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Garrido-Maraver J.,Pablo De Olavide University | Paz M.V.,Pablo De Olavide University | Cordero M.D.,University of Seville | Bautista-Lorite J.,Servicio de Neurologia | And 10 more authors.
Biochimica et Biophysica Acta - Molecular Basis of Disease | Year: 2015

MELAS syndrome is a mitochondrial disorder that is caused mainly by the m.3243A>G mutation in mitochondrial DNA. Here, we report on how the severity of pathophysiological alterations is differently expressed in fibroblasts derived from patients with MELAS disease. We evaluated mitophagy activation and mitochondrial biogenesis which are the main mechanisms regulating the degradation and genesis of mitochondrial mass in MELAS fibroblasts and transmitochondrial cybrids. Our results suggest a critical balance between mitophagy and mitochondrial biogenesis which leads to the expression of different degrees of pathological severity among MELAS fibroblast cell lines according to their heteroplasmy load and the activation of AMP-activated protein kinase (AMPK). AMPK-activators such as 5-aminoimidazole-4-carboxamide 1-ß-D-ribofuranoside (AICAR) or coenzyme Q10 (CoQ) increased peroxisome proliferator-activated receptor alpha (PGC-1a) nuclear translocation, mitochondrial biogenesis, antioxidant enzyme system response, autophagic flux and improved pathophysiological alterations in MELAS fibroblasts with the most severe phenotype. Our findings support the hypothesis that mitochondrial biogenesis, increased antioxidant response and autophagy clearance serve as compensatory mechanisms in response to mitophagic degradation of dysfunctional mitochondria and point out that AMPK is an important player in this balance. © 2015 Elsevier B.V. Source


de la Mata M.,Pablo De Olavide University | Garrido-Maraver J.,Pablo De Olavide University | Cotan D.,Pablo De Olavide University | Cordero M.D.,Pablo De Olavide University | And 8 more authors.
Neurotherapeutics | Year: 2012

Mitochondrial DNA mutations are an important cause of human disease for which there is no effective treatment. Myoclonic epilepsy with ragged-red fibers (MERRF) is a mitochondrial disease usually caused by point mutations in transfer RNA genes encoded by mitochondrial DNA. The most common mutation associated with MERRF syndrome, m.8344A > G in the gene MT-TK, which encodes transfer RNALysine, affects the translation of all mitochondrial DNA encoded proteins. This impairs the assembly of the electron transport chain complexes leading to decreased mitochondrial respiratory function. Here we report on how this mutation affects mitochondrial function in primary fibroblast cultures established from patients harboring the A8344G mutation. Coenzyme Q10 (CoQ) levels, as well as mitochondrial respiratory chain activity, and mitochondrial protein expression levels were significantly decreased in MERRF fibroblasts. Mitotracker staining and imaging analysis of individual mitochondria indicated the presence of small, rounded, depolarized mitochondria in MERRF fibroblasts. Mitochondrial dysfunction was associated with increased oxidative stress and increased degradation of impaired mitochondria by mitophagy. Transmitochondrial cybrids harboring the A8344G mutation also showed CoQ deficiency, mitochondrial dysfunction, and increased mitophagy activity. All these abnormalities in patient-derived fibroblasts and cybrids were partially restored by CoQ supplementation, indicating that these cell culture models may be suitable for screening and validation of novel drug candidates for MERRF disease. © 2012 The American Society for Experimental NeuroTherapeutics, Inc. Source


Oropesa-Avila M.,Pablo De Olavide University | Fernandez-Vega A.,Pablo De Olavide University | De La Mata M.,Pablo De Olavide University | Maraver J.G.,Pablo De Olavide University | And 10 more authors.
Cell Death and Disease | Year: 2013

Apoptotic microtubule network (AMN) is organized during apoptosis, forming a cortical structure beneath plasma membrane, which has an important role in preserving cell morphology and plasma membrane permeability. The aim of this study was to examine the role of AMN in maintaining plasma membrane integrity during the execution phase of apoptosis. We demonstrated in camptothecin-induced apoptosis in H460 cells that AMN delimits an active caspase free area beneath plasma membrane that permits the preservation of cellular cortex and transmembrane proteins. AMN depolymerization in apoptotic cells by a short exposure to colchicine allowed active caspases to reach the cellular cortex and cleave many key proteins involved in plasma membrane structural support, cell adhesion and ionic homeostasis. Cleavage of cellular cortex and plasma membrane proteins, such as a-spectrin, paxilin, focal adhesion kinase (FAK), E-cadherin and integrin subunit β4 was associated with cell collapse and cell detachment. Otherwise, cleavage-mediated inactivation of calcium ATPase pump (PMCA-4) and Na+/Ca2+ exchanger (NCX) involved in cell calcium extrusion resulted in calcium overload. Furthermore, cleavage of Na +/K+ pump subunit b was associated with altered sodium homeostasis. Cleavage of cell cortex and plasma membrane proteins in apoptotic cells after AMN depolymerization increased plasma permeability, ionic imbalance and bioenergetic collapse, leading apoptotic cells to secondary necrosis. The essential role of caspase-mediated cleavage in this process was demonstrated because the concomitant addition of colchicine that induces AMN depolymerization and the pan-caspase inhibitor z-VAD avoided the cleavage of cortical and plasma membrane proteins and prevented apoptotic cells to undergo secondary necrosis. Furthermore, the presence of AMN was also critical for proper phosphatidylserine externalization and apoptotic cell clearance by macrophages. These results indicate that AMN is essential to preserve an active caspase free area in the cellular cortex of apoptotic cells that allows plasma membrane integrity during the execution phase of apoptosis. © 2013 Macmillan Publishers Limited. Source


Rodriguez-Beltran J.,Institute Biomedicina Of Seville Ibis Csic | Rodriguez-Beltran J.,CSIC - National Center for Biotechnology | Tourret J.,French Institute of Health and Medical Research | Tourret J.,University Paris Diderot | And 13 more authors.
Molecular Biology and Evolution | Year: 2015

Homologous recombination promotes genetic diversity by facilitating the integration of foreign DNA and intrachromosomal gene shuffling. It has been hypothesized that if recombination is variable among strains, selection should favor higher recombination rates among pathogens, as they face additional selection pressures from host defenses. To test this hypothesis we have developed a plasmid-based method for estimating the rate of recombination independently of other factors such as DNA transfer, selective processes, and mutational interference. Our results with 160 human commensal and extraintestinal pathogenic Escherichia coli (ExPEC) isolates show that the recombinant frequencies are extremely diverse (ranging 9 orders of magnitude) and plastic (they are profoundly affected by growth in urine, a condition commonly encountered by ExPEC). We find that the frequency of recombination is biased by strain lifestyle, as ExPEC isolates display strikingly higher recombination rates than their commensal counterparts. Furthermore, the presence of virulence factors is positively associated with higher recombination frequencies. These results suggest selection for high homologous recombination capacity, which may result in a higher evolvability for pathogens compared with commensals. © The Author 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. Source

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