Deletion of Clusterin Protects Cochlear Hair Cells against Hair Cell Aging and Ototoxicity

Abstract

Hearing loss is a debilitating disease that affects 10% of adults worldwide. Most sensorineural hearing loss is caused by the loss of mechanosensitive hair cells in the cochlea, often due to aging, noise, and ototoxic drugs. The identification of genes that can be targeted to slow aging and reduce the vulnerability of hair cells to insults is critical for the prevention of sensorineural hearing loss. Our previous cell-specific transcriptome analysis of adult cochlear hair cells and supporting cells showed that Clu, encoding a secreted chaperone that is involved in several basic biological events, such as cell death, tumor progression, and neurodegenerative disorders, is expressed in hair cells and supporting cells. We generated Clu-null mice (C57BL/6) to investigate its role in the organ of Corti, the sensory epithelium responsible for hearing in the mammalian cochlea. We showed that the deletion of Clu did not affect the development of hair cells and supporting cells; hair cells and supporting cells appeared normal at 1 month of age. Auditory function tests showed that Clu-null mice had hearing thresholds comparable to those of wild-type littermates before 3 months of age. Interestingly, Clu-null mice displayed less hair cell and hearing loss compared to their wildtype littermates after 3 months. Furthermore, the deletion of Clu is protected against aminoglycoside-induced hair cell loss in both in vivo and in vitro models. Our findings suggested that the inhibition of Clu expression could represent a potential therapeutic strategy for the alleviation of age-related and ototoxic drug-induced hearing loss.

Figure 1

Construction of Clu knockout mice. (a) Schematic drawing showing the strategy used for Clu gene disruption. The target sites of clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 small guide RNAs (sgRNAs) in the Clu gene are indicated in red, and the deleted region of the Clu gene in knockout mice is indicated by dashes. The positions of RT-PCR primers are indicated by arrows. (b) PCR products were separated on a 1.5% agarose gel, and the expected band sizes for WT and knockout (KO) alleles were 1,063 and 480 bps, respectively. (c) Sequencing results for homozygotes. The black arrows represent wild-type sequences, and the red arrows represent sequences following the deleted fragment.

Figure 2

Clu expression in the organ of Corti of the cochlea. (a) RNA-seq in the adult CBA/J mouse cochlea showed Clu expression in OHCs, IHCs, pillar cells, Deiters' cells, and melanocytes, with strong expression in OHCs. (b) RNA-Seq examining gene expression during mouse inner ear development showed Clu expression in hair cells (GFP⁺) and supporting cells (GFP⁻) in the mouse cochlea, from E16 to P7. (c) Temporal expression patterns of Clu mRNA in the mouse inner ear. Clu mRNA transcripts were strongly expressed at P12, with slight but significantly decreased expression with aging. N = 4. (d) In situ hybridization performed in the WT cochlea at P21 showed Clu expression in OHCs and stronger expression in the pillar and Deiters' cells.

Figure 3

Hearing and basilar membrane morphology of Clu^−/− mice in one month. (a) ABR hearing thresholds and DPOAE hearing threshold were measured at 32, 16, and 8 kHz in Clu^−/− and WT mice at P1m. N = 6. (b) Cochlear basilar membrane morphology of Clu^−/− mice at P1m. (c) Organ of Corti in the basal cochlear regions of P1m Clu^−/− mice. No hair cells or supporting cell defects were observed.

Figure 4

Assessment of age-related auditory dysfunction. (a) ABR hearing thresholds were measured at 32, 16, and 8 kHz in WT and Clu^−/− mice at P2m, P4m, P6m, and P9m. Clu deficiency delayed ARHL in C57BL/6 mice. Data are presented as the mean ± SD. ^∗p < 0.05, ^∗∗p < 0.01. N = 6. (b) ABR thresholds of WT and Clu^−/− mice at P2m, P4m, P6m, and P9m. N = 6.

Figure 5

Assessment of age-related morphology of hair cells and supporting cells. (a) Cochlear basilar membrane immunostaining in P2m, P4m, P6m, and P9m Clu^−/− mice. (b) Cochlear basilar membrane immunostaining in P2m, P4m, P6m, and P9m WT mice. The number of remaining hair cells in Clu^−/− mice was significantly higher than that in WT mice at the same age. (c) Quantification of OHCs and IHCs in mice at P2m, P4m, P6m, and P9m across all tested frequencies in WT and Clu^−/− mice. Data are presented as the mean ± SD. ^∗p < 0.05, ^∗∗p < 0.01. N = 6. (d) Quantification of supporting cells at P4m, P6m, and P9m in WT and Clu^−/− mice. The red box shows a typical section after three-dimensional reconstruction. The percentages of supporting cells and hair cells showed no significant differences between WT and Clu^−/− mice. N = 6.

Figure 6

Clu deficiency protects hearing ability and inhibits sensory cell death induced by the coadministration of kanamycin and furosemide. (a) Assessment of auditory dysfunction in WT and Clu^−/− mice treated with furosemide plus kanamycin. N = 6. (b) Quantification of OHCs and IHCs in WT and Clu^−/− mice after treatment with furosemide plus kanamycin. Data are presented as the mean ± SD. ^∗p < 0.05, ^∗∗p < 0.01. N = 6. (c) Representative confocal microscopy images from WT and Clu^−/− mice after treatment with furosemide plus kanamycin.

Figure 7

Clu deficiency attenuates gentamycin-induced cochlear hair cell death in vitro. (a) Representative images of myosin-stained hair cells in the CBM after exposure to the gentamycin (GM). n = 6 CBMS per condition. (b) Quantification of myosin staining in IHCs and OHCs. Data are presented as the mean ± SD. ^∗p < 0.05, ^∗∗p < 0.01. N = 6. (c) The percentages of supporting cells and hair cells showed no significant differences between WT and Clu^−/− mice. N = 6.