Modeling Pathogenic Mutations of Human Twinkle in Drosophila Suggests an Apoptosis Role in Response to Mitochondrial Defects Alvaro Sanchez-Martinez1,2¤a, Manuel Calleja3, Susana Peralta1,2¤b, Yuichi Matsushima4¤c, Rosana Hernandez-Sierra1,2, Alexander J. Whitworth5, Laurie S. Kaguni4, Rafael Garesse1,2* 1 Departamento de Bioqu´ımica, Instituto de Investigaciones Biome´dicas ‘‘Alberto Sols’’ Universidad Auto´ noma de Madrid-Consejo Superior de Investigaciones Cient´ıficas, and Centro de Investigacio´ n Biome´dica en Red en Enfermedades Raras, Facultad de Medicina, Universidad Auto´ noma de Madrid, Madrid, Spain, 2 Instituto de Investigacio´ n Santitaria Hospital 12 de Octubre (i+12), Madrid, Spain, 3 Centro de Biolog´ıa Molecular ‘‘Severo Ochoa’’ Universidad Auto´ noma de Madrid-Consejo Superior de Investigaciones Cient´ıficas, Madrid, Spain, 4 Department of Biochemistry and Molecular Biology and Center for Mitochondrial Science and Medicine, Michigan State University, East Lansing, Michigan, United States of America, 5 Department of Biomedical Sciences, MRC Centre for Developmental and Biomedical Genetics, University of Sheffield, Sheffield, United Kingdom Abstract The human gene C10orf2 encodes the mitochondrial replicative DNA helicase Twinkle, mutations of which are responsible for a significant fraction of cases of autosomal dominant progressive external ophthalmoplegia (adPEO), a human mitochondrial disease caused by defects in intergenomic communication. We report the analysis of orthologous mutations in the Drosophila melanogaster mitochondrial DNA (mtDNA) helicase gene, d-mtDNA helicase. Increased expression of wild type d-mtDNA helicase using the UAS-GAL4 system leads to an increase in mtDNA copy number throughout adult life without any noteworthy phenotype, whereas overexpression of d-mtDNA helicase containing the K388A mutation in the helicase active site results in a severe depletion of mtDNA and a lethal phenotype. Overexpression of two d-mtDNA helicase variants equivalent to two human adPEO mutations shows differential effects. The A442P mutation exhibits a dominant negative effect similar to that of the active site mutant. In contrast, overexpression of d-mtDNA helicase containing the W441C mutation results in a slight decrease in mtDNA copy number during the third instar larval stage, and a moderate decrease in life span in the adult population. Overexpression of d-mtDNA helicase containing either the K388A or A442P mutations causes a mitochondrial oxidative phosphorylation (OXPHOS) defect that significantly reduces cell proliferation. The mitochondrial impairment caused by these mutations promotes apoptosis, arguing that mitochondria regulate programmed cell death in Drosophila. Our study of d-mtDNA helicase overexpression provides a tractable Drosophila model for understanding the cellular and molecular effects of human adPEO mutations. Citation: Sanchez-Martinez A, Calleja M, Peralta S, Matsushima Y, Hernandez-Sierra R, et al. (2012) Modeling Pathogenic Mutations of Human Twinkle in Drosophila Suggests an Apoptosis Role in Response to Mitochondrial Defects. PLoS ONE 7(8): e43954. doi:10.1371/journal.pone.0043954 Editor: Yidong Bai, University of Texas Health Science Center at San Antonio, United States of America Received June 13, 2012; Accepted July 27, 2012; Published August 28, 2012 Copyright: 2012 Sanchez-Martinez et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by grants from the Centre for Biomedical Research on Rare Diseases, Instituto de Salud Carlos III (grant number PI 07/0167), and the Comunidad de Madrid (grant number GEN-0269/2006) to RG, and the National Institutes of Health (grant number GM45295) to LSK. ASM was a Fellow from the Comunidad de Madrid. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: rafael.garesse@uam.es €a Current address: Department of Biomedical Sciences, MRC Centre for Developmental and Biomedical Genetics, University of Sheffield, Sheffield, United Kingdom €b Current address: Department of Neurology and Cell Biology and Anatomy, University of Miami Miller School of Medicine, Miami, Florida, United States of America €c Current address: Department of Mental Retardation & Birth Defect Research, National Institute of Neuroscience, National Center of Neurology & Psychiatry, 4-1- 1 Ogawahigashi, Kodaira, Tokyo, Japan Introduction The majority of cellular ATP is generated by oxidative phosphorylation carried out within the mitochondrial inner membrane. Animal mitochondrial DNA encodes 13 polypeptides involved in OXPHOS, whereas all the factors essential for mtDNA replication are encoded in the nuclear genome [1]. As a consequence, the biogenesis of the OXPHOS system is subject to a highly-coordinated, dual genetic control [2]. Mutations in genes encoding the factors essential for mtDNA replication results in both loss of mtDNA integrity via base substitution mutations, duplications and deletions, and in mtDNA depletion [3]. Autosomal dominant progressive external ophthalmoplegia is a human mitochondrial disease associated with the presence of multiple deletions in the mtDNA [3,4]. The disease has an adult- onset at 20–40 years of age. Its symptoms include muscle weakness, wasting, exercise intolerance, ataxia, hearing loss, cardiomyopathy and peripheral neuropathy [3]. Most adPEO families carry heterozygous mutations in one of three genes: ANT1 (adenine nucleotide translocator 1), POLG (mitochondrial DNA polymerase), or C10orf2/Twinkle (mtDNA helicase) [3,5,6,7]. Whereas the nuclear DNA replication machinery is very complex, the number of factors needed to replicate the mitochondrial DNA is comparatively small [8,9,10]. The human PLOS ONE | www.plosone.org 1 August 2012 | Volume 7 | Issue 8 | e43954