Long-term NAD+ supplementation prevents the progression of age-related hearing loss in mice

Abstract

Age-related hearing loss (ARHL) is the most common sensory disability associated with human aging. Yet, there are no approved measures for preventing or treating this debilitating condition. With its slow progression, continuous and safe approaches are critical for ARHL treatment. Nicotinamide Riboside (NR), a NAD+ precursor, is well tolerated even for long-term use and is already shown effective in various disease models including Alzheimer's and Parkinson's disease. It has also been beneficial against noise-induced hearing loss and in hearing loss associated with premature aging. However, its beneficial impact on ARHL is not known. Using two different wild-type mouse strains, we show that long-term NR administration prevents the progression of ARHL. Through transcriptomic and biochemical analysis, we find that NR administration restores age-associated reduction in cochlear NAD+ levels, upregulates biological pathways associated with synaptic transmission and PPAR signaling, and reduces the number of orphan ribbon synapses between afferent auditory neurons and inner hair cells. We also find that NR targets a novel pathway of lipid droplets in the cochlea by inducing the expression of CIDEC and PLIN1 proteins that are downstream of PPAR signaling and are key for lipid droplet growth. Taken together, our results demonstrate the therapeutic potential of NR treatment for ARHL and provide novel insights into its mechanism of action.

Keywords: NAD+; age-related hearing loss; nicotinamide riboside.

FIGURE 1

NAD+ supplementation using NR prevents the progression of age‐related hearing loss in WT mice (mtKeima). (a) The total NAD+ levels per ug of the cochlea (left panel) and relative NAD+/NADH levels (right panel) were measured in the cochlea of young (2‐month‐old), old (12‐month‐old), and NR‐treated old mice (12‐month‐old). N = 3 and ordinary one‐way ANOVA were used to determine significant differences. (b) Outline for NR treatment and ABR/DPOAE recordings in WT mice (mtKeima). (c) ABR thresholds for WT and NR‐treated WT mice at 2, 8, and 12 m of age. A total of 40 WT mice were tested for hearing capacity at the age of 2 m and then randomly split into two groups of NR‐treated (N = 25) and non‐treated (N = 15). NR treatment started at the age of 2 m. ABRs in both groups were measured again at the age of 8 and 12 m, which correspond to 6 and 10 m of NR treatment respectively. Mixed effect analysis with Sidak's multiple comparison test was used to determine significant differences. (d) Threshold shifts at 8, 16, and 32 kHz in NR‐treated and untreated groups. ABR data in (c) were used to calculate the hearing shift. Each dot represents a data point for an individual mouse. Two‐tailed t‐test was used to determine significant differences. Mean ± SE, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001 and n.s., not significant.

FIGURE 2

NR does not affect DPOAE but elevates wave I and III ABR amplitudes in WT mice (mtKeima). (a) Averaged ABR waveforms in NR‐treated and non‐treated mice resulting from a 32 kHz 40 dB SPL stimulus presented at 2, 8, and 12 m of age. (b) Quantification of wave I‐V amplitudes at 12 m. NR treatment significantly preserves wave I and III amplitudes. Each dot represents a data point for an individual mouse. Two‐tailed t‐test was used to determine significant differences. (c) DPOAE levels at 32 kHz are shown at the age of 2, 8, and 12 m of age in NR‐treated (N = 25) and non‐treated mice (N = 13). The area under the curve (above −10 on the y‐axis) is calculated for each sample and two‐way ANOVA with Tukey's post hoc test was used for statistical analysis. Mean ± SE. *p ≤ 0.05, ****p ≤ 0.0001 and n.s., not significant.

FIGURE 3

The transcriptomic analysis in NR‐treated and non‐treated cochlea in WT mice (mtKeima) (N = 5, 12‐month‐old). (a) The number of up‐ and down‐regulated genes with a p‐value ≤0.05 in NR‐treated and non‐treated mice cochlea. (b) Heatmap showing clustering of differentially expressed genes (DEG) (p ≤ 0.05) in NR‐treated and non‐treated mice cochlea. (c) Graph showing the top 50 up‐ or down‐regulated GO terms from the WT cochlea ±NR treatment. A padj‐value ≤0.05 were the cutoff used for significance. (d) Graph showing the up‐regulated KEGG pathways from the WT cochlea ±NR treatment. A padj‐value ≤0.05 were the cutoff used for significance. No significant down‐regulated KEGG pathways were detected. (e) The table shows a list of the top 10 genes with the highest value of fold change (up‐ or down‐regulated). A padj‐value ≤0.05 and fold‐change (log2) ≥3 were the cut‐offs used for significance.

FIGURE 4

NAD+ supplementation using NR modulates the expression of CIDEC and PLIN1, key proteins of lipid droplets dynamics in WT mice (mtKeima). (a, b) Quantitative RT‐PCR results demonstrate the relative fold change in Pck1, Cidec, Plin1, and PPARγ in the cochlea of young (2‐month‐old), old (12‐month‐old), and NR‐treated old mice (12‐month‐old). (N = 4 mice per group, one‐way ANOVA with Tukey's post hoc test was used for statistical analysis.) (c) Western blot depicts protein expression in the cochlea of young (2‐month‐old), old (12‐month‐old), and NR‐treated old mice (12‐month‐old). (d) The graph demonstrates the quantification of the average signal from the Western Blot in Figure 4c using the NIH ImageJ program. One‐way ANOVA with Tukey's post hoc test was used for statistical analysis. (e) Quantitative RT‐PCR results demonstrate the relative fold change in Pck1 and Cidec following NR treatment (1 mM, 24H) in HEI‐OC1 cells. Each dot represents a data point from independent biological repeats. (f) Western blot depicts protein expression from HEI‐OC1 cells following NR treatment (1 mM, 24H) from two independent biological repeats (lanes 1 and 3 demonstrate experiment #1, lanes 2 and 4 demonstrate Experiment #2). (g, h) Quantitative RT‐PCR results demonstrate the relative fold change in genes in the cochlea of young (2‐month‐old), old (12‐month‐old), and NR‐treated old mice (12‐month‐old). (N = 4 mice per group, one‐way ANOVA with Tukey's post hoc test was used for statistical analysis.) Mean ± SE. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and n.s., not significant.

FIGURE 5

NR delays hearing loss progression in aged female WT mice (mtKeima) at 24 kHz frequency. (a) Outline for NR treatment and ABR recordings in WT mice (mtKeima). (b) ABR thresholds for NR‐treated and non‐treated mice at 15, and 16.5 m of age. A total of 26 mice were tested for hearing capacity at the age of 15 m and then split into two groups of NR‐treated (N = 13; 6 M, 9F) and non‐treated (N = 13; 6 M, 9F). Groups are gender‐matched and hearing capacity‐matched at 32 kHz at the age of 15 m. NR treatment started at the age of 15 m. ABRs in both groups were measured again at the age of 16.5 m, which corresponds to 1.5 m of NR treatment. Mixed effect analysis with Sidak's multiple comparison test was used to determine significant differences. (c) NR treatment reduces the increased threshold shift at 24 kHz. ABR data in (b) were used to calculate the hearing shift. Two‐tailed t‐test was used to determine significant differences. Each dot represents a data point for an individual mouse. Mean ± SE. **p ≤ 0.01, and n.s., not significant.

FIGURE 6

NAD+ supplementation using NR reduces the progression of age‐related hearing loss in WT mice (CBA/CaJ). (a) Outline for NR treatment and ABR recordings in WT mice (CBA/CaJ). (b) The total NAD+ levels per ug of the cochlea (left panel) and relative NAD+/NADH levels (right panel) were measured in the cochlea of young (3‐month‐old), old (27‐month‐old), and NR‐treated old mice (27‐month‐old). N = 4. (c) ABR thresholds for WT and NR‐treated WT mice at 3, 9, 15, 21, and 27 m of age. A total of 29 WT mice were tested at the age of 3 m and then randomly split into two groups of NR‐treated (N = 15) and non‐treated (N = 14). NR treatment started at the age of 3 m. ABRs in both groups were measured again at the age of 9, 15, 21, and 27 m, which correspond to 6, 12, 18, and 24 m of NR treatment respectively. (d) NR treatment reduces the increased threshold shift at 24 and 32 kHz. ABR data in (c) were used to calculate the hearing shift. The left panel in (e–g) demonstrates the average synaptic ribbon count per IHC while the right panel shows a representative image of immunostaining for synaptic ribbons (violet, anti‐Ctbp2 (Ribeye) and post‐synaptic receptor (green, anti‐GluR2a) of cochlear middle, base, and apex segments. The anti‐Ctbp2 faintly stains hair cell nuclei. Zoom‐in images on the right‐top corners of the panels illustrate the juxtaposition of ribbon–receptor pairs in selected areas. (h) The graphs show the average number of orphan ribbons per inner cell. The violet puncta (anti‐Ctbp2) without green puncta (anti‐GluR2a) juxtaposition is considered an orphan ribbon. Mean ± SE. Mixed effect analysis with Sidak's multiple comparison test in (c), Two‐tailed t‐test in (d) and one‐way ANOVA with Tukey's post hoc test in (a and e–h) were used to determine significant differences. Each dot represents a data point for an individual mouse. Mean ± SE. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001 and n.s., not significant.