MELAS mitochondrial DNA mutation A3243G reduces glutamate transport in cybrids cell lines Jacopo C. DiFrancesco a,c,d, ⁎, J. Mark Cooper d , Amanda Lam d , Paul E. Hart d , Lucio Tremolizzo a,b , Carlo Ferrarese a,b,c , Antony H. Schapira d,e 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 article info abstract Article history: Received 20 November 2007 Revised 10 March 2008 Accepted 15 March 2008 Available online 26 March 2008 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 [ 3 H]-glutamate influx mediated by specific excitatory amino acid transporters (EAATs). The maximal rate (V max ) 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. © 2008 Elsevier Inc. All rights reserved. Keywords: MELAS A3243G Excitotoxicity Glutamate Mitochondria Cybrid Introduction Mitochondrial encephalomyopathies are a diverse range of dis- orders caused by a number of different mutations of either the mitochondrial or nuclear genomes (DiMauro and Schon, 2003; Schapira, 2006). The MELAS phenotype may be caused by several different mitochondrial DNA (mtDNA) mutations of which the A3243G is the most common (Goto et al., 1990). The clinical effects are diverse and encompass not only the full MELAS phenotype, but also mono- symptomatic features such as deafness or diabetes mellitus. The stroke- like episodes represent one of the most important clinical features of MELAS. Their pathogenesis is likely to be multifactorial and it is still debated: the ischemic vascular hypothesis suggests they are caused by “mitochondrial angiopathy” and the generalized cytopathic theory proposes that neuronal hyperexcitability may initiate, then maintain and develop the cascade of stroke-like events caused by “mitochondrial cytopathy”. Once neuronal hyperexcitability is developed in a localized brain region, this could depolarize the adjacent neurons, spreading at the surrounding cortex, in agreement with the non vascular distribu- tion of the stroke-like events (Iizuka and Sakai, 2005). The molecular consequences of the A3243G mutation are not completely understood, but may include effects on both transcription and translation of mtDNA. In fact, this mutation has been linked to a marked decrease in both the rates of synthesis and the steady-state levels of the mitochondrial translation products; moreover a small but consistent increase in the levels of an unprocessed RNA containing the tRNA Leu(UUR) sequence (RNA 19) has been reported and it could contribute to the observed inhibition of mitochondrial protein synthesis (King et al., 1992). Other authors have shown a diminished steady-state level of the tRNA Leu(UUR) in mutant cellular models. This has been linked to post-transcriptional modifications such as diminution of methylation, which could be responsible for a slower processing rate of the precursor transcript and may accelerate the rate Experimental Neurology 212 (2008) 152–156 Abbreviations: Cybrids, cytoplasmic hybrids; DHE, dihydroethidium; DHR, dihy- drorhodamine 123; EAATs, excitatory amino acid transporters; K m , 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; V max , glutamate uptake maximal rate. ⁎ Corresponding author. University of Milano-Bicocca, Department of Neuroscience – Section of Neurology, Via Cadore, 48 – 20052 Monza (MI) – Italy. Fax: +39 02 6448 8108. E-mail address: jacopo.difrancesco@unimib.it (J.C. DiFrancesco). 0014-4886/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.expneurol.2008.03.015 Contents lists available at ScienceDirect Experimental Neurology journal homepage: www.elsevier.com/locate/yexnr