Sodium-hydrogen exchanger 6 (NHE6) deficiency leads to hearing loss, via reduced endosomal signalling through the BDNF/Trk pathway

Abstract

Acid-base homeostasis is critical for normal growth, development, and hearing function. The sodium-hydrogen exchanger 6 (NHE6), a protein mainly expressed in early and recycling endosomes, plays an important role in regulating organellar pH. Mutations in NHE6 cause complex, slowly progressive neurodegeneration. Little is known about NHE6 function in the mouse cochlea. Here, we found that all NHE isoforms were expressed in wild-type (WT) mouse cochlea. Nhe6 knockout (KO) mice showed significant hearing loss compared to WT littermates. Immunohistochemistry in WT mouse cochlea showed that Nhe6 was localized in the organ of Corti (OC), spiral ganglion (SG), stria vascularis (SV), and afferent nerve fibres. The middle and the inner ears of WT and Nhe6 KO mice were not different morphologically. Given the putative role of NHE6 in early endosomal function, we examined Rab GTPase expression in early and late endosomes. We found no change in Rab5, significantly lower Rab7, and higher Rab11 levels in the Nhe6 KO OC, compared to WT littermates. Because Rabs mediate TrkB endosomal signalling, we evaluated TrkB phosphorylation in the OCs of both strains. Nhe6 KO mice showed significant reductions in TrkB and Akt phosphorylation in the OC. In addition, we examined genes used as markers of SG type I (Slc17a7, Calb1, Pou4f1, Cal2) and type II neurons (Prph, Plk5, Cacna1g). We found that all marker gene expression levels were significantly elevated in the SG of Nhe6 KO mice, compared to WT littermates. Anti-neurofilament factor staining showed axon loss in the cochlear nerves of Nhe6 KO mice compared to WT mice. These findings indicated that BDNF/TrkB signalling was disrupted in the OC of Nhe6 KO mice, probably due to TrkB reduction, caused by over acidification in the absence of NHE6. Thus, our findings demonstrated that NHEs play important roles in normal hearing in the mammalian cochlea.

Figure 1

Quantitative PCR results show SLC9A subgroup genes, Nhe1 – 9, expressed in the cochlea of 5-day-old WT and Nhe6 KO mice. (Top) In OC samples, Nhe3, 5, and 7 expression levels were significantly down-regulated in the OCs of KO compared to WT mice. In SV samples, no significant difference is observed between WT and KO mice. (Bottom) In SG samples, only in Nhe1 and Nhe8 was down-regulated in KO compared to WT mice. Results are the mean fold-change in transcript levels ± SD, compared to GAPDH, a housekeeping gene. qPCR was done in triplicate (n = 20 mice and 40 OC per strain was pooled) *p < 0.05 (Student’s t-test).

Figure 2

Light microscopy images of WT (a–e) and Nhe6 KO mice (f–j) mouse cochleae (sagittal sections) stained with haematoxylin/eosin. No differences were observed in the morphology of WT and Nhe6 KO mice. Arrows and abbreviations indicate the locations of inner hair cells (IHC), outer HCs (OHC), the spiral ganglion (SG), cochlear nerve (CoN) and stria vascularis (SV). (n = 3 mice per strain). Scale bars = 50 µm.

Figure 3

Immunohistochemistry images show NHE6 protein expression patterns in adult mouse cochleae. Cell nuclei are stained blue and NHE6 is stained red. Myosin VIIa (green) served as a marker for hair cells. (a) Cochlear section stained only with secondary antibodies (negative control). (b) Cochlea from a Nhe6 KO mouse shows no NHE6 expression. (c) NHE6 is abundant in the cochlea of a WT mouse. NHE6 is located in the SV and SG. (c,d) Strong labelling is observed in or around the IHC and OHC of the WT mice. (e) NHE6 expression in the SV at higher magnification. (f) NHE6 expression in the SG at higher magnification. OC, organ of Corti; SV, stria vascularis; SG spiral ganglion; IHC, inner hair cells; OHC, outer hair cells; (n = 3 mice per strain). Scale bars 50 µm.

Figure 4

Click-induced ABR thresholds in WT and Nhe6 KO mice at 3 months of age. (a) Clicks and frequency-specific (4 kHz, 12 kHz, and 32 kHz) sounds were delivered to determine ABR thresholds in 5 WT and 7 Nhe6 KO animals. Bars represent mean ± standard deviation. Click-induced ABR thresholds were significantly lower (about 15 dB; SPL) in WT mice than in Nhe6 KO mice (about 35 dB; SPL) *p < 0.05 (ANOVA). (b) Representative examples of click-induced ABRs in WT and Nhe6 KO. The peaks are indicated in Roman numerals (I–V). (c,d) Sounds 40 dB above the threshold at each frequency were delivered to WT and Nhe6 KO mice. Plots show (c) peak amplitudes and (d) latencies at different points on the ABR waveforms (P1-P5). The P1-P5 amplitudes were not significantly different between WT and Nhe6 KO mice. The raw latency shown for P1 and the corrected latencies of P2-P5 (corrected for P1 latency) were not significantly different between WT and Nhe6 KO mice. Error bars represent the mean ± standard deviation.

Figure 5

Western blot detection of BDNF, phosphorylated TrkB (p-Trk), and total TrkB (t-Trk) proteins in the OCs of WT and Nhe6 KO mouse cochleae. BDNF, p-Trk, and t-Trk proteins were detected in extracts from the OCs of WT and KO P5 mice. (a) BDNF levels were the same in both samples. (b) Quantification of Western blot signals show BDNF protein expression. Values are the mean ± SD expression levels relative to endogenous β-actin. (c) p-Trk levels were reduced in KO samples, compared to WT samples. t-Trk levels were similar in the two mouse strains. (d) Quantification of Western blot signals show the ratio of p-Trk/t-Trk signals. Values are the mean ± SD, normalized to ß-actin. n = 10 mice per strain, 20 explants per mouse group; *p < 0.035.

Figure 6

Western blots show expression of Akt and Rab5, 7, and 11 in the OC of WT and Nhe6 KO mice. (a, Left) Phosphorylated Akt (p-Akt) was reduced in KO samples compared to WT samples. (Right) Quantification of Western blot signals show the mean expression of p-Akt levels normalized to total Akt (t-Akt) levels. (b) Rab5 expression is the same in the both samples. In contrast, Rab7 is reduced and Rab 11 is elevated in Nhe6 KO compared to WT mice. (c) Quantification of Western blot signals show mean expression levels normalized to β-actin levels. n = 20 mice per strain and including pups of both sexes., 40 OCs per mouse group, p < 0.05 with the Student’s t-test.

Figure 7

Quantitative PCR results show expression of specific markers for SG neurons, types Ia-Ic and II, in WT and Nhe6 KO mice. To elucidate the mechanisms underlying the NHE6 influence on the BDNF-Trk signalling pathway, which regulates neuronal growth and branching, we exam  ined gene expression in SG neurons. Type I neurons were identified with specific genetic markers for 3 subclasses: Slc17a7, Calb1, Pou4f1, and Cal2. Type II neurons were identified with 3 specific genetic markers: Prph, Plk5, and Cacna1g. Almost all genes are significantly up-regulated in Nhe6 KO mice SGs compared to WT SGs. Values are the mean ± SD, n = 20 mice per strain and 40 OC per mouse group including pups of both sexes. **p < 0.01; ***p < 0.001; ****p < 0.001; n.s.: not significantly different.

Figure 8

Immunofluorescence microscopy detection of NF200 in the cochleae of WT and Nhe6 KO mice. (a) Cochlear sagittal sections of adult WT (left) and Nhe6 KO (right) mice labelled with anti-NF200 antibody (a-1) Negative control (in WT), blue staining is DAPI. (a-2,3) Visualization of afferent innervations with red NF200 staining in WT. (a-4,5) Visualization of afferent innervations with red NF200 staining in KO. Blue: nuclear marker (DAPI). Arrows point to neuronal fibres in cochlear nerves. (n = 3 mice per strain) Scale bars = 50 μm. (b) Quantifications of fluorescence intensities indicate the abundance of fibres with detectable neurofilaments in the cochlear nerves. WT mice had significantly more fibres than Nhe6 KO mice. Values are the mean ± SD *p < 0.0195.