Elsevier

Neuroscience

Volume 328, 22 July 2016, Pages 80-91
Neuroscience

Distinct roles of Eps8 in the maturation of cochlear and vestibular hair cells

https://doi.org/10.1016/j.neuroscience.2016.04.038Get rights and content

Highlights

  • Eps8 is required for proper stereocilia elongation in cochlear and vestibular hair cells.

  • Eps8-KO vestibular hair cells, unlike cochlear inner hair cells, express their normal pattern of basolateral ion channels.

  • Eps8 deletion similarly affects cochlear and vestibular hair bundles growth but impairs hearing only.

Abstract

Several genetic mutations affecting the development and function of mammalian hair cells have been shown to cause deafness but not vestibular defects, most likely because vestibular deficits are sometimes centrally compensated. The study of hair cell physiology is thus a powerful direct approach to ascertain the functional status of the vestibular end organs. Deletion of Epidermal growth factor receptor pathway substrate 8 (Eps8), a gene involved in actin remodeling, has been shown to cause deafness in mice. While both inner and outer hair cells from Eps8 knockout (KO) mice showed abnormally short stereocilia, inner hair cells (IHCs) also failed to acquire mature-type ion channels. Despite the fact that Eps8 is also expressed in vestibular hair cells, Eps8 KO mice show no vestibular deficits. In the present study we have investigated the properties of vestibular Type I and Type II hair cells in Eps8-KO mice and compared them to those of cochlear IHCs. In the absence of Eps8, vestibular hair cells show normally long kinocilia, significantly shorter stereocilia and a normal pattern of basolateral voltage-dependent ion channels. We have also found that while vestibular hair cells from Eps8 KO mice show normal voltage responses to injected sinusoidal currents, which were used to mimic the mechanoelectrical transducer current, IHCs lose their ability to synchronize their responses to the stimulus. We conclude that the absence of Eps8 produces a weaker phenotype in vestibular hair cells compared to cochlear IHCs, since it affects the hair bundle morphology but not the basolateral membrane currents. This difference is likely to explain the absence of obvious vestibular dysfunction in Eps8 KO mice.

Introduction

Hair cells are the sensory receptors of the auditory-vestibular system in all vertebrates. Sound or motion is transduced into electrical signals by the hair bundles, the mechanosensory actin-packed stereocilia protruding from the apical surface of hair cells. Stereocilia of different lengths are arranged in a staircase-like structure, the number of which changes depending on the inner ear organ. Hair bundle deflection, induced by sound or head motion, modulates the open probability of mechanoelectrical transducer channels localized at the tips of the stereocilia (Beurg et al., 2009), and as such generates a receptor potential. Cell voltage responses are then shaped by different types of voltage-dependent ion channels, among which Ca2+ channels are coupled to neurotransmitter (glutamate) exocytosis. Several genetic mutations that cause deafness in mice and humans have been shown to affect the development and/or function of cochlear hair cells (see Hereditary Hearing Loss Homepage http://hereditaryhearingloss.org). Given the common embryonic origins and biology of the auditory and vestibular hair cells, it is conceivable to postulate that single gene mutations known to cause inherited hearing loss would also lead to vestibular dysfunction. Instead, vestibular function is often retained even in the case of profound deafness (Jones and Jones, 2014). One possible explanation is that deficits in vestibular hair cells have sometimes gone undetected because of compensation or adaptation by the central nervous system.

Deletion of Epidermal growth factor receptor pathway substrate 8 (Eps8), a gene involved in actin remodeling (Di Fiore and Scita, 2002), hampers normal stereocilia growth (Manor et al., 2011, Zampini et al., 2011) and ion channel expression in mouse cochlear inner hair cells (IHCs) (Zampini et al., 2011). Despite the similar expression profile of Eps8 in cochlear and vestibular hair cells (Manor et al., 2011, Zampini et al., 2011), Eps8 knockout (KO) mice are deaf but show no obvious vestibular deficits. Similarly, it has been reported that a biallelic nonsense mutation of human EPS8, presumably coding a truncated non-functional protein or none (like the Eps8-KO mouse), results in deafness but no balance defects (Behlouli et al., 2014). In order to elucidate the importance of Eps8 in vestibular hair cells, we have investigated the bundle morphology and the biophysical properties of vestibular hair cells of the Eps8-KO mouse and compare them to those of WT mice. We provide evidence that the absence of Eps8 alters the growth of vestibular hair cells stereocilia. We have also found that, different from IHCs, the receptor potential of vestibular hair cells was not affected by the absence of Eps8. The above findings could explain why Eps8 deletion, and presumably EPS8 mutation, primarily affects the auditory function.

Section snippets

Experimental procedures

All procedures used were approved by the Ministero Italiano della Salute (Rome, Italy) and animal experiments were carried out in accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC). Eps8-KO mice were obtained by breeding heterozygote mice. All animals were genotyped as previously described (Offenhäuser et al., 2006). To increase the number of controls, and particularly for the hair cells dissociation protocol (see below), wild-type (WT) mice (C57 and

Morphological features of Eps8-KO vestibular hair cells

Eps8 is expressed at the stereocilia tips of both the cochlear and the vestibular hair cells (Manor et al., 2011). The hair bundles of Eps8-KO IHCs are shorter than normal, with the first (longest) row of stereocilia being the most affected (Zampini et al., 2011). Since no clear data are available concerning Eps8-KO vestibular hair bundles, we first investigated this aspect. Fig. 1A, B show the hair bundle populations in the intermediate region of the crista ampullaris from a WT and an Eps8-KO

Discussion

We found that in the absence of Eps8 the normal growth of the stereociliary bundle was prevented in vestibular hair cells, which is consistent with previous observations from cochlear hair cells (Manor et al., 2011, Zampini et al., 2011). Despite this morphological defect, Eps8-KO mice are deaf but do not show any obvious vestibular defects (Manor et al., 2011, Zampini et al., 2011). One possible explanation for this discrepancy is that vestibular hair cells retain the kinocilium and have much

Acknowledgments

Tavazzani Elisa, Spaiardi Paolo, Contini Donatella and Manca Marco performed and analyzed the experiments on vestibular hair cells; Zampini Valeria performed and analyzed the experiments on vestibular and cochlear hair cells and helped with the writing of the manuscript; Russo Giancarlo and Prigioni Ivo helped with the design of the experiments, the figures and the writing of the manuscript; Marcotti Walter helped with the experiments and the data analysis of experiments on cochlear hair cells,

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  • Cited by (0)

    Present address: Paris Descartes University, Biomedical and Fundamental Science Faculty, Neurophotonics Laboratory, CNRS UMR8250, 45, rue des Saints Pères, 75270 Paris Cedex 06, France.

    Present address: Department of Anatomy and Cell Biology, University of Illinois College of Medicine, 808 S. Wood St., Chicago, IL 60612, USA.

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