A deafness-associated tRNA^Hismutation alters the mitochondrial function, ROS production and membrane potential

Abstract

In this report, we investigated the molecular genetic mechanism underlying the deafness-associated mitochondrial tRNAHis 12201T>C mutation. The destabilization of a highly conserved base-pairing (5A-68U) by the m.12201T>C mutation alters structure and function of tRNAHis. Using cybrids constructed by transferring mitochondria from lymphoblastoid cell lines derived from a Chinese family into mtDNA-less (ρo) 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. The failure in tRNAHis metabolism was responsible for the variable reductions in seven mtDNA-encoded polypeptides in mutant cells, ranging from 37 to 81%, with the average of ∼46% reduction, as compared with those of control cells. The impaired mitochondrial translation caused defects in respiratory capacity in mutant cells. Furthermore, marked decreases in the levels of mitochondrial ATP and membrane potential were observed in mutant cells. These mitochondrial dysfunctions caused an increase in the production of reactive oxygen species in the mutant cells. The data provide the evidence for a mitochondrial tRNAHis mutation leading to deafness.

Contribution of the tRNAIle 4317A→G mutation to the phenotypic manifestation of the deafness-associated mitochondrial 12S rRNA 1555A→G mutation

“…Indeed, mitochondrial membrane potentials reflect the pumping of hydrogen ions across the inner membrane during the process of electron transport and oxidative phosphorylation (49,59). In this study, 24% and 41% reductions in mitochondrial membrane potential in lymphoblastoid cell lines carrying the only m.1555A>G mutation and both m.1555A>G and m.4317A>G mutations was much lower than those in cell lines carrying the m.12201T>C mutation (8). The abnormal oxidative phosphorylation and mitochondrial membrane potential led to the increased production of reactive oxygen species and the subsequent failure of cellular energetic processes in mutant cells carrying m.1555A>G and m.4317A>G mutations.…”

“…Populations of cells were incubated in the media in the presence of glucose, and 2-deoxy-D-glucose with pyruvate (8). As shown in Figure 8A, in the presence of glucose (total cellular levels of ATP), the average levels of ATP production in mutant cells carrying both m.1555A>G and m.4317A>G mutations and only m.1555A>G mutation were 92 % (P=0.729) and 98 % (P=0.243), relative to the mean value measured in the control cell lines, respectively.…”

“…The ratio of fluorescence intensities Ex/Em=490/590 and 490/530 nm (FL590/FL530) were recorded to delineate the ΔΨm of each sample. The relative ratios of FL590/FL530 geometric mean between mutant and control cell lines were calculated to represent the level of ΔΨm, as described elsewhere (8). As shown in Figure 9, the levels of ΔΨm in mutant cell lines carrying both m.1555A>G and m.4317A>G mutations and only m.1555A>G mutation were 59% (P<0.001) and 76% (P<0.001) of the mean value measured in the control cell lines, respectively.…”

“…As a result, cochlear hair cells bearing both m.1555A>G and m.4317A>G mutations may be particularly sensitive to increased ATP demand (1,2,58). Furthermore, the deficient activities of respiratory chain complexes often impaired mitochondrial membrane potentials, which is a key indicator of cellular viability (8,10). Indeed, mitochondrial membrane potentials reflect the pumping of hydrogen ions across the inner membrane during the process of electron transport and oxidative phosphorylation (49,59).…”

“…DIG-labeled oligodeoxynucleotides were generated by using DIG oligonucleotide Tailing kit (Roche). The hybridization and quantification of density in each band were performed as detailed previously (8)(9)(10).…”