlight the potential for targeting DNA repair/metabolic crosstalks for cancer therapy.

15.
Nat Commun ; 8: 15824, 2017 06 12.
Artigo em Inglês | MEDLINE | ID: mdl-28604674

RESUMO

Across a variety of Mendelian disorders, ∼50-75% of patients do not receive a genetic diagnosis by exome sequencing indicating disease-causing variants in non-coding regions. Although genome sequencing in principle reveals all genetic variants, their sizeable number and poorer annotation make prioritization challenging. Here, we demonstrate the power of transcriptome sequencing to molecularly diagnose 10% (5 of 48) of mitochondriopathy patients and identify candidate genes for the remainder. We find a median of one aberrantly expressed gene, five aberrant splicing events and six mono-allelically expressed rare variants in patient-derived fibroblasts and establish disease-causing roles for each kind. Private exons often arise from cryptic splice sites providing an important clue for variant prioritization. One such event is found in the complex I assembly factor TIMMDC1 establishing a novel disease-associated gene. In conclusion, our study expands the diagnostic tools for detecting non-exonic variants and provides examples of intronic loss-of-function variants with pathological relevance.


Assuntos
Perfilação da Expressão Gênica , Doenças Mitocondriais/genética , Análise de Sequência de RNA , Técnicas e Procedimentos Diagnósticos , Humanos , Processamento de RNA
16.
Brain ; 140(6): 1595-1610, 2017 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-28549128

RESUMO

Although mitochondrial disorders are clinically heterogeneous, they frequently involve the central nervous system and are among the most common neurogenetic disorders. Identifying the causal genes has benefited enormously from advances in high-throughput sequencing technologies; however, once the defect is known, researchers face the challenge of deciphering the underlying disease mechanism. Here we characterize large biallelic deletions in the region encoding the ATAD3C, ATAD3B and ATAD3A genes. Although high homology complicates genomic analysis of the ATAD3 defects, they can be identified by targeted analysis of standard single nucleotide polymorphism array and whole exome sequencing data. We report deletions that generate chimeric ATAD3B/ATAD3A fusion genes in individuals from four unrelated families with fatal congenital pontocerebellar hypoplasia, whereas a case with genomic rearrangements affecting the ATAD3C/ATAD3B genes on one allele and ATAD3B/ATAD3A genes on the other displays later-onset encephalopathy with cerebellar atrophy, ataxia and dystonia. Fibroblasts from affected individuals display mitochondrial DNA abnormalities, associated with multiple indicators of altered cholesterol metabolism. Moreover, drug-induced perturbations of cholesterol homeostasis cause mitochondrial DNA disorganization in control cells, while mitochondrial DNA aggregation in the genetic cholesterol trafficking disorder Niemann-Pick type C disease further corroborates the interdependence of mitochondrial DNA organization and cholesterol. These data demonstrate the integration of mitochondria in cellular cholesterol homeostasis, in which ATAD3 plays a critical role. The dual problem of perturbed cholesterol metabolism and mitochondrial dysfunction could be widespread in neurological and neurodegenerative diseases.


Assuntos
Adenosina Trifosfatases/genética , Cerebelo/anormalidades , DNA Mitocondrial/genética , Proteínas de Membrana/genética , Doenças Mitocondriais/genética , Proteínas Mitocondriais/genética , Malformações do Sistema Nervoso/genética , ATPases Associadas a Diversas Atividades Celulares , Adulto , Cerebelo/diagnóstico por imagem , Cerebelo/fisiopatologia , Consanguinidade , Deficiências do Desenvolvimento/diagnóstico por imagem , Deficiências do Desenvolvimento/genética , Deficiências do Desenvolvimento/fisiopatologia , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Doenças Mitocondriais/diagnóstico por imagem , Doenças Mitocondriais/fisiopatologia , Malformações do Sistema Nervoso/diagnóstico por imagem , Malformações do Sistema Nervoso/fisiopatologia
17.
Clin Neurophysiol ; 128(7): 1258-1263, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28535487

RESUMO

OBJECTIVE: To explore potential spreading to peripheral nerves of the mitochondrial dysfunction in chronic progressive external ophthalmoplegia (CPEO) by assessing axonal excitability. METHODS: CPEO patients (n=13) with large size deletion of mitochondrial DNA and matching healthy controls (n=22) were included in a case-control study. Muscle strength was quantified using MRC sum-score and used to define two groups of patients: CPEO-weak and CPEO-normal (normal strength). Nerve excitability properties of median motor axons were assessed with the TROND protocol and changes interpreted with the aid of a model. RESULTS: Alterations of nerve excitability strongly correlated with scores of muscle strength. CPEO-weak displayed abnormal nerve excitability compared to CPEO-normal and healthy controls, with increased superexcitability and responses to hyperpolarizing current. Modeling indicated that the CPEO-weak recordings were best explained by an increase in the 'Barrett-Barrett' conductance across the myelin sheath. CONCLUSION: CPEO patients with skeletal weakness presented sub-clinical nerve excitability changes, which were not consistent with axonal membrane depolarization, but suggested Schwann cell involvement. SIGNIFICANCE: This study provides new insights into the spreading of large size deletion of mitochondrial DNA to Schwann cells in CPEO patients.


Assuntos
Debilidade Muscular/diagnóstico , Debilidade Muscular/fisiopatologia , Condução Nervosa/fisiologia , Oftalmoplegia Externa Progressiva Crônica/diagnóstico , Oftalmoplegia Externa Progressiva Crônica/fisiopatologia , Adolescente , Estudos de Casos e Controles , Criança , DNA Mitocondrial/genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Debilidade Muscular/genética , Oftalmoplegia Externa Progressiva Crônica/genética , Estudos Prospectivos , Adulto Jovem
18.
J Inherit Metab Dis ; 40(3): 403-414, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-28303425

RESUMO

OBJECTIVES: The common data elements (CDE) project was developed by the National Institute of Neurological Disorders and Stroke (NINDS) to provide clinical researchers with tools to improve data quality and allow for harmonization of data collected in different research studies. CDEs have been created for several neurological diseases; the aim of this project was to develop CDEs specifically curated for mitochondrial disease (Mito) to enhance clinical research. METHODS: Nine working groups (WGs), composed of international mitochondrial disease experts, provided recommendations for Mito clinical research. They initially reviewed existing NINDS CDEs and instruments, and developed new data elements or instruments when needed. Recommendations were organized, internally reviewed by the Mito WGs, and posted online for external public comment for a period of eight weeks. The final version was again reviewed by all WGs and the NINDS CDE team prior to posting for public use. RESULTS: The NINDS Mito CDEs and supporting documents are publicly available on the NINDS CDE website ( https://commondataelements.ninds.nih.gov/ ), organized into domain categories such as Participant/Subject Characteristics, Assessments, and Examinations. CONCLUSION: We developed a comprehensive set of CDE recommendations, data definitions, case report forms (CRFs), and guidelines for use in Mito clinical research. The widespread use of CDEs is intended to enhance Mito clinical research endeavors, including natural history studies, clinical trial design, and data sharing. Ongoing international collaboration will facilitate regular review, updates and online publication of Mito CDEs, and support improved consistency of data collection and reporting.


Assuntos
Elementos de Dados Comuns/normas , Doenças Mitocondriais/patologia , Doenças do Sistema Nervoso/patologia , Acidente Vascular Cerebral/patologia , Pesquisa Biomédica/normas , Coleta de Dados/normas , Humanos , National Institute of Neurological Disorders and Stroke (USA) , Projetos de Pesquisa/normas , Estados Unidos
19.
Cell Stem Cell ; 20(5): 659-674.e9, 2017 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-28132834

RESUMO

Mitochondrial DNA (mtDNA) mutations frequently cause neurological diseases. Modeling of these defects has been difficult because of the challenges associated with engineering mtDNA. We show here that neural progenitor cells (NPCs) derived from human induced pluripotent stem cells (iPSCs) retain the parental mtDNA profile and exhibit a metabolic switch toward oxidative phosphorylation. NPCs derived in this way from patients carrying a deleterious homoplasmic mutation in the mitochondrial gene MT-ATP6 (m.9185T>C) showed defective ATP production and abnormally high mitochondrial membrane potential (MMP), plus altered calcium homeostasis, which represents a potential cause of neural impairment. High-content screening of FDA-approved drugs using the MMP phenotype highlighted avanafil, which we found was able to partially rescue the calcium defect in patient NPCs and differentiated neurons. Overall, our results show that iPSC-derived NPCs provide an effective model for drug screening to target mtDNA disorders that affect the nervous system.


Assuntos
DNA Mitocondrial/genética , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Mitocôndrias/genética , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Cálcio/metabolismo , Linhagem Celular , Descoberta de Drogas/métodos , Humanos , Mutação
20.
ACS Infect Dis ; 3(3): 216-224, 2017 03 10.
Artigo em Inglês | MEDLINE | ID: mdl-28086019

RESUMO

Theileria annulata infects bovine leukocytes, transforming them into invasive, cancer-like cells that cause the widespread disease called tropical theileriosis. We report that in Theileria-transformed leukocytes hexokinase-2 (HK2) binds to B cell lymphoma-2-associated death promoter (BAD) only when serine (S) 155 in BAD is phosphorylated. We show that HK2 recruitment to BAD is abolished by a cell-penetrating peptide that acts as a nonphosphorylatable BAD substrate that inhibits endogenous S155 phosphorylation, leading to complex dissociation and ubiquitination and degradation of HK2 by the proteasome. As HK2 is a critical enzyme involved in Warburg glycolysis, its loss forces Theileria-transformed macrophages to switch back to HK1-dependent oxidative glycolysis that down-regulates macrophage proliferation only when they are growing on glucose. When growing on galactose, degradation of HK2 has no effect on Theileria-infected leukocyte proliferation, because metabolism of this sugar is independent of hexokinases. Thus, targeted disruption of the phosphorylation-dependent HK2/BAD complex may represent a novel approach to control Theileria-transformed leukocyte proliferation.


Assuntos
Doenças dos Bovinos/parasitologia , Hexoquinase/metabolismo , Theileria annulata/patogenicidade , Theileriose/metabolismo , Proteína de Morte Celular Associada a bcl/genética , Animais , Bovinos , Doenças dos Bovinos/metabolismo , Proliferação de Células , Peptídeos Penetradores de Células/farmacologia , Glicólise , Leucócitos/citologia , Leucócitos/metabolismo , Leucócitos/parasitologia , Fosforilação , Regiões Promotoras Genéticas , Proteólise , Serina/metabolismo , Theileriose/parasitologia
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