Hearing in NR-treated CS mouse genotypes

We previously reported that reduced NAD+ levels are associated with CS pathology.7,17 Given that hearing loss is a cardinal symptom of CS, we assayed NAD+ levels in the cochlea of CSBm/m mice. We found that total NAD+ and relative NAD+/NADH levels were lower in the cochlea of the CSBm/m mice compared to WT (Fig. 1a). In this study, CS mice were dosed with NR, an NAD+ precursor, to assess the effect of NAD+ supplementation on progressive hearing loss in CS mouse genotypes. We began treating mice with NR just after 5 weeks of age and assessed hearing capacity by measuring ABRs (see Methods for details) in response to sound at multiple frequencies (Fig. 1b). We observed that CSBm/m mice at 6.5 weeks of age develop a progressive hearing impairment and have a hearing loss of more than 35 dB at 32 kHz compared to WT (Fig. 1c and Supplementary Fig. 1). Strikingly, only 10 days of exposure to NR (12 mM) significantly prevented the hearing loss in CSBm/m mice and reduced the high-frequency hearing threshold from 77 to 40 dB (Fig. 1c, compare top and lower panels). We then compared the hearing threshold shift in individual mice in NR-treated and non-treated groups. We observed that non-treated CSBm/m mice at 6.5 weeks of age, on average, lost 12 dB over 10 days and the treatment with NR not only prevented hearing loss, but improved hearing by an average of 15 dB (Fig. 1d).

Fig. 1: NAD+ supplementation prevents progressive hearing loss in CSBm/m mice. a Total NAD+ levels per mg of the cochlea and relative NAD+/NADH levels were measured in the cochlea of CSBm/m and WT (6-month-old) mice (N = 3). Two-tailed t-tests were used to determine significant difference. b Outline for NR treatment and ABR recordings in CSBm/m mice. c ABR thresholds for CSBm/m and WT mice following a 10-day treatment with NR (12 mM) delivered via drinking water. A total of 21 mice (CSBm/m (N = 7); CSBm/m + NR (N = 6); WT (N = 4); WT + NR (N = 4)) were tested by ABR at 5 weeks of age and again following the 10-day treatment (6.5 weeks of age). Multiple graphs are used to visualize error bars on each dataset. Two-way ANOVA with Tukey’s post-hoc test was used to determine significant difference. d Treatment with NR (12 mM) prevents the increased threshold shift observed in CSBm/m mice between 5 weeks and 6.5 weeks at 32 kHz. Note: ABR data in c at 5 and 6.5 weeks of age were used to calculate the hearing shift. Two-way ANOVA with uncorrected Fisher's LSD post-hoc test was used to determine significant difference. Full size image

Like CSBm/m, CSA−/− mice also manifested progressive hearing loss although less severe6 (Supplementary Fig. 2a). This less severe phenotype has also been reported in CSA patients.4,6 We found that NAD+/NADH levels were also lower in the cochlea of CSA−/− mice (Fig. 2a) and tested the effect of NAD+ supplementation on the hearing defect in CSA−/− mice (Fig. 2b). Initial treatment with 12 mM dose of NR did not attenuate the hearing loss in these mice (Supplementary Fig. 2b) and thus we increased the dose of NR (24 mM) over a 4-week period from 5 to 9 weeks of age. After exposure to 24 mM NR, the ABR threshold at 32 kHz for CSA−/− decreased by ~25 dB (Fig. 2c, compare top and lower panels). During this 4-week period, non-treated CSA−/− mice developed an average of 28 dB of hearing loss while the hearing threshold in NR-treated CSA−/− mice was elevated only 6 dB, which is not considered as hearing loss (<10 dB) (Fig. 2d). These results collectively suggest that NAD+ supplementation strongly reduces hearing loss in both CSA−/− and CSBm/m mice.

Fig. 2: NAD+ supplementation prevents progressive hearing loss in CSA−/− mice. a NAD+/NADH levels were measured in CSA−/− (7-month-old) mice (N = 3). Two-tailed t-tests were used to determine significant difference. b Outline for NR treatment and ABR recordings in CSA−/− mice. c ABR thresholds of CSA−/− and WT mice following 4 weeks of NR treatment (24 mM) in their drinking water. Total of 19 mice (CSA−/− (N = 6); CSA−/− + NR (N = 5); WT (N = 4); WT + NR (N = 4)) were used to measure ABR at the age of 5 weeks (before NR treatment) and 9 weeks (after NR treatment). CSA−/− mice do not show a significant threshold shift at any frequency at 5 weeks of age. However, at 9 weeks, CSA−/− mice show an approximately 20-dB shift by comparison with WT. Multiple graphs are used to visualize error bars on each dataset. Two-way ANOVA with Tukey’s post-hoc test was used in to determine significant difference. d A significant threshold shift is present in CSA−/− mice and WT at 32 kHz. Treatment with NR (24 mM) induced a significant rescue of the age-related threshold shift in CSA−/− and WT mice. ABR data in c at 5 and 9 weeks of age were used to calculate the hearing shift. Two-way ANOVA with uncorrected Fisher's LSD post-hoc test was used to determine significant difference. Full size image

Outer hair cells in NR-treated CS mouse genotypes

To gain more insight into the mechanisms by which NR reduces hearing loss in CS mouse genotypes, we assessed auditory capacity with DPOAE assay, an approach that provides information about cochlear integrity and outer hair cell function. The results show that CSBm/m mice at 12 weeks of age have lower DPOAE signals than WT mice at 16 kHz (Fig. 3a, left panel), but not at 10 and 12 kHz (Supplementary Fig. 3a). This decrease at 16 kHz was normalized by exposure to NR (Fig. 3a, middle panel), suggesting that NAD+ supplementation improves outer hair cell function in CSBm/m mice at 16 kHz. In the CSA−/− mice at 9 weeks of age, the DPOAE response was similar to WT mice for all frequencies tested, suggesting that outer hair cell function in 9-week-old CSA−/− mice are intact and functional (Fig. 3b and Supplementary Fig. 3b).

Fig. 3: Short-term NR supplementation restores reduced DPOAE levels in CSBm/m mice at 16 kHz. a NR intervention (12 mM of NR for 7 weeks) corrects reduced DPOAEs in CSBm/m mice at 12 weeks of age at 16 kHz (CSBm/m (N = 5); CSBm/m + NR (N = 3); WT (N = 7); WT + NR (N = 6)). Multiple graphs are used to visualize error bars in each dataset. b DPOAE levels in CSA−/− and WT at 9 weeks of age (CSA−/− (N = 6); CSA−/− + NR (N = 5); WT (N = 3); WT + NR (N = 4)) were recorded at 16 kHz following 4 weeks of NR treatment (24 mM) in their drinking water. Mean ± S.E. is shown in all graphs. Multiple graphs are used to visualize error bars on each dataset. The input in dB/output in dB functions in the range from 50 dB SPL to 60 dB SPL input were used to perform statistical analysis on groups, followed by a linear mixed-effect model on repeated-measures of DPOAEs in each group. Full size image

Sensorineural hearing loss arises specifically from degenerative and functional changes in cochlear hair and auditory nerve cells, and loss of their synaptic connections.18,19 Next, we assessed the degenerative changes in cochlear hair cells in CSBm/m and CSA−/− mice via cochlear histology analysis. The cochlea is composed of base, middle, and apex regions; the base region senses high-frequency and the apex region senses low-frequency sounds. Since hearing loss in CS mice was restricted to high frequencies (Fig. 2), we focused on the basal region of the cochlea, although middle and apical regions were also analyzed (see Methods for details). Hair cell numbers in each area correlate with the hearing capacity for specific frequency ranges. To investigate the influence of NR on hair cell number and function, cochleae were isolated and stained with an antibody to Myo7a, a hair cell marker. There were significantly fewer outer hair cells in the basal cochlear region in CSBm/m mice than in WT (Fig. 4a, b), while middle and apex regions were largely unaffected (Supplementary Fig. 4). This observation is consistent with the ABR data showing high-frequency hearing loss in CSBm/m mice. Exposure to NR normalized outer hair cell numbers in the base cochlear region in CSBm/m mice (Fig. 4a, b). The outer hair cell number was also reduced in the base cochlear region in CSA−/− mice but did not increase significantly after treatment with NR (Fig. 4c, d). As was the case for CSBm/m, the numbers of outer hair cells in the apical and middle regions of the cochleae in CSA−/− mice were not affected, which is consistent with our DPOAE and ABR results at lower frequencies (Supplementary Fig. 5). These results suggest that NAD+ supplementation restores outer hair cell number specifically in CSBm/m mice.

Fig. 4: Outer hair cells are reduced in CSA−/− and CSBm/m mice, and short-term NR supplementation rescues outer hair cell loss in CSBm/m mice. a Summary data for the average number of outer (left) and inner (right) hair cells per 100 μm in the basal region of the cochlea in WT or CSBm/m mice with and without NR treatment (12 mM in drinking water for 10 days) at 6.5 weeks of age. The decreased number of outer hair cells observed in CSBm/m mice is prevented by NR treatment. In contrast, no changes in the number of inner hair cells were observed between conditions. N = 4; mean ± S.E.; two-way ANOVA with Tukey’s post-hoc test was used to determine significant difference. b Representative images of outer and inner hair cells in the cochlear base region of CSBm/m and WT mice following NR treatment as described in a. Myo7a staining is used to identify hair cells. c Outer hair cells were reduced in base region in CSA−/− mice (CSA−/− (N = 4); CSA−/− + NR (N = 3); WT (N = 4); WT + NR (N = 5)). Mean ± S.E.; two-way ANOVA with Tukey’s post-hoc test was used to determine significant difference. d Representative images of base outer and inner hair cells of CSA−/− and WT mice following NR treatment as described in c. Myo7a staining is used to identify hair cells. Full size image

Ribbon synapses in NR-treated CS mouse genotypes

The number of inner hair cells in all regions of the cochlea was similar in WT, CSA−/−, and CSBm/m mice (Fig. 4a, c, right panels). We therefore next investigated the integrity of synaptic transmission between the inner hair cells and afferent neurons affecting the hearing loss in CS mouse genotypes. Inner hair cells form a specialized synapse, ribbon synapse, which provides rapid and sustained synaptic transmission.15,20,21 The function of ribbon synapses relies on the formation of unique electron-dense structures called synaptic ribbons, which are regarded as an accurate metric of inner hair cell afferent innervation.15,20,22 Interestingly, the major component of the synaptic ribbons is the protein Ribeye, which is important for the function and physical integrity of ribbon synapses, and it possesses a binding pocket for NAD(H).23 It has been shown that NAD(H) binding to Ribeye modulates the dimerization of Ribeye monomers to build the synaptic ribbon.16,24 Therefore, we tested whether NAD+ supplementation impacts ribbon formation in CS. We isolated cochleae and stained them with anti-Ctbp2 (Ribeye). Immunolabeling was quantified by counting ribbon-forming puncta in inner hair cells of WT, CSA−/−, and CSBm/m mice. Compared to WT mice, CSA−/− and CSBm/m mice exhibited a significant reduction in ribbon counts of inner hair cells in the basal region of the cochlea, suggesting a defect in synaptic transmission in both genotypes of CS mouse (Figs. 5a, b and 6a, b). Remarkably, treatment with NR normalized ribbon counts in the same cochlear region of CSA−/− and CSBm/m mice (Figs. 5a, b and 6a, b). Synaptic ribbons were also reduced in the middle and apex cochlear regions in CSA−/− and CSBm/m mice, respectively (Figs. 5e, f and 6c, d). After intervention with NR, synaptic ribbons were normalized in the apex region in CSBm/m but not in CSA−/− mice. NR had no impact on the middle cochlear ribbons in either mouse strain (Figs. 5c, d and 6c, d). Although NR reduced hearing loss in WT mice (Fig. 2d), intervention with NR did not alter inner hair cell number/synaptic ribbon count in WT mice (Fig. 6a, b). Collectively, these results suggest that NR intervention restores impaired ribbon synapse formation, thereby improving synaptic transmission during the cochlear response to auditory stimuli to rescue high-frequency hearing loss in both genotypes of CS mouse.

Fig. 5: NR enhances synaptic ribbon count per inner hair cell in the base and apex regions in CSBm/m mice cochlea. a, c, e The average synaptic ribbon count per inner hair cell in the cochlea base region (a), middle region (c), and apex region (e). The average number of ribbons is reduced in the base turn of the cochlea in CSBm/m mice relative to WT at 6.5 weeks of age. However, this effect is prevented by NR treatment. A similar change is observed in the apical turn but not in the middle region of the cochlea. b, d, f Representative image of immunostaining for synaptic ribbons (red, anti-Ctbp2) of cochlear base segments (b), middle segments (d), and apex segments (f). A magnification of 40× was used for all images that are oriented such that the base of each hair cell is located on the right side of the image. Hair cell nuclei, which are also labeled with anti-Ctbp2, are located on the left side. Each individual puncta represents a single synaptic ribbon (arrows). Fifteen cells per region per mouse are used for quantification; 12 mM NR in drinking water for 10 days was used for intervention (CSBm/m (N = 4); CSBm/m + NR (N = 5); WT (N = 4); WT + NR (N = 4); mean ± S.E.; two-way ANOVA with Tukey’s post-hoc test was used to determine significant difference). Full size image