gene C10orf2 encodes the essential mitochondrial replicative DNA helicase, Twinkle. Twinkle shares high homology with the bacteriophage T7 gene 4 protein (T7 gp4), which contains both helicase and primase catalytic activities located in its carboxyl- and amino-terminal halves, respectively [6]. The amino acid sequence of the T7 gp4 helicase domain is well conserved in Twinkle, but varies substantially in the primase domain. Twinkle co-localizes with mtDNA structures designated as mitochondrial nucleoids [6], and it has been shown to modulate mtDNA copy number in vivo [11]. Studies in human cell culture and in a transgenic mouse expressing a Twinkle variant with an in-frame duplication of amino acids 353–365 that is analogous to a human adPEO mutation showed the accumulation of multiple mtDNA deletions and mitochondrial dysfunction [12,13]. Twinkle mutations are mainly associated with adPEO, but have recently been linked to SANDO [14] and Infantile-Onset Spinocerebellar Ataxia (IOSCA) [15]. Surprisingly, neither mtDNA deletions nor point mutations have been found, suggest- ing that IOSCA mutations in Twinkle affect mtDNA stability in a physiological and tissue specific manner. Recently, a novel protein with nuclease/helicase activity localized within the mitochondria has been described and designated as DNA2. In humans, hDNA2 forms a complex with the mitochondrial DNA polymerase to stimulate its DNA poly- merase activity. Although little is known about this protein, it appears to play a role in processing mtDNA intermediates during replication and repair [16]. However, only a single mtDNA helicase (d-mtDNA helicase) has been identified in Drosophila. Several studies have demonstrated that mitochondria play a crucial role in the apoptotic pathway in mammals [17], and thus mutations that affect mitochondrial function such as adPEO, could critically impact this pathway. Despite the fact that almost all of the proteins implicated in cell death are highly conserved in metazoans, the role of mitochondria in apoptosis in Drosophila remains controversial [18,19,20]. However, one study indicates a significant role for mitochondria in programmed cell death in the fly, where upon apoptosis, the Reaper and HID proteins cause mitochondrial fragmentation and release of cytochrome c in both cultured S2 cells and in the developing fly embryo [21]. It is known that factors involved in mitochondrial dynamics play a key role in the segregation of dysfunctional mitochondria [22], and deregulation of these pathways could trigger mitochondrially- mediated apoptosis. In this study, we overexpressed various adPEO mutations of d- mtDNA helicase in Drosophila melanogaster to characterize their effects in a tractable animal model, and to analyze the role of dysfunctional mitochondria in apoptosis. Our data show that overexpression of adPEO mutations in the fly causes a severe depletion of mtDNA and as a consequence, an increase in programed cell death. Taken together, our previous [23,24] and present results give evidence in vivo of the crucial role of mitochondria in apoptosis in Drosophila melanogaster, providing some clues for the understanding of the pathophysiology of human mitochondrial diseases. Results A Drosophila Model to Study adPEO Mutations Genomic analysis has identified a number of mutations associated with adPEO in the human mtDNA helicase gene, C10orf2 [6]. The Drosophila genome encodes a highly conserved orthologue in the CG5924 gene, with the two proteins sharing 54.6% identity and 73% similarity (as evaluated by Mobyle Pasteur MATCHER). Most of the amino acid residues mutated in Drosophila adPEO Mutations adPEO patients are conserved in the Drosophila protein [24], raising the possibility to establish Drosophila as a tractable animal model to study them. With this aim, we used the inducible UAS- GAL4 system to express d-mtDNA helicase variants containing human adPEO mutations. UAS lines with four versions of the gene were generated: the wild type (wt) version was used as a control, and three versions containing the ‘‘Walker A’’ [27] K388A active site mutation, analogous to K318 in the helicase domain of T7 gp4, and the W441C and A442P mutations, which are analogous to the W474C and A475P mutations found in adPEO patients [6]. These mutations map within the linker region and helicase domain, respectively (Figure 1). Previous results in Drosophila S2 cells showed severe mtDNA depletion when d- mtDNA helicase containing the K388A or A442P mutations were overexpressed, whereas cells that overexpressed d-mtDNA helicase containing the W441C mutation did not show a significant phenotype. In contrast to patients harbouring these mutations, multiple deletions in mtDNA were not observed in cultured fly cells [24]. To characterize the effects of overexpression of the variants of d-mtDNA helicase in Drosophila, the daughterless-GAL4 driver line was used to induce constitutive and ubiquitous expression. Overexpression of the K388A and A442P d-mtDNA helicase mutations caused complete lethality in third larval instar and pupal stages, respectively (Figure 2C–E). However, the over- expression of W441C variant and the wild type version did not show any observable phenotype (Figure 2, D). These results were quantified (Figure 2F) and corroborated with multiple independent lines for each mutation. Immunoblot analysis of third instar larvae expressing the d-mtDNA helicase variant and control lines showed relatively similar levels of d-mtDNA helicase protein among the different lines; in contrast, d-mtDNA helicase was undetectable in non-transgenic control animals under the same conditions indicating normally low levels of endogenous expression and the relatively high levels of transgene expression (Figure 2G). Constitutive Overexpression of Mutant d-mtDNA Helicase Induces mtDNA Depletion and OXPHOS Impairment in Drosophila The effect of overexpression of d-mtDNA helicase variants on mtDNA levels was evaluated by quantitative real time PCR analysis. We found a significant decrease in mtDNA copy number in third instar larvae of the lines overexpressing K388A, W441C and A442P mutations as compared with the control lines (Figure 3A). Overexpression of wild type d-mtDNA helicase did not cause decrease in mtDNA. However, despite the presence of multiple deletions in mtDNA being a common feature in skeletal muscle in adPEO patients, we found no evidence by Southern Blot (Figure 3B) and long range-PCR analysis (data not shown) of multiple deletions in the mutant or control lines. These results are comparable with data analysing overexpression of d-mtDNA helicase active site mutants in Drosophila Schneider cells [24]. However, in contrast to the in vitro study [24], we also observed significantly decreased levels of mitochondrial transcripts in vivo (Figure 3C). To analyze the biochemical consequence of the mtDNA depletion and the decrease in mtDNA-encoded tran- scripts caused by mutated d-mtDNA helicase, we measured the enzymatic activity of cytochrome c oxidase (complex IV) in larvae of the control and mutant lines. As expected, we observed a significant decrease in complex IV activity in the K388A and A442P expressing lines (Figure 4A), which showed a clear impairment of the OXPHOS function. Complex IV activity in the W441C line was similar to the control line, consistent with the PLOS ONE | www.plosone.org 2 August 2012 | Volume 7 | Issue 8 | e43954