Drosophila adPEO Mutations Figure 1. Sequence alignment and location of amino acid substitutions in mtDNA helicase. Schematic diagram of the sequence organization of mtDNA helicase; eight amino acid sequence motifs common to ring primases and helicases are indicated in gray. The bacteriophage T7 gp4 linker region is indicated in the middle of the diagram. Mutations in the human mtDNA helicase gene (Twinkle/C10orf2) found in adPEO are shown above the scheme (reviewed in [46]), the orthologous adPEO mutations W441C and A442P in the Drosophila mtDNA helicase gene (d-mtDNA helicase), and the ‘‘Walker A’’ mutation K388A are shown below the scheme. The underlined mutations indicate autosomal recessive PEO mutations. The positions of mutations in d-mtDNA helicase used in this study are shown in bold. Sequence alignment of the regions containing altered amino acids is shown in the lower panel. Dm, fly; Ag, mosquito; Mm, mouse; Hs, human; T7, bacteriophage T7. doi:10.1371/journal.pone.0043954.g001 lack of an apparent phenotype produced by the moderate decrease in mtDNA levels. To evaluate if the OXPHOS impairment produces any change in the levels of reactive oxygen species (ROS), we employed two different approaches: we used the MitoSox probe that binds specifically to the mitochondrial superoxide anion, and reactive dihydroethidium (DHE), which permeates cell membranes freely, as indicators of cytosolic superoxide production. Neither approach revealed any significant different between the control lines and the overexpression of the d-mtDNA helicase variants, even in the presence of OXPHOS impairment (Figure 4B, C). This finding is consistent with that observed in adPEO patients, where no increase of ROS was found. Constitutive Overexpression of W441C d-mtDNA Helicase does not Impair OXPHOS Function, but Slightly Reduces Life Span Patients carrying adPEO mutations in the Twinkle gene have adult onset symptoms between the ages of 20 and 40 years, suggesting that these mutations are only moderately deleterious for enzyme function. Expression of the W441C mutation at levels comparable to those of the other pathogenic mutations did not produce development defects, despite causing mild mtDNA depletion in the third instar larval stage. Hence, this line allowed us to study the effects of mutant d-mtDNA helicase in adult Drosophila. First, we analyzed its effects on lifespan, where we observed a slight decrease in longevity in the W441C flies as compared to those overexpressing wild type d-mtDNA helicase (Figure 5A). Despite observing a decrease in mtDNA in the larval stage, mtDNA depletion was not observed in 5, 25 or 50 day-old adults expressing the W441C mutation, arguing that normal mtDNA levels are restored in somatic cells. In contrast, we found a slight increase in the level of mtDNA throughout adult life in the wild type overexpressing line (Figure 5B), an observation consistent with the results reported upon overexpression or Twinkle in mice [11], reinforcing the idea that the mtDNA helicase is limiting for mtDNA replication in animals. d-mtDNA Helicase Mutations Decrease Cell Proliferation and Increase Apoptosis in vivo Various mutations in the d-mtDNA helicase gene cause a significant depletion of mtDNA. We have shown previously that impairment of mtDNA replication and/or maintenance in Drosophila causes mtDNA depletion and an increase in apoptosis [23,28]. To address this in our d-mtDNA helicase mutant lines, we analysed the levels of apoptosis and cell proliferation in the wing imaginal discs of third instar larvae, in lines overexpressing the variants of d-mtDNA helicase under the control of different GAL4-drivers. We used anti-caspase3 immunocytochemistry to highlight cells undergoing apoptosis, and anti-phosphohistone antibody to label proliferating cells. Compared with controls, wing imaginal discs from the K338A and A442P mutant lines showed an increase in apoptosis and a decrease in cell proliferation (Figure 6A–L). In contrast, the phenotypes of the lines overexpressing the wild type version and W441C mutant were similar to that of controls (Figure 6K, L). To corroborate these results, we overexpressed the variants of the d-mtDNA helicase in the posterior compartment of wing discs using the driver en-GAL4 and evaluated apoptosis such that the anterior compartment could be used as an internal control with the same cellular background. As shown in Figure 6, we observed an increase in apoptotic clusters in the posterior compartment (Figure 6N,P) as compared to the anterior compartment, suggesting that the elevated levels of apoptosis are due specifically to the documented mitochondrial dysfunction, arguing that mitochondria are key players in this pathway. PLOS ONE | www.plosone.org 3 August 2012 | Volume 7 | Issue 8 | e43954