Introduction

The sodium–hydrogen exchangers NHE6 and NHE 9, members of the solute carrier (SLC) gene superfamily, are associated with endosomes. NHE6 is involved in early and recycling endosomes and NHE9 with late recycling endosomes1,2, with NHE6 also localizing to the cell surface. Variants of NHE6 (which is encoded at the Xq26.3 locus by SLC9A6) have been linked to features of Angelman syndrome in males, including ataxia, behavioural differences, epilepsy, intellectual disability, and microcephaly3. However, variants also have been identified that delineate three apparent subtypes outside of the classic Angelman presentation: two that are Angelman-like, including Christianson syndrome4, and a suite of features that includes autistic behaviours with severe intellectual disability, along with tau deposition and corticobasal degeneration5,6.

NHE6 is integral component of early, and recycling endosomes, and the cell surface in different phenotypes7. It is a candidate regulator of neuronal endolysosomal pathways. This protein has been suggested to regulate receptor recycling at the cell surface, along with endosomal volume and endosomal pH through sodium–hydrogen exchange8,9,10. Even after internalization, recycled receptors – including neurotrophin receptors – can continue to signal. The pH of endosomal populations represents a crucial regulator of how long these signals persist11, and the internal pH can be increased by proton exchange for cytoplasmic sodium or potassium, mediated by NHE proteins in the endosome.

Many members of the Rab family of small GTPases play central roles in compartment-specific processes; thus, they are commonly used as markers of vesicular identity12,13. The small GTPases, Rab5, Rab7, and Rab11, are key markers of early and late endosomes; these GTPases organize effector proteins into specific membrane subdomains14,15,16.

The regulation of pH creates a chain of associated events involving the kidney, bone, and inner ear that may triangulate on a cochlear role for NHEs. Although NHE activity in the inner ear remains something of a black box, NHEs 6 and 9 both are enriched in vestibular hair bundle plasma membranes, where they probably release H+ into the cytoplasm in exchange for K+. This process allows hair cells to dump protons in an ATP-independent manner following proton ingress via calcium pump activity17. At the kidney, coupling of bicarbonate reabsorption with acid secretion maintain the body's acid–base balance over the long term18, but in cases of failure, the result is distal renal tubular acidosis (dRTA). If acidosis persists, bone dissolution can follow, with attendant diseases such as osteomalacia or rickets. Progressive bilateral sensorineural hearing loss is a common comorbidity of dRTA18, possibly because of disrupted pH homeostasis in the inner ear19.

Tying these events together is the possibility that endosome acidification underlies some relevant neuronal defects. Neurotrophin signalling through brain-derived neurotrophic factor (BDNF) is required for central nervous system (CNS) development and maintenance20. Neuronal signalling using neurotrophin receptors (Trks) relies on endosomal pathways, which maintain neuron survival and correct targeting in the peripheral nervous system21,22. Survival of cochlear and vestibular neurons of the inner ear requires both BDNF and neurotrophin 3 (Nt3; now Ntf3), along with their receptors TrkB (Ntrk2) and TrkC (Ntrk3)23. In addition, these signalling molecules are responsible for outer hair cell (OHC) innervation23. NHE6 loss leads to smaller synapse populations and defects in dendrite and axon branching and dendritic spines, diminishing connectivity24.

In humans, sound perception has a major role in communication. Via mechanoreception, cochlear hair cells innervated by type I and II afferent neurons transduce incoming signals and pass them to the CNS through chemical synapses with the dendrites of spiral ganglion (SG) neurons25,26. Based on their molecular profiles, adult SG neurons are categorized into four types: three novel type I subclasses (Ia, Ib, and Ic neurons) and type II neurons27.

We hypothesized that the absence of NHE6 might disturb endosomal–lysosomal function, which might lead to a lack of BDNF/TrkB signalling within the cochlea and afferent cochlear nerve. Therefore, we examined the role of NHE6 in mouse cochlea by studying hearing loss in Nhe6 knockout (KO) mice. We used various methods for assessing the consequences of hearing loss. We also studied secondary interruptions in BDNF/TrkB signalling and endosomal-lysosomal dysfunction. We next hypothesized that abnormal endosomal acidification might perturb endosomal signalling mechanisms relevant to neuronal development and arborization. We focused on endosomal-lysosomal dysfunction and found differences in Rab5, 7, and 11 expression in the SGs of WT and KO mice. In addition, we found that Nhe6 KO mice had significant changes in gene expression specific to SG types I and II nerves and a reduction in cochlear afferent nerve fibres.

Results

NHE1 – 9 genes are expressed in postnatal WT and Nhe6 KO mouse cochleae

The NHE proteins belong to a large family of transporters known as the solute carrier (SLC) gene superfamily28,29. We performed quantitative PCR (qPCR) to investigate NHE expression in cochlear samples from postnatal day 5 (P5) WT and Nhe6 KO mice. We found that all NHE isoforms were expressed in the inner ears of both strains (except NHE6 KO mice lacked Nhe6). However, Nhe3, Nhe5, and Nhe7 expression in the OC, and Nhe1 and Nhe8 expression in the SG were down-regulated in Nhe6 KO mice, compared to WT mice (Fig. 1). Considering these differences in gene expression, we have tested the protein expression levels of NHE3 and NHE6 and found no significant differences between WT and NHE6 KO (Supplementary Fig. 2).

Figure 1
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 mice56. 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).

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Adult Nhe6 KO and WT mice show similar cochlear microanatomy

Recently, physiological and pharmacological studies have indicated that NHEs participate in ion homeostasis in the inner ears of guinea pigs and gerbils30,31. We stained sections of the temporal bones of adult WT and Nhe6 KO mice with haematoxylin and eosin to study potential differences in cochlear microanatomy. We found no significant differences in inner ear microanatomy between WT (Fig. 2a–e) and Nhe6 KO mice (Fig. 2f–j). All displayed OCs with three rows of OHCs and one row of inner hair cells (IHCs). The basilar membrane, SG neurons, and the SV also showed similar morphology between mouse strains. Finally, the middle ear microanatomy was not significantly different between WT and Nhe6 KO mice (Supplementary Fig. 1).

Figure 2
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.

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NHE6 protein expression patterns in adult mouse cochlea

We first investigated the expression and distribution of NHE6 in the mouse cochlea. Previously, NHE activity was demonstrated in the guinea pig cochlea32. We stained formalin-fixed paraffin-embedded adult mouse cochlear sections with specific fluorescently-labelled antibodies against NHE6. Cochlear section of WT mice stained only with secondary antibodies served as negative control (Fig. 3a). As expected, we detected no NHE6 staining in KO mouse cochleae (Fig. 3b). In WT mouse cochleae, the NHE6 protein was found in the IHCs, OHCs, SV, and SG (Fig. 3c). Higher-magnification images revealed strong NHE6 staining in IHCs and OHCs (Fig. 3d). NHE6 staining was also detected in the SV and SG (Fig. 3e,f). Split single channel exposure to myosin VIIa, DAPI and NHE6 could be found in Supplementary Fig. 3.

Figure 3
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.

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Adult NHE6 KO mice show elevated auditory thresholds compared to WT littermates

To extend these studies, we tested hearing function in vivo in WT and Nhe6 KO mice. Mice were tested with broadband click stimuli and pure tones to determine hearing thresholds. Nhe6 KO mice exhibited significantly elevated auditory brainstem response33,34,35,36 thresholds for click stimuli and sound stimuli delivered at 4 kHz, 12 kHz, and 32 kHz frequencies, compared to WT littermates (Fig. 4a). Representative click-induced ABR recordings from WT and Nhe6 KO mice are shown in Fig. 4b. No differences were observed in the ABR waveforms, amplitudes (Fig. 4c), or inter-peak latencies (Fig. 4d) between WT and Nhe6 KO mice.

Figure 4
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.

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Phospho-Trk and Akt levels are reduced in Nhe6 KO, compared to WT mice

BDNF regulates many facets of central neurons, including neuronal survival and differentiation, neuronal growth, synaptogenesis, plasticity, and the maintenance of neuronal circuits37. BDNF mainly mediates neuronal growth effects through its tyrosine kinase receptor, TrkB38. Activation first causes TrkB dimerization and autophosphorylation, which then leads to the activation of the three main signal pathways: Ras/Raf/MAPK, PI3K/AKT, and PLC-γ39. Neurotrophin receptors use endosomal pathways for signalling in neurons. Post-endocytic trafficking of endocytic receptors are mainly regulated by Rab monomeric GTPases. Rabs regulate vesicular trafficking by controlling the transport, anchoring, and coupling of vesicles through effector binding40. Rab5, Rab7, and Rab11 are among the key GTPases known to be involved in BDNF/TrkB signalling41.

We detected no significant difference in BDNF protein contents between KO and WT mice (Fig. 5a). Quantification of Western blot signals show BDNF protein expression in OC explants of KO and WT mice (Fig. 5b). Western blot analyses indicated that phosphorylated TrkB (p-Trk) levels in the OC of Nhe6 KO mice neurons were significantly reduced compared to WT mice, while levels of total TrkB (t-TrkB) remain similar (Fig. 5c). These findings suggested that the absence of NHE6 promoted significant differences between WT and KO mice (Fig. 5d).

Figure 5
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.

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To investigate Akt activation, we performed Western blots with OC lysates from WT and KO animals. The phosphorylated Akt signal (p-Akt) was significantly lower in the OC of KO mice compared to WT mice (Fig. 6a), indicated by the ratio of p-Akt to total Akt (t-Akt). To characterize the NHE6 endosomal compartment further, we quantified the expression of Rab5 (early endosomes), Rab7 (late endosome), and Rab11 (recycling endosome). Rab5 expression was not significantly different between the strains. Furthermore, Rab7 protein levels were reduced and Rab11 levels were increased in Nhe6 KO mice compared to WT littermates (Fig. 6b,c).

Figure 6
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.

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Gene expression in SG neurons I and II in WT and Nhe6 KO mice

Petipré et al. identified four distinct types of adult SG neurons, including three novel subclasses of type I neurons (Ia, Ib, and Ic neurons) and type II neurons. These types can be distinguished based on unique and combinatorial molecular profiles27. We next explored the expression of specific genes in types I and II SG neurons dissected from WT and Nhe6 KO cochleae. We performed qPCR in three biological replicates to investigate differences in gene expression. SG II neurons were specifically identified by the expression of peripherin (Prph), Plk5, and Cacna1g. SG Ia neurons were identified by the expression of Slc17a7, Calb1, and Pou4f1. SG Ib neurons were identified by the expression of Lypd1 and Pou4f1. SG Ic neurons were identified by the expression of Calb2. All these genes, except Cacna1g, were differentially expressed in Nhe6 KO mice, compared to WT mice (Fig. 7).

Figure 7
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.

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Nhe6 KO mice exhibit a reduction in afferent cochlear neurons

Neurotrophin signalling controls cochlear innervation. A lack of neurotrophin signalling in the cochlea has been well documented in early postnatal animals. This condition results in the loss of cochlear sensory neurons and a region-specific reduction in target innervation along the tonotopic axis42. Disruptions in BDNF/Trkb signalling lead to impaired neural growth. Here, we sought to evaluate neural growth by staining cochlear sagittal sections of WT and Nhe6 KO adult mice with an anti-neurofilament (anti-NF200) antibody to visualize afferent innervations (Fig. 8a). Signal intensity quantifications revealed a significant reduction in NF200-positive fibres in the cochlear nerves of Nhe6 KO mice, compared to observations in WT mice (Fig. 8b).

Figure 8
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.

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Discussion

NHE6 participates in regulating cytosolic and organellar pH and cell volume. NHE6 also contributes to whole body volume and acid–base homeostasis. Mutations in the NHE6 genes cause neurological disease and contribute to the pathophysiology of multiple human diseases3. Little is currently known about NHEs in the inner ear. In this study, we investigated the expression and function of NHE6 in mouse cochlea with the Nhe6 KO mouse. We found that NHE6 mRNA was expressed in all compartments of the mouse cochlea (OC, SG, SV). These results were consistent with those from studies performed in guinea pigs and gerbils30,31. Despite the fact that we did not find any morphological differences in the middle or inner ears of WT and Nhe6 KO mice, the Nhe6 KO mice displayed significant hearing loss compared to WT littermates. Nhe6 KO mice had elevated hearing thresholds in click-induced and frequency-specific ABR measurements, compared to WT littermates. The ABR measurements were, on average, about 15 dB different between Nhe6 KO and WT mice, in the acoustic spectrum of 4 kHz to 32 kHz. Interestingly, our data did not show any differences in the latencies or amplitudes of the evoked waves between strains. This finding suggested that the auditory stimuli could travel normally along the successive nuclei in the central auditory pathway, once sound levels surpassed the elevated threshold. These findings indicated that hearing loss in Nhe6 KO animals was due to cochlear damage.

As noted, NHE6 loss can lead to a too-steep decrease in endosomal pH and reduced signalling24,43 which in turn can reshape neuronal arborization through changes in endosomal neurotrophin signalling44,45,46. Embryonic cochlear innervation relies on neutrophins and their respective Trks42,47, and in model organisms, cochlear sensory neurons are lost without appropriate signalling. Loss of signalling also is associated with region-specific reductions in innervation, decreases that track with the axis of frequency detection42. Here, mice lacking Nhe6 had reduced levels of phosphorylated Trk and Akt proteins in the OC compared to WT animals, yet knockout animals had no differences in BDNF protein levels versus WT mice. BDNF, like other neutrophins, is secreted from both the soma and distal neuronal regions, so that signalling to the nucleus can be either local or long distance. One group proposed that the OC does not itself produce BDNF but rather acquires it via the systemic circulation, explaining how levels could still be the same despite strain differences47. The PI3K/AKT signalling pathway controls neuronal growth through its effects on axonal and dendritic protein production and cytoskeleton dynamics38, and NHE6-associated endosomes have been identified in growing axons and dendrites24.

To characterize the NHE6 endosomal compartment in mouse cochlea further, we quantified the levels of Rab5 (early endosomes), Rab7 (late endosomes), and Rab11 (recycling endosomes) in the OCs of both strains. Rab5, 7, and 11 were previously shown to be important for normal neuronal migration and maturation in the cortex, through the regulation of N-cadherin trafficking48. That finding revealed that the endocytic pathway played a physiological role in development. We found that Rab5 expression was similar in both strains, but the level of Rab7 protein was reduced in KO, compared to WT animals. These two endosomal proteins mediate endocytosis and promote neuronal growth through BDNF/Trk signalling. The reduction in Rab7 signalling was a consequence of interrupted signalling. In contrast, Rab11 expression was increased in KO samples, compared to WT samples. This result might be explained by findings from Rink et al., who showed that Rab11 was overexpressed in injured neurons, which led to neuronal autophagy. That study showed that neuronal exosomes enriched with miR-21-5p could inhibit neuronal autophagy by targeting Rab11a, which suppressed trauma-induced, autophagy-mediated nerve injury in vitro13.

Auditory nerve sound signal processing was hypothesized to originate from the diverse biophysical properties of class I and II SG neuronal fibres. Therefore, we examined the expression of specific markers for types I and II SG neurons. Petipré at al identified four types of SG neurons (i.e., three type I subclass neurons and one type II class), along with numerous new marker genes. They also described a comprehensive genetic framework that could shape synaptic communication patterns. In addition, they characterized the differential projection patterns of distinct of type I subclasses to hair cells27. In our study, genes specific to both types I and II neurons were up-regulated in the SGs of Nhe6 KO mice. In contrast, immunohistochemistry showed a reduced number of neuronal fibres in the cochlear nerve of Nhe6 KO mice. Additionally, mouse brains with disrupted NHE6 gene displayed reduced axonal and dendritic branching, reduced synapse numbers, and reduced circuit strength24,49. We assumed that these findings might be explained by the fact that the SG samples were derived from postnatal mice, and there is evidence that perinatal neuronal diversification occurs before the onset of hearing27,50. We showed that BDNF/Trk signalling was impaired in the cochlea of Nhe6 KO mice, which resulted in reductions in neuronal production and development. We found that the other genes described by Petipré et al. were up-regulated in the present study, which might reflect a compensatory effect in the SG of Nhe6 KO mice.

In conclusion, our study showed that Nhe6 KO mice displayed significant, moderate hearing loss. NHE6 was expressed in all WT cochlear tissues. Our findings indicated that the depletion of NHE6 had an effect on Trk protein turnover and endosomal signalling. These results suggested that the failure of dendritic and axonal growth and branching in NHE6 KO mice might have occurred, in part, due to deficiencies in BDNF/TrkB signalling. This novel finding suggested that NHE6 is important for normal hearing and function.

Materials and Methods

Animal care and handling

We isolated cochleae from Nhe6 KO mice and their WT littermates. Steven Walkley of Albert Einstein College of Medicine (Bronx, NY) provided animals with a targeted Nhe6 gene disruption (Jackson Laboratories Stock# 005843, strain name B6.129P2-Slc9a65tm1Dgen). All mice described here were backcrossed for >10 generations into a C57bl6/j (WT) background. We performed genotyping as described in Strømme et al.51. Animals had ad libitum access to water and standard mouse diet and were maintained under 12-h light/12-h dark at the animal experimental station in the Department of Biomedicine Research, which provides housing, feeding, and in-house breeding for colony maintenance. After a week of acclimation, we placed one male with two females in a single cage for breeding. For this work, we used both adult and postnatal-day-5 mice.

All animal procedures were conducted in compliance with the European Communities Council Directive of 24 November 1986 (86/609/EEC) and approved by the Kantonales Veterinäramt, Basel, Switzerland. Animals were inspected regularly for health status, and maintenance protocols were subject to the provisos of the animal welfare ordinance.

Cochlear microdissection

For the microdissections, we decapitated P5 Nhe6 KO and WT male and female mice and performed microdissection under light microscopy to isolate the OC, SV, and SG. Tissues were placed initially in ice-cold PBS, and OCs were prepared as described52.

RNA isolation and quantitative PCR

Tissues for RNA isolation were placed in RNAlater (Ambion, USA), and RNA was isolated as described53, using the Direct-Zol RNA MiniPrep kit (Zymo Research, USA) and following manufacturer instructions. RNA quantity and quality were determined using a NanoDrop 1000 (Thermo Scientific), and all samples had 260/280-nm absorbance ratios between 1.8 and 2.1. Reverse-transcription of total RNA (1000 ng) was performed using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, USA), and qPCR was run with the ABI Prism 7900HT Sequence Detection System (Applied Biosystems, USA) and with Power Sybr Green Master Mix (Applied Biosystems, USA) or TaqMan protocol (for NHE gene expression). qPCR primers (250 nM per reaction) were obtained from Microsynth (St. Gallen, Switzerland) and are listed in Table 1 in the supplementary information. The cycling steps were as follows: 10 min at 95 °C, followed by 40 cycles of 95 °C for 15 s and 60 °C for 60 s. To control for nonspecific amplification and contamination, we ran template-free controls. We used the comparative threshold cycle method to calculate relative cDNA quantities, with GAPDH as reference.

Western blotting

We have previously described preparation of protein samples from the cochlea of P5 pups53. Briefly, samples were placed in cell lysis buffer with a protease inhibitor cocktail (Sigma C3228, P8340), followed by homogenization on ice for 1 min. We used the NanoDrop 1000 (Thermo Scientific) to measure proteins, with mouse brain lysate serving as control. Lysates then were mixed with Laemmli sample buffer (Sigma) in equal amounts and heated for 5 min at 95 °C. After sample resolution on SDS-PAGE gels (10 μg protein per lane), gels were blotted onto polyvinylidene fluoride membranes. Following blockage of nonspecific sites with 5% nonfat dry milk in PBS for 1 h at room temperature (RT), we incubated membranes with primary antibodies in PBS-Tween. Primary antibodies were as follows: mouse monoclonal anti-p-TrkB (1:1000, Santa Cruz Biotechnology, sc-8058), mouse monoclonal anti-t-TrkB (1:1000, Invitrogen, MA5-14903), rabbit polyclonal anti-p-Akt 1:1000 (Cell Signalling, #9275), rabbit polyclonal anti-t-Akt 1:1000 (Cell Signalling, #9272), rabbit polyclonal anti-BDNF (1:1000, Sigma AG, AB1779SP), and rabbit monoclonal anti-Rab5, 7, 11 (1:1000, Cell Signalling, #3547; #9367; #5589). Secondary antibodies were anti-mouse (1:3000, Cell Signalling, #7076P2) and horseradish peroxidase–linked anti-rabbit (1:2000, Cell Signalling, #7074P2). Membranes were incubated overnight with primary antibodies at 4 °C, washed with PBS-Tween (3 × 10 min), and incubated with an appropriate secondary antibody at RT for 1 h. After washings, Super Signal West Dura Extended Duration Substrate (Thermo Scientific, Switzerland) was used to visualize bands, with rabbit polyclonal β-actin (1:1000, Cell Signalling, #4967) to control for protein loading.

Paraffin cochlear sections for morphological staining and immunofluorescence

To assess middle and inner ear morphology and HNE6 and N200 expression levels, we used cochleae from adult male and female WT and Nhe6 KO mice. After animals (n = 4) were killed via sodium pentobarbital (100 mg/kg), they were transcardially perfused (50 ml phosphate-buffered 4% paraformaldehyde, pH 7.4; 4 °C). The inner ear was removed and decalcified for 10 days in a light-protected flask containing 120 mM EDTA (Merck, New Jersey, USA) in distilled water (pH 6.8). Cochleae then were dehydrated in graded ethanols (70, 80, 95, and 3 × 100%, 1 h each; 3× xylol for 1 h; 2× Paraplast at −60 °C for 1 h; and 1× Paraplast at −60 °C for 10 h), following by paraffin embedding at 56°C.

For histological evaluations, 10-µm sections were made using a Leitz microtome and mounted on Superfrost-Plus slides (Menzel, Braunschweig, Germany). Immunohistochemistry has been described before54, but briefly, sections were deparaffinized, rehydrated, and washed in PBS for 5 min, followed by incubation in blocking solution (TBS: 50 mM Tris, 0.9% NaCl; 0.5% Triton X-100, pH 7.35; 3% normal goat serum [NGS]) for 1 h at RT. For antibody binding, we incubated the sections with a mouse monoclonal antibody against myosin VIIa (1:350, Abcam, ab150386), primary anti-NF200 (1:500, Sigma AG, N4142), and rabbit anti-NHE6 (1:1000). To generate a rabbit polyclonal antibody targeting the NHE6 carboxyl terminus, we used cysteine bonding to couple maleimide-activated KLH with a synthetic peptide containing the last 13 amino acids of mouse NHE6. Rabbit immunization took place at UT Southwestern, following IACUC-approved protocols55. After overnight dilution of antibodies in TBS with 1% NGS at 4 °C and then three TBS washes, sections were incubated with the appropriate Alexa-conjugated secondary antibodies (1:250; Molecular Probes, Lubio Science, Switzerland). Antibodies were diluted in TBS with 1% NGS, and incubations were done at RT for 2 h. Following another TBS wash and DAPI counterstain, sections were mounted on glass slides using Mowiol. Negative controls were samples of WT tissues. To visualize binding, we used an Olympus AX-70 microscope equipped with a spot digital camera and adjusted recorded images for brightness and contrast using Image-Pro Plus and Photoshop.

Haematoxylin and eosin staining also was performed on some paraffin-embedded sections. As we previously described52, haematoxylin staining (Sigma Aldrich, Switzerland) was performed on sections for 5 min, followed by a 15-min rinse in tap water and immersion in eosin 1% aqueous (Eosin Y disodium salt, in deionized water; Sigma Aldrich, Switzerland) for 1–2 min. Before use, the solutions were filtered (Baxter grade 363, qualitative), and sections were rinsed in tap water until the water was clear. After dehydration in ascending alcohol concentrations (50%, 70%, 80%, 2× 95%, and 2× 100%), xylene clearance (2 times), and mounting with Eukitt (Fluka, Sigma Aldrich, Switzerland), sections were imaged using an Olympus IX50 microscope with a spot digital camera. As above, images were adjusted for brightness and contrast using Image-Pro plus and Photoshop.

Hearing function tests

We administered the ABR test to all animals before treatment, as described previously53 (Tucker-Davis Technologies; TDT -RZ6-A-P1 hardware and software; Alachua, FL, USA), followed by their anesthetization with intraperitoneal ketamine (80 mg/kg body weight [BW]; Graeub AG, Switzerland), 12 mg/kg BW xylazine (Graeub AG, Switzerland), and 2 mg/kg BW acepromazine (Arovet AG, Switzerland). Testing was performed in sound-attenuating acoustic chambers, with infrared warming pads (Kent Scientific Corporation, USA) to keep animal body temperature at 38 °C. BioSig software was used to synthesize the tone burst acoustic stimuli (duration, 10 ms; rise/fall time, 0.5 ms; Blackman window), with an SA1 audio amplifier and a Multi Field Speaker-Stereo (MF1-S) as transducer. For testing, we placed an inverting electrode over the mastoid of the target ear, a non-inverting needle electrode at the midline vertex, and a ground electrode on the upper hindlimb. The speaker (MF1-S) was placed in the ear canal. Electrodes collected the signals, which then were amplified 20× with band-pass filters (100 Hz to 5 kHz) and input into a real-time processor (RA4PA) for software processing (BioSig RP software; TDT). We evaluated thresholds at 4, 12, or 32 kHz. Signals were preamplified with a gain of 20, and for each trial, we averaged 1000 sweeps. The starting level for stimuli presentation was 90 dB, which was incrementally decreased by 5 dB to threshold (the lowest intensity at which a visible, repeatable ABR wave could be observed in two averaged runs) and the ABR wave disappeared for each frequency. We calculated the amplitude and latency growth input–output function slopes (i.e., amplitude and latency as a function of stimulus intensity), as described before53,54,55.

Statistical analysis

Statistical analyses were performed with the Student's t-test for unpaired samples. When more than two groups were compared, we performed a one-way analysis of variance (ANOVA), followed by the Bonferroni post-hoc test. A p value <0.05 was considered statistically significant. All statistical tests were two-sided. Data were analysed with GraphPad Prism 7 software (La Jolla, CA, USA).