Mitochondrial oxygen consumption in the central nervous system and in the musculature of Twinkle mutant Drosophila lines

Abstract

More than 30 mutations have been described in the human TWNK gene, which encodes the Twinkle mitochondrial replicative helicase. These mutations have heterogeneous clinical consequences, but generally affect the neurolocomotor system. Autosomal dominant progressive external ophthalmoplegia (adPEO), often caused by mutations in TWNK, is a late-onset neuromuscular disorder in which patients may present with progressive weakness of the external eye muscles and/or skeletal muscles, sensory axonal neuropathy, ataxia, parkinsonism, and depression. adPEO is mainly characterized by the large-scale accumulation of several deletions in the mitochondrial DNA (mtDNA) of muscle fibers during the life of patients, but deletions have also been observed in different regions of the central nervous system. Although adPEO is a relatively rare disease, it represents an important model, as mtDNA deletions increase in frequency in neurons and muscle fibers of normal human subjects as they age. Keeping the mitochondrial genome intact and at the proper level requires a number of proteins involved in replication, transcription, repair and recombination. Although Twinkle may participate in recombinational repair, its roles in the mtDNA replication process are better understood. When mtDNA is being copied, Twinkle translocates ahead of the replication fork, hydrolyzing NTPs and opening the parental mtDNA double strand. Unpublished data from our laboratory indicate that Twinkle has tissue-specific functions in drosophila, which has been widely used as a model organism for understanding the processes related to mtDNA replication. The aim of this project is to measure mitochondrial oxygen consumption in heads and thoraxes of drosophila strains with mutations in Twinkle, following a protocol already well established in our laboratory. The measurement of oxygen consumption associated with the oxidation of mitochondrial substrates will be performed in real time using the high-resolution respirometry system Oxygraph-2k (O2k, Oroboros Instruments). We will evaluate oxidative phosphorylation, maximum electron transfer capacity and respiration leak. The level of coupling between oxygen consumption and ADP phosphorylation will be estimated through the respiratory control rate, for which the respiration value in the OXPHOS state will be divided by the leak value. (AU)