Mitochondrial Calcium Transporters Mediate Sensitivity to Noise-Induced Losses of Hair Cells and Cochlear Synapses

Abstract

Mitochondria modulate cellular calcium homeostasis by the combined action of the mitochondrial calcium uniporter (MCU), a selective calcium entry channel, and the sodium calcium exchanger (NCLX), which extrudes calcium from mitochondria. In this study, we investigated MCU and NCLX in noise-induced hearing loss (NIHL) using adult CBA/J mice and noise-induced alterations of inner hair cell (IHC) synapses in MCU knockout mice. Following noise exposure, immunoreactivity of MCU increased in cochlear sensory hair cells of the basal turn, while immunoreactivity of NCLX decreased in a time- and exposure-dependent manner. Inhibition of MCU activity via MCU siRNA pretreatment or the specific pharmacological inhibitor Ru360 attenuated noise-induced loss of sensory hair cells and synaptic ribbons, wave I amplitudes, and NIHL in CBA/J mice. This protection was afforded, at least in part, through reduced cleavage of caspase 9 (CC9). Furthermore, MCU knockout mice on a hybrid genetic CD1 and C57/B6 background showed resistance to noise-induced seizures compared to wild-type littermates. Owing to the CD1 background, MCU knockouts and littermates suffer genetic high frequency hearing loss, but their IHCs remain intact. Noise-induced loss of IHC synaptic connections and reduction of auditory brainstem response (ABR) wave I amplitude were recovered in MCU knockout mice. These results suggest that cellular calcium influx during noise exposure leads to mitochondrial calcium overload via MCU and NCLX. Mitochondrial calcium overload, in turn, initiates cell death pathways and subsequent loss of hair cells and synaptic connections, resulting in NIHL.

Keywords: auditory threshold shifts; mitochondrial calcium uniporter (MCU); mouse model; noise-exposure; ribbon synapses; sensory hair cell.

Figure 1

Inhibition of mitochondrial calcium uniporter (MCU) via siRNA silencing or the pharmacological inhibitor Ru360 attenuated noise-induced hair cell loss and hearing loss. (A) Noise-induced outer hair cell (OHC) loss was reduced by siMCU pretreatment as well as by Ru360 treatment. The distance along the cochlear duct correlating with the frequencies of 8, 16, and 32 kHz is indicated. Data are presented as means ± SD; ***p < 0.001. (B) Pretreatment with siMCU attenuated 101-dB-noise-induced auditory threshold shifts measured 14 days after the exposure. Data are presented as individual points and means ± SD, ***p < 0.001. (C) Treatment with Ru360 also attenuated 101-dB-noise-induced auditory threshold shifts measured 14 days after the exposure. Data are presented as individual points and means ± SD, **p < 0.01. (D) Treatment with Ru360 attenuated 108-dB-noise-induced inner hair cell (IHC) loss. The distance along the cochlear duct correlating with the frequencies of 8, 16, and 32 kHz is indicated. Data are presented as means ± SD, *p < 0.05, **p < 0.01. (E) Treatment with Ru360 attenuated 108-dB-noise-induced OHC loss at 3 and 3.5 mm from the apex. Data are presented as means ± SD, *p < 0.05. (F) Treatment with Ru360 did not attenuate 108-dB-noise-induced auditory threshold shifts measured 14 days after the exposure. Data are presented as individual points and means ± SD, n in all figures indicates the number of mice per group; left cochlea was assessed per mouse.

Figure 2

Inhibition of MCU via Ru360 or siMCU attenuates noise-induced loss of synaptic ribbons and wave I amplitudes after the completion of noise exposure. (A) Representative images revealed immunolabeling for CtBP2 examined 14 days after noise exposure. Images are comprised of 40 Z-stack projections taken from the middle turn corresponding to sensitivity to 16 kHz. Blue: myosin-VIIa labeled IHCs, red: CtBP2-labeled synaptic ribbons and nuclei of IHCs; scale bar = 10 μm. (B) Quantification of CtBP2-immunolabeled ribbon particles in IHCs corresponding to 5, 8, 16, 22, and 32 kHz showed significant reduction examined 14 days after noise exposure at all frequencies except 5 kHz (see Supplementary Table S1 for detailed statistical values). Treatment with Ru360 prevented noise-induced synaptic ribbon loss at 8 and 16 kHz. The distance along the cochlear duct correlating with the frequency regions is indicated. Data are presented as means + SEM. **p < 0.01, ***p < 0.001 for 101 dB + Saline vs. 101 dB + Ru360. (C) CtBP2-immunolabeled ribbon particles in IHCs at 16 kHz region also decrease examined 1 h after noise exposure that partially prevented with siMCU pretreatment; n = 4 mice per group with one cochlea used per mouse. Treatment with Ru360 also attenuated higher intensity noise sound pressure level (108-dB-SPL)-induced synaptic ribbon loss; n = 6 mice per group with one cochlea used per mouse. Data are presented as means + SD. ***p < 0.001. (D) Ru360 treatment alone increased wave I amplitudes at sound intensities of 90 dB SPL. Noise-reduced wave I amplitudes at sound intensities of 80 and 90 dB SPL were rescued by treatment with Ru360. Data are presented as means + SEM, *p < 0.05, ***p < 0.001 corresponds to 101 dB + Saline vs. 101 dB + Ru360. In panels (B,D) n indicates the number of mice per group; the left cochlea was used from each mouse for these experiments.

Figure 3

Noise exposure increased immunolabeling for MCU in OHCs and the stria vascularis of the basal turn. (A) Paraffin sections of the adult CBA/J mouse inner ear revealed an increase in immunolabeling for MCU in DAB-stained OHCs (arrows and enlarged image inserts) in the organ of Corti (OC) and the stria vascularis (arrow) in the lateral wall, and no obvious change in spiral ganglion neurons (SGNs) examined 1 h after completion of the 101-dB noise exposure. These images were taken with 40×-magnification lens and are representative of five individual mice per group; scale bar = 10 μm. (B) Representative images for MCU in OHCs of surface preparations stained with phalloidin when processed 1 h after completion of the noise exposure. An enlarged image of three OHCs better illustrates the immunolabeling for MCU. Images were taken from the area of the basal turn corresponding to 22–32 kHz; OHC1, 2, 3 indicate the three rows of OHCs, scale bar = 10 μm. (C) Quantification of immunolabeling for MCU in OHCs in the 22–32 kHz region showed a significant increase when processed 1 h after and 24 h after completion of the exposure. Data are presented as individual points and means ± SD; *p < 0.05, **p < 0.001. Control: n = 8, 101-dB 1 h post: n = 8, 101-dB 24 h post: n = 6 with one cochlea from each mouse in the group. (D) Immunoblots using total cochlear homogenates of CBA/J mice revealed no difference in MCU band densities between control (Ctrl) and noise-exposed mice processed 1 h after completion of the noise exposure (101-dB 1 h post). GAPDH was used as a loading control; n = 8 mice per group.

Figure 4

Noise exposure decreased NCLX immunoreactivity in OHCs of the basal turn in a time-dependent and intensity-dependent manner. (A) Representative images of surface preparations revealed co-localization of NCLX (green) and MitoTracker (red) in OHCs (merged, yellow). An enlarged image of three OHCs better illustrates the co-localization; scale bar = 10 μm, n = 3 per group with one cochlea used per mouse. (B) Representative images for NCLX in OHCs 1 h and 24 h after completion of the exposure. Green: phalloidin-stained OHCs. Images were taken from the 22–32 kHz region of the surface preparations using a Leica SP5 confocal microscope; scale bar = 10 μm. (C) Quantitative analysis of NCLX immunolabeling in OHCs showed a significant decrease in a time-dependent manner. Data are presented as individual points and means ± SD; *p < 0.05, ***p < 0.001, ****p < 0.0001. Control: n = 9, 101-dB 1 h post: n = 6, 101-dB 24 h post: n = 4 with one cochlea used per mouse. (D) Representative immunoblots of total cochlear homogenates from CBA/J mice showed no difference in NCLX band densities between control and noise exposed mice when examined 1 h and 24 h after completion of the exposure. GAPDH served as the sample loading control; n = 6 mice per group.

Figure 5

Inhibition of MCU via the pharmacological inhibitor Ru360 or siMCU attenuated noise-induced increases in cleaved caspase 9 (CC9) in OHCs of the basal turn. (A) Representative images show an increase in immunoreactivity for CC9 (red) in OHCs stained with phalloidin 1 h after completion of the exposure (panel 2) compared to control mice without exposure (panel 1). Treatment with Ru360 attenuated noise-induced CC9 in OHCs (panel 3). Images were taken from the region of the surface preparations corresponding to sensitivity to 22–32 kHz using a Leica SP5 confocal microscope; scale bar = 10 μm. (B) Quantification of CC9 in OHCs confirmed a significant increase after noise exposure and attenuation of this increase with Ru360 treatment; n = 4 per group with one cochlea used per mouse. Data are presented as means + SD, **p < 0.01. (C) Representative images show that pretreatment with siMCU decreases immunoreactivity for CC9 (red) in OHCs stained with phalloidin (green) 1 h after completion of the exposure compared to siControl treatment. Images were taken from the region of the surface preparations corresponding to sensitivity to 22–32 kHz using a Zeiss confocal microscope; scale bar = 10 μm. (D) Pretreatment with siMCU also significantly reduced noise-increased immunolabeling for CC9 in OHCs compared to mice exposed to scrambled siRNA (siControl). Data are presented as means + SD, n = 4 per group with one cochlea used per mouse, *p < 0.05.

Figure 6

MCU knockout mice (MCU^−/−) and wild-type littermates (MCU^+/+) on a hybrid CD1 and C57/B6 background had OHC loss in the basal turn and high-frequency hearing loss. (A) Auditory brainstem response (ABR) thresholds at 8 kHz were not significantly elevated and not different between MCU^−/− mice and MCU^+/+ from weeks 4 to 7 weeks of age but displayed wide variations at 16 kHz. Data are presented as individual points and means ± SD. (B) ABR thresholds at 32 kHz were greatly elevated in 4-week-old MCU^−/− and MCU^+/+ mice without a significant difference between these two groups. Data are presented as individual points and means ± SD. (C) Body weights of MCU^−/− mice were significantly lower than that of littermates at the age of 4–5 weeks, but were the same as wild-type littermates by 6 weeks. Data are presented as individual points and means ± SEM, *p < 0.05. In panels (A,B) n indicates the number of mice per group with one cochlea used per mouse, n in panel (C) indicates the number of mice.

Figure 7

Noise-induced losses of synapses were attenuated in MCU knockout mice at 14 days after the completion of noise exposure. (A,B) Representative images of immunolabeling for IHC synapses in the apical region corresponding to 8 kHz of MCU^+/+ or MCU^−/− mice examined 14 days after the noise exposure. The images were projected from Z sections. Red: CtBP2, green: GluA2, blue: myosin-VIIa-labeled IHCs; scale bar = 10 μm. (C) The number of synapses per IHC was similar between MCU^+/+ and MCU^−/− mice without noise exposure. (D) The number of synapses had recovered significantly in MCU knockout mice but not in littermates when examined 14 days after the noise exposure. (E) Noise-induced loss of ribbons was also similar between MCU^−/− mice and littermates when examined 1 h after the completion of noise exposure. (F) Ribbons were not different between MCU^−/− mice and MCU^+/+ when examined 24 h after the completion of noise exposure. Data are shown as means ± SEM in panels (C–F), n indicates the number of mice with one cochlea used per mouse, *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 8

MCU knockout mice were resistant to reduction in their ABR wave I amplitudes at 8 kHz measured 14 days after noise exposure. (A) There was no significant difference in wave I amplitudes between MCU^−/− and MCU^+/+ mice without noise exposure. (B) MCU knockouts were significantly resistant to reduction in ABR wave I amplitudes after noise in comparison to wild-type littermates at sound intensities of 90 and 100 dB SPL. Data are shown as means ± SEM, *p < 0.05, **p < 0.01. n indicates the number of mice; one cochlea was used per mouse.