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. 2024 Jan-Feb;38(1):196-204.
doi: 10.21873/invivo.13426.

Mitophagy Decreases in the Peripheral Vestibular System of Aged C57BL/6J Mice

Sung Il Cho  1 , Eu-Ri Jo  2   3 , Suhyun Lee  4
Affiliations

Mitophagy Decreases in the Peripheral Vestibular System of Aged C57BL/6J Mice

Sung Il Cho et al. In Vivo. 2024 Jan-Feb.

Abstract

Background/aim: Mitophagy is a cardinal process for maintaining healthy and functional mitochondria. A decline in mitophagy has been associated with age-related pathologies. We aimed to investigate mitophagy changes in age-related balance problems using an animal model.

Materials and methods: C57BL/6J mice were divided into young (1 month old) and aged (12 months old) groups. Balance performance, mitochondrial DNA integrity, ATP content, mitophagic process, and mitophagy-related genes and proteins were investigated in both groups.

Results: Balance and motor performance were reduced in the aged group. Mitochondrial DNA integrity and ATP content, and mRNA levels of PINK1, Parkin, BNIP3, AMBRA1, MUL1, NIX, Bcl2-L-13, Atg3, Atg5, Atg12, and Atg13 in the vestibule were significantly lower in aged mice compared with those in young mice. The protein levels of PINK1, Parkin, BNIP3, LC3B, and OXPHOS subunits were significantly decreased in the aged vestibule. Mitophagosome and mitophagolysosome counts and the immunohistochemical expression of Parkin and BNIP3 were also decreased in the saccule, utricle, and crista ampullaris in the aged group.

Conclusion: A general decrease in mitophagy with aging might be attributed to a decrease in cellular function in the aged vestibule during the development of age-related balance problems.

Keywords: Mitophagy; aging; mice; vestibular system.

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Conflict of interest statement

The Authors have no conflicts of interest to declare in relation to this study.

Figures

Figure 1
Figure 1. Locomotor performance of young and aged mice on rotarod test. (A) The latency and speed to fall on an acceleration rotarod in the aged group both significantly decreased in comparison to those of the young group. (B) The latency to fall on the rotarod at a constant speed was investigated between 4 and 40 rpm. The aged group showed a significant decrease in latency to fall at 4, 16, 28, and 40 rpm. The data are shown as the mean±SEM of five independent experimental results (1 m: 1 month; 12 m: 12 months; *p<0.05; **p<0.01).
Figure 2
Figure 2. Changes in mitochondrial DNA integrity, ATP content, and the expression of mitophagy-related genes in the aged vestibular system. (A) Mitochondrial DNA integrity in the peripheral vestibular system of mice in the aged group was significantly lower in comparison to that of the young group. (B) ATP content in the peripheral vestibular system of aged mice was significantly lower than that of young mice. (C) Relative mRNA levels of PINK1, Parkin, BNIP3, AMBRA1, MUL1, NIX, BCL2-L-13, Atg3, Atg5, Atg12, and Atg13 were all significantly decreased in the peripheral vestibular system of mice in the aged group compared with those in the young group. The data are shown as the mean±SEM of five independent experimental results (1 m: 1 month; 12 m: 12 months; *p<0.05; **p<0.01).
Figure 3
Figure 3. Changes in the expression of mitophagy-related and oxidative phosphorylation (OXPHOS) proteins in the aged vestibular system. (A) Western blot analysis of PINK1, Parkin, BNIP3, COX4, LC3B, and OXPHOS subunits. β-actin was used as a loading control. (B) Relative protein levels of PINK1, Parkin, BNIP3, COX4, LC3B, and OXPHOS (I, III, IV, and V) all significantly decreased in the peripheral vestibular system of mice in the aged group compared with those in the young group. The data are shown as the mean±SEM of five independent experimental results (1 m: 1 month; 12 m: 12 months; **p<0.01).
Figure 4
Figure 4. Impaired mitophagy in the aged vestibular organs of C57BL/6J mice. (A) Autophagosomes and mitochondria were investigated using immunofluorescence for LC3B (green) and TOM20 (red), respectively. Mitophagosomes were analyzed by their colocalization (yellow, the overlap color) and the colocalization significantly decreased in the saccule, utricle, and crista ampullaris of mice in the aged group than in those of the young mice. (B) The fusion of lysosomes and mitophagosomes was investigated using immunofluorescence for LAMP1 (green) and TOM20 (red), respectively. Mitophagolysosomes were analyzed by their colocalization (yellow, the overlap color) and the colocalization significantly decreased in the saccule, utricle, and crista ampullaris of mice in the aged group than in those of the young group. The data are shown as the mean±SEM of five independent experimental results (1 m: 1 month; 12 m: 12 months; **p<0.01).
Figure 5
Figure 5. Decreased Parkin and BNIP3 in the aged vestibular organs of C57BL/6J mice. Immunohistochemistry (IHC) scores of Parkin and BNIP3 in the saccule, utricle, and crista ampullaris of mice in the aged group were significantly lower than those of the young group. The data are shown as the mean±SEM of five independent experimental results (1 m: 1 month; 12 m: 12 months; *p<0.05; **p<0.01).
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