Distinct Roles of the Mitochondrial DNA Helicase Twinkle in Drosophila Neurons and Muscles

Abstract

Mutations in the human gene TWNK, which encodes the mitochondrial DNA (mtDNA) helicase Twinkle, frequently affect the neurolocomotor system. Patients may present accumulation of multiple large-scale mtDNA deletions in muscle fibers, cerebral cortex, basal ganglia, and cerebellum throughout lifetime, as observed in cases of autosomal dominant progressive external ophthalmoplegia (adPEO). Specially in high energy demanding tissues such as muscles and neurons, mtDNA deletions and/or depletion can affect mitochondrial function, since this genome plays a central role in ATP production by means of oxidative phosphorylation (OXPHOS). Maintaining this genome intact and at the appropriate level requires an array of proteins involved in replication, transcription, repair, and recombination. Although Twinkle may participate in recombinational repair, its roles in the replication process is best understood. We have been using Drosophila melanogaster as a model to understand the role of Twinkle in mtDNA maintenance in a tissue-specific pattern, with focus on the central nervous system and the musculature. We have shown that defective versions of Twinkle that are ubiquitously expressed cause lethality at the larval stage, but directing the expression only to neurons or to muscles does not impact development. Furthermore, our results indicate that neuron-specific, but not muscle-specific, expression of ATPase-deficient versions of Twinkle leads to a dramatic shortening of adult lifespan, which is accompanied by severe locomotor problems. The lack of phenotype upon Twinkle dysfunction in muscles, however, is unexpected considering the high OXPHOS activity and high mtDNA copy number of the Drosophila flight muscles. We also overexpressed Twinkle mutants in Drosophila S2 cells in culture to get a first insight into how mtDNA maintenance may be disturbed. We have previously shown that overexpressing wild-type (WT) Twinkle leads to a decrease in intermediates at two important replication pausing regions in the mitochondrial genome. Interestingly, our preliminary findings indicate that the overexpression of an ATPase-deficient Twinkle mutant causes severe accumulation of replication intermediates at the same pausing sites. Because in whole adult fly samples the vast majority of mitochondria (and therefore mtDNA) come from the flight musculature, it is safe to assume that the mode of mtDNA replication previously described for adult individuals is in fact the mode for muscle mtDNA. In this case, it is intriguing that mtDNA maintenance in S2 cells and in adult flight muscles is so similar, and yet a severely defective Twinkle enzyme causes a strong mtDNA depletion in the former and no detectable phenotype in the latter. Since there is no available effective treatment for adPEO and other mtDNA diseases, determining if, how and when Twinkle participates in DNA maintenance and repair inside mitochondria may provide insights into how to regulate Twinkle's functions to improve conditions of mitochondrial dysfunction.