Drosophila adPEO Mutations Figure 3. Differential changes in mtDNA copy number and mitochondrial transcript levels. (A) Relative mtDNA copy number was measured by qRT-PCR in third instar larvae of Drosophila lines expressing mutant d-mtDNA helicase. (B) Total DNA (10 mg) was fractionated in an 0.8% agarose gel, blotted to nylon membrane, and hybridized with radiolabeled probes for the mtDNA to observe multiple deletions. (C) Relative mitochondrial mRNA levels of the ND5 and COX I genes was measured by qRT-PCR. Data represent the mean 6 s.d., *P#0.05, **P#0.005 as compared with wild-type (w1118). doi:10.1371/journal.pone.0043954.g003 we employed the UAS-GAL4 system to examine the effect of the K338A, W441C, and A442P mutations in d-mtDNA helicase, and have established a Drosophila model of nucleo-mitochondrial communication defect, in a system in which the physiological conditions of the whole organism can be evaluated to understand pathophysiological disorders. We observed a severe decrease in mtDNA copy number and mtDNA-encoded transcripts in the K388A and A442P mutant lines, which caused lethality at the third larval instar and pupal stages, respectively. However, the overexpression of the mutant W441C caused only slight mtDNA depletion without apparent developmental effects. Interestingly, unlike the findings reported for adPEO patients [29], and in line with the observations in human cells [13], in the Drosophila system, mtDNA deletions were not found either in S2 cell lines or in animals. A likely explanation might be that the generation of deleted molecules in Drosophila cells is slower than in mammals. Another explication for the findings observed in this and previous studies [13] could be that replication stalling caused by overexpression of mutant versions of Twinkle might induce an increased rate of mtDNA turnover, via mitophagy [12,30]. The molecular mechanism by which a dominant mutation in mtDNA helicase generates multiple mtDNA deletions is unknown [31], and the accumulation of deleted mtDNA molecules varies widely in different postmitotic tissues in adPEO patients [29,32]. Similarly, in a transgenic mouse model expressing a pathogenic adPEO duplication in the Twinkle/C10orf2 gene, mtDNA deletions were detected only after 18 months of life [12] suggesting that mtDNA defects may be a direct consequence of age-associated deterioration of the repair mechanisms (discussed in [13]). Despite of this discrepancy, and in contrast to that observed in human patients, the absence of mtDNA deletions is a common observa- tion in other genetics defects affecting the nDNA-mtDNA intergenomic communication [33,34,35]. In adPEO patients, many of the mutations in the Twinkle gene map within a small region, which corresponds to the linker region of bacteriophage T7 gp4. The linker region separates the primase and helicase domains and is important for hexamer formation [36]. We found that the A442P mutant displays a dominant negative effect like the helicase active site mutant K338A. Lys338 is analogous to Lys318 in T7 gp4, and is not essential for T7 gp4 PLOS ONE | www.plosone.org 5 August 2012 | Volume 7 | Issue 8 | e43954