MELAS mitochondrial DNA mutation A3243G reduces glutamate transport in cybrids cell lines |
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Authors: | DiFrancesco Jacopo C Cooper J Mark Lam Amanda Hart Paul E Tremolizzo Lucio Ferrarese Carlo Schapira Antony H |
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Institution: | a Department of Neuroscience and Biomedical Technologies, University of Milano-Bicocca, Monza, Italy b Department of Neurology, San Gerardo Hospital, Monza, Italy c Scientific Institute “E. Medea”, Bosisio Parini (LC), Italy d University Department of Clinical Neurosciences, Royal Free and University College Medical School, London, UK e Institute of Neurology, University College London, London UK |
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Abstract: | MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes) is commonly associated with the A3243G mitochondrial DNA (mtDNA) mutation encoding the transfer RNA of leucine (UUR) (tRNA Leu(UUR)). The pathogenetic mechanisms of this mutation are not completely understood. Neuronal functions are particularly vulnerable to alterations in oxidative phosphorylation, which may affect the function of the neurotransmitter glutamate, leading to excitotoxicity. In order to investigate the possible effects of A3243G upon glutamate homeostasis, we assessed glutamate uptake in osteosarcoma-derived cytoplasmic hybrids (cybrids) expressing high levels of this mutation. High-affinity Na+-dependent glutamate uptake was assessed as radioactive 3H]-glutamate influx mediated by specific excitatory amino acid transporters (EAATs). The maximal rate (Vmax) of Na+-dependent glutamate uptake was significantly reduced in all the mutant clones. Although the defect did not relate to either the mutant load or magnitude of oxidative phosphorylation defect, we found an inverse relationship between A3243G mutation load and mitochondrial ATP synthesis, without any evidence of increased cellular or mitochondrial free radical production in these A3243G clones. These data suggest that a defect of glutamate transport in MELAS neurons may be due to decreased energy production and might be involved in mediating the pathogenic effects of the A3243G mtDNA mutation. |
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Keywords: | Cybrids cytoplasmic hybrids DHE dihydroethidium DHR dihydrorhodamine 123 EAATs excitatory amino acid transporters Km glutamate uptake affinity LHON Leber's hereditary optic neuropathy MELAS mitochondrial myopathy encephalopathy lactic acidosis and stroke-like episodes mtDNA mitochondrial genome ROS reactive oxygen species THA l-(-)-threo-3-hydroxyaspartic acid Vmax glutamate uptake maximal rate |
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