Roles of Nedd4-2 in the auditory portion of mouse inner ear

Hearing loss is the most common neurosensory disorder with more than 466 million people affected worldwide. In most cases, hearing loss is caused by the dysfunction or the loss of the sensory cells (the hair cells) and/or their afferent neurons (spiral ganglion neurons) within the ventral portion of the inner ear, called the cochlea. To date, many genetic mutations have been associated with congenital deafness or early-onset hearing loss, allowing for the discovery of genes involved in the development or maintenance of the cochlea. Given the importance of protein ubiquitination in controlling a variety of important biological processes during development, together with a recent report identifying deafness-associated mutations in the human gene encoding the E3 Ubiquitin-ligase NEDD4L (Neurally Expressed and Developmentally Downregulated 4-like), we plan to uncover its implication in cochlear development and function.
We first characterized the spatio-temporal expression profile of Nedd4-2, the mouse ortholog of Nedd4L, together with the closely related Nedd4-1 gene. In situ hybridization experiments allowed us to confirm the presence of Nedd4-1 and Nedd4-2 transcripts during the embryonic stages of cochlear development. More specifically, both genes are expressed in the sensory epithelium, comprising the hair cells, and in the spiral ganglion. After birth, Nedd4-1 expression persists in these tissues while Nedd4-2 transcripts were undetectable. Next, we generated conditional knockout animals (cKO), in which Nedd4-2 gene is specifically invalidated in the otic vesicle at embryonic day 8.5 (E8.5), in order to decipher its role during mouse cochlear development. Histological analyses, Scanning Electron Microscopy and specific immunostainings indicate that Nedd4-2 cKO cochleae develop normally during embryonic and early postnatal stages. However, similar analyses performed at postnatal days 30, 45, 60 and 90 (P30, P45, P60 and P90) suggest an early degeneration of the hair cells and their innervating spiral ganglion neurons. Interestingly, cochlear cell loss in Nedd4-2 cKO animals follows a gradient from the basal to the apical turn of the spiraled cochlea, similarly to what occurs in age-related or sound-induced hearing loss. At P90, Nedd4-2 cKO mice are likely to be profoundly deaf as we do not observe any hair cells remaining in the cochlear epithelium. Altogether, our results suggest a major contribution of the ubiquitin ligase NEDD4-2 in the maintenance of the cochlea integrity. In future experiments, we will combine the use of transgenic mouse models, gain- and loss-of function experiments in tissue cultures and biochemical assays to uncover the specific substrates of Nedd4-2 involved in cochlea preservation. These findings may provide future therapeutic perspectives against hearing loss.