c.753A>G genome editing of a Cdh23ahl allele delays age-related hearing loss and degeneration of cochlear hair cells in C57BL/6J mice.

C57BL/6J mice have long been studied as a model of age-related hearing loss (ARHL). In C57BL/6J mice, ARHL begins in the high-frequency range at 3 months of age and spreads toward low frequencies by 10 months of age. We previously confirmed that c.753A>G genome editing of an ahl allele (c.753A) in the cadherin 23 gene (Cdh23) suppressed the onset of ARHL until 12 months of age. We further investigated the hearing phenotypes of the original and genome-edited C57BL/6J-Cdh23+/+ (c.753G/G) mice until 24 months of age. The hearing tests revealed that most of the C57BL/6J mice maintained good hearing levels until 14 months of age following genome editing of a Cdh23ahl allele. However, the hearing levels of the C57BL/6J-Cdh23+/+ mice gradually declined, and severe ARHL developed with increasing age. ARHL in the C57BL/6J mice was correlated with degeneration of the stereocilia in cochlear hair cells. The stereocilia degeneration was rescued in the C57BL/6J-Cdh23+/+ mice at 12 months of age, but the stereocilia bundles exhibited abnormal phenotypes similar to those of the original C57BL/6J mice at more advanced ages. Therefore, genome editing of Cdh23ahl did not completely suppress ARHL in C57BL/6J mice. We also compared the hearing levels of C57BL/6J-Cdh23+/+ mice with those of C3H/HeN and MSM/Ms mice, which carry the Cdh23+ allele. The severity and onset patterns of ARHL in the C57BL/6J-Cdh23+/+ mice differed from those observed in other Cdh23+/+ mice. Therefore, we hypothesize that other susceptible and/or resistant alleles of ARHL exist in the genetic backgrounds of these mice.

A novel splice site mutation of myosin VI in mice leads to stereociliary fusion caused by disruption of actin networks in the apical region of inner ear hair cells.

An unconventional myosin encoded by the myosin VI gene (MYO6) contributes to hearing loss in humans. Homozygous mutations of MYO6 result in nonsyndromic profound congenital hearing loss, DFNB37. Kumamoto shaker/waltzer (ksv) mice harbor spontaneous mutations, and homozygous mutants exhibit congenital defects in balance and hearing caused by fusion of the stereocilia. We identified a Myo6c.1381G>A mutation that was found to be a p.E461K mutation leading to alternative splicing errors in Myo6 mRNA in ksv mutants. An analysis of the mRNA and protein expression in animals harboring this mutation suggested that most of the abnormal alternatively spliced isoforms of MYO6 are degraded in ksv mice. In the hair cells of ksv/ksv homozygotes, the MYO6 protein levels were significantly decreased in the cytoplasm, including in the cuticular plates. MYO6 and stereociliary taper-specific proteins were mislocalized along the entire length of the stereocilia of ksv/ksv mice, thus suggesting that MYO6 attached to taper-specific proteins at the stereociliary base. Histological analysis of the cochlear hair cells showed that the stereociliary fusion in the ksv/ksv mutants, developed through fusion between stereociliary bundles, raised cuticular plate membranes in the cochlear hair cells and resulted in incorporation of the bundles into the sheaths of the cuticular plates. Interestingly, the expression of the stereociliary rootlet-specific TRIO and F-actin binding protein (TRIOBP) was altered in ksv/ksv mice. The abnormal expression of TRIOBP suggested that the rootlets in the hair cells of ksv/ksv mice had excessive growth. Hence, these data indicated that decreased MYO6 levels in ksv/ksv mutants disrupt actin networks in the apical region of hair cells, thereby maintaining the normal structure of the cuticular plates and rootlets, and additionally provided a cellular basis for stereociliary fusion in Myo6 mutants.

Heterozygous mutation of Ush1g/Sans in mice causes early-onset progressive hearing loss, which is recovered by reconstituting the strain-specific mutation in Cdh23.

Most clinical reports have suggested that patients with congenital profound hearing loss have recessive mutations in deafness genes, whereas dominant alleles are associated with progressive hearing loss (PHL). Jackson shaker (Ush1gjs) is a mouse model of recessive deafness that exhibits congenital profound deafness caused by the homozygous mutation of Ush1g/Sans on chromosome 11. We found that C57BL/6J-Ush1gjs/+ heterozygous mice exhibited early-onset PHL (ePHL) accompanied by progressive degeneration of stereocilia in the cochlear outer hair cells. Interestingly, ePHL did not develop in mutant mice with the C3H/HeN background, thus suggesting that other genetic factors are required for ePHL development. Therefore, we performed classical genetic analyses and found that the occurrence of ePHL in Ush1gjs/+ mice was associated with an interval in chromosome 10 that contains the cadherin 23 gene (Cdh23), which is also responsible for human deafness. To confirm this mutation effect, we generated C57BL/6J-Ush1gjs/+, Cdh23c.753A/G double-heterozygous mice by using the CRISPR/Cas9-mediated Cdh23c.753A>G knock-in method. The Cdh23c.753A/G mice harbored a one-base substitution (A for G), and the homozygous A allele caused moderate hearing loss with aging. Analyses revealed the complete recovery of ePHL and stereocilia degeneration in C57BL/6J-Ush1gjs/+ mice. These results clearly show that the development of ePHL requires at least two mutant alleles of the Ush1g and Cdh23 genes. Our results also suggest that because the SANS and CDH23 proteins form a complex in the stereocilia, the interaction between these proteins may play key roles in the maintenance of stereocilia and the prevention of ePHL.

Quantitative trait loci on chromosome 5 for susceptibility to frequency-specific effects on hearing in DBA/2J mice.

The DBA/2J strain is a model for early-onset, progressive hearing loss in humans, as confirmed in the present study. DBA/2J mice showed progression of hearing loss to low-frequency sounds from ultrasonic-frequency sounds and profound hearing loss at all frequencies before 7 months of age. It is known that the early-onset hearing loss of DBA/2J mice is caused by affects in the ahl (Cdh23(ahl)) and ahl8 (Fscn2(ahl8)) alleles of the cadherin 23 and fascin 2 genes, respectively. Although the strong contributions of the Fscn2(ahl8) allele were detected in hearing loss at 8- and 16-kHz stimuli with LOD scores of 5.02 at 8 kHz and 8.84 at 16 kHz, hearing loss effects were also demonstrated for three new quantitative trait loci (QTLs) for the intervals of 50.3-54.5, 64.6-119.9, and 119.9-137.0 Mb, respectively, on chromosome 5, with significant LOD scores of 2.80-3.91 for specific high-frequency hearing loss at 16 kHz by quantitative trait loci linkage mapping using a (DBA/2J × C57BL/6J) F1 × DBA/2J backcross mice. Moreover, we showed that the contribution of Fscn2(ahl8) to early-onset hearing loss with 32-kHz stimuli is extremely low and raised the possibility of effects from the Cdh23(ahl) allele and another dominant quantitative trait locus (loci) for hearing loss at this ultrasonic frequency. Therefore, our results suggested that frequency-specific QTLs control early-onset hearing loss in DBA/2J mice.

Compound heterozygosity of the functionally null Cdh23(v-ngt) and hypomorphic Cdh23(ahl) alleles leads to early-onset progressive hearing loss in mice.

The waltzer (v) mouse mutant harbors a mutation in Cadherin 23 (Cdh23) and is a model for Usher syndrome type 1D, which is characterized by congenital deafness, vestibular dysfunction, and prepubertal onset of progressive retinitis pigmentosa. In mice, functionally null Cdh23 mutations affect stereociliary morphogenesis and the polarity of both cochlear and vestibular hair cells. In contrast, the murine Cdh23(ahl) allele, which harbors a hypomorphic mutation, causes an increase in susceptibility to age-related hearing loss in many inbred strains. We produced congenic mice by crossing mice carrying the v niigata (Cdh23(v-ngt)) null allele with mice carrying the hypomorphic Cdh23(ahl) allele on the C57BL/6J background, and we then analyzed the animals' balance and hearing phenotypes. Although the Cdh23(v-ngt/ahl) compound heterozygous mice exhibited normal vestibular function, their hearing ability was abnormal: the mice exhibited higher thresholds of auditory brainstem response (ABR) and rapid age-dependent elevation of ABR thresholds compared with Cdh23(ahl/ahl) homozygous mice. We found that the stereocilia developed normally but were progressively disrupted in Cdh23(v-ngt/ahl) mice. In hair cells, CDH23 localizes to the tip links of stereocilia, which are thought to gate the mechanoelectrical transduction channels in hair cells. We hypothesize that the reduction of Cdh23 gene dosage in Cdh23(v-ngt/ahl) mice leads to the degeneration of stereocilia, which consequently reduces tip link tension. These findings indicate that CDH23 plays an important role in the maintenance of tip links during the aging process.

Advantages of a mouse model for human hearing impairment.

Hearing is a major factor in human quality of life. Mouse models are important tools for discovering the genes that are responsible for genetic hearing loss, and these models often allow the processes that regulate the onset of deafness in humans to be analyzed. Thus far, in the study of hearing and deafness, at least 400 mutants with hearing impairments have been identified in laboratory mouse populations. Analysis of through a combination of genetic, morphological, and physiological studies is revealing valuable insights into the ontogenesis, morphogenesis, and function of the mammalian ear. This review discusses the advantages of the mouse models of human hearing impairment and highlights the identification of the molecules required for stereocilia development in the inner ear hair cells by analysis of various mouse mutants.