Functional characterization of the mitochondrial 12S rRNA C1494T mutation associated with aminoglycoside-induced and non-syndromic hearing loss

Abstract

In this study, we report the biochemical characterization of the deafness-associated mitochondrial 12S rRNA C1494T mutation using 27 cybrid cell lines constructed by transferring mitochondria from 9 lymphoblastoid cell lines derived from a Chinese family into human mitochondrial DNA (mtDNA)-less (rho degrees) cells. Six cybrids derived from two asymptomatic members, and nine cybrids derived from three symptomatic members of the Chinese family carrying the C1494T mutation exhibited approximately 38 and 43% decrease in the rate of mitochondrial protein labeling, respectively, compared with twelve cybrids derived from four Chinese control individuals. These defects are apparently a primary contributor to significant reductions in the rate of overall respiratory capacity or the rate of malate/glutamate promoted respiration, or succinate/G3P-promoted respiration, or TMPD/ascorbate-promoted respiration in mutant cybrid cell lines derived from either symptomatic or asymptomatic individuals. Furthermore, the very significant/nearly identical increase in the ratio of doubling times in DMDM medium in the presence/absence of high concentration of paromomycin was observed in symptomatic or asymptomatic cybrid cell lines carrying the C1494T mutation as compared with the average rate in control cell lines. These observations provide the direct biochemical evidences that the C1494T mutation is a pathogenic mtDNA mutation associated with aminoglycoside-induced and non-syndromic hearing loss. In addition, these data provide the first biochemical evidence that nuclear background plays a critical role in the phenotypic manifestation of non-syndromic hearing loss and aminoglycoside toxicity associated with the C1494T mutation.

Figure 1

The location of the C1494T mutation in the decoding region of small ribosomal RNAs. The A-site of human mitochondrial 12S rRNA is shown as the wild type version (A) and the version containing the A1555G mutation (B) and C1494T mutation (C), respectively. The sites for the A1555G or C1494T mutation are indicated by arrows.

Figure 2

Electrophoretic patterns of the mitochondrial translation products of cybrids and of 143B.TK⁻ cells labeled for 30 min with [³⁵S]methionine in the presence of 100 μg/ml of emetine. Samples containing equal amounts of protein (20 μg) were run in SDS/polyacrylamide gradient gels. The two panels represent electrophoretic patterns obtained in separate gel runs, each one including the 143B.TK⁻ control for normalization purposes. COI, COII and COIII, subunits I, II and III of cytochrome c oxidase; ND1, ND2, ND3, ND4, ND4L, ND5 and ND6, subunits 1, 2, 3, 4, 4L, 5 and 6 of the respiratory chain NADH dehydrogenase; A6 and A8, subunits 6 and 8 of the H⁺-ATPase; and CYTb, apocytochrome b.

Figure 3

Quantification of the rates of labeling of the mitochondrial translation products, after a 30 min [³⁵S]methionine pulse, in cybrids derived from different donor lymphoblastoid cell lines. The rates of mitochondrial protein labeling, determined as detailed in Materials and Methods, are expressed as percentages of the value for 143B.TK⁻ in each gel, with error bars representing two standard errors of the mean (SE). A total of 2–3 independent labeling experiments and 2–4 electrophoretic analyses of each labeled preparation were carried out on three cybrids derived from each donor lymphoblastoid cell line. C, control; AS, asymptomatic individuals; and S, symptomatic individuals. The horizontal dashed lines represent the average value for each group, and the vertical arrows refer to two SE; P indicates the significance, according to the Student's t-test, of the differences between AS mean and C mean, and between S mean and C mean.

Figure 4

Average rates of endogenous O₂ consumption per cell measured in different cybrids are shown, with error bars representing two SE. A total of 4–6 determinations were made on each of three cybrids derived from each donor lymphoblastoid cell line. Graph details and symbols are explained in the legend to Figure 3.

Figure 5

Polarographic analysis of O₂ consumption in digitonin-permeabilized cells of the various cell lines using different substrates and inhibitors. The activities of the various components of the respiratory chain were investigated by measuring on ∼5 × 10⁶ digitonin-permeabilized cells the respiration dependent on malate/glutamate, on succinate/G3P and on TMPD/ascorbate. A total of 2–4 determinations were made on each of the three cybrid cell lines derived from each donor lymphoblastoid cell line. Graph details and symbols are explained in the legend to Figure 3. mal/glu, malate/glutamate-dependent respiration; succ/G-3-P, succinate/G3P-dependent respiration; and asc/TMPD, TMPD/ascorbate-dependent respiration.

Figure 6

Growth properties of cybrid cell lines. The population DTs during 4 days of growth was determined in the DMEM medium in the presence and absence of paromomycin. The ratios of DTs in the presence and absence of 2 mg paromomycin per ml are shown. A total of 4–6 determinations were made on each of the three cybrids derived from each donor lymphoblastoid cell line. Graph details and symbols are explained in the legend to Figure 3.