BRAIN A JOURNAL OF NEUROLOGY Mitochondrial DNA mutations affect calcium handling in differentiated neurons Andrew J. Trevelyan, 1,2,  Denise M. Kirby, 1,3,  ,† Tora K. Smulders-Srinivasan, 1 Marco Nooteboom, 1 Rebeca Acin-Perez, 4 Jose ´ Antonio Enriquez, 5 Miles A. Whittington, 2 Robert N. Lightowlers 1,2 and Doug M. Turnbull 1 1 Mitochondrial Research Group, Institute for Ageing and Health, Medical School, Newcastle University, Framlington Place, Newcastle upon Tyne, UK 2 Institute of Neuroscience, Medical School, Newcastle University, Framlington Place, Newcastle upon Tyne, UK 3 Mitochondrial and Metabolic Research, Murdoch Children’s Research Institute, Royal Children’s Hospital, Parkville,Melbourne, Australia 4 Department of Neurology and Neuroscience, Weill Medical School of Cornell University, New York, USA 5 Departamento de Bioquimica, Universidad de Zaragoza, Miguel Servet, Zaragoza, Spain. 6 Centro Nacional de Investigacio ´ nes Cardiovasculares Carlos III (CNIC), Melchor Fernandez Almagro, Madrid, Spain  These authors contributed equally to this work. † Sadly, Denise Kirby died after a short illness during the writing of this article and we would like to dedicate this publication to her memory. Correspondence to: Dr A. J. Trevelyan, Mitochondrial Research Group, Institute for Ageing and Health Medical School, Newcastle University, Framlington Place, Newcastle upon Tyne, NE2 4HH, England E-mail: a.j.trevelyan@gmail.com Correspondence may also be addressed to: Prof. D. M. Turnbull, E-mail: d.m.turnbull@ncl.ac.uk Mutations in the mitochondrial genome are associated with a wide range of neurological symptoms, but many aspects of the basic neuronal pathology are not understood. One candidate mechanism, given the well-established role of mitochondria in calcium buffering, is a deficit in neuronal calcium homoeostasis. We therefore examined calcium responses in the neurons derived from various ‘cybrid’ embryonic stem cell lines carrying different mitochondrial DNA mutations. Brief (50 ms), focal glutamatergic stimuli induced a transient rise in intracellular calcium concentration, which was visualized by bulk loading the cells with the calcium dye, Oregon Green BAPTA-1. Calcium entered the neurons through N-methyl-D-aspartic acid and voltage-gated calcium channels, as has been described in many other neuronal classes. Intriguingly, while mitochondrial muta- tions did not affect the calcium transient in response to single glutamatergic stimuli, they did alter the responses to repeated stimuli, with each successive calcium transient decaying ever more slowly in mitochondrial mutant cell lines. A train of stimuli thus caused intracellular calcium in these cells to be significantly elevated for many tens of seconds. These results suggest that calcium-handling deficits are likely to contribute to the pathological phenotype seen in patients with mitochondrial DNA mutations. Keywords: calcium; mitochondrial disease; SERCA; ATP; ageing; neurodegeneration Abbreviations: mtDNA = mitochondrial DNA; NMDA = N-methyl-D-aspartic acid; OGB = Oregon Green 488 BAPTA doi:10.1093/brain/awq023 Brain 2010: 133; 787–796 | 787 Received November 30, 2009. Revised January 12, 2010. Accepted January 13, 2010 ß The Author(s) 2010. Published by Oxford University Press on behalf of Brain. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.