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ABSTRACT: In this minireview, we briefly survey the molecular processes that lead to reactive oxygen species (ROS) production by the respiratory complex III (CIII or cytochrome bc1). In particular, we discuss the "forward" and "reverse" electron transfer pathways that lead to superoxide generation at the quinol oxidation (Qo) site of CIII, and the components that affect these reactions. We then describe and compare the properties of a bacterial (Rhodobacter capsulatus) mutant enzyme producing ROS with its mitochondrial (human cybrids) counterpart associated with a disease. The mutation under study is located at a highly conserved tyrosine residue of cytochrome b (Y302 in R. capsulatus and Y278 in human mitochondria) that is at the heart of the quinol oxidation (Qo) site of CIII. Similarities of the major findings of bacterial and human mitochondrial cases, including decreased catalytic activity of CIII, enhanced ROS production and ensuing cellular responses and damages, are remarkable. This case illustrates the usefulness of undertaking parallel and complementary studies using biologically different yet evolutionarily related systems, such as α-proteobacteria and human mitochondria. It progresses our understanding of CIII mechanism of function and ROS production, and underlines the possible importance of supra molecular organization of bacterial and mitochondrial respiratory chains (i. e., respirasomes) and their potential disease-associated protective roles. This article is part of a Special Issue entitled: Respiratory complex III and related bc complexes.
Biochimica et Biophysica Acta 03/2013; · 4.66 Impact Factor
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Anna Ghelli,
Concetta V Tropeano,
Maria Antonietta Calvaruso,
Alessandra Marchesini,
Luisa Iommarini,
Anna Maria Porcelli,
Claudia Zanna,
Vera De Nardo,
Andrea Martinuzzi,
Flemming Wibrand,
John Vissing,
Ivana Kurelac,
Giuseppe Gasparre,
Nur Selamoglu,
Fevzi Daldal, Michela Rugolo
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ABSTRACT: Cytochrome b is the only mtDNA-encoded subunit of the mitochondrial complex III (CIII), the functional bottleneck of the respiratory chain. Previously, the human cytochrome b missense mutation m.15579A>G, which substitutes the Tyr 278 with Cys (p.278Y>C), was identified in a patient with severe exercise intolerance and multisystem manifestations. In this study, we characterized the biochemical properties of cybrids carrying this mutation and report that the homoplasmic p.278Y>C mutation caused a dramatic reduction in the CIII activity and in CIII-driven mitochondrial ATP synthesis. However, the CI, CI + CIII and CII + CIII activities and the rate of ATP synthesis driven by the CI or CII substrate were only partially reduced or unaffected. Consistent with these findings, mutated cybrids maintained the mitochondrial membrane potential in the presence of oligomycin, indicating that it originated from the respiratory electron transport chain. The p.278Y>C mutation enhanced superoxide production, as indicated by direct measurements in mitochondria and by the imbalance of glutathione homeostasis in intact cybrids. Remarkably, although the assembly of CI or CIII was not affected, the examination of respiratory supercomplexes revealed that the amounts of CIII dimer and III2IV1 were reduced, whereas those of I1III2IVn slightly increased. We therefore suggest that the deleterious effects of p.278Y>C mutation on cytochrome b are palliated when CIII is assembled into the supercomplexes I1III2IVn, in contrast to when it is found alone. These findings underline the importance of supramolecular interactions between complexes for maintaining a basal respiratory chain activity and shed light to the molecular basis of disease manifestations associated with this mutation.
Human Molecular Genetics 02/2013; · 7.64 Impact Factor
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ABSTRACT: Significance: The maintenance of mitochondrial genome integrity is a major challenge for cells in order to sustain energy production by respiration. Recent Advances: Recently, mitochondrial membrane dynamics has emerged as a key process contributing to prevent mitochondrial DNA alterations. Indeed, both fundamental and clinical data suggest that disruption of mitochondrial fusion, related to mutations in the OPA1, MFN2, PINK1 and Parkin genes, leads to the accumulation of mutations in the mitochondrial genome. Critical Issues: We discuss here the possibility that mitochondrial fusion acts as a direct mechanism to prevent the generation of altered mitochondrial DNA and to eliminate mutated deleterious genomes either by trans-complementation or by mitophagy. Future Directions: Finally, we conclude this review with a short evolutionary comparison between the mechanisms involved in mitochondrial and bacterial modes of genome distribution and plasticity, highlighting possible common conserved processes required for the maintenance of their genome integrity, which should inspire our future investigations.
Antioxidants & Redox Signaling 01/2013; · 8.20 Impact Factor
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ABSTRACT: We have studied the effects of idebenone on mitochondrial function in cybrids derived from one normal donor (HQB17) and one patient harboring the G3460A/MT-ND1 mutation of Leber's Hereditary Optic Neuropathy (RJ206); and in XTC.UC1 cells bearing a premature stop codon at amino acid 101 of MT-ND1 that hampers complex I assembly. Addition of idebenone to HQB17 cells caused mitochondrial depolarization and NADH depletion, which were inhibited by cyclosporin (Cs) A and decylubiquinone, suggesting an involvement of the permeability transition pore (PTP). On the other hand, addition of dithiothreitol together with idebenone did not cause PTP opening and allowed maintenance of the mitochondrial membrane potential even in the presence of rotenone. Addition of dithiothreitol plus idebenone, or of idebenol, to HQB17, RJ206 and XTC.UC1 cells sustained membrane potential in intact cells and ATP synthesis in permeabilized cells even in the presence of rotenone and malonate, and restored a good level of coupled respiration in complex I-deficient XTC.UC1 cells. These findings demonstrate that idebenol can feed electrons at complex III. If the quinone is maintained in the reduced state, a task that in some cell types appears to be performed by dicoumarol-sensitive NAD(P)H:quinone oxidoreductase 1 [Haefeli et al. (2011) PLoS One 6, e17963], electron transfer to complex III may allow reoxidation of NADH in complex I deficiencies.
Biochimica et Biophysica Acta 11/2011; 1817(2):363-9. · 4.66 Impact Factor
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Giuseppe Gasparre,
Ivana Kurelac,
Mariantonietta Capristo,
Luisa Iommarini,
Anna Ghelli,
Claudio Ceccarelli,
Giordano Nicoletti,
Patrizia Nanni,
Carla De Giovanni,
Katia Scotlandi,
Christine M Betts,
Valerio Carelli,
Pier Luigi Lollini,
Giovanni Romeo, Michela Rugolo,
Anna Maria Porcelli
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ABSTRACT: The oncogenic versus suppressor roles of mitochondrial genes have long been debated. Peculiar features of mitochondrial genetics such as hetero/homoplasmy and mutation threshold are seldom taken into account in this debate. Mitochondrial DNA (mtDNA) mutations generally have been claimed to be protumorigenic, but they are also hallmarks of mostly benign oncocytic tumors wherein they help reduce adaptation to hypoxia by destabilizing hypoxia-inducible factor-1α (HIF1α). To determine the influence of a disassembling mtDNA mutation and its hetero/homoplasmy on tumorigenic and metastatic potential, we injected mice with tumor cells harboring different loads of the gene MTND1 m.3571insC. Cell cultures obtained from tumor xenografts were then analyzed to correlate energetic competence, apoptosis, α-ketoglutarate (α-KG)/succinate (SA) ratio, and HIF1α stabilization with the mutation load. A threshold level for the antitumorigenic effect of MTND1 m.3571insC mutation was defined, above which tumor growth and invasiveness were reduced significantly. Notably, HIF1α destabilization and downregulation of HIF1α-dependent genes occurred in cells and tumors lacking complex I (CI), where there was an associated imbalance of α-KG/SA despite the presence of an actual hypoxic environment. These results strongly implicate mtDNA mutations as a cause of oncocytic transformation. Thus, the antitumorigenic and antimetastatic effects of high loads of MTND1 m.3571insC, following CI disassembly, define a novel threshold-regulated class of cancer genes. We suggest these genes be termed oncojanus genes to recognize their ability to contribute either oncogenic or suppressive functions in mitochondrial settings during tumorigenesis.
Cancer Research 08/2011; 71(19):6220-9. · 7.86 Impact Factor
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ABSTRACT: A prominent role for mitochondrial genes and metabolism has been recently characterized in oncocytic transformation of cancer cells. From mitochondrial ultrastructure alterations to respiratory complexes disruption and mutations within mitochondrial genes, oncocytic tumors present with a plethora of features that have helped understand the role that these organelles and their fundamental metabolic functions may play in cancer development. The history of this under-diagnosed subset of tumors and the bioenergetic implications of their mitochondrial derangement are discussed in this review along with the opportunities that oncocytic tumors offer to draw general conclusions on the involvement of mitochondria in cancer.
Biochimica et Biophysica Acta 06/2011; 1807(6):633-42. · 4.66 Impact Factor
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Valerio Carelli,
Simone Schimpf,
Nico Fuhrmann,
Maria Lucia Valentino,
Claudia Zanna,
Luisa Iommarini,
Monika Papke,
Simone Schaich,
Sabine Tippmann,
Britta Baumann, [......],
Richard J Youle,
Laura Bucchi,
Rosanna Carroccia,
Maria Pia Giannoccaro,
Caterina Tonon,
Raffaele Lodi,
Giovanna Cenacchi,
Pasquale Montagna,
Rocco Liguori,
Bernd Wissinger
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ABSTRACT: Dominant optic atrophy (DOA) is genetically heterogeneous and pathogenic mutations have been identified in the OPA1 and OPA3 genes, both encoding for mitochondrial proteins. We characterized clinical and laboratory features in a large OPA1-negative family with complicated DOA. Search for mitochondrial dysfunction was performed by studying muscle biopsies, fibroblasts, platelets and magnetic resonance (MR) spectroscopy. Genetic investigations included mitochondrial DNA (mtDNA) analysis, linkage analysis, copy number variation (CNV) analysis and candidate gene screening. Optic neuropathy was undistinguishable from that in OPA1-DOA and frequently associated with late-onset sensorineural hearing loss, increases of central conduction times at somato-sensory evoked potentials and various cardiac abnormalities. Serum lactic acid after exercise, platelet respiratory complex activities, adenosine triphosphate (ATP) content in fibroblasts and muscle phosphorus MR spectroscopy all failed to reveal a mitochondrial dysfunction. However, muscle biopsies and their mtDNA analysis showed increased mitochondrial biogenesis. Furthermore, patient's fibroblasts grown in the galactose medium were unable to increase ATP content compared with controls, and exhibited abnormally high rate of fusion activity. Genome-wide linkage revealed a locus on chromosome 16q21-q22 with a maximum two-point LOD score of 8.84 for the marker D16S752 and a non-recombinant interval of ∼ 6.96 cM. Genomic screening of 45 genes in this interval including several likely candidate genes (CALB2, CYB5B, TK2, DHODH, PLEKHG4) revealed no mutation. Moreover, we excluded the presence of CNVs using array-based comparative genome hybridization. The identification of a new OPA locus (OPA8) in this pedigree demonstrates further genetic heterogeneity in DOA, and our results indicate that the pathogenesis may still involve mitochondria.
Human Molecular Genetics 02/2011; 20(10):1893-905. · 7.64 Impact Factor
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Ghizlane Elachouri,
Sara Vidoni,
Claudia Zanna,
Alexandre Pattyn,
Hassan Boukhaddaoui,
Karen Gaget,
Patrick Yu-Wai-Man,
Giuseppe Gasparre,
Emmanuelle Sarzi,
Cécile Delettre,
Aurélien Olichon,
Dominique Loiseau,
Pascal Reynier,
Patrick F Chinnery,
Agnès Rotig,
Valerio Carelli,
Christian P Hamel, Michela Rugolo,
Guy Lenaers
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ABSTRACT: Eukaryotic cells harbor a small multiploid mitochondrial genome, organized in nucleoids spread within the mitochondrial network. Maintenance and distribution of mitochondrial DNA (mtDNA) are essential for energy metabolism, mitochondrial lineage in primordial germ cells, and to prevent mtDNA instability, which leads to many debilitating human diseases. Mounting evidence suggests that the actors of the mitochondrial network dynamics, among which is the intramitochondrial dynamin OPA1, might be involved in these processes. Here, using siRNAs specific to OPA1 alternate spliced exons, we evidenced that silencing of the OPA1 variants including exon 4b leads to mtDNA depletion, secondary to inhibition of mtDNA replication, and to marked alteration of mtDNA distribution in nucleoid and nucleoid distribution throughout the mitochondrial network. We demonstrate that a small hydrophobic 10-kDa peptide generated by cleavage of the OPA1-exon4b isoform is responsible for this process and show that this peptide is embedded in the inner membrane and colocalizes and coimmunoprecipitates with nucleoid components. We propose a novel synthetic model in which a peptide, including two trans-membrane domains derived from the N terminus of the OPA1-exon4b isoform in vertebrates or from its ortholog in lower eukaryotes, might contribute to nucleoid attachment to the inner mitochondrial membrane and promotes mtDNA replication and distribution. Thus, this study places OPA1 as a direct actor in the maintenance of mitochondrial genome integrity.
Genome Research 10/2010; 21(1):12-20. · 13.61 Impact Factor
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Anna Maria Porcelli,
Anna Ghelli,
Claudio Ceccarelli,
Martin Lang,
Giovanna Cenacchi,
Mariantonietta Capristo,
Lucia Fiammetta Pennisi,
Isabella Morra,
Enrica Ciccarelli,
Antonio Melcarne,
Anna Bartoletti-Stella,
Nunzio Salfi,
Giovanni Tallini,
Andrea Martinuzzi,
Valerio Carelli,
Marcella Attimonelli, Michela Rugolo,
Giovanni Romeo,
Giuseppe Gasparre
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ABSTRACT: We previously showed that disruptive complex I mutations in mitochondrial DNA are the main genetic hallmark of oncocytic tumors of the thyroid and kidney. We here report a high frequency of homoplasmic disruptive mutations in a large panel of oncocytic pituitary and head-and-neck tumors. The presence of such mutations implicates disassembly of respiratory complex I in vivo which in turn contributes to the inability of oncocytic tumors to stabilize HIF1alpha and to display pseudo-hypoxia. By utilizing transmitochondrial cytoplasmic hybrids (cybrids), we induced the shift to homoplasmy of a truncating mutation in the mitochondria-coded MTND1 gene. Such shift is associated with a profound metabolic impairment leading to the imbalance of alpha-ketoglutarate and succinate, the Krebs cycle metabolites which are the main responsible for HIF1alpha stabilization. We conclude that the main hallmarks of oncocytic transformation, namely the occurrence of homoplasmic disruptive mutations and complex I disassembly, may explain the benign nature of oncocytic neoplasms through lack of HIF1alpha stabilization.
Human Molecular Genetics 12/2009; 19(6):1019-32. · 7.64 Impact Factor
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Anna Ghelli,
Anna Maria Porcelli,
Claudia Zanna,
Sara Vidoni,
Stefano Mattioli,
Anna Barbieri,
Luisa Iommarini,
Maria Pala,
Alessandro Achilli,
Antonio Torroni, Michela Rugolo,
Valerio Carelli
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ABSTRACT: Leber's hereditary optic neuropathy (LHON) is a maternally inherited blinding disease due to mitochondrial DNA (mtDNA) point mutations in complex I subunit genes, whose incomplete penetrance has been attributed to both genetic and environmental factors. Indeed, the mtDNA background defined as haplogroup J is known to increase the penetrance of the 11778/ND4 and 14484/ND6 mutations. Recently it was also documented that the professional exposure to n-hexane might act as an exogenous trigger for LHON. Therefore, we here investigate the effect of the n-hexane neurotoxic metabolite 2,5-hexanedione (2,5-HD) on cell viability and mitochondrial function of different cell models (cybrids and fibroblasts) carrying the LHON mutations on different mtDNA haplogroups. The viability of control and LHON cybrids and fibroblasts, whose mtDNAs were completely sequenced, was assessed using the MTT assay. Mitochondrial ATP synthesis rate driven by complex I substrates was determined with the luciferine/luciferase method. Incubation with 2,5-HD caused the maximal loss of viability in control and LHON cells. The toxic effect of this compound was similar in control cells irrespective of the mtDNA background. On the contrary, sensitivity to 2,5-HD induced cell death was greatly increased in LHON cells carrying the 11778/ND4 or the 14484/ND6 mutation on haplogroup J, whereas the 11778/ND4 mutation in association with haplogroups U and H significantly improved cell survival. The 11778/ND4 mutation on haplogroup U was also more resistant to inhibition of complex I dependent ATP synthesis by 2,5-HD. In conclusion, this study shows that mtDNA haplogroups modulate the response of LHON cells to 2,5-HD. In particular, haplogroup J makes cells more sensitive to its toxic effect. This is the first evidence that an mtDNA background plays a role by interacting with an environmental factor and that 2,5-HD may be a risk element for visual loss in LHON. This proof of principle has broad implications for other neurodegenerative disorders such as Parkinson's disease.
PLoS ONE 01/2009; 4(11):e7922. · 4.09 Impact Factor
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Giuseppe Gasparre,
Luisa Iommarini,
Anna Maria Porcelli,
Martin Lang,
Gian Gaetano Ferri,
Ivana Kurelac,
Roberta Zuntini,
Elisa Mariani,
Lucia Fiammetta Pennisi,
Ernesto Pasquini,
Gianandrea Pasquinelli,
Anna Ghelli,
Elena Bonora,
Claudio Ceccarelli, Michela Rugolo,
Nunzio Salfi,
Giovanni Romeo,
Valerio Carelli
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ABSTRACT: A disruptive frameshift mtDNA mutation affecting the ND5 subunit of complex I is present in homoplasmy in a nasopharyngeal oncocytic tumor and inherited as a heteroplasmic germline mutation recurring in two of the patient's siblings. Homoplasmic ND5 mutation in the tumor correlates with lack of the ND6 subunit, suggesting complex I disassembly. A few oncocytic areas, expressing ND6 and heteroplasmic for the ND5 mutation, harbor a de novo homoplasmic ND1 mutation. Since shift to homoplasmy of ND1 and ND5 mutations occurs exclusively in tumor cells, we conclude that complex I mutations may have a selective advantage and accompany oncocytic transformation.
Human Mutation 01/2009; 30(3):391-6. · 5.69 Impact Factor
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ABSTRACT: We have studied mitochondrial bioenergetics in HL180 cells (a cybrid line harboring the T14484C/ND6 and G14279A/ND6 mtDNA mutations of Leber hereditary optic neuropathy, leading to an approximately 50% decrease of ATP synthesis) and XTC.UC1 cells (derived from a thyroid oncocytoma bearing a disruptive frameshift mutation in MT-ND1, which impairs complex I assembly). The addition of rotenone to HL180 cells and of antimycin A to XTC.UC1 cells caused fast mitochondrial membrane depolarization that was prevented by treatment with cyclosporin A, intracellular Ca2+ chelators, and antioxidant. Both cell lines also displayed an anomalous response to oligomycin, with rapid onset of depolarization that was prevented by cyclosporin A and by overexpression of Bcl-2. These findings indicate that depolarization by respiratory chain inhibitors and oligomycin was due to opening of the mitochondrial permeability transition pore (PTP). A shift of the threshold voltage for PTP opening close to the resting potential may therefore be the underlying cause facilitating cell death in diseases affecting complex I activity. This study provides a unifying reading frame for previous observations on mitochondrial dysfunction, bioenergetic defects, and Ca2+ deregulation in mitochondrial diseases. Therapeutic strategies aimed at normalizing the PTP voltage threshold may be instrumental in ameliorating the course of complex I-dependent mitochondrial diseases.
Journal of Biological Chemistry 01/2009; 284(4):2045-52. · 4.77 Impact Factor
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Patrizia Amati-Bonneau,
Maria Lucia Valentino,
Pascal Reynier,
Maria Esther Gallardo,
Belén Bornstein,
Anne Boissière,
Yolanda Campos,
Henry Rivera,
Jesús González de la Aleja,
Rosanna Carroccia, [......],
Bernd Wissinger,
Christophe Verny,
Robert Schwarzenbacher,
Miguel Angel Martín,
Joaquín Arenas,
Carmen Ayuso,
Rafael Garesse,
Guy Lenaers,
Dominique Bonneau,
Valerio Carelli
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ABSTRACT: Mutations in OPA1, a dynamin-related GTPase involved in mitochondrial fusion, cristae organization and control of apoptosis, have been linked to non-syndromic optic neuropathy transmitted as an autosomal-dominant trait (DOA). We here report on eight patients from six independent families showing that mutations in the OPA1 gene can also be responsible for a syndromic form of DOA associated with sensorineural deafness, ataxia, axonal sensory-motor polyneuropathy, chronic progressive external ophthalmoplegia and mitochondrial myopathy with cytochrome c oxidase negative and Ragged Red Fibres. Most remarkably, we demonstrate that these patients all harboured multiple deletions of mitochondrial DNA (mtDNA) in their skeletal muscle, thus revealing an unrecognized role of the OPA1 protein in mtDNA stability. The five OPA1 mutations associated with these DOA 'plus' phenotypes were all mis-sense point mutations affecting highly conserved amino acid positions and the nuclear genes previously known to induce mtDNA multiple deletions such as POLG1, PEO1 (Twinkle) and SLC25A4 (ANT1) were ruled out. Our results show that certain OPA1 mutations exert a dominant negative effect responsible for multi-systemic disease, closely related to classical mitochondrial cytopathies, by a mechanism involving mtDNA instability.
Brain 03/2008; 131(Pt 2):338-51. · 9.46 Impact Factor
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ABSTRACT: To use different paradigms of oxidative and metabolic stress in a cellular model of Leber hereditary optic neuropathy (LHON), with the aim of evaluating the efficacy of potentially therapeutic molecules for the treatment of this disease.
Cybrids bearing one of the three most common LHON pathogenic mutations (11778/ND4, 3460/ND1, 14484/ND6) were incubated with two compounds known to induce oxidative injury, tert-butyl hydroperoxide (t-BH) and rotenone. To mimic metabolic stress, cells were incubated in a glucose-free medium containing galactose. Cell viability was determined using the MTT assay. To identify the apoptotic type of cell death, nuclear morphology was examined after cell loading with Hoechst. Cellular glutathione (GSH), and oxidized glutathione (GSSG) levels were measured enzymatically.
Incubation with t-BH caused apoptotic cell death of control and LHON cybrids, whereas only LHON cybrids were damaged by rotenone concentrations up to 2.5 muM. Both types of stress caused a marked imbalance in the glutathione levels, but an increase in the GSSG/GSH+GSSG ratio was detected only after rotenone treatment. The efficacy of several antioxidant and antiapoptotic compounds was then assessed in cells exposed to these two oxidative paradigms. Only exogenous GSH remarkably protected the t-BH- and rotenone-treated cybrids from cell death. In contrast, GSH was unable to increase the viability of cybrids exposed to metabolic stress.
These results suggest that GSH is an effective antioxidant compound to be tested as a potential treatment for LHON.
Investigative Ophthalmology & Visual Science 03/2008; 49(2):671-6. · 3.60 Impact Factor
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Claudia Zanna,
Anna Ghelli,
Anna Maria Porcelli,
Mariusz Karbowski,
Richard J Youle,
Simone Schimpf,
Bernd Wissinger,
Marcello Pinti,
Andrea Cossarizza,
Sara Vidoni,
Maria Lucia Valentino, Michela Rugolo,
Valerio Carelli
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ABSTRACT: Dominant optic atrophy (DOA) is characterized by retinal ganglion cell degeneration leading to optic neuropathy. A subset of DOA is caused by mutations in the OPA1 gene, encoding for a dynamin-related GTPase required for mitochondrial fusion. The functional consequences of OPA1 mutations in DOA patients are still poorly understood. This study investigated the effect of five different OPA1 pathogenic mutations on the energetic efficiency and mitochondrial network dynamics of skin fibroblasts from patients. Although DOA fibroblasts maintained their ATP levels and grew in galactose medium, i.e. under forced oxidative metabolism, a significant impairment in mitochondrial ATP synthesis driven by complex I substrates was found. Furthermore, balloon-like structures in the mitochondrial reticulum were observed in galactose medium and mitochondrial fusion was completely inhibited in about 50% of DOA fibroblasts, but not in control cells. Respiratory complex assembly and the expression level of complex I subunits were similar in control and DOA fibroblasts. Co-immunoprecipitation experiments revealed that OPA1 directly interacts with subunits of complexes I, II and III, but not IV and with apoptosis inducing factor. The results disclose a novel link between OPA1, apoptosis inducing factor and the respiratory complexes that may shed some light on the pathogenic mechanism of DOA.
Brain 03/2008; 131(Pt 2):352-67. · 9.46 Impact Factor
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ABSTRACT: alpha1-Adrenergic stimulation triggers glucose transport in the heart through the translocation of glucose transporter (GLUT) 1 and GLUT4 to plasma membranes, mediated by protein kinase C (PKC) isoforms. Evidence is emerging that dietary polyphenolic compounds may act not only as antioxidants but also by modulating PKC-mediated signaling. This study evaluated the ability of a green tea extract (GTE) to modulate alpha1-adrenoceptor-mediated glucose transport in rat cardiomyocytes. GTE supplementation decreased phenylephrine (PhE)-stimulated glucose uptake and GLUT4 recruitment. PhE stimulation activated PKC alpha, beta, delta, and epsilon, while GTE supplementation decreased the translocation of beta and delta isoforms, but not alpha and epsilon, supporting the notion that GTE directly affects PKC activation and is a beta and delta isoform-selective PKC inhibitor. Due to reactive oxygen species (ROS) involvement in pathological heart alterations, the observation that GTE is able to both inhibit effects originated by some PKC isoforms and counteract ROS deleterious effects could be important in the prevention/counteraction of these diseases.
Journal of Agricultural and Food Chemistry 10/2007; 55(18):7553-8. · 2.82 Impact Factor
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Giuseppe Gasparre,
Anna Maria Porcelli,
Elena Bonora,
Lucia Fiammetta Pennisi,
Matteo Toller,
Luisa Iommarini,
Anna Ghelli,
Massimo Moretti,
Christine M Betts,
Giuseppe Nicola Martinelli,
Alberto Rinaldi Ceroni,
Francesco Curcio,
Valerio Carelli, Michela Rugolo,
Giovanni Tallini,
Giovanni Romeo
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ABSTRACT: Oncocytic tumors are a distinctive class of proliferative lesions composed of cells with a striking degree of mitochondrial hyperplasia that are particularly frequent in the thyroid gland. To understand whether specific mitochondrial DNA (mtDNA) mutations are associated with the accumulation of mitochondria, we sequenced the entire mtDNA in 50 oncocytic lesions (45 thyroid tumors of epithelial cell derivation and 5 mitochondrion-rich breast tumors) and 52 control cases (21 nononcocytic thyroid tumors, 15 breast carcinomas, and 16 gliomas) by using recently developed technology that allows specific and reliable amplification of the whole mtDNA with quick mutation scanning. Thirteen oncocytic lesions (26%) presented disruptive mutations (nonsense or frameshift), whereas only two samples (3.8%) presented such mutations in the nononcocytic control group. In one case with multiple thyroid nodules analyzed separately, a disruptive mutation was found in the only nodule with oncocytic features. In one of the five mitochondrion-rich breast tumors, a disruptive mutation was identified. All disruptive mutations were found in complex I subunit genes, and the association between these mutations and the oncocytic phenotype was statistically significant (P=0.001). To study the pathogenicity of these mitochondrial mutations, primary cultures from oncocytic tumors and corresponding normal tissues were established. Electron microscopy and biochemical and molecular analyses showed that primary cultures derived from tumors bearing disruptive mutations failed to maintain the mutations and the oncocytic phenotype. We conclude that disruptive mutations in complex I subunits are markers of thyroid oncocytic tumors.
Proceedings of the National Academy of Sciences 06/2007; 104(21):9001-6. · 9.68 Impact Factor
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ABSTRACT: The aim of this work was to investigate the role of cytosolic calcium and calmodulin-dependent systems in the activation of glucose uptake in the human megakaryocytic cell line M07e. Glucose uptake was significantly raised by elevation of cytosolic Ca(2+) concentration ([Ca(2+)](c)) with thapsigargin, this effect being additive to the activation induced by cytokines (SCF, GM-CSF and TPO) and hydrogen peroxide. Intracellular Ca(2+) chelation by BAPTA decreased basal and activated glucose uptake in a dose-dependent manner. BAPTA reduced the GLUT1 translocation induced by SCF and H(2)O(2), suggesting a major role for Ca(2+) in GLUT1 intracellular trafficking. In the absence of extracellular Ca(2+), 2-aminoethoxydiphenyl-borate (2-APB) abolished the activation of glucose uptake induced by cytokines and H(2)O(2) suggesting an involvement in GLUT1 regulation in responses related to InsP(3)-induced Ca(2+) release. Under our experimental conditions, all the stimuli inducing glucose uptake activation failed to increase [Ca(2+)](c) suggesting that cytosolic Ca(2+) plays a permissive role in the regulation of GLUT1. The calmodulin antagonist W-7 and the inhibitor of Ca(2+)-calmodulin dependent protein kinase II (CAMK II) KN-62 removed the glucose transport activation by all the tested stimuli. These results suggest that in M07e cells calmodulin and CAMKII are involved in GLUT1 stimulation by cytokines and ROS.
Cell Calcium 11/2006; 40(4):373-81. · 3.77 Impact Factor
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Elena Bonora,
Anna Maria Porcelli,
Giuseppe Gasparre,
Annalisa Biondi,
Anna Ghelli,
Valerio Carelli,
Alessandra Baracca,
Giovanni Tallini,
Andrea Martinuzzi,
Giorgio Lenaz, Michela Rugolo,
Giovanni Romeo
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ABSTRACT: Oncocytic tumors are characterized by cells with an aberrant accumulation of mitochondria. To assess mitochondrial function in neoplastic oncocytic cells, we studied the thyroid oncocytic cell line XTC.UC1 and compared it with other thyroid non-oncocytic cell lines. Only XTC.UC1 cells were unable to survive in galactose, a condition forcing cells to rely solely on mitochondria for energy production. The rate of respiration and mitochondrial ATP synthesis driven by complex I substrates was severely reduced in XTC.UC1 cells. Furthermore, the enzymatic activity of complexes I and III was dramatically decreased in these cells compared with controls, in conjunction with a strongly enhanced production of reactive oxygen species. Osteosarcoma-derived transmitochondrial cell hybrids (cybrids) carrying XTC.UC1 mitochondrial DNA (mtDNA) were generated to discriminate whether the energetic failure depended on mitochondrial or nuclear DNA mutations. In galactose medium, XTC.UC1 cybrid clones showed reduced viability and ATP content, similarly to the parental XTC.UC1, clearly pointing to the existence of mtDNA alterations. Sequencing of XTC.UC1 mtDNA identified a frameshift mutation in ND1 and a nonconservative substitution in cytochrome b, two mutations with a clear pathogenic potential. In conclusion, this is the first demonstration that mitochondrial dysfunction of XTC.UC1 is due to a combined complex I/III defect associated with mtDNA mutations, as proven by the transfer of the defective energetic phenotype with the mitochondrial genome into the cybrids.
Cancer Research 07/2006; 66(12):6087-96. · 7.86 Impact Factor
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ABSTRACT: A study on Ca2+ transport by mitochondria isolated from Jerusalem artichoke (Helianthus tuberosus L. cv. OB1) tubers is presented. By following the distribution of Ca2+ under respiratory conditions, we have been able to show that Ca2+ accumulation into the matrix space depends on membrane potential (ΔΨ) since the uptake is not affected by the protonophore nigericin but fully blocked by valinomycin and carbonyl cyanide-p-trifluoromethoxy phenylhydrazone (FCCP). Ca2+ uptake requires phosphate (Pi) and is inhibited by mersalyl and by ruthenium red (RR). In addition to a Ca2+ influx route, mitochondria from H. tuberosus possess an RR-insensitive Ca2+ efflux pathway which is not stimulated by external Na+, Ca2+ is rapidly released from Ca2+-loaded mitochondria in the presence of ionophores such as A23187 and valinomycin and of the uncoupler FCCP. The Pi-transport inhibitor mersalyl also induces a massive Ca2+ release through reversal of the uptake route, the latter process being blocked by RR. Thus Jerusalem artichoke mitochondria possess a Ca2+ cycling mechanism which is different from that of animal mitochondria and certain other plant species.
Physiologia Plantarum 04/2006; 79(2):297 - 302. · 3.11 Impact Factor
