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. 2020 Sep 29;21(19):7202.
doi: 10.3390/ijms21197202.

Age-Related Hearing Loss in C57BL/6J Mice Is Associated with Mitophagy Impairment in the Central Auditory System

Cha Kyung Youn  1 , Yonghyun Jun  2 , Eu-Ri Jo  3 , Sung Il Cho  3
Affiliations

Age-Related Hearing Loss in C57BL/6J Mice Is Associated with Mitophagy Impairment in the Central Auditory System

Cha Kyung Youn et al. Int J Mol Sci. .

Abstract

Aging is associated with functional and morphological changes in the sensory organs, including the auditory system. Mitophagy, a process that regulates the turnover of dysfunctional mitochondria, is impaired with aging. This study aimed to investigate the effect of aging on mitophagy in the central auditory system using an age-related hearing loss mouse model. C57BL/6J mice were divided into the following four groups based on age: 1-, 6-, 12-, and 18-month groups. The hearing ability was evaluated by measuring the auditory brainstem response (ABR) thresholds. The mitochondrial DNA damage level and the expression of mitophagy-related genes, and proteins were investigated by real-time polymerase chain reaction and Western blot analyses. The colocalization of mitophagosomes and lysosomes in the mouse auditory cortex and inferior colliculus was analyzed by immunofluorescence analysis. The expression of genes involved in mitophagy, such as PINK1, Parkin, and BNIP3 in the mouse auditory cortex and inferior colliculus, was investigated by immunohistochemical staining. The ABR threshold increased with aging. In addition to the mitochondrial DNA integrity, the mRNA levels of PINK1, Parkin, NIX, and BNIP3, as well as the protein levels of PINK1, Parkin, BNIP3, COX4, LC3B, mitochondrial oxidative phosphorylation (OXPHOS) subunits I-IV in the mouse auditory cortex significantly decreased with aging. The immunofluorescence analysis revealed that the colocalization of mitophagosomes and lysosomes in the mouse auditory cortex and inferior colliculus decreased with aging. The immunohistochemical analysis revealed that the expression of PINK1, Parkin, and BNIP3 decreased in the mouse auditory cortex and inferior colliculus with aging. These findings indicate that aging-associated impaired mitophagy may contribute to the cellular changes observed in an aged central auditory system, which result in age-related hearing loss. Thus, the induction of mitophagy can be a potential therapeutic strategy for age-related hearing loss.

Keywords: age-related hearing loss; auditory cortex; mitochondria; mitophagy; presbycusis.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
The effect of aging on auditory brainstem response (ABR) thresholds and mitochondrial DNA (mtDNA) integrity. (A) The hearing thresholds of mice at frequencies of 8 kHz, 16 kHz, and 32 kHz increased with aging (fifteen mice per group). (B) The mtDNA integrity was assessed by measuring the expression level of 10 kb amplicon. The mtDNA integrity in the mouse auditory cortex significantly decreased with aging. The data are shown as mean ± standard error of mean (five mice per group; 1 m, 1 month; 6 m, 6 months; 12 m, 12 months; 18 m, 18 months; SPL, sound pressure level). * p < 0.05, ** p < 0.01.
Figure 2
Figure 2
The effect of aging on the expression of mitophagy-related genes, proteins, and oxidative phosphorylation (OXPHOS) subunits in the mouse auditory cortex. (A) The mRNA expression levels of PINK1, Parkin, BNIP3, and NIX in the mouse auditory cortex significantly decreased with aging. (B) The Western blotting analysis of PINK1, Parkin, BNIP3, COX4, LC3B, and OXPHOS subunits. β-actin was used as the loading control. (C) The relative protein expression levels of PINK1, Parkin, BNIP3, COX4, LC3B, and OXPHOS subunits I–IV in the mouse auditory cortex significantly decreased with aging. The data are shown as mean ± standard error of mean (five mice per group; 1 m, 1 month; 6 m, 6 months; 12 m, 12 months; 18 m, 18 months). * p < 0.05, ** p < 0.01, ns: not significant.
Figure 3
Figure 3
Impairment of mitophagy in the mouse auditory cortex with aging. (A) Colocalization analysis of autophagosomes and mitochondria. Immunofluorescence analysis revealed that the colocalization (yellow puncta, the overlap color) of LC3B (green) and TOM20 (red) in the mouse auditory cortex significantly decreased with aging. (B) Colocalization analysis of lysosomes and mitophagosomes. Immunofluorescence analysis revealed that the colocalization (yellow puncta, the overlap color) of LAMP1 (green) and TOM20 (red) in the mouse auditory cortex significantly decreased with aging. The data are shown as mean ± standard error of mean (five mice per group; 1 m, 1 month; 6 m, 6 months; 12 m, 12 months; 18 m, 18 months; DAPI, 4′,6-diamidino-2-phenylindole). * p < 0.05, ** p < 0.01, ns: not significant.
Figure 4
Figure 4
Impairment of mitophagy in the mouse inferior colliculus with aging. (A) Colocalization analysis of autophagosomes and mitochondria. Immunofluorescence analysis revealed that the colocalization (yellow puncta, the overlap color) of LC3B (green) and TOM20 (red) in the mouse inferior colliculus significantly decreased with aging. (B) Colocalization analysis of lysosomes and mitophagosomes. Immunofluorescence analysis revealed that the colocalization (yellow puncta, the overlap color) of LAMP1 (green) and TOM20 (red) in the mouse inferior colliculus significantly decreased with aging. The data are shown as mean ± standard error of mean (five mice per group; 1 m, 1 month; 6 m, 6 months; 12 m, 12 months; 18 m, 18 months; DAPI, 4′,6-diamidino-2-phenylindole). * p < 0.05, ** p < 0.01.
Figure 5
Figure 5
The protein expression levels of PINK1, Parkin, and BNIP3 in the mouse auditory cortex decrease with aging. Immunohistochemical (IHC) scores of PINK1, Parkin, and BNIP3 in the mouse auditory cortex significantly decreased with aging. The data are shown as mean ± standard error of mean (five mice per group; 1 m, 1 month; 6 m, 6 months; 12 m, 12 months; 18 m, 18 months). * p < 0.05, ** p < 0.01, ns: not significant.
Figure 6
Figure 6
The protein expression levels of PINK1, Parkin, and BNIP3 in the mouse inferior colliculus decrease with age. Immunohistochemical (IHC) scores of PINK1, Parkin, and BNIP3 in the mouse inferior colliculus significantly decreased with aging. The data are shown as mean ± standard error of mean (five mice per group; 1 m, 1 month; 6 m, 6 months; 12 m, 12 months; 18 m, 18 months). * p < 0.05, ** p < 0.01, ns: not significant.
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