Drosophila adPEO Mutations Figure 5. Characterization during adult life of the wild type and W441C mutant d-mtDNA helicase. (A) Longevity curve of lines overexpressing the wild type and W441C mutants using a constitutive and ubiquitous driver (da-GAL4). 100 adult males (0–2 days old) were collected in an empty tube with standard medium and the number of surviving flies was counted every 2 days. Kaplan-Meier survival curve was performed (p = 0.045, n = 3 biological experiments). (B) Quantitation of mtDNA copy number by qRT-PCR at 5, 25 and 50 days of life in both lines. doi:10.1371/journal.pone.0043954.g005 span of these animals was slightly reduced as compared to animals overexpressing the wild type form. In the T7 gp4 enzyme, the amino acid orthologous to W441C in flies is not predicted to interact directly with the linker region and in theory, this mutant is able to form hexamers. However, in vitro helicase activity of the equivalent W474C mutant in human cells was reduced by 70%, leading to a decrease in mtDNA levels in vivo [6,13,39]. 2D-PAGE analysis showed that overexpression of this mutant produces an altered pattern of replication intermediates, suggestive of a specific effect in initiation. Furthermore, nucleoid morphology was perturbed in the W474C expressing cells, suggesting a mtDNA segregation defect [13]. The most obvious phenotype in the Drosophila lines over- expressing the K388A and A442P mutations is premature death in the larval and pupal stages, respectively, with a decrease in mtDNA copy number as compared with control lines. We explored the effect on mitochondrial function of this mtDNA depletion, and found reduced activity of complex IV, indicating an impairment of OXPHOS function. Increased levels of ROS have been documented to be a direct consequence of OXPHOS impairment [40]. Nonetheless, we found no evidence of an increase of ROS or of oxidative damage in any of the Drosophila lines overexpressing the d-mtDNA helicase mutants, consistent with observations in adPEO patients [41]. In fact, there are notable differences in comparing the effects produced by other mitochondrial-associated mutations in Drosophila. For instance, overexpression of the catalytic core of pol c, the product of the tamas gene, interferes with mtDNA replication leading to mtDNA depletion, OXPHOS defects and an increase in oxidative stress [28]. It is known that the mitochondrial quality control pathway is regulated strictly by various factors [22], such that defective mitochondria may be removed by mitophagy before the accumu- lation of ROS occurs, suggesting that different alterations in respiratory chain function may affect ROS production in diverse ways. A direct consequence of the overexpression of the K388A and A442P helicase mutants and likely due to the mtDNA depletion is an increase in apoptosis and a decrease in cell proliferation. We reported previously that silencing or overexpression of genes involved in replication and expression of mtDNA in Drosophila increases significantly the number of apoptotic cells [23,42]. It has also been shown that the mitochondria play a central role in the PLOS ONE | www.plosone.org 7 August 2012 | Volume 7 | Issue 8 | e43954