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.
Highlights
The sodium–hydrogen exchangers NHE6 and NHE 9, members of the solute carrier (SLC) gene superfamily, are associated with endosomes
NHE1 – 9 genes are expressed in postnatal WT and Nhe[6] KO mouse cochleae
We found that all NHE isoforms were expressed in the inner ears of both strains
Summary
The sodium–hydrogen exchangers NHE6 and NHE 9, members of the solute carrier (SLC) gene superfamily, are associated with endosomes. NHE6 is integral component of early, and recycling endosomes, and the cell surface in different phenotypes[7] It is a candidate regulator of neuronal endolysosomal pathways. 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. 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+. Survival of cochlear and vestibular neurons of the inner ear requires both BDNF and neurotrophin 3 (Nt3; Ntf3), along with their receptors TrkB (Ntrk2) and TrkC (Ntrk3)[23] These signalling molecules are responsible for outer hair cell (OHC) innervation[23]. NHE6 loss leads to smaller synapse populations and defects in dendrite and axon branching and dendritic spines, diminishing connectivity[24]
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