In the present study, to contribute to the understanding of the evolutionary history of sheep, the mitochondrial (mt) DNA polymorphisms occurring in modern Turkish native domestic (n = 628), modern wild (Ovis gmelinii anatolica) (n = 30) and ancient domestic sheep from Oylum Höyük in Kilis (n = 33) were examined comparatively with the accumulated data in the literature. The lengths (75 bp/76 bp) of the second and subsequent repeat units of the mtDNA control region (CR) sequences differentiated the five haplogroups (HPGs) observed in the domestic sheep into two genetic clusters as was already implied by other mtDNA markers: the first cluster being composed of HPGs A, B, D and the second cluster harboring HPGs C, E. To manifest genetic relatedness between wild Ovis gmelinii and domestic sheep haplogroups, their partial cytochrome B sequences were examined together on a median-joining network. The two parallel but wider aforementioned clusters were observed also on the network of Ovis gmelenii individuals, within which domestic haplogroups were embedded. The Ovis gmelinii wilds of the present day appeared to be distributed on two partially overlapping geographic areas parallel to the genetic clusters that they belong to (the first cluster being in the western part of the overall distribution). Thus, the analyses suggested that the domestic sheep may be the products of two maternally distinct ancestral Ovis gmelinii populations. Furthermore, Ovis gmelinii anatolica individuals exhibited a haplotype of HPG A (n = 22) and another haplotype (n = 8) from the second cluster which was not observed among the modern domestic sheep. HPG E, with the newly observed members (n = 11), showed signs of expansion. Studies of ancient and modern mtDNA suggest that HPG C frequency increased in the Southeast Anatolia from 6% to 22% some time after the beginning of the Hellenistic period, 500 years Before Common Era (BCE).

The mTOR inhibitor rapamycin ameliorates the clinical and biochemical phenotype of mouse, worm, and cellular models of mitochondrial disease, via an unclear mechanism. Here, we show that prolonged rapamycin treatment improved motor endurance, corrected morphological abnormalities of muscle, and increased cytochrome c oxidase (COX) activity of a muscle‐specific Cox15 knockout mouse (Cox15^sm/sm). Rapamycin treatment restored autophagic flux, which was impaired in naïve Cox15^sm/sm muscle, and reduced the number of damaged mitochondria, which accumulated in untreated Cox15^sm/sm mice. Conversely, rilmenidine, an mTORC1‐independent autophagy inducer, was ineffective on the myopathic features of Cox15^sm/sm animals. This stark difference supports the idea that inhibition of mTORC1 by rapamycin has a key role in the improvement of the mitochondrial function in Cox15^sm/sm muscle. In contrast to rilmenidine, rapamycin treatment also activated lysosomal biogenesis in muscle. This effect was associated with increased nuclear localization of TFEB, a master regulator of lysosomal biogenesis, which is inhibited by mTORC1‐dependent phosphorylation. We propose that the coordinated activation of autophagic flux and lysosomal biogenesis contribute to the effective clearance of dysfunctional mitochondria by rapamycin. Synopsis Mitochondrial diseases are a large family of genetic disorders for which no cure is currently available. Rapamycin, a TORC1‐dependent autophagy activator, ameliorates the phenotype a muscle‐specific Cox15^sm/sm mouse model of severe mitochondrial myopathy. Rapamycin increases the reduced autophagic flux in the skeletal muscle of Cox15^sm/sm mice. Rilmenidine, a mTORC1‐independent autophagy inducer, increases autophagy without rescuing the phenotype of Cox15^sm/sm mice. By inhibiting mTORC1, rapamycin induces the translocation of TFEB to the nucleus, inducing lysosomal biogenesis. The coordinated activation of autophagy and lysosomal biogenesis contributes to the beneficial effects of rapamycin in Cox15^sm/sm mice.

Alternative oxidases (AOXs) bypass respiratory complexes III and IV by transferring electrons from coenzyme Q directly to O2. They have therefore been proposed as a potential therapeutic tool for mitochondrial diseases. We crossed the severely myopathic skeletal muscle-specific COX15 knockout (KO) mouse with an AOX-transgenic mouse. Surprisingly, the double KO-AOX mutants had decreased lifespan and a substantial worsening of the myopathy compared with KO alone. Decreased ROS production in KO-AOX versus KO mice led to impaired AMPK/PGC-1α signaling and PAX7/MYOD-dependent muscle regeneration, blunting compensatory responses. Importantly, the antioxidant N-acetylcysteine had a similar effect, decreasing the lifespan of KO mice. Our findings have major implications for understanding pathogenic mechanisms in mitochondrial diseases and for the design of therapies, highlighting the benefits of ROS signaling and the potential hazards of antioxidant treatment.

Although mitochondrial dysfunction is often accompanied by excessive reactive oxygen species (ROS) production, we previously showed that an increase in random somatic mtDNA mutations does not result in increased oxidative stress. Normal levels of ROS and oxidative stress could also be a result of an active compensatory mechanism such as a mild increase in proton leak. Uncoupling protein 2 (UCP2) was proposed to play such a role in many physiological situations. However, we show that upregulation of UCP2 in mtDNA mutator mice is not associated with altered proton leak kinetics or ROS production, challenging the current view on the role of UCP2 in energy metabolism. Instead, our results argue that high UCP2 levels allow better utilization of fatty acid oxidation resulting in a beneficial effect on mitochondrial function in heart, postponing systemic lactic acidosis and resulting in longer lifespan in these mice. This study proposes a novel mechanism for an adaptive response to mitochondrial cardiomyopathy that links changes in metabolism to amelioration of respiratory chain deficiency and longer lifespan. Mitochondria produce the majority of the energy needed for numerous cell functions through oxidative phosphorylation. However, this comes with the cost in the form of potentially harmful reactive oxygen species (ROS) that could damage all kinds of biological macromolecules. Changes in mitochondrial membrane potential through mild uncoupling could alter ROS production in the cell (“uncoupling to survive”). Mitochondrial uncoupling proteins (UCPs) are believed to play a central role in this process. We detected increased amounts of UCP2 in mtDNA mutator mice, a model for premature aging. Depletion of UCP2 in mtDNA mutator mice led to further shortening of the lifespan with earlier signs of mitochondrial cardiomyopathy accompanied with high systemic lactic acidosis, often used as a marker of mitochondrial diseases. Remarkably, our results demonstrate that the presence of UCP2 wields beneficial effect on respiratory deficient mitochondria without affecting ROS production or uncoupling. Instead, UCP2 protein seems to mediate a valuable upregulation of fatty acid metabolism detected in mtDNA mutator hearts. Our results provide a novel mechanism of adaptation of mitochondria to respiratory deficiency mediated by UCP2 that clearly argues against the “uncoupling to survive” theory.