44 results on '"Javier Torres-Torronteras"'
Search Results
2. RRM1 variants cause a mitochondrial DNA maintenance disorder via impaired de novo nucleotide synthesis
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Jonathan Shintaku, Wolfgang M. Pernice, Wafaa Eyaid, Jeevan B. GC, Zuben P. Brown, Marti Juanola-Falgarona, Javier Torres-Torronteras, Ewen W. Sommerville, Debby M.E.I. Hellebrekers, Emma L. Blakely, Alan Donaldson, Ingrid van de Laar, Cheng-Shiun Leu, Ramon Marti, Joachim Frank, Kurenai Tanji, David A. Koolen, Richard J. Rodenburg, Patrick F. Chinnery, H.J.M. Smeets, Gráinne S. Gorman, Penelope E. Bonnen, Robert W. Taylor, and Michio Hirano
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Genetics ,Medicine - Abstract
Mitochondrial DNA (mtDNA) depletion/deletions syndromes (MDDS) encompass a clinically and etiologically heterogenous group of mitochondrial disorders caused by impaired mtDNA maintenance. Among the most frequent causes of MDDS are defects in nucleoside/nucleotide metabolism, which is critical for synthesis and homeostasis of the deoxynucleoside triphosphate (dNTP) substrates of mtDNA replication. A central enzyme for generating dNTPs is ribonucleotide reductase, a critical mediator of de novo nucleotide synthesis composed of catalytic RRM1 subunits in complex with RRM2 or p53R2. Here, we report 5 probands from 4 families who presented with ptosis and ophthalmoplegia as well as other clinical manifestations and multiple mtDNA deletions in muscle. We identified 3 RRM1 loss-of-function variants, including a dominant catalytic site variant (NP_001024.1: p.N427K) and 2 homozygous recessive variants at p.R381, which has evolutionarily conserved interactions with the specificity site. Atomistic molecular dynamics simulations indicate mechanisms by which RRM1 variants affect protein structure. Cultured primary skin fibroblasts of probands manifested mtDNA depletion under cycling conditions, indicating impaired de novo nucleotide synthesis. Fibroblasts also exhibited aberrant nucleoside diphosphate and dNTP pools and mtDNA ribonucleotide incorporation. Our data reveal that primary RRM1 deficiency and, by extension, impaired de novo nucleotide synthesis are causes of MDDS.
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- 2022
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3. Age-related metabolic changes limit efficacy of deoxynucleoside-based therapy in thymidine kinase 2-deficient miceResearch in context
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Cora Blázquez-Bermejo, David Molina-Granada, Ferran Vila-Julià, Daniel Jiménez-Heis, Xiaoshan Zhou, Javier Torres-Torronteras, Anna Karlsson, Ramon Martí, and Yolanda Cámara
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Medicine ,Medicine (General) ,R5-920 - Abstract
Background: Thymidine kinase 2 (TK2) catalyses the phosphorylation of deoxythymidine (dThd) and deoxycytidine (dCtd) within mitochondria. TK2 deficiency leads to mtDNA depletion or accumulation of multiple deletions. In patients, TK2 mutations typically manifest as a rapidly progressive myopathy with infantile onset, leading to respiratory insufficiency and encephalopathy in the most severe clinical presentations. TK2-deficient mice develop the most severe form of the disease and die at average postnatal day 16. dThd+dCtd administration delayed disease progression and expanded lifespan of a knockin murine model of the disease. Methods: We daily administered TK2 knockout mice (Tk2KO) from postnatal day 4 with equimolar doses of dThd+dCtd, dTMP+dCMP, dThd alone or dCtd alone. We monitored body weight and survival and studied different variables at 12 or 29 days of age. We determined metabolite levels in plasma and target tissues, mtDNA copy number in tissues, and the expression and activities of enzymes with a relevant role in mitochondrial dNTP anabolism or catabolism. Findings: dThd+dCtd treatment extended average lifespan of Tk2KO mice from 16 to 34 days, attenuated growth retardation, and rescued mtDNA depletion in skeletal muscle and other target tissues of 12-day-old mice, except in brain. However, the treatment was ineffective in 29-day-old mice that still died prematurely. Bioavailability of dThd and dCtd markedly decreased during mouse development. Activity of enzymes catabolizing dThd and dCtd increased with age in small intestine. Conversely, the activity of the anabolic enzymes decreased in target tissues during mouse development. We also found that administration of dThd alone had the same impact on survival to that of dThd+dCtd, whereas dCtd alone had no influence on lifespan. Interpretation: dThd+dCtd treatment recruits alternative cytosolic salvage pathways for dNTP synthesis, suggesting that this therapy would be of benefit for any Tk2 mutation. dThd accounts for the therapeutic effect of the combined treatment in mice. During the first weeks after birth, mice experience marked tissue-specific metabolic regulations and ontogenetic changes in dNTP metabolism-related enzymes that limit therapeutic efficacy to early developmental stages. Fund: This study was funded by grants from the Spanish Ministry of Industry, Economy and Competitiveness, the Spanish Instituto de Salud Carlos III, the Fundación Inocente, Inocente, AFM Téléthon and the Generalitat de Catalunya. The disclosed funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Keywords: TK2, Deoxynucleoside therapy, mtDNA depletion, Encephalomyopathy, Thymidine, Deoxycytidine
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- 2019
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4. Efficacy of adeno-associated virus gene therapy in a MNGIE murine model enhanced by chronic exposure to nucleosides
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Ferran Vila-Julià, Raquel Cabrera-Pérez, Yolanda Cámara, Miguel Molina-Berenguer, Silvia Lope-Piedrafita, Michio Hirano, Federico Mingozzi, Javier Torres-Torronteras, and Ramon Martí
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MNGIE ,Gene therapy ,Nucleosides ,Mitochondrial disease ,Thymidine phosphorylase ,Medicine ,Medicine (General) ,R5-920 - Abstract
Background: Preclinical studies have shown that gene therapy is a feasible approach to treat mitochondrial neurogastrointestinal encephalomyopathy (MNGIE). However, the genetic murine model of the disease (Tymp/Upp1 double knockout, dKO) has a limited functional phenotype beyond the metabolic imbalances, and so the studies showing efficacy of gene therapy have relied almost exclusively on demonstrating correction of the biochemical phenotype. Chronic oral administration of thymidine (dThd) and deoxyuridine (dUrd) to dKO mice deteriorates the phenotype of the animals, providing a better model to test therapy approaches. Methods: dKO mice were treated with both dThd and dUrd in drinking water from weaning until the end of the study. At 8 - 11 weeks of age, mice were treated with several doses of adeno-associated virus (AAV) serotype 8 vector carrying the human TYMP coding sequence under the control of different liver-specific promoters (TBG, AAT, or HLP). The biochemical profile and functional phenotype were studied over the life of the animals. Findings: Nucleoside exposure resulted in 30-fold higher plasma nucleoside levels in dKO mice compared with non-exposed wild type mice. AAV-treatment provided elevated TP activity in liver and lowered systemic nucleoside levels in exposed dKO mice. Exposed dKO mice had enlarged brain ventricles (assessed by magnetic resonance imaging) and motor impairment (rotarod test); both were prevented by AAV treatment. Among all promoters tested, AAT showed the best efficacy. Interpretation: Our results show that AAV-mediated gene therapy restores the biochemical homeostasis in the murine model of MNGIE and, for the first time, demonstrate that this treatment improves the functional phenotype. Funding: This work was funded in part by the Spanish Instituto de Salud Carlos III, and the Generalitat de Catalunya. The disclosed funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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- 2020
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5. Preclinical Efficacy and Safety Evaluation of Hematopoietic Stem Cell Gene Therapy in a Mouse Model of MNGIE
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Rana Yadak, Raquel Cabrera-Pérez, Javier Torres-Torronteras, Marianna Bugiani, Joost C. Haeck, Marshall W. Huston, Elly Bogaerts, Steffi Goffart, Edwin H. Jacobs, Merel Stok, Lorena Leonardelli, Luca Biasco, Robert M. Verdijk, Monique R. Bernsen, George Ruijter, Ramon Martí, Gerard Wagemaker, Niek P. van Til, and Irenaeus F.M. de Coo
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MNGIE ,thymidine phosphorylase ,hematopoietic stem cells ,lentiviral vectors ,gene therapy ,Genetics ,QH426-470 ,Cytology ,QH573-671 - Abstract
Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive disorder caused by thymidine phosphorylase (TP) deficiency resulting in systemic accumulation of thymidine (d-Thd) and deoxyuridine (d-Urd) and characterized by early-onset neurological and gastrointestinal symptoms. Long-term effective and safe treatment is not available. Allogeneic bone marrow transplantation may improve clinical manifestations but carries disease and transplant-related risks. In this study, lentiviral vector-based hematopoietic stem cell gene therapy (HSCGT) was performed in Tymp−/−Upp1−/− mice with the human phosphoglycerate kinase (PGK) promoter driving TYMP. Supranormal blood TP activity reduced intestinal nucleoside levels significantly at low vector copy number (median, 1.3; range, 0.2–3.6). Furthermore, we covered two major issues not addressed before. First, we demonstrate aberrant morphology of brain astrocytes in areas of spongy degeneration, which was reversed by HSCGT. Second, long-term follow-up and vector integration site analysis were performed to assess safety of the therapeutic LV vectors in depth. This report confirms and supplements previous work on the efficacy of HSCGT in reducing the toxic metabolites in Tymp−/−Upp1−/− mice, using a clinically applicable gene transfer vector and a highly efficient gene transfer method, and importantly demonstrates phenotypic correction with a favorable risk profile, warranting further development toward clinical implementation.
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- 2018
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6. Therapy Prospects for Mitochondrial DNA Maintenance Disorders
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Javier Ramón, Ferran Vila-Julià, David Molina-Granada, Miguel Molina-Berenguer, Maria Jesús Melià, Elena García-Arumí, Javier Torres-Torronteras, Yolanda Cámara, and Ramon Martí
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mitochondria ,mtDNA ,replication ,depletion ,multiple deletions ,therapy ,Biology (General) ,QH301-705.5 ,Chemistry ,QD1-999 - Abstract
Mitochondrial DNA depletion and multiple deletions syndromes (MDDS) constitute a group of mitochondrial diseases defined by dysfunctional mitochondrial DNA (mtDNA) replication and maintenance. As is the case for many other mitochondrial diseases, the options for the treatment of these disorders are rather limited today. Some aggressive treatments such as liver transplantation or allogeneic stem cell transplantation are among the few available options for patients with some forms of MDDS. However, in recent years, significant advances in our knowledge of the biochemical pathomechanisms accounting for dysfunctional mtDNA replication have been achieved, which has opened new prospects for the treatment of these often fatal diseases. Current strategies under investigation to treat MDDS range from small molecule substrate enhancement approaches to more complex treatments, such as lentiviral or adenoassociated vector-mediated gene therapy. Some of these experimental therapies have already reached the clinical phase with very promising results, however, they are hampered by the fact that these are all rare disorders and so the patient recruitment potential for clinical trials is very limited.
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- 2021
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7. Identification and Characterization of New RNASEH1 Mutations Associated With PEO Syndrome and Multiple Mitochondrial DNA Deletions
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Lidia Carreño-Gago, Cora Blázquez-Bermejo, Jordi Díaz-Manera, Yolanda Cámara, Eduard Gallardo, Ramon Martí, Javier Torres-Torronteras, and Elena García-Arumí
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mtDNA ,mitochondrial disease ,PEO ,multiple mtDNA deletions ,RNASEH1 ,Genetics ,QH426-470 - Abstract
Mitochondrial DNA (mtDNA) depletion and deletion syndrome encompasses a group of disorders caused by mutations in genes involved in mtDNA replication and maintenance. The clinical phenotype ranges from fatal infantile hepatocerebral forms to mild adult onset progressive external ophthalmoplegia (PEO). We report the case of a patient with PEO and multiple mtDNA deletions, with two new homozygous mutations in RNASEH1. The first mutation (c.487T>C) is located in the same catalytic domain as the four previously reported mutations, and the second (c.258_260del) is located in the connection domain, where no mutations have been reported. In silico study of the mutations predicted only the first mutation as pathogenic, but functional studies showed that both mutations cause loss of ribonuclease H1 activity. mtDNA replication dysfunction was demonstrated in patient fibroblasts, which were unable to recover normal mtDNA copy number after ethidium bromide-induced mtDNA depletion. Our results demonstrate the pathogenicity of two new RNASEH1 variants found in a patient with PEO syndrome, multiple deletions, and mild mitochondrial myopathy.
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- 2019
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8. The G1/S Specific Cyclin D2 Is a Regulator of HIV-1 Restriction in Non-proliferating Cells.
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Roger Badia, Maria Pujantell, Eva Riveira-Muñoz, Teresa Puig, Javier Torres-Torronteras, Ramón Martí, Bonaventura Clotet, Rosa M Ampudia, Marta Vives-Pi, José A Esté, and Ester Ballana
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Immunologic diseases. Allergy ,RC581-607 ,Biology (General) ,QH301-705.5 - Abstract
Macrophages are a heterogeneous cell population strongly influenced by differentiation stimuli that become susceptible to HIV-1 infection after inactivation of the restriction factor SAMHD1 by cyclin-dependent kinases (CDK). Here, we have used primary human monocyte-derived macrophages differentiated through different stimuli to evaluate macrophage heterogeneity on cell activation and proliferation and susceptibility to HIV-1 infection. Stimulation of monocytes with GM-CSF induces a non-proliferating macrophage population highly restrictive to HIV-1 infection, characterized by the upregulation of the G1/S-specific cyclin D2, known to control early steps of cell cycle progression. Knockdown of cyclin D2, enhances HIV-1 replication in GM-CSF macrophages through inactivation of SAMHD1 restriction factor by phosphorylation. Co-immunoprecipitation experiments show that cyclin D2 forms a complex with CDK4 and p21, a factor known to restrict HIV-1 replication by affecting the function of the downstream cascade that leads to SAMHD1 deactivation. Thus, we demonstrate that cyclin D2 acts as regulator of cell cycle proteins affecting SAMHD1-mediated HIV-1 restriction in non-proliferating macrophages.
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- 2016
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9. A transcriptomic approach to search for novel phenotypic regulators in McArdle disease.
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Gisela Nogales-Gadea, Inés Consuegra-García, Juan C Rubio, Joaquin Arenas, Marc Cuadros, Yolanda Camara, Javier Torres-Torronteras, Carmen Fiuza-Luces, Alejandro Lucia, Miguel A Martín, Elena García-Arumí, and Antoni L Andreu
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Medicine ,Science - Abstract
McArdle disease is caused by lack of glycogen phosphorylase (GP) activity in skeletal muscle. Patients experience exercise intolerance, presenting as early fatigue and contractures. In this study, we investigated the effects produced by a lack of GP on several genes and proteins of skeletal muscle in McArdle patients. Muscle tissue of 35 patients and 7 healthy controls were used to identify abnormalities in the patients' transcriptomic profile using low-density arrays. Gene expression was analyzed for the influence of variables such as sex and clinical severity. Differences in protein expression were studied by immunoblotting and 2D electrophoresis analysis, and protein complexes were examined by two-dimensional, blue native gel electrophoresis (BN-PAGE). A number of genes including those encoding acetyl-coA carboxylase beta, m-cadherin, calpain III, creatine kinase, glycogen synthase (GS), and sarcoplasmic reticulum calcium ATPase 1 (SERCA1), were found to be downregulated in patients. Specifically, compared to controls, GS and SERCA1 proteins were reduced by 50% and 75% respectively; also, unphosphorylated GS and SERCA1 were highly downregulated. On BN-PAGE analysis, GP was present with GS in two muscle protein complexes. Our findings revealed some issues that could be important in understanding the physiological consequences of McArdle disease: (i) SERCA1 downregulation in patients could result in impaired calcium transport in type II (fast-twitch) muscle fibers, leading to early fatigability during exercise tasks involving type II fibers (which mostly use glycolytic metabolism), i.e. isometric exercise, lifting weights or intense dynamic exercise (stair climbing, bicycling, walking at a very brisk pace), (ii) GP and GS were found together in two protein complexes, which suggests a new regulatory mechanism in the activity of these glycogen enzymes.
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- 2012
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10. Limited dCTP availability accounts for mitochondrial DNA depletion in mitochondrial neurogastrointestinal encephalomyopathy (MNGIE).
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Emiliano González-Vioque, Javier Torres-Torronteras, Antoni L Andreu, and Ramon Martí
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Genetics ,QH426-470 - Abstract
Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is a severe human disease caused by mutations in TYMP, the gene encoding thymidine phosphorylase (TP). It belongs to a broader group of disorders characterized by a pronounced reduction in mitochondrial DNA (mtDNA) copy number in one or more tissues. In most cases, these disorders are caused by mutations in genes involved in deoxyribonucleoside triphosphate (dNTP) metabolism. It is generally accepted that imbalances in mitochondrial dNTP pools resulting from these mutations interfere with mtDNA replication. Nonetheless, the precise mechanistic details of this effect, in particular, how an excess of a given dNTP (e.g., imbalanced dTTP excess observed in TP deficiency) might lead to mtDNA depletion, remain largely unclear. Using an in organello replication experimental model with isolated murine liver mitochondria, we observed that overloads of dATP, dGTP, or dCTP did not reduce the mtDNA replication rate. In contrast, an excess of dTTP decreased mtDNA synthesis, but this effect was due to secondary dCTP depletion rather than to the dTTP excess in itself. This was confirmed in human cultured cells, demonstrating that our conclusions do not depend on the experimental model. Our results demonstrate that the mtDNA replication rate is unaffected by an excess of any of the 4 separate dNTPs and is limited by the availability of the dNTP present at the lowest concentration. Therefore, the availability of dNTP is the key factor that leads to mtDNA depletion rather than dNTP imbalances. These results provide the first test of the mechanism that accounts for mtDNA depletion in MNGIE and provide evidence that limited dNTP availability is the common cause of mtDNA depletion due to impaired anabolic or catabolic dNTP pathways. Thus, therapy approaches focusing on restoring the deficient substrates should be explored.
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- 2011
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11. Uridine metabolism in HIV-1-infected patients: effect of infection, of antiretroviral therapy and of HIV-1/ART-associated lipodystrophy syndrome.
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Pere Domingo, Javier Torres-Torronteras, Virginia Pomar, Marta Giralt, Joan Carles Domingo, Maria Del Mar Gutierrez, José M Gallego-Escuredo, Maria Gracia Mateo, Pedro Cano-Soldado, Irene Fernandez, Marçal Pastor-Anglada, Francesc Vidal, Francesc Villarroya, Antoni Andreu, and Ramon Marti
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Medicine ,Science - Abstract
BackgroundUridine has been advocated for the treatment of HIV-1/HAART-associated lipodystrophy (HALS), although its metabolism in HIV-1-infected patients is poorly understood.MethodsPlasma uridine concentrations were measured in 35 controls and 221 HIV-1-infected patients and fat uridine in 15 controls and 19 patients. The diagnosis of HALS was performed following the criteria of the Lipodystrophy Severity Grading Scale. Uridine was measured by a binary gradient-elution HPLC method. Analysis of genes encoding uridine metabolizing enzymes in fat was performed with TaqMan RT-PCR.ResultsMedian plasma uridine concentrations for HIV-1-infected patients were 3.80 µmol/l (interquartile range: 1.60), and for controls 4.60 µmol/l (IQR: 1.8) (P = 0.0009). In fat, they were of 6.0 (3.67), and 2.8 (4.65) nmol/mg of protein, respectively (P = 0.0118). Patients with a mixed HALS form had a median plasma uridine level of 4.0 (IC95%: 3.40-4.80) whereas in those with isolated lipoatrophy it was 3.25 (2.55-4.15) µmol/l/l (P = 0.0066). The expression of uridine cytidine kinase and uridine phosphorylase genes was significantly decreased in all groups of patients with respect to controls. A higher expression of the mRNAs for concentrative nucleoside transporters was found in HIV-1-infected patients with respect to healthy controls.ConclusionsHIV-1 infection is associated with a decrease in plasma uridine and a shift of uridine to the adipose tissue compartment. Antiretroviral therapy was not associated with plasma uridine concentrations, but pure lipoatrophic HALS was associated with significantly lower plasma uridine concentrations.
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- 2010
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12. Dysfunctional mitochondrial translation and combined oxidative phosphorylation deficiency in a mouse model of hepatoencephalopathy due to Gfm1 mutations
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Miguel Molina‐Berenguer, Ferran Vila‐Julià, Sandra Pérez‐Ramos, Maria Teresa Salcedo‐Allende, Yolanda Cámara, Javier Torres‐Torronteras, and Ramon Martí
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Mice, Knockout ,Mutation, Missense ,Mitochondria, Liver ,Peptide Elongation Factor G ,Biochemistry ,Oxidative Phosphorylation ,Electron Transport Complex IV ,Mitochondrial Proteins ,Disease Models, Animal ,Mice ,Amino Acid Substitution ,Hepatic Encephalopathy ,Protein Biosynthesis ,Genetics ,Animals ,Molecular Biology ,Metabolism, Inborn Errors ,Biotechnology - Abstract
Hepatoencephalopathy due to combined oxidative phosphorylation deficiency type 1 (COXPD1) is a recessive mitochondrial translation disorder caused by mutations in GFM1, a nuclear gene encoding mitochondrial elongation factor G1 (EFG1). Patients with COXPD1 typically present hepatoencephalopathy early after birth with rapid disease progression, and usually die within the first few weeks or years of life. We have generated two different mouse models: a Gfm1 knock-in (KI) harboring the p.R671C missense mutation, found in at least 10 patients who survived more than 1 year, and a Gfm1 knock-out (KO) model. Homozygous KO mice (Gfm1
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- 2021
13. Therapy Prospects for Mitochondrial DNA Maintenance Disorders
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Yolanda Cámara, Ferran Vila-Julià, David Molina-Granada, Maria Jesús Melià, Javier Torres-Torronteras, Miguel Molina-Berenguer, Ramon Martí, Elena García-Arumí, Javier Ramón, Institut Català de la Salut, [Ramón J, Vila-Julià F, Molina-Granada D, Molina-Berenguer M, Melià MJ, García-Arumí E, Torres-Torronteras J, Cámara Y, Martí R] Grup de Recerca en Malalties Neuromusculars i Mitocondrials, Vall d’Hebron Institut de Recerca, Barcelona, Spain. Universitat Autònoma de Barcelona, Bellaterra, Spain. Biomedical Network Research Centre on Rare Diseases (CIBERER), Instituto de Salud Carlos III, 28029 Madrid, Spain, and Vall d'Hebron Barcelona Hospital Campus
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0301 basic medicine ,Mitochondrial Diseases ,Genetic enhancement ,medicine.medical_treatment ,Genetic Phenomena::DNA Replication [PHENOMENA AND PROCESSES] ,Multiple deletions ,Review ,Nutritional and Metabolic Diseases::Metabolic Diseases::Mitochondrial Diseases [DISEASES] ,Mitochondrion ,Liver transplantation ,Bioinformatics ,0302 clinical medicine ,Nucleoside ,Biology (General) ,Otros calificadores::/terapia [Otros calificadores] ,Spectroscopy ,depletion ,mtDNA ,Disease Management ,General Medicine ,multiple deletions ,Combined Modality Therapy ,gene therapy ,Computer Science Applications ,Mitochondria ,mitochondria ,Chemistry ,Mitocondris - Malalties - Tractament ,Disease Susceptibility ,Stem cell ,enfermedades nutricionales y metabólicas::enfermedades metabólicas::enfermedades mitocondriales [ENFERMEDADES] ,DNA Replication ,Mitochondrial DNA ,replication ,ADN - Duplicació ,QH301-705.5 ,Replication ,Dysfunctional family ,nucleoside ,DNA, Mitochondrial ,Catalysis ,Mitochondrial Proteins ,Inorganic Chemistry ,03 medical and health sciences ,Depletion ,fenómenos genéticos::replicación del ADN [FENÓMENOS Y PROCESOS] ,Gene therapy ,medicine ,Animals ,Humans ,Physical and Theoretical Chemistry ,QD1-999 ,Molecular Biology ,therapy ,business.industry ,Organic Chemistry ,Other subheadings::/therapy [Other subheadings] ,Mtdn ,Transplantation ,Clinical trial ,030104 developmental biology ,Gene Expression Regulation ,Mutation ,Therapy ,business ,030217 neurology & neurosurgery - Abstract
Esgotament; Teràpia gènica; Mitocondris Mitochondria; Depletion; Gene therapy Agotamiento; Terapia génica; Mitocondrias Mitochondrial DNA depletion and multiple deletions syndromes (MDDS) constitute a group of mitochondrial diseases defined by dysfunctional mitochondrial DNA (mtDNA) replication and maintenance. As is the case for many other mitochondrial diseases, the options for the treatment of these disorders are rather limited today. Some aggressive treatments such as liver transplantation or allogeneic stem cell transplantation are among the few available options for patients with some forms of MDDS. However, in recent years, significant advances in our knowledge of the biochemical pathomechanisms accounting for dysfunctional mtDNA replication have been achieved, which has opened new prospects for the treatment of these often fatal diseases. Current strategies under investigation to treat MDDS range from small molecule substrate enhancement approaches to more complex treatments, such as lentiviral or adenoassociated vector-mediated gene therapy. Some of these experimental therapies have already reached the clinical phase with very promising results, however, they are hampered by the fact that these are all rare disorders and so the patient recruitment potential for clinical trials is very limited.
- Published
- 2021
14. Effect of Resveratrol Content in Red Wine on Circulating Sex Hormone‐Binding Globulin: Lessons from a Pilot Clinical Trial
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Laura Briansó‐Llort, Olga Simó‐Servat, Lorena Ramos‐Perez, Javier Torres‐Torronteras, Cristina Hernandez, Rafael Simó, and David M. Selva
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Male ,Cholesterol ,Cardiovascular Diseases ,Resveratrol ,Sex Hormone-Binding Globulin ,Humans ,Female ,Pilot Projects ,Wine ,Food Science ,Biotechnology - Abstract
Low sex hormone-binding globulin (SHBG) levels are associated with higher risk of developing cardiovascular disease. Epidemiological studies have shown that red wine has beneficial effects on cardiovascular disease. In this work if resveratrol content in red wine increases SHBG levels is explored.A pilot study aims at testing the effect of drinking for 14 days two types of red wine with different resveratrol content is conducted in 26 healthy volunteers. SHBG levels and several biochemical parameters are measured at the beginning and the end of every period. Results show that consumption of both wines does not change body mass index or biochemical markers of liver injury. The low resveratrol wine does not modify the lipid profile or SHBG levels. By contrast, red wine with high resveratrol content significantly reduces total cholesterol in both men and women. Finally, red wine with high resveratrol content increases circulating SHBG in women but not in men.Red wine rich in resveratrol reduces total cholesterol in men and women and increases SHBG only in women. Further research aims at investigating the potential SHBG role enhancement mediated by resveratrol regarding cardiovascular protection that presents women in comparison with men seems warranted.
- Published
- 2022
15. Efficacy of adeno-associated virus gene therapy in a MNGIE murine model enhanced by chronic exposure to nucleosides
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Ramon Martí, Miguel Molina-Berenguer, Javier Torres-Torronteras, Raquel Cabrera-Pérez, Yolanda Cámara, Ferran Vila-Julià, Michio Hirano, Silvia Lope-Piedrafita, and Federico Mingozzi
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0301 basic medicine ,Mitochondrial Diseases ,Genetic enhancement ,Gene Dosage ,lcsh:Medicine ,Gene Expression ,Pharmacology ,medicine.disease_cause ,chemistry.chemical_compound ,Mice ,0302 clinical medicine ,Adeno-associated virus ,Mice, Knockout ,lcsh:R5-920 ,Ophthalmoplegia ,Nucleosides ,General Medicine ,Dependovirus ,Phenotype ,Combined Modality Therapy ,Treatment Outcome ,Liver ,030220 oncology & carcinogenesis ,MNGIE ,lcsh:Medicine (General) ,Research Paper ,Genetic Vectors ,General Biochemistry, Genetics and Molecular Biology ,Virus ,03 medical and health sciences ,Gene therapy ,Muscular Dystrophy, Oculopharyngeal ,medicine ,Animals ,Humans ,Thymidine phosphorylase ,business.industry ,lcsh:R ,Intestinal Pseudo-Obstruction ,Genetic Therapy ,Deoxyuridine ,Mitochondrial disease ,Enzyme Activation ,Disease Models, Animal ,030104 developmental biology ,chemistry ,business ,Thymidine ,Nucleoside - Abstract
Background Preclinical studies have shown that gene therapy is a feasible approach to treat mitochondrial neurogastrointestinal encephalomyopathy (MNGIE). However, the genetic murine model of the disease (Tymp/Upp1 double knockout, dKO) has a limited functional phenotype beyond the metabolic imbalances, and so the studies showing efficacy of gene therapy have relied almost exclusively on demonstrating correction of the biochemical phenotype. Chronic oral administration of thymidine (dThd) and deoxyuridine (dUrd) to dKO mice deteriorates the phenotype of the animals, providing a better model to test therapy approaches. Methods dKO mice were treated with both dThd and dUrd in drinking water from weaning until the end of the study. At 8 - 11 weeks of age, mice were treated with several doses of adeno-associated virus (AAV) serotype 8 vector carrying the human TYMP coding sequence under the control of different liver-specific promoters (TBG, AAT, or HLP). The biochemical profile and functional phenotype were studied over the life of the animals. Findings Nucleoside exposure resulted in 30-fold higher plasma nucleoside levels in dKO mice compared with non-exposed wild type mice. AAV-treatment provided elevated TP activity in liver and lowered systemic nucleoside levels in exposed dKO mice. Exposed dKO mice had enlarged brain ventricles (assessed by magnetic resonance imaging) and motor impairment (rotarod test); both were prevented by AAV treatment. Among all promoters tested, AAT showed the best efficacy. Interpretation Our results show that AAV-mediated gene therapy restores the biochemical homeostasis in the murine model of MNGIE and, for the first time, demonstrate that this treatment improves the functional phenotype. Funding This work was funded in part by the Spanish Instituto de Salud Carlos III, and the Generalitat de Catalunya. The disclosed funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
- Published
- 2020
16. Preclinical Efficacy and Safety Evaluation of Hematopoietic Stem Cell Gene Therapy in a Mouse Model of MNGIE
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Ramon Martí, Niek P. van Til, Joost C. Haeck, Lorena Leonardelli, Luca Biasco, Elly Bogaerts, Irenaeus F.M. de Coo, Merel Stok, Raquel Cabrera-Pérez, Gerard Wagemaker, Steffi Goffart, Marshall W. Huston, Marianna Bugiani, George J G Ruijter, Edwin H. Jacobs, Javier Torres-Torronteras, Monique R. Bernsen, Rana Yadak, Robert M. Verdijk, Pathology, Amsterdam Neuroscience - Cellular & Molecular Mechanisms, Neurology, Radiology & Nuclear Medicine, and Clinical Genetics
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0301 basic medicine ,lcsh:QH426-470 ,Genetic enhancement ,lentiviral vectors ,thymidine phosphorylase ,Article ,Viral vector ,03 medical and health sciences ,chemistry.chemical_compound ,Gene therapy ,Genetics ,Medicine ,Vector (molecular biology) ,lcsh:QH573-671 ,Thymidine phosphorylase ,Molecular Biology ,Phosphoglycerate kinase ,lcsh:Cytology ,business.industry ,Hematopoietic stem cell ,Lentiviral vectors ,gene therapy ,humanities ,Deoxyuridine ,hematopoietic stem cells ,3. Good health ,lcsh:Genetics ,030104 developmental biology ,medicine.anatomical_structure ,chemistry ,MNGIE ,Cancer research ,Molecular Medicine ,business ,Thymidine ,Hematopoietic stem cells - Abstract
Altres ajuts: The authors acknowledge the financial support for this study by Join4energy, Ride4Kids, the Sophia Foundation (SSW0645), Stichting NeMo, in the context of funding provided by the European Commission's 5th, 6th, and 7th Framework Programs(contracts QLK3-CT-2001-00427-INHERINET, LSHB-CT-2004-005242-CONSERT, LSHB-CT-2006-19038 Magselectofection, and grant agreements 222878-PERSIST and 261387 CELL-PID), and by the Netherlands Health Research and Development Organization ZonMw (Translational Gene Therapy program projects 43100016 and 43400010). We thank Dr. Michio Hirano (Department of Neurology, Columbia University Medical Center, New York, USA) for providing the murine model, Louis Boon (Epirus Biopharmaceuticals, Utrecht, the Netherlands) for kindly providing anti-B220 antibody, Prof. Peter A.E. Sillevis Smitt (Department of Neurology, Erasmus MC, Rotterdam, the Netherlands), Pier.G. Mastroberardino and Chiara Milanese (Department of Molecular Genetics, Erasmus MC), Kees Schoonderwoerd (Department of Clinical Genetics, Erasmus MC), and Jeroen de Vrij (Department of Neurosurgery, Erasmus MC) for valuable discussions, Lidia Hussaarts (Department of Clinical Genetics, Erasmus MC) for technical support, King Lam (Department of Pathology, Erasmus MC) for pathology evaluation, and F. Dionisio and A. Aiuti from HSR-TIGET, Milan, for the support to the integration site analysis. Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive disorder caused by thymidine phosphorylase (TP) deficiency resulting in systemic accumulation of thymidine (d-Thd) and deoxyuridine (d-Urd) and characterized by early-onset neurological and gastrointestinal symptoms. Long-term effective and safe treatment is not available. Allogeneic bone marrow transplantation may improve clinical manifestations but carries disease and transplant-related risks. In this study, lentiviral vector-based hematopoietic stem cell gene therapy (HSCGT) was performed in Tymp −/− Upp1 −/− mice with the human phosphoglycerate kinase (PGK) promoter driving TYMP. Supranormal blood TP activity reduced intestinal nucleoside levels significantly at low vector copy number (median, 1.3; range, 0.2-3.6). Furthermore, we covered two major issues not addressed before. First, we demonstrate aberrant morphology of brain astrocytes in areas of spongy degeneration, which was reversed by HSCGT. Second, long-term follow-up and vector integration site analysis were performed to assess safety of the therapeutic LV vectors in depth. This report confirms and supplements previous work on the efficacy of HSCGT in reducing the toxic metabolites in Tymp −/− Upp1 −/− mice, using a clinically applicable gene transfer vector and a highly efficient gene transfer method, and importantly demonstrates phenotypic correction with a favorable risk profile, warranting further development toward clinical implementation.
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- 2018
17. Polyphosphate is a key factor for cell survival after DNA damage in eukaryotic cells
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Marta Rafel, Ramon Martí, Samuel Bru, Eva Quandt, Joan M. Martínez-Láinez, Bàrbara Samper-Martín, Josep Clotet, Eloi Garí, Sara Hernández-Ortega, Javier Jiménez, Javier Torres-Torronteras, and Mariana P.C. Ribeiro
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0301 basic medicine ,DNA Repair ,Cell Survival ,DNA repair ,DNA damage ,Deoxyribonucleotides ,Mutant ,Saccharomyces cerevisiae ,Biochemistry ,Saccharomyces ,03 medical and health sciences ,chemistry.chemical_compound ,Polyphosphates ,Polyphosphate ,otorhinolaryngologic diseases ,Pi ,Humans ,Human dermal fibroblasts ,Molecular Biology ,Genetics ,030102 biochemistry & molecular biology ,biology ,HEK 293 cells ,Mammalian cells ,DNA ,Cell Biology ,biology.organism_classification ,digestive system diseases ,Yeast ,Cell biology ,HEK293 Cells ,surgical procedures, operative ,030104 developmental biology ,chemistry ,Repair ,DNA Damage - Abstract
Cells require extra amounts of dNTPs to repair DNA after damage. Polyphosphate (polyP) is an evolutionary conserved linear polymer of up to several hundred inorganic phosphate (Pi) residues that is involved in many functions, including Pi storage. In the present article, we report on findings demonstrating that polyP functions as a source of Pi when required to sustain the dNTP increment essential for DNA repair after damage. We show that mutant yeast cells without polyP produce less dNTPs upon DNA damage and that their survival is compromised. In contrast, when polyP levels are ectopically increased, yeast cells become more resistant to DNA damage. More importantly, we show that when polyP is reduced in HEK293 mammalian cell line cells and in human dermal primary fibroblasts (HDFa), these cells become more sensitive to DNA damage, suggesting that the protective role of polyP against DNA damage is evolutionary conserved. In conclusion, we present polyP as a molecule involved in resistance to DNA damage and suggest that polyP may be a putative target for new approaches in cancer treatment or prevention. We would like to thank all the members of our group (E. Bállega, O. Mirallas, M. Ribeiro, A. Sánchez, B. Semper, and R. Carballar) for day-to-day talks, and Marta Pérez for technical assistance. The yeast ppn1Δ, ppn2Δ, ppx1Δ strain is a kind gift from A Mayer (Université de Lausanne). This work was supported by funding from the Spanish Government, with a MINECO grant (Ref: BFU 2013-44189-P) awarded to J. Clotet and a MICINN grant (Ref: BFU 2013-42895-P) awarded to E. Garí. J.M.M. was the recipient of a post-graduate Junior Faculty Fellowship from the UIC and l’Obra Social la Caixa.
- Published
- 2017
18. Alpha-1-Antitrypsin Promoter Improves the Efficacy of an Adeno-Associated Virus Vector for the Treatment of Mitochondrial Neurogastrointestinal Encephalomyopathy
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Ramon Martí, Ferran Vila-Julià, Raquel Cabrera-Pérez, Federico Mingozzi, Javier Torres-Torronteras, Michio Hirano, Universitat Autònoma de Barcelona (UAB), Instituto de Salud Carlos III [Madrid] (ISC), Columbia University Medical Center (CUMC), Columbia University [New York], Immunologie moléculaire et biothérapies innovantes (IMBI), École pratique des hautes études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Généthon, Généthon, Bitoun, Marc, and École Pratique des Hautes Études (EPHE)
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[SDV]Life Sciences [q-bio] ,Genetic Vectors ,Gene Expression ,Alpha (ethology) ,Mitochondria, Liver ,Mitochondrion ,Biology ,medicine.disease_cause ,thymidine ,Mice ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Mitochondrial Encephalomyopathies ,Transduction, Genetic ,Gene Order ,Genetics ,medicine ,Animals ,Homeostasis ,Vector (molecular biology) ,Thymidine phosphorylase ,Promoter Regions, Genetic ,Molecular Biology ,Adeno-associated virus ,Research Articles ,030304 developmental biology ,Mice, Knockout ,Thymidine Phosphorylase ,0303 health sciences ,promoter ,AAV ,Genetic Therapy ,Dependovirus ,Molecular biology ,mitochondria ,[SDV] Life Sciences [q-bio] ,Disease Models, Animal ,Liver ,chemistry ,alpha 1-Antitrypsin ,030220 oncology & carcinogenesis ,MNGIE ,Molecular Medicine ,Thymidine - Abstract
International audience; Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is a devastating disease caused by mutations in TYMP, which encodes thymidine phosphorylase (TP). In MNGIE patients, TP dysfunction results in systemic thymidine and deoxyuridine overload, which interferes with mitochondrial DNA replication. Preclinical studies have shown that gene therapy using a lentiviral vector targeted to hematopoietic stem cells or an adeno-associated virus (AAV) vector transcriptionally targeted to liver are feasible approaches to treat MNGIE. Here, we studied the effect of various promoters (thyroxine-binding globulin [TBG], phosphoglycerate kinase [PGK], hybrid liver-specific promoter [HLP], and alpha-1-antitrypsin [AAT]) and DNA configuration (single stranded or self complementary) on expression of the TYMP transgene in the AAV8 serotype in a murine model of MNGIE. All vectors restored liver TP activity and normalized nucleoside homeostasis in mice. However, the liver-specific promoters TBG, HLP, and AAT were more effective than the constitutive PGK promoter, and the self-complementary DNA configuration did not provide any therapeutic advantage over the single-stranded configuration. Among all constructs, only AAV-AAT was effective in all mice treated at the lowest dose (5 × 1010 vector genomes/kg). As use of the AAT promoter will likely minimize the dose needed to achieve clinical efficacy as compared to the other promoters tested, we propose using the AAT promoter in the vector eventually designed for clinical use.
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- 2019
19. Deoxynucleoside Therapy for Thymidine Kinase 2-Deficient Myopathy
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Francisco Javier Aguirre‐Rodríguez, Susana G. Kalko, Elena Martín-Hernández, Fabiola Mavillard, Michio Hirano, Javier Torres-Torronteras, Bruce Levin, Marcos Madruga-Garrido, Cecilia Jimenez-Mallebrera, Yuqi Tu, Juan P. Morealejo‐Aycinena, Yuelin Long, Karin Kleinsteuber, Ramon Martí, Itxaso Marti, Jasim Uddin, Olga Serrano, Caterina Garone, Concepcion Álvarez del Vayo, M. Alice Donati, Francina Munell, John L.P. Thompson, Carmen Paradas, Cristina Domínguez-González, Andrés Nascimento, M. Dolores Sardina, Kristen Engelstad, Dominguez-Gonzalez C., Madruga-Garrido M., Mavillard F., Garone C., Aguirre-Rodriguez F.J., Donati M.A., Kleinsteuber K., Marti I., Martin-Hernandez E., Morealejo-Aycinena J.P., Munell F., Nascimento A., Kalko S.G., Sardina M.D., Alvarez del Vayo C., Serrano O., Long Y., Tu Y., Levin B., Thompson J.L.P., Engelstad K., Uddin J., Torres-Torronteras J., Jimenez-Mallebrera C., Marti R., Paradas C., Hirano M., Instituto de Salud Carlos III, Generalitat de Catalunya, European Commission, Muscular Dystrophy Association (US), and Arturo Estopinan TK2 Research Fund
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0301 basic medicine ,Adult ,Compassionate Use Trials ,Male ,medicine.medical_specialty ,Neurology ,Side effect ,medicine.medical_treatment ,Deoxyribonucleosides ,Walk Test ,Gastroenterology ,Thymidine Kinase ,Article ,03 medical and health sciences ,0302 clinical medicine ,Muscular Diseases ,Internal medicine ,tk2, myopathy, nucleosides, therapy ,medicine ,Humans ,Myopathy ,Child ,Feeding tube ,Mechanical ventilation ,business.industry ,Discontinuation ,Diarrhea ,030104 developmental biology ,Child, Preschool ,Female ,Neurology (clinical) ,GDF15 ,medicine.symptom ,business ,030217 neurology & neurosurgery - Abstract
[Objective] Thymidine kinase 2, encoded by the nuclear gene TK2, is required for mitochondrial DNA maintenance. Autosomal recessive TK2 mutations cause depletion and multiple deletions of mtDNA that manifest predominantly as a myopathy usually beginning in childhood and progressing relentlessly. We investigated the safety and efficacy of deoxynucleoside monophosphate and deoxynucleoside therapies., [Methods] We administered deoxynucleoside monophosphates and deoxynucleoside to 16 TK2‐deficient patients under a compassionate use program., [Results] In 5 patients with early onset and severe disease, survival and motor functions were better than historically untreated patients. In 11 childhood and adult onset patients, clinical measures stabilized or improved. Three of 8 patients who were nonambulatory at baseline gained the ability to walk on therapy; 4 of 5 patients who required enteric nutrition were able to discontinue feeding tube use; and 1 of 9 patients who required mechanical ventilation became able to breathe independently. In motor functional scales, improvements were observed in the 6‐minute walk test performance in 7 of 8 subjects, Egen Klassifikation in 2 of 3, and North Star Ambulatory Assessment in all 5 tested. Baseline elevated serum growth differentiation factor 15 levels decreased with treatment in all 7 patients tested. A side effect observed in 8 of the 16 patients was dose‐dependent diarrhea, which did not require withdrawal of treatment. Among 12 other TK2 patients treated with deoxynucleoside, 2 adults developed elevated liver enzymes that normalized following discontinuation of therapy., [Interpretation] This open‐label study indicates favorable side effect profiles and clinical efficacy of deoxynucleoside monophosphate and deoxynucleoside therapies for TK2 deficiency. ANN NEUROL 2019;86:293–303, This work was supported in part by grants from the Spanish Carlos III Health Institute (PMP15/00025 for C.P., F.Ma., and R.M.; PI16/00579 and CP09/00011 for C.J.‐M.), Muscular Dystrophy Association (577391), Arturo Estopinan TK2 Research Fund, Generalitat de Catalunya PERIS program (SLT002/16/00370 for J.T‐T.), and European Regional Development Fund.
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- 2019
20. Increased dNTP pools rescue mtDNA depletion in human POLG-deficient fibroblasts
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Ramon Martí, Raquel Cabrera-Pérez, Anne Lombès, David Molina-Granada, Javier Torres-Torronteras, Xavier de la Cruz, Elena García-Arumí, Cora Blázquez-Bermejo, Lidia Carreño-Gago, Javier Ramón, Miguel Martín, Cristina Domínguez-González, Josu Aguirre, and Yolanda Cámara
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0301 basic medicine ,Adult ,DNA Replication ,Male ,Models, Molecular ,Mitochondrial DNA ,Deoxyribonucleoside triphosphate ,Genotype ,Protein Conformation ,Mitochondrial disease ,Deoxyribonucleotides ,Mutation, Missense ,Mitochondrion ,Biology ,Real-Time Polymerase Chain Reaction ,Biochemistry ,DNA, Mitochondrial ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Deoxyadenosine ,Catalytic Domain ,Ethidium ,Genetics ,medicine ,Humans ,Point Mutation ,Molecular Biology ,Gene ,Cells, Cultured ,Sequence Deletion ,Point mutation ,Adenine ,Fibroblasts ,medicine.disease ,Molecular biology ,DNA Polymerase gamma ,Mitochondria, Muscle ,030104 developmental biology ,Phenotype ,chemistry ,Female ,030217 neurology & neurosurgery ,Biotechnology ,Mitochondrial DNA replication - Abstract
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.-Blazquez-Bermejo, C., Carreno-Gago, L., Molina-Granada, D., Aguirre, J., Ramon, J., Torres-Torronteras, J., Cabrera-Perez, R., Martin, M. A., Dominguez-Gonzalez, C., de la Cruz, X., Lombes, A., Garcia-Arumi, E., Marti, R., Camara, Y. Increased dNTP pools rescue mtDNA depletion in human POLG-deficient fibroblasts.
- Published
- 2019
21. Age-related metabolic changes limit efficacy of deoxynucleoside-based therapy in thymidine kinase 2-deficient mice
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Anna Karlsson, Ramon Martí, Xiaoshan Zhou, Ferran Vila-Julià, David Molina-Granada, Javier Torres-Torronteras, Yolanda Cámara, Daniel Jiménez-Heis, Cora Blázquez-Bermejo, [Blázquez-Bermejo C, Molina-Granada D, Vila-Julià F, Torres-Torronteras J, Martí R, Cámara Y] Grup de Recerca en Patologia Neuromuscular i Mitocondrial, Vall d'Hebron Institut de Recerca (VHIR), Universitat Autònoma de Barcelona (UAB), Barcelona, Spain. Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Instituto de Salud Carlos III (ISCIII), Madrid, Spain. [Jiménez-Heis D] Grup de Recerca en Patologia Neuromuscular i Mitocondrial, Vall d'Hebron Institut de Recerca (VHIR), Universitat Autònoma de Barcelona (UAB), Barcelona, Spain. [Zhou X, Karlsson A] Division of Clinical Microbiology, Department of Laboratory Medicine, Karolinska Institute, Karolinska University Hospital, Stockholm, Sweden, and Vall d'Hebron Barcelona Hospital Campus
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0301 basic medicine ,Mtdna, mitochondrial DNA ,dCK, deoxycytidine kinase ,Dtmp, deoxythymidine monophosphate ,Research paper ,Anabolism ,Dck, deoxycytidine kinase ,ADN mitocondrial ,Mitochondrion ,Deoxycytidine ,chemistry.chemical_compound ,dN, deoxynucleoside ,0302 clinical medicine ,nucleótidos y nucleósidos de ácidos nucleicos::ácidos nucleicos::ADN::ADN circular::ADN mitocondrial [COMPUESTOS QUÍMICOS Y DROGAS] ,Longevitat ,TK2 ,Dn, deoxynucleoside ,Deoxynucleoside therapy ,Mtdna depletion ,TK1, thymidine kinase 1 ,General Medicine ,Deoxycytidine kinase ,dCtd, deoxycytidine ,dThd, deoxythymidine ,030220 oncology & carcinogenesis ,Knockout mouse ,Enzymes and Coenzymes::Enzymes::Transferases::Phosphotransferases::Phosphotransferases (Alcohol Group Acceptor)::Thymidine Kinase [CHEMICALS AND DRUGS] ,medicine.medical_specialty ,Dnmp, deoxynucleoside monophosphate ,Tymp, thymidine phosphorylase gene ,Ndna, nuclear DNA ,Encephalomyopathy ,Timidina ,CNS, central nervous system ,General Biochemistry, Genetics and Molecular Biology ,TK2, thymidine kinase 2 ,03 medical and health sciences ,PBS, phosphate buffered saline ,Nucleic Acids, Nucleotides, and Nucleosides::Nucleic Acids::DNA::DNA, Circular::DNA, Mitochondrial [CHEMICALS AND DRUGS] ,Internal medicine ,medicine ,Dctd, deoxycytidine ,Dntp, deoxynucleoside triphosphate ,Thymidine phosphorylase ,Thymidine kinase 1 ,dTMP, deoxythymidine monophosphate ,Dthd, deoxythymidine ,Physiological Phenomena::Growth and Development::Aging::Longevity [PHENOMENA AND PROCESSES] ,dNMP, deoxynucleoside monophosphate ,KO, knockout ,Thy, thymine ,TP, thymidine phosphorylase ,Catabolism ,business.industry ,mtDNA depletion ,WT, wild-type ,mtDNA, mitochondrial DNA ,030104 developmental biology ,Endocrinology ,chemistry ,fenómenos fisiológicos::crecimiento y desarrollo::envejecimiento::longevidad [FENÓMENOS Y PROCESOS] ,enzimas y coenzimas::enzimas::transferasas::fosfotransferasas::fosfotransferasas (grupo alcohol aceptor)::timidina cinasa [COMPUESTOS QUÍMICOS Y DROGAS] ,dNTP, deoxynucleoside triphosphate ,Dcmp, deoxycytidine monophosphate ,nDNA, nuclear DNA ,Thymidine ,business ,dCMP, deoxycytidine monophosphate ,CDA, cytidine deaminase - Abstract
Deoxycytidine; Deoxynucleoside therapy; Encephalomyopathy Desoxicitidina; Terapia basada en desoxinucleósido; Encefalomiopatía Desoxicitidina; Teràpia basada en desoxinucleòsid; Encefalomiopatia BACKGROUND: Thymidine kinase 2 (TK2) catalyses the phosphorylation of deoxythymidine (dThd) and deoxycytidine (dCtd) within mitochondria. TK2 deficiency leads to mtDNA depletion or accumulation of multiple deletions. In patients, TK2 mutations typically manifest as a rapidly progressive myopathy with infantile onset, leading to respiratory insufficiency and encephalopathy in the most severe clinical presentations. TK2-deficient mice develop the most severe form of the disease and die at average postnatal day 16. dThd+dCtd administration delayed disease progression and expanded lifespan of a knockin murine model of the disease. METHODS: We daily administered TK2 knockout mice (Tk2KO) from postnatal day 4 with equimolar doses of dThd+dCtd, dTMP+dCMP, dThd alone or dCtd alone. We monitored body weight and survival and studied different variables at 12 or 29 days of age. We determined metabolite levels in plasma and target tissues, mtDNA copy number in tissues, and the expression and activities of enzymes with a relevant role in mitochondrial dNTP anabolism or catabolism. FINDINGS: dThd+dCtd treatment extended average lifespan of Tk2KO mice from 16 to 34 days, attenuated growth retardation, and rescued mtDNA depletion in skeletal muscle and other target tissues of 12-day-old mice, except in brain. However, the treatment was ineffective in 29-day-old mice that still died prematurely. Bioavailability of dThd and dCtd markedly decreased during mouse development. Activity of enzymes catabolizing dThd and dCtd increased with age in small intestine. Conversely, the activity of the anabolic enzymes decreased in target tissues during mouse development. We also found that administration of dThd alone had the same impact on survival to that of dThd+dCtd, whereas dCtd alone had no influence on lifespan. INTERPRETATION: dThd+dCtd treatment recruits alternative cytosolic salvage pathways for dNTP synthesis, suggesting that this therapy would be of benefit for any Tk2 mutation. dThd accounts for the therapeutic effect of the combined treatment in mice. During the first weeks after birth, mice experience marked tissue-specific metabolic regulations and ontogenetic changes in dNTP metabolism-related enzymes that limit therapeutic efficacy to early developmental stages. This study was funded by grants from the Spanish Ministry of Industry, Economy and Competitiveness [grant BFU2014-52618-R and SAF2017-87506-R to YC], the Spanish Instituto de Salud Carlos III [grant PI15/00465 and grant PMP 15/00025 to RM, co-financed with ERDF], the Fundacion Inocente, Inocente [grant 2017 to YC], and AFMTelethon [grant 19,965 to YC]. JT was funded by a fellowship granted by the Generalitat de Catalunya (PERIS program, SLT002/16/00370). The disclosed funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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- 2019
22. Polyphosphate is involved in cell cycle progression and genomic stability inSaccharomyces cerevisiae
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Javier Torres-Torronteras, Ramon Martí, Joaquín Ariño, Eva Quandt, Samuel Bru, Sushma Sharma, Josep Clotet, Joan M. Martínez-Láinez, Sara Hernández-Ortega, Javier Jiménez, and David Canadell
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0301 basic medicine ,chemistry.chemical_classification ,Genome instability ,biology ,Cell division ,Mutant ,Saccharomyces cerevisiae ,biology.organism_classification ,Microbiology ,digestive system diseases ,03 medical and health sciences ,chemistry.chemical_compound ,surgical procedures, operative ,030104 developmental biology ,chemistry ,Biochemistry ,Organelle ,Gene duplication ,otorhinolaryngologic diseases ,Nucleotide ,neoplasms ,Molecular Biology ,DNA - Abstract
Polyphosphate (polyP) is a linear chain of up to hundreds of inorganic phosphate residues that is necessary for many physiological functions in all living organisms. In some bacteria, polyP supplies material to molecules such as DNA, thus playing an important role in biosynthetic processes in prokaryotes. In the present study, we set out to gain further insight into the role of polyP in eukaryotic cells. We observed that polyP amounts are cyclically regulated in Saccharomyces cerevisiae, and those mutants that cannot synthesise (vtc4Δ) or hydrolyse polyP (ppn1Δ, ppx1Δ) present impaired cell cycle progression. Further analysis revealed that polyP mutants show delayed nucleotide production and increased genomic instability. Based on these findings, we concluded that polyP not only maintains intracellular phosphate concentrations in response to fluctuations in extracellular phosphate levels, but also muffles internal cyclic phosphate fluctuations, such as those produced by the sudden demand of phosphate to synthetize deoxynucleotides just before and during DNA duplication. We propose that the presence of polyP in eukaryotic cells is required for the timely and accurate duplication of DNA.
- Published
- 2016
23. Inhibition of herpes simplex virus type 1 by the CDK6 inhibitor PD-0332991 (palbociclib) through the control of SAMHD1
- Author
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Ramon Martí, Guillem Angulo, Teresa Puig, Ester Ballana, Roger Badia, Eva Riveira-Muñoz, Javier Torres-Torronteras, Maria Pujantell, Eduardo Pauls, Bonaventura Clotet, José A. Esté, and Cristina Ramirez
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0301 basic medicine ,Microbiology (medical) ,Pyridines ,viruses ,Herpesvirus 1, Human ,Palbociclib ,Virus Replication ,Piperazines ,SAM Domain and HD Domain-Containing Protein 1 ,03 medical and health sciences ,Cyclin-dependent kinase ,Animals ,Humans ,Pharmacology (medical) ,Cells, Cultured ,Monomeric GTP-Binding Proteins ,Original Research ,Pharmacology ,biology ,Kinase ,Cyclin-dependent kinase 4 ,Chemistry ,Macrophages ,Cyclin-Dependent Kinase 6 ,Cell cycle ,Virology ,Molecular biology ,030104 developmental biology ,Infectious Diseases ,Viral replication ,biology.protein ,Cyclin-dependent kinase 6 ,SAMHD1 - Abstract
OBJECTIVES Sterile α motif and histidine-aspartate domain-containing protein 1 (SAMHD1) has been shown to restrict retroviruses and DNA viruses by decreasing the pool of intracellular deoxynucleotides. In turn, SAMHD1 is controlled by cyclin-dependent kinases (CDK) that regulate the cell cycle and cell proliferation. Here, we explore the effect of CDK6 inhibitors on the replication of herpes simplex virus type 1 (HSV-1) in primary monocyte-derived macrophages (MDM). METHODS MDM were treated with palbociclib, a selective CDK4/6 inhibitor, and then infected with a GFP-expressing HSV-1. Intracellular deoxynucleotide triphosphate (dNTP) content was determined using a polymerase-based method. RESULTS CDK6 inhibitor palbociclib blocked SAMHD1 phosphorylation, intracellular dNTP levels and HSV-1 replication in MDM at subtoxic concentrations. Treatment of MDM with palbociclib reduced CDK2 activation, measured as the phosphorylation of the T-loop at Thr160. The antiviral activity of palbociclib was lost when SAMHD1 was degraded by viral protein X. Similarly, palbociclib did not block HSV-1 replication in SAMHD1-negative Vero cells at subtoxic concentrations, providing further evidence for a role of SAMHD1 in mediating the antiviral effect. CONCLUSIONS SAMHD1-mediated HSV-1 restriction is controlled by CDK and points to a preferential role for CDK6 and CDK2 as mediators of SAMHD1 activation. Similarly, the restricting activity of SAMHD1 against DNA viruses suggests that control of dNTP availability is the major determinant of its antiviral activity. This is the first study describing the anti-HSV-1 activity of palbociclib.
- Published
- 2015
24. Adipocyte MTERF4 regulates non-shivering adaptive thermogenesis and sympathetic-dependent glucose homeostasis
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Ramon Martí, Inés Pedriza, Maria Vilà, Anna Castillo, Edgar Martín, Rubén Nogueiras, Daniel Beiroa, Josep A. Villena, Yolanda Cámara, Marta Oteo, Rosario Pardo, Rafael Simó, Miguel Ángel Morcillo, and Javier Torres-Torronteras
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0301 basic medicine ,Male ,Mitochondrion ,Biology ,Oxidative Phosphorylation ,Electron Transport Complex IV ,Mitochondrial Proteins ,03 medical and health sciences ,chemistry.chemical_compound ,Electron Transport Complex III ,Mice ,0302 clinical medicine ,Insulin resistance ,Adipose Tissue, Brown ,Adipocyte ,Brown adipose tissue ,medicine ,Adipocytes ,Glucose homeostasis ,Animals ,Homeostasis ,Humans ,Insulin ,Molecular Biology ,Transcription factor ,Mice, Knockout ,Electron Transport Complex I ,Organelle Biogenesis ,Thermogenesis ,Adrenergic beta-Agonists ,medicine.disease ,Cell biology ,Mitochondria ,Cold Temperature ,030104 developmental biology ,medicine.anatomical_structure ,Glucose ,chemistry ,Mitochondrial biogenesis ,Gene Expression Regulation ,Molecular Medicine ,Insulin Resistance ,030217 neurology & neurosurgery ,Signal Transduction ,Transcription Factors - Abstract
In humans, low brown adipose tissue (BAT) mass and activity have been associated with increased adiposity and fasting glucose levels, suggesting that defective BAT-dependent thermogenesis could contribute to the development of obesity and/or type 2 diabetes. The thermogenic function of BAT relies on a vast network of mitochondria exclusively equipped with UCP1. Mitochondrial biogenesis is exquisitely regulated by a well-defined network of transcription factors that coordinate the expression of nuclear genes required for the formation of functional mitochondria. However, less is known about the mitochondrial factors that control the expression of the genes encoded by the mitochondrial genome. Here, we have studied the role of mitochondrial transcription termination factor-4 (MTERF4) in BAT by using a new mouse model devoid of MTERF4 specifically in adipocytes (MTERF4-FAT-KO mice). Lack of MTERF4 in BAT leads to reduced OxPhos mitochondrial protein levels and impaired assembly of OxPhos complexes I, III and IV due to deficient translation of mtDNA-encoded proteins. As a result, brown adipocytes lacking MTERF4 exhibit impaired respiratory capacity. MTERF4-FAT-KO mice show a blunted thermogenic response and are unable to maintain body temperature when exposed to cold. Despite impaired BAT function, MTERF4-FAT-KO mice do not develop obesity or insulin resistance. Still, MTERF4-FAT-KO mice became resistant to the insulin-sensitizing effects of β3-specific adrenergic receptor agonists. Our results demonstrate that MTERF4 regulates mitochondrial protein translation and is essential for proper BAT thermogenic activity. Our study also supports the notion that pharmacological activation of BAT is a plausible therapeutic target for the treatment of insulin resistance.
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- 2018
25. Author Correction: Resveratrol Increases Hepatic SHBG Expression through Human Constitutive Androstane Receptor: a new Contribution to the French Paradox
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Geoffrey L. Hammond, Laura Brianso-Llort, David M. Selva, Cristina Saez-Lopez, Javier Torres-Torronteras, and Rafael Simó
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Male ,medicine.medical_specialty ,Alcohol Drinking ,Biopsy ,lcsh:Medicine ,Receptors, Cytoplasmic and Nuclear ,Mice, Transgenic ,Wine ,Biology ,Resveratrol ,chemistry.chemical_compound ,Mice ,Sex hormone-binding globulin ,Genes, Reporter ,Internal medicine ,Sex Hormone-Binding Globulin ,Constitutive androstane receptor ,medicine ,French paradox ,Animals ,Humans ,lcsh:Science ,Author Correction ,Luciferases ,Constitutive Androstane Receptor ,Metabolic Syndrome ,Multidisciplinary ,lcsh:R ,Hep G2 Cells ,Culture Media ,Mice, Inbred C57BL ,Endocrinology ,chemistry ,Expression (architecture) ,Diabetes Mellitus, Type 2 ,Liver ,Cardiovascular Diseases ,biology.protein ,ComputingMethodologies_DOCUMENTANDTEXTPROCESSING ,lcsh:Q ,Female - Abstract
Sex hormone-binding globulin (SHBG) carries sex steroids in blood regulating their bioavailability. Red wine consumption increases plasma SHBG levels, and we have discovered that resveratrol, a polyphenol enriched in red wine, acts specifically through the human constitutive androstane receptor (CAR), a drug/xenobiotic detoxification gene regulator, to increase hepatic SHBG production. Chromatin immunoprecipitation and luciferase reporter gene assays show that human CAR binds to a typical direct repeat 1 nuclear hormone receptor-binding element in the human SHBG proximal promoter. Resveratrol also increased hepatic SHBG production in humanized SHBG/CAR transgenic mice. Moreover, SHBG expression correlated significantly with CAR mRNA levels in human liver biopsies. We conclude that the beneficial effects of red wine on the metabolic syndrome and it associated co-morbidities, including cardiovascular disease and type 2 diabetes, may be mediated in part by resveratrol acting via CAR to increase plasma SHBG levels.
- Published
- 2018
26. Long-Term Sustained Effect of Liver-Targeted Adeno-Associated Virus Gene Therapy for Mitochondrial Neurogastrointestinal Encephalomyopathy
- Author
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Ramon Martí, Javier Torres-Torronteras, Raquel Cabrera-Pérez, Carlo Viscomi, Yolanda Cámara, Michio Hirano, Ferran Vila-Julià, Massimo Zeviani, Viscomi, Carlo [0000-0001-6050-0566], and Apollo - University of Cambridge Repository
- Subjects
0301 basic medicine ,Male ,Time Factors ,Carcinogenesis ,Genetic enhancement ,Genetic Vectors ,Gene Dosage ,Mitochondria, Liver ,Kaplan-Meier Estimate ,Oculopharyngeal ,Mitochondrion ,Biology ,medicine.disease_cause ,thymidine phosphorylase ,03 medical and health sciences ,Mice ,Muscular Dystrophy, Oculopharyngeal ,medicine ,Genetics ,Animals ,Muscular Dystrophy ,Transgenes ,Thymidine phosphorylase ,Gene ,Adeno-associated virus ,Molecular Biology ,Research Articles ,AAV, liver/metabolic, MNGIE, thymidine phosphorylase, mitochondria ,Molecular Medicine ,chemistry.chemical_classification ,Ophthalmoplegia ,Intestinal Pseudo-Obstruction ,AAV ,Genetic Therapy ,Dependovirus ,Deoxyuridine ,Mitochondria ,liver/metabolic ,030104 developmental biology ,Enzyme ,chemistry ,Liver ,MNGIE ,Cancer research ,Female ,Thymidine ,Thymidine Phosphorylase - Abstract
Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is caused by mutations in TYMP, the gene encoding the enzyme thymidine phosphorylase (TP). TP dysfunction results in systemic accumulation of the noxious TP substrates thymidine and deoxyuridine. Gene therapy using either a lentiviral vector or adeno-associated vector (AAV) has proven to be a feasible strategy, as both vectors restore biochemical homeostasis in a murine model of the disease. This study shows that the effect of an AAV containing the TYMP coding sequence transcriptionally targeted to the liver persists long term in mice. Although the vector copy number was diluted and AAV-mediated liver TP activity eventually reduced or lost after 21 months at the lowest vector doses, the effect was sustained (with a negligible decrease in TP activity) and fully effective on nucleoside homeostasis for at least 21 months at a dose of 2 × 10(12) vg/kg. Macroscopic visual inspection of the animals' organs at completion of the study showed no adverse effects associated with the treatment. These results further support the feasibility of gene therapy for MNGIE.
- Published
- 2018
27. Prospective therapeutic approaches in mitochondrial neurogastrointestinal encephalomyopathy (MNGIE)
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Yolanda Cámara, Ferran Vila-Julià, Javier Torres-Torronteras, Raquel Cabrera-Pérez, Francisco J. Ortega, Ramon Martí, and Jordi Barquinero
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business.industry ,Health Policy ,Genetic enhancement ,medicine.medical_treatment ,Disease ,Hematopoietic stem cell transplantation ,Mitochondrion ,Virology ,Deoxyuridine ,chemistry.chemical_compound ,chemistry ,medicine ,Cancer research ,Pharmacology (medical) ,Thymidine phosphorylase ,Adverse effect ,business ,Pharmacology, Toxicology and Pharmaceutics (miscellaneous) ,Mitochondrial DNA replication - Abstract
Introduction: Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is caused by mutations in TYMP, which encodes thymidine phosphorylase (TP). TP dysfunction leads to systemic overload of thymidine (dThd) and deoxyuridine (dUrd), and altered mitochondrial deoxyribonucleotide homeostasis, which interferes with mitochondrial DNA replication and results in mitochondrial dysfunction. In MNGIE, the clinical phenotype is the consequence of an accumulation of noxious metabolites.Areas covered: Knowledge gained about the pathomechanisms involved in MNGIE has allowed the design of plausible treatments aimed to clear the systemic dThd and dUrd overload. This article describes these strategies, from the first attempts to treat the disease through dialysis, to allogeneic hematopoietic stem cell transplantation (allo-HSCT), which has been the most successful treatment in the long term to date. This option, however, is associated with a high risk of severe adverse effects so safer alternatives with long-term e...
- Published
- 2015
28. Cyclin D3-dependent control of the dNTP pool and HIV-1 replication in human macrophages
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Ramon Martí, Bonaventura Clotet, Alba Ruiz, Ester Ballana, Javier Torres-Torronteras, Eva Riveira-Muñoz, Eduardo Pauls, José A. Esté, and Roger Badia
- Subjects
Cyclin D ,Deoxyribonucleotides ,Cyclin A ,Virus Replication ,Models, Biological ,SAM Domain and HD Domain-Containing Protein 1 ,Cyclin-dependent kinase ,Report ,Humans ,Cyclin D3 ,Phosphorylation ,Molecular Biology ,Monomeric GTP-Binding Proteins ,biology ,Macrophages ,Cyclin-Dependent Kinase 6 ,Cell Biology ,Cell cycle ,Cell biology ,enzymes and coenzymes (carbohydrates) ,Biochemistry ,Gene Knockdown Techniques ,HIV-1 ,biology.protein ,RNA Interference ,Cyclin-dependent kinase 6 ,Cyclin A2 ,Developmental Biology ,SAMHD1 - Abstract
Cyclins control the activation of cyclin-dependent kinases (CDK), which in turn, control the cell cycle and cell division. Intracellular availability of deoxynucleotides (dNTP) plays a fundamental role in cell cycle progression. SAM domain and HD domain-containing protein 1 (SAMHD1) degrades nucleotide triphosphates and controls the size of the dNTP pool. SAMHD1 activity appears to be controlled by CDK. Here, we show that knockdown of cyclin D3 a partner of CDK6 and E2 a partner of CDK2 had a major impact in SAMHD1 phosphorylation and inactivation and led to decreased dNTP levels and inhibition of HIV-1 at the reverse transcription step in primary human macrophages. The effect of cyclin D3 RNA interference was lost after degradation of SAMHD1 by HIV-2 Vpx, demonstrating the specificity of the mechanism. Cyclin D3 inhibition correlated with decreased activation of CDK2. Our results confirm the fundamental role of the CDK6-cyclin D3 pair in controlling CDK2-dependent SAMHD1 phosphorylation and dNTP pool in primary macrophages.
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- 2015
29. Thymidine phosphorylase is both a therapeutic and a suicide gene in a murine model of mitochondrial neurogastrointestinal encephalomyopathy
- Author
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Jordi Barquinero, Sergio López-Estévez, Javier Torres-Torronteras, María José Mansilla, Sílvia Casacuberta-Serra, Lluís Martorell, Ramon Martí, G Ferrer, and Michio Hirano
- Subjects
Male ,Genetic Vectors ,Biology ,Deoxycytidine ,Gene Knockout Techniques ,Mice ,Muscular Dystrophy, Oculopharyngeal ,Mitochondrial Encephalomyopathies ,Cell Line, Tumor ,Genetics ,Animals ,Humans ,Thymidine phosphorylase ,Molecular Biology ,Capecitabine ,Thymidine Phosphorylase ,Ophthalmoplegia ,Intestinal Pseudo-Obstruction ,Lentivirus ,Genes, Transgenic, Suicide ,Genetic Therapy ,Suicide gene ,Combined Modality Therapy ,Mice, Inbred C57BL ,Disease Models, Animal ,Biochemistry ,Murine model ,Cancer research ,Molecular Medicine ,Fluorouracil - Abstract
Suicide gene therapy (SGT) is a promising strategy for treating cancer. In this work, we show that thymidine phosphorylase (TP) deficiency, the underlying genetic defect in mitochondrial neurogastrointestinal encephalomyopathy (MNGIE), presents an opportunity to apply SGT using capecitabine, a commonly used prodrug that is converted into 5-fluorouracil by TP. Using an immortalised B-lymphoblastoid cell line from a patient with MNGIE, the tumourigenic EL-4 cell line, lentiviral vectors encoding TP and a double knockout (Tymp(-/-)Upp1(-/-)) murine model, we found that EL-4 cell-derived TP(+) tumours were exquisitely sensitive to capecitabine and generated a significant local bystander effect. In addition, we detected a spontaneous cytolytic immune response in a significant fraction of the animals surviving more than 20 days after termination of the therapy. These data indicate that, in individuals lacking TP expression, TP is a highly specific suicide gene, which can be used to treat tumours that could hypothetically arise in MNGIE patients undergoing gene therapy, as these tumours will likely originate from the gene-modified cells and will be selectively targeted by capecitabine. These observations have important implications for gene therapy for MNGIE.
- Published
- 2014
30. SAMHD1 is active in cycling cells permissive to HIV-1 infection
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Javier Torres-Torronteras, Eva Riveira-Muñoz, Ester Ballana, Ramon Martí, Eduardo Pauls, Bonaventura Clotet, Roger Badia, Luis Menéndez-Arias, Maria Pujantell, Albert Ruzo, José A. Esté, Ministerio de Economía y Competitividad (España), and Instituto de Salud Carlos III
- Subjects
0301 basic medicine ,DNA Replication ,030106 microbiology ,SAMHDI ,Integrase inhibitor ,Gene Expression ,dNTPase activity ,HIV Infections ,Biology ,Virus Replication ,Cell Line ,SAM Domain and HD Domain-Containing Protein 1 ,03 medical and health sciences ,Zidovudine ,Virology ,medicine ,Humans ,Clustered Regularly Interspaced Short Palindromic Repeats ,Viral Regulatory and Accessory Proteins ,Phosphorylation ,Pharmacology ,Gene Editing ,Wild type ,virus diseases ,Reverse Transcription ,Raltegravir ,SAMHD1 ,Cell biology ,030104 developmental biology ,HEK293 Cells ,Viral replication ,Cell culture ,CRISPR ,Gene Knockdown Techniques ,HIV-2 ,Host-Pathogen Interactions ,HIV-1 ,Nucleoside ,medicine.drug ,Genome editing - Abstract
SAMHD1 is a triphosphohydrolase that restricts HIV-1 by limiting the intracellular dNTP pool required for reverse transcription. Although SAMHD1 is expressed and active/unphosphorylated in most cell lines, its restriction activity is thought to be relevant only in non-cycling cells. However, an in depth evaluation of SAMHD1 function and relevance in cycling cells is required. Here, we show that SAMHD1-induced degradation by HIV-2 Vpx affects the dNTP pool and HIV-1 replication capacity in the presence of the 3′-azido-3′-deoxythymidine (AZT) in cycling cells. Similarly, in SAMHD1 knockout cells, HIV-1 showed increased replicative capacity in the presence of nucleoside inhibitors, especially AZT, that was reverted by re-expression of wild type SAMHD1. Sensitivity to non-nucleoside inhibitors (nevirapine and efavirenz) or the integrase inhibitor raltegravir was not affected by SAMHD1. Combination of three mutations (S18A, T21A, T25A) significantly prevented SAMHD1 phosphorylation but did not significantly affect HIV-1 replication in the presence of AZT. Our results demonstrate that SAMHD1 is active in HIV-1 permissive cells, does not modify susceptibility to HIV-1 infection but strongly affects sensitivity to nucleoside inhibitors., Spanish Ministerio de Economía y Competitividad (MINECO) project BFU2015-63800-R and BIO2013-48788-C2-1-R and Instituto de Salud Carlos III, Fondo de Investigación Sanitaria (FIS) PI13/01083, PI15/00492 and PI16/00103 cofinanced by FEDER
- Published
- 2016
31. Long-Term Restoration of Thymidine Phosphorylase Function and Nucleoside Homeostasis Using Hematopoietic Gene Therapy in a Murine Model of Mitochondrial Neurogastrointestinal Encephalomyopathy
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Javier Torres-Torronteras, Antoni L. Andreu, Raquel Cabrera-Pérez, Ignasi Barba, Jordi Barquinero, Yolanda Cámara, Noemi de Luna, Michio Hirano, Ramon Martí, and Carme Martínez Costa
- Subjects
0301 basic medicine ,Male ,Genetic enhancement ,medicine.medical_treatment ,Genetic Vectors ,Hematopoietic stem cell transplantation ,Biology ,03 medical and health sciences ,chemistry.chemical_compound ,Mice ,0302 clinical medicine ,Muscular Dystrophy, Oculopharyngeal ,Mitochondrial Encephalomyopathies ,Genetics ,medicine ,Animals ,Homeostasis ,Thymidine phosphorylase ,Molecular Biology ,Research Articles ,Mice, Knockout ,Thymidine Phosphorylase ,Ophthalmoplegia ,Intestinal Pseudo-Obstruction ,Lentivirus ,Hematopoietic Stem Cell Transplantation ,Hematopoietic stem cell ,Nucleosides ,Genetic Therapy ,Combined Modality Therapy ,Mice, Inbred C57BL ,Disease Models, Animal ,030104 developmental biology ,medicine.anatomical_structure ,Biochemistry ,chemistry ,Cancer research ,Molecular Medicine ,Ectopic expression ,Female ,Thymidine ,030217 neurology & neurosurgery ,Mitochondrial DNA replication - Abstract
Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is a metabolic disorder caused by mutations in TYMP, encoding thymidine phosphorylase (TP). In MNGIE patients, TP dysfunction produces systemic thymidine and deoxyuridine accumulation, which ultimately impairs mitochondrial DNA replication and results in mitochondrial dysfunction. To date, only allogeneic hematopoietic stem cell transplantation has demonstrated long-term clinical efficacy, but high morbidity and mortality associated with this procedure necessitate the search for safer alternatives. In a previous study, we demonstrated that hematopoietic stem cell gene therapy using a lentiviral vector containing the coding sequence of TYMP restored the biochemical homeostasis in an animal model of MNGIE. In the present follow-up study, we show that ectopic expression of TP in the hematopoietic system restores normal nucleoside levels in plasma, as well as in tissues affected in MNGIE such as small intestine, skeletal muscle, brain, and liver. Mitochondrial dNTP pool imbalances observed in liver of the animal model were also corrected by the treatment. The biochemical effects were maintained at least 20 months even with low levels of chimerism. No alterations in the blood cell counts or other toxic effects were observed in association with the lentiviral transduction or TP overexpression. These results further support the notion that gene therapy is a feasible treatment option for MNGIE.
- Published
- 2016
32. Polyphosphate is involved in cell cycle progression and genomic stability in Saccharomyces cerevisiae
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Samuel, Bru, Joan Marc, Martínez-Laínez, Sara, Hernández-Ortega, Eva, Quandt, Javier, Torres-Torronteras, Ramón, Martí, David, Canadell, Joaquin, Ariño, Sushma, Sharma, Javier, Jiménez, and Josep, Clotet
- Subjects
Organelles ,Prokaryotic Cells ,Polyphosphates ,Cell Cycle Checkpoints ,Saccharomyces cerevisiae ,Cell Division ,Genomic Instability - Abstract
Polyphosphate (polyP) is a linear chain of up to hundreds of inorganic phosphate residues that is necessary for many physiological functions in all living organisms. In some bacteria, polyP supplies material to molecules such as DNA, thus playing an important role in biosynthetic processes in prokaryotes. In the present study, we set out to gain further insight into the role of polyP in eukaryotic cells. We observed that polyP amounts are cyclically regulated in Saccharomyces cerevisiae, and those mutants that cannot synthesise (vtc4Δ) or hydrolyse polyP (ppn1Δ, ppx1Δ) present impaired cell cycle progression. Further analysis revealed that polyP mutants show delayed nucleotide production and increased genomic instability. Based on these findings, we concluded that polyP not only maintains intracellular phosphate concentrations in response to fluctuations in extracellular phosphate levels, but also muffles internal cyclic phosphate fluctuations, such as those produced by the sudden demand of phosphate to synthetize deoxynucleotides just before and during DNA duplication. We propose that the presence of polyP in eukaryotic cells is required for the timely and accurate duplication of DNA.
- Published
- 2015
33. Mitochondrial Neurogastrointestinal Encephalomyopathy (MNGIE): Biochemical Features and Therapeutic Approaches
- Author
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Michio Hirano, Javier Torres-Torronteras, Maria Lucia Valentino, Ramon Martí, M. C. Lara, Lara, M.C., Valentino, M.L., Torres-Torronteras, J., Hirano, M., and Martí, R.
- Subjects
TP ,Mitochondrial DNA ,Gastrointestinal Diseases ,Genetic enhancement ,Biophysics ,mitochondrial DNA ,Biology ,medicine.disease_cause ,DNA, Mitochondrial ,Biochemistry ,chemistry.chemical_compound ,Mitochondrial Encephalomyopathies ,medicine ,Humans ,Thymidine phosphorylase ,Molecular Biology ,Gene ,Thymidine Phosphorylase ,Mutation ,mitochondrial neurogastrointestinal encephalomyopathy ,mtDNA ,Cell Biology ,Molecular biology ,Deoxyuridine ,Human genetics ,Transplantation ,chemistry ,MNGIE ,Cancer research - Abstract
Over the last 15 years, important research has expanded our knowledge of the clinical, molecular genetic, and biochemical features of mitochondrial neurogastrointestinal encephalomyopathy (MNGIE). The characterization of mitochondrial involvement in this disorder and the seminal determination of its genetic cause, have opened new possibilities for more detailed and deeper studies on the pathomechanisms in this progressive and fatal disease. It has been established that MNGIE is caused by mutations in the gene encoding thymidine phosphorylase (TP), which lead to absolute or nearly complete loss of its catalytic activity, producing systemic accumulations of its substrates, thymidine (dThd) and deoxyuridine (dUrd). Findings obtained from in vitro and in vivo studies indicate that the biochemical imbalances specifically impair mitochondrial DNA (mtDNA) replication, repair, or both leading to mitochondrial dysfunction. We have proposed that therapy for MNGIE should be aimed at reducing the concentrations of these toxic nucleosides to normal or nearly normal levels. The first treatment, allogeneic stem-cell transplantation (alloSCT) reported in 2006, produced a nearly full biochemical correction of the dThd and dUrd imbalances in blood. Clinical follow-up of this and other patients receiving alloSCT is necessary to determine whether this and other therapies based on a permanent restoration of TP will be effective treatment for MNGIE. © 2007 The Biochemical Society.
- Published
- 2007
34. Palbociclib, a selective inhibitor of cyclin-dependent kinase4/6, blocks HIV-1 reverse transcription through the control of sterile α motif and HD domain-containing protein-1 (SAMHD1) activity
- Author
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Roger Badia, Alba Ruiz, José A. Esté, Marc Permanyer, Javier Torres-Torronteras, Eduardo Pauls, Bonaventura Clotet, Ester Ballana, Eva Riveira-Muñoz, and Ramon Martí
- Subjects
Anti-HIV Agents ,Pyridines ,Immunology ,Palbociclib ,Real-Time Polymerase Chain Reaction ,Piperazines ,SAM Domain and HD Domain-Containing Protein 1 ,Cyclin-dependent kinase ,Immunology and Allergy ,Humans ,Protein Kinase Inhibitors ,Cells, Cultured ,Monomeric GTP-Binding Proteins ,biology ,Kinase ,Cyclin-dependent kinase 2 ,Cyclin-Dependent Kinase 2 ,Cyclin-Dependent Kinase 6 ,Reverse Transcription ,Molecular biology ,Reverse transcriptase ,Infectious Diseases ,DNA, Viral ,biology.protein ,HIV-1 ,Leukocytes, Mononuclear ,Phosphorylation ,Cyclin-dependent kinase 6 ,SAMHD1 - Abstract
Background: Sterile α motif and HD domain-containing protein-1 (SAMHD1) inhibits HIV-1 reverse transcription by decreasing the pool of intracellular deoxynucleotides. SAMHD1 is controlled by cyclin-dependent kinase (CDK)-mediated phosphorylation. However, the exact mechanism of SAMHD1 regulation in primary cells is unclear. We explore the effect of palbociclib, a CDK6 inhibitor, in HIV-1 replication. Methods: Human primary monocytes were differentiated into macrophages with monocyte-colony stimulating factor and CD4+ T lymphocytes stimulated with phytohaemagglutinin (PHA)/interleukin-2. Cells were treated with palbociclib and then infected with a Green fluorescent protein-expressing HIV-1 or R5 HIV-1 BaL. Viral DNA was measured by quantitative PCR and infection assessed by flow cytometry. Deoxynucleotide triphosphate (dNTP) content was determined using a polymerase-based method. Results: Pan-CDK inhibitors AT7519, roscovitine and purvalanol A reduced SAMHD1 phosphorylation. HIV-1 replication was blocked by AT7519 (66.4 ± 3.8%; n = 4), roscovitine (47.3 ± 3.9%; n = 4) and purvalanol A (55.7 ± 15.7%; n = 4) at subtoxic concentrations. Palbociclib, a potent and selective CDK6 inhibitor, blocked SAMHD1 phosphorylation, intracellular dNTP levels, HIV-1 reverse transcription and HIV-1 replication in primary macrophages and CD4+ T lymphocytes. Notably, treatment of macrophages with palbociclib led to reduced CDK2 activation, measured as the phosphorylation of the T-loop at the Thr160. The antiviral effect was lost when SAMHD1 was degraded by Vpx, providing further evidence for a role of SAMHD1 in mediating the antiretroviral effect. Conclusions: Our results indicate that SAMHD1-mediated HIV-1 restriction is controlled by CDK as previously suggested but point to a preferential role for CDK2 and CDK6 as mediators of SAMHD1 activation. Our study provides a new signaling pathway susceptible for the development of new therapeutic approaches against HIV-1 infection.
- Published
- 2014
35. Cell cycle control and HIV-1 susceptibility are linked by CDK6-dependent CDK2 phosphorylation of SAMHD1 in myeloid and lymphoid cells
- Author
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Ramon Martí, Alba Ruiz, Francesc Posas, Mar Álvarez, Javier Torres-Torronteras, Albert Gubern, Manel Crespo, Christian Brander, Beatriz Mothe, Roger Badia, Luis Menéndez-Arias, José A. Esté, Eduardo Pauls, Eva Riveira-Muñoz, Ester Ballana, Oliver T. Keppler, Marc Permanyer, and Bonaventura Clotet
- Subjects
CD4-Positive T-Lymphocytes ,Benzylamines ,Receptors, CXCR4 ,Immunology ,HIV Infections ,Cyclams ,Lymphocyte Activation ,SAM Domain and HD Domain-Containing Protein 1 ,Cyclin-dependent kinase ,Heterocyclic Compounds ,Immunology and Allergy ,Humans ,Myeloid Cells ,Lymphocytes ,Phosphorylation ,RNA, Small Interfering ,Cells, Cultured ,Monomeric GTP-Binding Proteins ,biology ,Kinase ,Macrophages ,Cyclin-dependent kinase 2 ,HEK 293 cells ,Cell Cycle ,Cyclin-Dependent Kinase 2 ,Cell Cycle Checkpoints ,Cyclin-Dependent Kinase 6 ,Cell cycle ,Molecular biology ,HEK293 Cells ,biology.protein ,HIV-1 ,RNA Interference ,Cyclin-dependent kinase 6 ,Intracellular ,SAMHD1 - Abstract
Proliferating cells are preferentially susceptible to infection by retroviruses. Sterile α motif and HD domain–containing protein-1 (SAMHD1) is a recently described deoxynucleotide phosphohydrolase controlling the size of the intracellular deoxynucleotide triphosphate (dNTP) pool, a limiting factor for retroviral reverse transcription in noncycling cells. Proliferating (Ki67+) primary CD4+ T cells or macrophages express a phosphorylated form of SAMHD1 that corresponds with susceptibility to infection in cell culture. We identified cyclin-dependent kinase (CDK) 6 as an upstream regulator of CDK2 controlling SAMHD1 phosphorylation in primary T cells and macrophages susceptible to infection by HIV-1. In turn, CDK2 was strongly linked to cell cycle progression and coordinated SAMHD1 phosphorylation and inactivation. CDK inhibitors specifically blocked HIV-1 infection at the reverse transcription step in a SAMHD1-dependent manner, reducing the intracellular dNTP pool. Our findings identify a direct relationship between control of the cell cycle by CDK6 and SAMHD1 activity, which is important for replication of lentiviruses, as well as other viruses whose replication may be regulated by intracellular dNTP availability.
- Published
- 2014
36. Gene Therapy Using a Liver-targeted AAV Vector Restores Nucleoside and Nucleotide Homeostasis in a Murine Model of MNGIE
- Author
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Alberto Auricchio, Michio Hirano, Jordi Barquinero, Yolanda Cámara, Ivano Di Meo, Raquel Cabrera-Pérez, Ramon Martí, Javier Torres-Torronteras, Massimo Zeviani, Carlo Viscomi, Giuseppe Pizzorno, Torres Torronteras, J, Viscomi, C, Cabrera P?rez, R, C?mara, Y, Di Meo, I, Barquinero, J, Auricchio, Alberto, Pizzorno, G, Hirano, M, Zeviani, M, and Mart?, R.
- Subjects
Mitochondrial DNA ,Genetic enhancement ,Genetic Vectors ,Oculopharyngeal ,Biology ,medicine.disease_cause ,DNA, Mitochondrial ,chemistry.chemical_compound ,Mice ,Muscular Dystrophy, Oculopharyngeal ,Mitochondrial Encephalomyopathies ,Drug Discovery ,Genetics ,medicine ,Animals ,Dependovirus ,Disease Models, Animal ,Homeostasis ,Humans ,Intestinal Pseudo-Obstruction ,Liver ,Mutation ,Thymidine ,Thymidine Phosphorylase ,Genetic Therapy ,Muscular Dystrophy ,Thymidine phosphorylase ,Molecular Biology ,Pharmacology ,Ophthalmoplegia ,Deoxycytidine triphosphate ,Animal ,DNA ,Molecular biology ,Deoxyuridine ,3. Good health ,Mitochondrial ,chemistry ,Disease Models ,Molecular Medicine ,Original Article ,Nucleoside - Abstract
Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive disorder caused by mutations in TYMP , enconding thymidine phosphorylase (TP). TP deficiency results in systemic accumulation of thymidine and deoxyuridine, which interferes with mitochondrial DNA (mtDNA) replication and leads to mitochondrial dysfunction. To date, the only treatment available for MNGIE patients is allogeneic hematopoietic stem cell transplantation, which is associated with high morbidity and mortality. Here, we report that AAV2/8-mediated transfer of the human TYMP coding sequence (hcTYMP) under the control of a liver-specific promoter prevents the biochemical imbalances in a murine model of MNGIE. hcTYMP expression was restricted to liver, and a dose as low as 2 × 10 11 genome copies/kg led to a permanent reduction in systemic nucleoside levels to normal values in about 50% of treated mice. Higher doses resulted in reductions to normal or slightly below normal levels in virtually all mice treated. The nucleoside reduction achieved by this treatment prevented deoxycytidine triphosphate (dCTP) depletion, which is the limiting factor affecting mtDNA replication in this disease. These results demonstrate that the use of AAV to direct TYMP expression in liver is feasible as a potentially safe gene therapy strategy for MNGIE.
- Published
- 2014
37. Administration of deoxyribonucleosides or inhibition of their catabolism as a pharmacological approach for mitochondrial DNA depletion syndrome
- Author
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Ramon Martí, Yolanda Cámara, Michio Hirano, Javier Torres-Torronteras, Emiliano González-Vioque, Mauro Scarpelli, and Andrea Caballero
- Subjects
Male ,Mitochondrial DNA ,DNA Copy Number Variations ,Purine nucleoside phosphorylase ,Deoxyribonucleosides ,deoxyribonucleosides ,catabolism inhibition ,mitochondrial DNA depletion syndrome ,pharmacological approach ,Mitochondrion ,Biology ,DNA, Mitochondrial ,chemistry.chemical_compound ,Muscular Dystrophy, Oculopharyngeal ,Mitochondrial Encephalomyopathies ,Genetics ,medicine ,Deoxyguanosine ,Animals ,Humans ,Molecular Biology ,Genetics (clinical) ,Cells, Cultured ,Mice, Knockout ,Ophthalmoplegia ,Catabolism ,Intestinal Pseudo-Obstruction ,General Medicine ,Cytidine deaminase ,Articles ,medicine.disease ,Molecular biology ,Mitochondria ,Deoxyribonucleoside ,chemistry ,Mitochondrial DNA depletion syndrome - Abstract
Mitochondrial DNA (mtDNA) depletion syndrome (MDS) is characterized by a reduction in mtDNA copy number and consequent mitochondrial dysfunction in affected tissues. A subgroup of MDS is caused by mutations in genes that disrupt deoxyribonucleotide metabolism, which ultimately leads to limited availability of one or several deoxyribonucleoside triphosphates (dNTPs), and subsequent mtDNA depletion. Here, using in vitro experimental approaches (primary cell culture of deoxyguanosine kinase-deficient cells and thymidine-induced mtDNA depletion in culture as a model of mitochondrial neurogastrointestinal encephalomyopathy, MNGIE), we show that supplements of those deoxyribonucleosides (dNs) involved in each biochemical defect (deoxyguanosine or deoxycytidine, dCtd) prevents mtDNA copy number reduction. Similar effects can be obtained by specific inhibition of dN catabolism using tetrahydrouridine (THU; inhibitor of cytidine deaminase) or immucillin H (inhibitor of purine nucleoside phosphorylase). In addition, using an MNGIE animal model, we provide evidence that mitochondrial dNTP content can be modulated in vivo by systemic administration of dCtd or THU. In spite of the severity associated with diseases due to defects in mtDNA replication, there are currently no effective therapeutic options available. Only in the case of MNGIE, allogeneic hematopoietic stem cell transplantation has proven efficient as a long-term therapeutic strategy. We propose increasing cellular availability of the deficient dNTP precursor by direct administration of the dN or inhibition of its catabolism, as a potential treatment for mtDNA depletion syndrome caused by defects in dNTP metabolism.
- Published
- 2013
38. 279. Efficient and Safe Lentiviral Vector-Mediated Hematopoietic Stem Cell Gene Therapy in MNGIE Mice
- Author
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Rana Yadak, Elly Bogaerts, Jordi Barquinero, George de Ruijter, René F.M. de Coo, Ramon Martí, Javier Torres Torronteras, Cabrera Pérez Raquel, Gerard Wagemaker, Niek P. van Til, Bert Smeets, and Marshall W. Huston
- Subjects
Pharmacology ,Genetic enhancement ,Hematopoietic stem cell ,Biology ,Total body irradiation ,Molecular biology ,Viral vector ,Transplantation ,medicine.anatomical_structure ,Immunology ,Drug Discovery ,medicine ,Genetics ,Molecular Medicine ,Bone marrow ,Stem cell ,Thymidine phosphorylase ,Molecular Biology - Abstract
Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) patients are deficient in thymidine phosphorylase(TP) resulting in systemic thymidine(Thd) and deoxyuridine(dUrd) accumulation affecting mtDNA replication and causing mitochondrial dysfunction. Common symptoms are gastrointestinal dysmotility, progressive ophthalmoplegia and leukoencephalopathy. Allogenic hematopoietic stem cell (HSC) transplantation has been shown to reduce disease symptoms, but is not well tolerated due to the inherent toxicity of the procedure. Therefore, syngeneic ex vivo lentiviral vector HSC gene therapy overexpressing the native cDNA or the codon optimized (TPco) sequence driven by the phosphoglycerate kinase (PGK) or spleen focus forming virus (SFFV) promoters in Tp−/-Upp−/- double knockout mice, a model for MNGIE disease, was investigated. At 1 month post transplantation after sublethal total body irradiation, very low TP activity was detected in blood of control wild type mice (0.07±0.03nmoles/h/mg), but enzyme activities in PGK treated mice were at least 90-fold higher (PGK-TP = 150±4 and PGK-TPco = 96±4 nmoles/h/mg), and in SFFV recipient mice 400-fold higher (450±5 nmoles/h/mg). Consequently, a significant reduction of plasma and urine Thd and dUrd levels was observed. Long-term follow up (14 months) showed on average 1.2-fold wild type TP activity levels increase in LV-PGK-TP and LV-PGK-TPco and 36-fold in SFFV-TPco treated mice. This was sufficient for sustained reduction of plasma and urine nucleoside levels, which was achieved at 76.5±8.2% donor chimerism levels with low LV vector copy numbers (1.0±1.1VCN/donor cell). The LV integration profile in bone marrow cells of primary recipients was analyzed; LVs displayed the expected tendency to integrate within highly expressed genes and the integration pattern did not differ from that of other SIN-LV vectors in other disease models (primary immune deficiencies and lysosomal enzyme storage disorders). Overall, stem cell gene therapy provided stable TP expression and long-term biochemical correction in MNGIE mice without genotoxicity or apparent phenotoxicity, which will be further evaluated for somatic and neurological phenotype correction and optimized to develop a clinical protocol to treat MNGIE patients.
- Published
- 2015
- Full Text
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39. Feeding the deoxyribonucleoside salvage pathway to rescue mitochondrial DNA
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Ramon Martí, Javier Torres-Torronteras, Emiliano González-Vioque, Mauro Scarpelli, and Yolanda Cámara
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deoxyribonucleoside ,Mitochondrial DNA ,Deoxyribonucleosides ,Mitochondrial Diseases ,Somatic cell ,Mitochondrial disease ,Biology ,DNA, Mitochondrial ,catabolism inhibition ,chemistry.chemical_compound ,mitochondrial disorders ,therapeutic strategy ,Drug Discovery ,medicine ,Animals ,Humans ,Gene ,Nucleotide salvage ,Pharmacology ,Genetics ,Point mutation ,Genetic Therapy ,medicine.disease ,chemistry ,DNA ,Gene Deletion - Abstract
Mutations in an increasing number of nuclear genes involved in deoxyribonucleotide homeostasis cause disorders associated with somatic mitochondrial DNA (mtDNA) abnormalities. Dysfunction of the products of these genes leads to limited availability of substrates for mtDNA replication and results in mtDNA depletion, multiple deletions or point mutations; mtDNA depletion is the molecular feature linked to greatest clinical severity. In this review, we discuss recent results demonstrating that enhancement of the salvage pathways by increasing the availability of deoxyribonucleosides needed for each specific genetic defect prevents mtDNA depletion. Hence, we propose administration of selected deoxyribonucleosides and/or inhibitors of their catabolism as a pharmacological strategy to treat these diseases.
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- 2013
40. The G1/S Specific Cyclin D2 Is a Regulator of HIV-1 Restriction in Non-proliferating Cells
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Marta Vives-Pi, Teresa Puig, Bonaventura Clotet, Eva Riveira-Muñoz, Javier Torres-Torronteras, Rosa Maria Ampudia, Ester Ballana, Roger Badia, Maria Pujantell, José A. Esté, and Ramon Martí
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RNA viruses ,0301 basic medicine ,Cyclin D ,Cyclin A ,Cyclin B ,Gene Expression ,HIV Infections ,Pathology and Laboratory Medicine ,Biochemistry ,Mice ,White Blood Cells ,0302 clinical medicine ,Immunodeficiency Viruses ,Animal Cells ,Medicine and Health Sciences ,Cyclin D2 ,Cell Cycle and Cell Division ,Post-Translational Modification ,Phosphorylation ,lcsh:QH301-705.5 ,biology ,Cell Differentiation ,Cell biology ,Cell Processes ,Medical Microbiology ,Viral Pathogens ,030220 oncology & carcinogenesis ,Viruses ,Cellular Types ,Pathogens ,Restriction point ,Research Article ,lcsh:Immunologic diseases. Allergy ,Cyclin-Dependent Kinase Inhibitor p21 ,Immune Cells ,Immunology ,Microbiology ,SAM Domain and HD Domain-Containing Protein 1 ,03 medical and health sciences ,Cyclin-dependent kinase ,Cyclins ,Virology ,Retroviruses ,VIH (Virus) ,Genetics ,Animals ,Humans ,Microbial Pathogens ,Cell Cycle Inhibitors ,Molecular Biology ,Cell Proliferation ,Monomeric GTP-Binding Proteins ,Cyclin-dependent kinase 1 ,Blood Cells ,Macrophages ,Lentivirus ,Organisms ,Cyclin-Dependent Kinase 4 ,Granulocyte-Macrophage Colony-Stimulating Factor ,Biology and Life Sciences ,HIV ,Proteins ,Cell Biology ,030104 developmental biology ,lcsh:Biology (General) ,HIV-1 ,biology.protein ,Cancer research ,Parasitology ,lcsh:RC581-607 ,Cyclin A2 ,Developmental Biology - Abstract
Macrophages are a heterogeneous cell population strongly influenced by differentiation stimuli that become susceptible to HIV-1 infection after inactivation of the restriction factor SAMHD1 by cyclin-dependent kinases (CDK). Here, we have used primary human monocyte-derived macrophages differentiated through different stimuli to evaluate macrophage heterogeneity on cell activation and proliferation and susceptibility to HIV-1 infection. Stimulation of monocytes with GM-CSF induces a non-proliferating macrophage population highly restrictive to HIV-1 infection, characterized by the upregulation of the G1/S-specific cyclin D2, known to control early steps of cell cycle progression. Knockdown of cyclin D2, enhances HIV-1 replication in GM-CSF macrophages through inactivation of SAMHD1 restriction factor by phosphorylation. Co-immunoprecipitation experiments show that cyclin D2 forms a complex with CDK4 and p21, a factor known to restrict HIV-1 replication by affecting the function of the downstream cascade that leads to SAMHD1 deactivation. Thus, we demonstrate that cyclin D2 acts as regulator of cell cycle proteins affecting SAMHD1-mediated HIV-1 restriction in non-proliferating macrophages., Author Summary Macrophages are a heterogeneous population of immune cells that provide crucial innate immune defense against pathogens, including HIV-1. The molecular biology of HIV-1 infection in macrophages is influenced by the presence of the host cell restriction factor SAMHD1, which is regulated by phosphorylation by cyclin dependent kinases, the catalytic proteins responsible for cell cycle progression. This study shows that differentiation stimuli strongly influence macrophage cell cycle and proliferation characteristics as well as susceptibility to HIV-1 infection through modulation of SAMHD1 activation. We have identified cyclin D2 as the key step controlling susceptibility to HIV-1 infection by modulation of the signaling pathway leading to SAMHD1 phosphorylation. We show that a complex formed by cyclin D2-CDK4-p21 in GM-CSF macrophages is responsible for the lack of the active CDK, which phosphorylates SAMHD1. This situation is reversed in the absence of cyclin D2, leading to the activation of CDKs and subsequent phosphorylation of its substrates, including SAMHD1. Thus, we propose that the differential expression of the G1/S-specific cyclin D2 controls the HIV-1 restriction pathway in primary macrophages.
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- 2016
41. A Transcriptomic Approach to Search for Novel Phenotypic Regulators in McArdle Disease
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Yolanda Cámara, Inés Consuegra-García, Elena García-Arumí, Antoni L. Andreu, Juan C. Rubio, Alejandro Lucia, Gisela Nogales-Gadea, Joaquín Arenas, Javier Torres-Torronteras, Marc Cuadros, Carmen Fiuza-Luces, and Miguel A. Martín
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Proteomics ,Muscle tissue ,medicine.medical_specialty ,Anatomy and Physiology ,lcsh:Medicine ,Exercise intolerance ,Genética humana ,Sarcoplasmic Reticulum Calcium-Transporting ATPases ,Glycogen phosphorylase ,chemistry.chemical_compound ,Genome Analysis Tools ,Internal medicine ,Genetics ,medicine ,Humans ,lcsh:Science ,Glycogen synthase ,Biology ,Musculoskeletal System ,Multidisciplinary ,biology ,Glycogen ,Glycogen Phosphorylase ,lcsh:R ,Computational Biology ,Skeletal muscle ,Genomics ,medicine.disease ,Glycogen Synthase ,Phenotype ,medicine.anatomical_structure ,Endocrinology ,Gene Expression Regulation ,chemistry ,Metabolic Disorders ,Case-Control Studies ,Multiprotein Complexes ,Muscle Fibers, Fast-Twitch ,biology.protein ,Muscle ,Medicine ,Glycogen Storage Disease Type V ,lcsh:Q ,Creatine kinase ,medicine.symptom ,Transcriptome ,Glycogen storage disease type V ,Research Article - Abstract
McArdle disease is caused by lack of glycogen phosphorylase (GP) activity in skeletal muscle. Patients experience exercise intolerance, presenting as early fatigue and contractures. In this study, we investigated the effects produced by a lack of GP on several genes and proteins of skeletal muscle in McArdle patients. Muscle tissue of 35 patients and 7 healthy controls were used to identify abnormalities in the patients' transcriptomic profile using low-density arrays. Gene expression was analyzed for the influence of variables such as sex and clinical severity. Differences in protein expression were studied by immunoblotting and 2D electrophoresis analysis, and protein complexes were examined by two-dimensional, blue native gel electrophoresis (BN-PAGE). A number of genes including those encoding acetyl-coA carboxylase beta, m-cadherin, calpain III, creatine kinase, glycogen synthase (GS), and sarcoplasmic reticulum calcium ATPase 1 (SERCA1), were found to be downregulated in patients. Specifically, compared to controls, GS and SERCA1 proteins were reduced by 50% and 75% respectively; also, unphosphorylated GS and SERCA1 were highly downregulated. On BN-PAGE analysis, GP was present with GS in two muscle protein complexes. Our findings revealed some issues that could be important in understanding the physiological consequences of McArdle disease: (i) SERCA1 downregulation in patients could result in impaired calcium transport in type II (fast-twitch) muscle fibers, leading to early fatigability during exercise tasks involving type II fibers (which mostly use glycolytic metabolism), i.e. isometric exercise, lifting weights or intense dynamic exercise (stair climbing, bicycling, walking at a very brisk pace), (ii) GP and GS were found together in two protein complexes, which suggests a new regulatory mechanism in the activity of these glycogen enzymes. 3.730 JCR (2012) Q1, 7/56 Multidisciplinary sciences UEM
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- 2012
42. A novel nonstop mutation in TYMP does not induce nonstop mRNA decay in a MNGIE patient with severe neuropathy
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Antoni L. Andreu, Anna Accarino, Agustí Rodríguez-Palmero, Guillem Pintos-Morell, Javier Torres-Torronteras, Ramon Martíí, and Tomàs Pinós
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Adolescent ,RNA Stability ,media_common.quotation_subject ,Molecular Sequence Data ,Nonsense ,Biology ,medicine.disease_cause ,NonStop ,Mitochondrial Encephalomyopathies ,In vivo ,Genetics ,medicine ,Humans ,RNA, Messenger ,Thymidine phosphorylase ,Gene ,Genetics (clinical) ,media_common ,Thymidine Phosphorylase ,Mutation ,Messenger RNA ,Base Sequence ,Homozygote ,RNA ,Molecular biology ,Female - Abstract
The cellular quality control systems enable surveillance and selective degradation of nonsense, nonstop, and no-go mRNAs. In the case of nonstop mRNA, different mechanisms of nonstop-mediated decay (NSD) have been described for bacteria, yeast and mammals, but the molecular consequences of nonstop mutations have been examined in only few cases of human disease. We describe a novel homozygous nonstop mRNA mutation (c.1416delC) in the TYMP gene encoding thymidine phosphorylase, in a patient with mitochondrial neurogastrointestinal encephalomyopathy (MNGIE). In contrast to previous reports showing selective decay of pathogenic nonstop mRNAs, quantitative real-time PCR and 3'-RACE-RFLP analysis revealed unreduced nonstop mRNA levels in our patient and 2 heterozygous carriers of the mutation. The absence of thymidine phosphorylase protein in the homozygous patient, together with the partial decrease in levels of this protein in 2 carriers suggest that the main control system in this case resides at the translational or post-translational levels rather than through NSD. This is the first report showing an absence of NSD in a human disease, revealing that this surveillance mechanism has exceptions in vivo.
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- 2011
43. Uridine metabolism in HIV-1-infected patients: effect of infection, of antiretroviral therapy and of HIV-1/ART-associated lipodystrophy syndrome
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Antoni L. Andreu, Joan Carles Domingo, Francesc Vidal, Marta Giralt, Maria del Mar Gutierrez, José M. Gallego-Escuredo, Ramon Martí, Marçal Pastor-Anglada, Maria Gracia Mateo, Pedro Cano-Soldado, Virginia Pomar, Pere Domingo, Javier Torres-Torronteras, Irene Fernández, Francesc Villarroya, and Universitat de Barcelona
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Male ,Gene Expression ,Adipose tissue ,HIV Infections ,Uridine kinase ,chemistry.chemical_compound ,Antiretroviral Therapy, Highly Active ,Blood plasma ,Lipoatrophy ,Uridine Phosphorylase ,Multidisciplinary ,Reverse Transcriptase Polymerase Chain Reaction ,Chemistry ,HIV-Associated Lipodystrophy Syndrome ,Antiretrovirals ,virus diseases ,Genetics and Genomics/Gene Expression ,Middle Aged ,Infectious Diseases/HIV Infection and AIDS ,Lipids ,Síndrome de lipodistròfia associada a VIH ,Metabolisme ,Adipose Tissue ,Uridine phosphorylase ,Medicine ,Female ,Lipodystrophy ,Research Article ,Adult ,medicine.medical_specialty ,Science ,Nucleoside Transport Proteins ,Internal medicine ,VIH (Virus) ,medicine ,Humans ,Chemistry/Biochemistry ,Uridine ,HIV (Viruses) ,HIV-associated lipodystrophy syndrome ,medicine.disease ,Antiretroviral agents ,Metabolism ,Endocrinology ,Immunology ,HIV-1 ,Uridine Kinase ,Nucleoside - Abstract
BackgroundUridine has been advocated for the treatment of HIV-1/HAART-associated lipodystrophy (HALS), although its metabolism in HIV-1-infected patients is poorly understood.MethodsPlasma uridine concentrations were measured in 35 controls and 221 HIV-1-infected patients and fat uridine in 15 controls and 19 patients. The diagnosis of HALS was performed following the criteria of the Lipodystrophy Severity Grading Scale. Uridine was measured by a binary gradient-elution HPLC method. Analysis of genes encoding uridine metabolizing enzymes in fat was performed with TaqMan RT-PCR.ResultsMedian plasma uridine concentrations for HIV-1-infected patients were 3.80 µmol/l (interquartile range: 1.60), and for controls 4.60 µmol/l (IQR: 1.8) (P = 0.0009). In fat, they were of 6.0 (3.67), and 2.8 (4.65) nmol/mg of protein, respectively (P = 0.0118). Patients with a mixed HALS form had a median plasma uridine level of 4.0 (IC95%: 3.40-4.80) whereas in those with isolated lipoatrophy it was 3.25 (2.55-4.15) µmol/l/l (P = 0.0066). The expression of uridine cytidine kinase and uridine phosphorylase genes was significantly decreased in all groups of patients with respect to controls. A higher expression of the mRNAs for concentrative nucleoside transporters was found in HIV-1-infected patients with respect to healthy controls.ConclusionsHIV-1 infection is associated with a decrease in plasma uridine and a shift of uridine to the adipose tissue compartment. Antiretroviral therapy was not associated with plasma uridine concentrations, but pure lipoatrophic HALS was associated with significantly lower plasma uridine concentrations.
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- 2010
44. Limited dCTP Availability Accounts for Mitochondrial DNA Depletion in Mitochondrial Neurogastrointestinal Encephalomyopathy (MNGIE)
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Ramon Martí, Emiliano González-Vioque, Antoni L. Andreu, and Javier Torres-Torronteras
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DNA Replication ,Cancer Research ,Mitochondrial DNA ,Mitochondrial Diseases ,Mitochondrial Myopathy ,Deoxyribonucleoside triphosphate ,lcsh:QH426-470 ,Cell Culture Techniques ,Encephalomyopathy ,Mitochondria, Liver ,Mitochondrion ,Biology ,DNA, Mitochondrial ,Biochemistry ,Mice ,chemistry.chemical_compound ,Mitochondrial Encephalomyopathies ,Myoneurogenic Gastrointestinal Encephalopathy ,Genetics ,Animals ,Humans ,Thymine Nucleotides ,heterocyclic compounds ,Thymidine phosphorylase ,Molecular Biology ,Gene ,Genetics (clinical) ,Ecology, Evolution, Behavior and Systematics ,Nucleotides ,DNA replication ,Human Genetics ,DNA ,Neuromuscular Diseases ,Fibroblasts ,Cell biology ,Nucleic acids ,lcsh:Genetics ,enzymes and coenzymes (carbohydrates) ,Neurology ,chemistry ,Deoxycytosine Nucleotides ,Medicine ,Research Article - Abstract
Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is a severe human disease caused by mutations in TYMP, the gene encoding thymidine phosphorylase (TP). It belongs to a broader group of disorders characterized by a pronounced reduction in mitochondrial DNA (mtDNA) copy number in one or more tissues. In most cases, these disorders are caused by mutations in genes involved in deoxyribonucleoside triphosphate (dNTP) metabolism. It is generally accepted that imbalances in mitochondrial dNTP pools resulting from these mutations interfere with mtDNA replication. Nonetheless, the precise mechanistic details of this effect, in particular, how an excess of a given dNTP (e.g., imbalanced dTTP excess observed in TP deficiency) might lead to mtDNA depletion, remain largely unclear. Using an in organello replication experimental model with isolated murine liver mitochondria, we observed that overloads of dATP, dGTP, or dCTP did not reduce the mtDNA replication rate. In contrast, an excess of dTTP decreased mtDNA synthesis, but this effect was due to secondary dCTP depletion rather than to the dTTP excess in itself. This was confirmed in human cultured cells, demonstrating that our conclusions do not depend on the experimental model. Our results demonstrate that the mtDNA replication rate is unaffected by an excess of any of the 4 separate dNTPs and is limited by the availability of the dNTP present at the lowest concentration. Therefore, the availability of dNTP is the key factor that leads to mtDNA depletion rather than dNTP imbalances. These results provide the first test of the mechanism that accounts for mtDNA depletion in MNGIE and provide evidence that limited dNTP availability is the common cause of mtDNA depletion due to impaired anabolic or catabolic dNTP pathways. Thus, therapy approaches focusing on restoring the deficient substrates should be explored., Author Summary Mitochondria are subcellular organelles that constitute the main energy supply within the cell. They contain their own DNA, which should be continuously replicated to ensure the correct mitochondrial function. Several mitochondrial diseases are caused by genetic defects that compromise this replication and result in mitochondrial DNA depletion. In most cases, these genetic defects block the synthesis of dATP, dGTP, dCTP, and dTTP, the 4 nucleotides needed for mitochondrial DNA replication. However, for one of these disorders (mitochondrial neurogastrointestinal encephalomyopathy, MNGIE), the biochemical pathways needed to synthesize them are intact, but degradation of dTTP is genetically blocked, leading to dTTP accumulation. We investigated the biochemical mechanisms through which the dTTP excess leads to mitochondrial DNA depletion in MNGIE, and we found that the delay of mitochondrial DNA replication rate observed when dTTP is in excess is not caused by this excess in itself. Instead, the dTTP overload produces a secondary dCTP depletion that actually delays mitochondrial DNA replication. Therefore, the common factor accounting for mitochondrial DNA depletion in these disorders is the limited availability of one or more nucleotides. This indicates that strategies to provide nucleotides to patients' mitochondria should be explored as a possible treatment for these fatal disorders.
- Published
- 2011
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