| Deafness is one of the major public health problems, affecting360million persons worldwide. Mitochondrial tRNA genes are one of the hot spot mutation area in mitochondrial DNA associated with maternally inherited deafness. In our previous study, we found a large Han Chinese pedigree subjected with maternally transmitted non-syndromic deafness carrying the m.12201T>C mutation in the tRNAHis gene. The m.12201T>C mutation destabilizes a highly conservative base pairing (5A-68U) on the acceptor stem of tRNAHis. The paper further explored the molecular pathogenesis of m.12201T>C mutation, involved how m.12201T>C mutation altered mitochondrial function, and how m.12201T>C mutation affected autophagy and mitophagy.Using cytoplasmic hybrid (cybrids) constructed by transferring mitochondria from lymphoblastoid cell lines derived from a Chinese family into mtDNA less (p0) cells. We showed-70%decrease in the steady-state level of tRNAHis in mutant cybrids, compared with control cybrids. The mutation changed the conformation of tRNAHis, as suggested by slower electrophoretic mobility of mutated tRNA with respect to the wild-type molecule. However,-60%increase in aminoacylated level of tRNAHis was observed in mutant cells that may be due to the instability of the mutant tRNA, where aminoacylation may provide some level of stabilization by compensatory effect. Alternatively, the mutant tRNAHis improperly charged by cognate amino acid(s) may contribute to the increasing aminoacylation level of tRNAHis.The failure in tRNAHis metabolism was responsible for the variable reductions in mtDNA-encoded polypeptides in mutant cells, ranging from37%to81%, with the average of-46%reduction, as compared with those of control cells. The impaired mitochondrial translation caused defects in stability of mitochondrial respiratory chain complexes and mitochondrial oxidative phosphorylation capacity in mutant cells. The basal OCR, ATP-linked OCR and maximal OCR in mutant cell lines were-58,56, and57%, relative to the control cell lines.31%drop in mitochondrial ATP production in cybrid carrying the m.12201T>C mutation. Furthermore, the deficient activities of respiratory chain complexes caused by tRNA mutations altered mitochondrial membrane potentials with80%reduction, also32%increase the ROS production.Autophagy serves as an essential cellular quality control mechanism, function in cells for the selective degradation of large-scale protein aggregates and organelles, including mitochondria. The only autophagy pathway needed to form a bilayer structure autophagosome and through the fusion of lysosomes to degrade the substrate called macroautophagy, which the major pathway of mitophagy. In this study, we examined autophagic status in human cybrid cell lines carrying deafness associated m.12201T>C mutation. Obvious decreases of macroautophagy level (-48%) and mitophagy level (-52%) were observed in the mutant cybrids compared with control cybrids. In addition, protein expression levels of key mitophagy protein PINK1and Parkin in the mutant cells were decreased by38%and33%, respectively. When macroautophagy is inhibited by lysosomal proteases such as E64d and pepstatin A, the accumulation rate of autophagosome protein in mutants was faster than the controls, consistent with the protein encoded by the mitochondria. We speculate that, on the one hand, in order to avoid excessive autophagy leading to apoptosis or necrosis, macroautophagy and mitophagy levels were reduced in the mutant cells; On the other hand, as to control the quality of the mitochondria, the mutant cells accelerated the rate of degradation of the mitochondria with protein synthesis defect, maintaining intracellular homeostasis.In summary, this study elaborated the molecular mechanism of m.12201T>C mutation causing mitochondrial dysfunction, while the introduction of mitophagy research provides a new perspective for the pathogenesis of maternally inherited deafness and other mitochondrial diseases. |
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