Genetic background effects on age-related hearing loss associated with Cdh23 variants in mice

Abstract

Inbred strain variants of the Cdh23 gene have been shown to influence the onset and progression of age-related hearing loss (AHL) in mice. In linkage backcrosses, the recessive Cdh23 allele (ahl) of the C57BL/6J strain, when homozygous, confers increased susceptibility to AHL, while the dominant allele (Ahl+) of the CBA/CaJ strain confers resistance. To determine the isolated effects of these alleles on different strain backgrounds, we produced the reciprocal congenic strains B6.CBACa-Cdh23(Ahl)(+) and CBACa.B6-Cdh23(ahl) and tested 15-30 mice from each for hearing loss progression. ABR thresholds for 8 kHz, 16 kHz, and 32 kHz pure-tone stimuli were measured at 3, 6, 9, 12, 15 and 18 months of age and compared with age-matched mice of the C57BL/6J and CBA/CaJ parental strains. Mice of the C57BL/6N strain, which is the source of embryonic stem cells for the large International Knockout Mouse Consortium, were also tested for comparisons with C57BL/6J mice. Mice of the C57BL/6J and C57BL/6N strains exhibited identical hearing loss profiles: their 32 kHz ABR thresholds were significantly higher than those of CBA/CaJ and congenic strain mice by 6 months of age, and their 16 kHz thresholds were significantly higher by 12 months. Thresholds of the CBA/CaJ, the B6.CBACa-Cdh23(Ahl)(+), and the CBACa.B6-Cdh23(ahl) strain mice differed little from one another and only slightly increased throughout the 18-month test period. Hearing loss, which corresponded well with cochlear hair cell loss, was most profound in the C57BL/6J and C57BL/6NJ strains. These results indicate that the CBA/CaJ-derived Cdh23(Ahl)(+) allele dramatically lessens hearing loss and hair cell death in an otherwise C57BL/6J genetic background, but that the C57BL/6J-derived Cdh23(ahl) allele has little effect on hearing loss in an otherwise CBA/CaJ background. We conclude that although Cdh23(ahl) homozygosity is necessary, it is not by itself sufficient to account for the accelerated hearing loss of C57BL/6J mice.

Figure 1. Hearing loss progression

Mice of the B6J, B6N, B6.CBA-Ahl+, CBA.B6-ahl, and CBA strains were tested at 3, 6, 9, 12, 15, and 18 months of age, and ABR thresholds were obtained for pure-tone 8 kHz, 16 kHz and 32 kHz auditory stimuli. For each auditory stimulus, plots of ABR threshold means (dB SPL) for each strain at successive test ages are shown on the left and corresponding plots of hearing loss means (dB) on the right. Numerical values, variance estimates, and numbers of mice tested are given in Table 1, and statistical analyses of mean differences among strains are presented in Table 2 and in Supplementary Figure 2.

Figure 2. Cochlear hair cell loss

Representative photomicrograph of surface preparations from a nineteen-month-old B6.CBA-Ahl+ mouse taken from (A) apical, (B) middle and (C) basal region of the cochlea (400X magnification). Focal plane of the microscope set near nuclei outer hair cells (OHC) and inner hair cells (IHC). Jagged arrows point to missing OHC and IHC.

Figure 3. Strain-specific patterns of hair cell loss

Representative photomicrographs (400X magnification) taken 40-60% (10-20 kHz region) of the distance from apex of the cochleae of (A) CBA, (B) CBA.B6-ahl, (C) B6.CBA-Ahl+ and (D) B6J mouse strains examined at 18-19 months of age. Most OHCs (yellow bracket) were present in CBA, CBA.B6-ahl and B6.CBA-Ahl+ mice, whereas most OHCs were missing in B6J mice (jagged, gold arrowhead). Nearly all IHCs were present in CBA and CBA.B6-ahl mice, whereas many IHCs were missing in B6J mice and B6.CBA-Ahl+ mice (red arrowhead). Note that there is little if any IHC or OHC loss in CBA or CBA.B6-ahl mice, that B6.CBA-Ahl+ mice show much less OHC loss than B6J mice, but more than CBA or CBA.B6-ahl mice, and that IHC loss is similar in B6.CBA-Ahl+ and B6J mice.

Figure 4. Quantification of cochlear hair cell loss

Mice of the CBA, B6J, CBA.B6-ahl, and B6.CBA-Ahl+ strains were examined at 18-19 months of age. A-D. For each strain, mean percent loss (± SEM) of IHC and OHC was plotted as a function of percent distance from the apex. E. Comparisons of mean (± SEM) percent OHC loss as a function of percent distance from the apex among strains. F. Comparisons of mean (± SEM) percent IHC loss as a function of percent distance from the apex of the cochlea among the strains. Statistical analyses of these data are presented in Table 3. Lower x-axis shows the relationship of test frequency to cochlear place (Muller et al., 2005).