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

Materials and Methods

Mice and animal care. All animal experiments were approved by the Institutional Animal Care and Use Committee of Chosun University (approval no. CIACUC2022-A0006) and performed in accordance with the ARRIVE guidelines and strict regulations approved by the committee. C57BL/6J mice (Mus musculus) were obtained from the Animal Facility of Aging Science at the KBSI Gwangju Center (Gwangju, South Korea). Mice were randomly divided into two groups (young: 1 month of age, n=5; age: 12 months, n=5). All mice were euthanized by cervical dislocation under 5% isoflurane anesthesia after the rotarod test and the peripheral vestibular structures were isolated from the mice.

Rotarod test. The rotarod test was performed using a rotarod apparatus (Panlab/Harvard Apparatus, Barcelona, Spain) to evaluate sensorimotor coordination and balance in the rodents. C57BL/6J mice were first habituated to stay on the rod for 60 s at a constant speed of five rotations per minute (rpm). This habituation procedure was repeated for a total of three trials per day separated by 10 min intervals for three days. After training, mice underwent two test sessions. They were first placed in separate lanes on the rod and subjected to an accelerating speed (from 4 to 40 rpm) of over 300 s. Next, mice were tested on the rotarod at constant speeds (4, 16, 28, and 40 rpm). The latency to fall (s) and speed at which they fell (rpm) were recorded for statistical analysis. Five trials were conducted for each mouse.

Real-time PCR analysis. To test mitochondrial DNA integrity, 20 ng of total DNA was isolated from whole vestibular organ tissues, except for the cochlea of the mouse inner ear. The mtDNA long fragment (10.1 kb) was amplified using the following primers: 5′-GCCAGCCTGACCCATAGCCATAATAT-3′ and 5′-GAGAGATT TTATGGGTGTAATGCGG-3′. The short mtDNA fragment (117 bp) was amplified using the following primers: 5′- CCCAGCTACTA CCATCATTCAAGT-3′ and 5′-GATGGT TTGGGAGATTGGTT GATGT-3′. Real-time PCR was performed using the SYBR Premix Ex Taq kit (TaKaRa Bio, Shiga, Japan) according to the manufacturer’s instructions. The short mtDNA fragment was used as a normalization factor for mtDNA copies.

To analyze mitophagy-related genes, total RNA was extracted from whole vestibular organ tissues, except for the cochlea of the mouse inner ear. RNA was reverse-transcribed using an M-MLV cDNA Synthesis Kit (Enzynomics, Daejeon, Republic of Korea) according to manufacturer’s instructions. Real-time PCR was performed using the SYBR Premix Ex Taq kit (TaKaRa Bio) according to the manufacturer’s instructions. Primers used for real-time PCR were as follows: PINK1 forward, 5′-GCTTGCCAATCCCTTCTATG-3′, reverse, 5′-CTCTCGCTGGAGCAGTGAC-3′; Parkin forward, 5′-AAACCGGATGAGTGGTGAGT-3′, reverse, 5′-AGCTACCGACG TGTCCTTGT-3′; AMBRA1 forward, 5′-CTACTGGGACCAGCTAA GTGAAA-3′, reverse, 5′-ACGTGGCTCTGCTGGTTC-3′; MUL1 forward, 5′-GTCATCGAAGGAGCTGTGC-3′, reverse, 5′-GTAGTT CGGTTCCACACCATC-3′; NIX forward, 5′-GCAGGGACTAGCT CTCAG-3′, reverse, 5′-TGCTCAGTCGCTTTCCAATA-3′; BNIP3 forward, 5′-GCTCCCAGACACCACAAGAT-3′, reverse, 5′-TGA GAGTAGCTGTGCGCTTC-3′; BCL2-L-13 forward, 5′- AGCAGAA TTCATGGCGTCCTCTACGACTGC-3′, reverse, 5′-AGCAGAATTC TTACTTTCTTCTTAAAGCCAGTGCA-3′; Atg3 forward, 5′-CCA TTGAAAACCATCCTCATCTC-3′, reverse, 5′-GCCTTCTGCAAC TGTCTCAATAATT-3′; Atg5 forward, 5′-GGACAGCTGCACACA CTTGG-3′, reverse, 5′-TGGCTCTATCCCGTGAATCAT-3′; Atg12 forward, 5′-TGAATCAGTCCTTTGCCCCT-3′, reverse, 5′-CATGC CTGGGATTTGCAGT-3′; Atg13 forward, 5′-TGGCGGAAGATTT GGACTCC-3′, reverse, 5′-GGGTTTCCACAAAGGCATCG-3′; and GAPDH forward, 5′-GTATTGGGCGCCTGGTCACC-3′, reverse, 5′-CGCTCCTGGAAGATGGTGATGG-3′.

The threshold cycle number (Ct) for the examined genes was normalized to the Ct value for the housekeeping gene, GAPDH. The relative mRNA level of the gene of interest was determined and expressed as the ratio of the mRNA level in the aged group mouse tissues to that in the young group mouse tissues. The fold induction values for gene expression levels were calculated using the ΔΔCt method.

ATP contents. ATP concentration in the tissues was evaluated using an ATP Assay Kit (Biovision, Milpitas, CA, USA) following the manufacturer’s protocol. The amount of ATP was measured using a BioTek ELz800 Microplate reader (BioTek, Winooski, VT, USA) and quantified based on the amount of light produced (absorbance at 570 nm). ATP levels were determined based on a standard curve, calculated as μmol/g (wet weight), and expressed as a relative percentage of the control (young group).

Western blot analysis. Total protein was extracted from the whole vestibular organ tissues, except for the cochlea of the mouse inner ear. The expression of each protein was determined using western blotting. Primary polyclonal PINK1, Parkin, BNIP3 (1:1,000; Thermo Fisher Scientific, Waltham, MA, USA), COX4, LC3B (1:1,000; Cell Signaling Technology, Danvers, MA, USA), OXPHOS Rodent WB Antibody Cocktail (6 μg/ml; Thermo Fisher Scientific), and β-actin (1:4,000; Santa Cruz Biotechnology, Dallas, TX, USA) antibodies were used together with anti-mouse in sheep or anti-rabbit in donkeys secondary antibodies (1:4,000; Jackson Immuno Research, West Grove, PA, USA). The relative expression levels of all proteins were determined by densitometry and normalized to that of β-actin.

Immunostaining. The inner ears of young and aged mice were fixed in 4% paraformaldehyde overnight at 4°C. The fixed inner ear was decalcified using a Calci-Clear Rapid (National Diagnostics, Atlanta, GA, USA) for 7 days at 4°C. After washing with PBS, tissues were dehydrated and embedded in paraffin. Paraffin-embedded tissues were sectioned into 4 μm sections at the level of the saccule, utricle, and crista ampullaris. Representative sections were used for experiments. For immunohistochemistry and immunofluorescence, previously characterized polyclonal antibodies against the following proteins were used: LC3B (1:100; Cell Signaling Technology), LAMP1, TOM20 (1:100; Santa Cruz Biotechnology, Dallas, TX, USA), Parkin, and BNIP3 (1:100; Thermo Fisher Scientific).

Sections for immunofluorescence were immunolabelled with Alexa Fluor 488 conjugated chicken anti-rabbit or Alexa Fluor 594 conjugated chicken anti-mouse antibody (1:200; Invitrogen, Carlsbad, CA, USA). Confocal laser microscopy was performed using a Carl Zeiss LSM800 laser confocal microscope (Carl Zeiss, Oberkochen, Germany), and imaged using the Zeiss microscope image software ZEN (Carl Zeiss). The colocalization coefficient levels were quantified using ImageJ software (NIH, Bethesda, MD, USA).

Immunohistochemical sections were visualized using a biotinylated anti-rabbit IgG and avidin-biotin-peroxidase (ABC) detection system (Vector Laboratories, Burlingame, CA, USA). Novocastra Hematoxylin (Leica Biosystems, Nussloch, Deutschland) was used to visualize the cell nucleus. The immunoreactivity of Parkin and BNIP3 was calculated by scoring the staining intensity (0, none; 1, weak; 2, moderate; 3, strong) and the percentage of positive cells (0: ≤5%, 1: 6-25%, 2: 26-50%, 3: 51-75%, 4: ≥76%). The results are expressed as the product of both scores.

Statistical analysis. The results were statistically analyzed using the SPSS 25.0 software (SPSS Inc., Chicago, IL, USA). The Mann-Whitney U-test was used to analyze the data. A p-value of <0.05 was considered statistically significant.