Increased burden of mitochondrial DNA deletions and point mutations in early-onset age-related hearing loss in mitochondrial mutator mice

Abstract

Mitochondrial DNA (mtDNA) mutations are thought to have a causal role in a variety of age-related neurodegenerative diseases, including age-related hearing loss (AHL). In the current study, we investigated the roles of mtDNA deletions and point mutations in AHL in mitochondrial mutator mice (Polg^mut/mut) that were backcrossed onto CBA/CaJ mice, a well-established model of late-onset AHL. mtDNA deletions accumulated significantly with age in the inner ears of Polg^mut/mut mice, while there were no differences in mtDNA deletion frequencies in the inner ears between 5 and 17 months old Polg^+/+ mice or 5 months old Polg^+/+ and Polg^mut/mut mice. mtDNA deletions also accumulated significantly in the inner ears of CBA/CaJ mice during normal aging. In contrast, 5 months old Polg^mut/mut mice displayed a 238-fold increase in mtDNA point mutation frequencies in the inner ears compared to age-matched Polg^+/+ mice, but there were no differences in mtDNA point mutation frequencies in the inner ears between 5 and 17 months old Polg^mut/mut mice. Seventeen-month-old Polg^mut/mut mice also displayed early-onset severe hearing loss associated with a significant reduction in neural output of the cochlea, while age-matched Polg^+/+ mice displayed little or no hearing impairment. Consistent with the physiological and mtDNA deletion test result, 17-month-old Polg^mut/mut mice displayed a profound loss of spiral ganglion neurons in the cochlea. Thus, our data suggest that a higher burden of mtDNA point mutations from a young age and age-related accumulation of mtDNA deletions likely contribute to early-onset AHL in mitochondrial mutator mice.

Keywords: Aging; Hearing loss; Mitochondrial DNA mutations; Mitochondrial disease.

Fig. 1.

Genotyping of Polg^+/+ and Polg^mut/mut mice. (A) PCR products were separated on 2% agarose gel. The expected band sizes for the wild-type and mutant alleles were 296 and 468 bps, respectively. (B) The Cdh23 gene in three Polg^+/+ and three Polg^mut/mut mice was sequenced. All the mice examined had the same wild-type Cdh23^753G/753G genotype.

Fig. 2.

Assessment of body weight in Polg^+/+ and Polg^mut/mut mice. (A) Eighteen-month-old WT and Polg^mut/mut mice. B-C: The body weight of male (B) and female (C) WT and Polg^mut/mut mice was measured every month from 4 months of age until 18 months of age. (N=3–5). Data are shown as means ± SEM.

Fig. 3.

Assessment of ABRs in Polg^+/+ and Polg^mut/mut mice. A-B: ABR thresholds were measured with a tone burst stimulus at 4, 8, 16, 32, 48, and 64 kHz in male (A) and female (B) WT and Polg^mut/mut mice at 5, 12, and 17 months of age (N=3–6). Data are shown as means ± SEM. *p < 0.05, 5 mo mut/mut vs. 17 mo mut/mut, **p < 0.05, 17 mo +/+ vs. 17 mo mut/mut. C-D: ABR latencies for wave I were measured with a click stimulus of 100 dB SPL in male (C) and female (D) WT and Polg^mut/mut mice at 5, 12, and 17 months of age (N=3–6). E-F: ABR amplitudes for wave I were measured with a click stimulus of 100 dB SPL in male (E) and female (F) WT and Polg^mut/mut mice at 5, 12, and 17 months of age (N=3–6). Data are shown as means ± SEM. *p < 0.05, 5 mo vs. 17 mo, **p < 0.05, 17 mo +/+ vs. 17 mo mut/mut.

Fig. 4.

Assessment of CAP and SP in Polg^+/+ and Polg^mut/mut mice. A-B: CAP amplitudes were measured at 80 dB SPL at 2, 4, 6, 8, 12, 16, 20, 24, 30, 35, 40, 45, 50, 55, 60, and 65 kHz in male (A) and female (B) WT and Polg^mut/mut mice at 17 months of age (N=3–5). C-D: CAP thresholds were measured at 2, 4, 6, 8, 12, 16, 20, 24, 30, 35, 40, 45, 50, 55, 60, and 65 kHz in male (C) and female (D) WT and Polg^mut/mut mice at 17 months of age (N=3–5). E-F: SP amplitudes were measured at 80 dB SPL at 12, 16, 20, 24, 30, 35, and 40 kHz in male (E) and female (F) WT and Polg^mut/mut mice at 17 months of age (N=3–5). Data are shown as means ± SEM. *p < 0.05, 17 mo +/+ vs. 17 mo mut/mut.

Fig. 5.

Assessment of SGN pathology in the cochlea of Polg^+/+ and Polg^mut/mut mice. (A) SGN densities were measured in the apical, middle, and basal regions in the cochlea of female WT and Polg^mut/mut mice at 5 and 17 months of age (N=3–4). Data are shown as means ± SEM. *p < 0.05, 5 mo mut/mut vs. 17 mo mut/mut, **p < 0.05, 17 mo +/+ vs. 17 mo mut/mut. B-M: SGN regions in the apical (B-E), middle (F-I), and basal (J-M) cochlear tissues from female WT and Polg^mut/mut mice at 5 and 17 months of age. Scale bar = 20 μm.

Fig. 6.

Assessment of HC pathology in the cochlea of Polg^+/+ and Polg^mut/mut mice. A-B: Cochleograms were recorded and averaged in the cochlea of female WT and Polg^mut/mut mice at 5 and 17 months of age (N=3–4). Graphs show percent loss of IHCs (A) and OHCs (B) as a function of percent distance from the apex of the cochlea. Lower x-axes show the frequency-place map for mouse cochlea. Data are shown as means ± SEM. *p < 0.05, 5 mo mut/mut vs. 17 mo mut/mut, **p < 0.05, 17 mo +/+ vs. 17 mo mut/mut. C-N: HC regions in the apical (C-F), middle (G-J), and basal (K-N) turns of cochlear basilar membrane from female WT and Polg^mut/mut mice at 5 and 17 months of age. Arrows indicate missing HCs. Scale bar = 50 μm.

Fig. 7.

Assessment of SV atrophy in the cochlea of Polg^+/+ and Polg^mut/mut mice. (A) SV thicknesses were measured in the apical, middle, and basal regions in the cochlea of female WT and Polg^mut/mut mice at 5 and 17 months of age (N=3–4). Data are shown as means ± SEM. *p < 0.05, 5 mo vs. 17 mo. B-M: SV regions in the apical (B-E), middle (F-I), and basal (J-M) cochlear tissues from female WT and Polg^mut/mut mice at 5 and 17 months of age. Scale bar = 20 μm.

Fig. 8.

Assessment of mtDNA mutations in the inner ears of Polg^+/+, Polg^mut/mut, and CBA/CaJ mice. A-C: mtDNA point mutation frequencies were measured in the inner ears of male WT and Polg^mut/mut mice at 5 and 17 months of age (N=3–6) and male CBA/CaJ mice at 5 and 25 months of age (N=3). D-F: mtDNA deletion frequencies were measured in the inner ears of male WT and Polg^mut/mut mice at 5 and 17 months of age (N=5–8) and male CBA/CaJ mice at 5 and 25 months of age (N=6). G-I: mtDNA copy numbers were measured in the inner ears of male WT and Polg^mut/mut mice at 5 and 17 months of age (N=5–8) and male CBA/CaJ mice at 5 and 25 months of age (N=6). Data are shown as means ± SEM. *p < 0.05, 5 mo mut/mut vs. 17 mo mut/mut, **p < 0.05, +/+ vs. mut/mut, ***p < 0.05, 5 mo CBA/CaJ vs. 25 mo CBA/CaJ, ****p < 0.05, 5 mo +/+ vs. 5 mo CBA/CaJ. J-M: Immunostaining for COI and Porin in SGNs of the cochlear tissues from 17-month-old WT and Polg^mut/mut mice. Normal SGNs showed blue/purple staining indicating presence of both COI and Porin (C). COI was not detected in some SGNs in the cochlear tissues from 17-month-old Polg^mut/mut mice (black arrow) (D). Nuclei were counterstained with methyl green. Scale bar = 10 μm.