Isabelle Perfettini

Targeted ablation of connexin26 in the inner ear epithelial gap junction network causes hearing impairment and cell death

Mutations in the gene encoding the gap junction protein connexin26 (Cx26) are responsible for the autosomal recessive isolated deafness, DFNB1, which accounts for half of the cases of prelingual profound hereditary deafness in Caucasian populations. To date, in vivo approaches to decipher the role of Cx26 in the inner ear have been hampered by the embryonic lethality of the Cx26 knockout mice. To overcome this difficulty, we performed targeted ablation of Cx26 specifically in one of the two cellular networks that it underlies in the inner ear, namely, the epithelial network. We show that homozygous mutant mice, Cx26(OtogCre), have hearing impairment, but no vestibular dysfunction. The inner ear developed normally. However, on postnatal day 14 (P14), i.e., soon after the onset of hearing, cell death appeared and eventually extended to the cochlear epithelial network and sensory hair cells. Cell death initially affected only the supporting cells of the genuine sensory cell (inner hair cell, IHC), thus suggesting that it could be triggered by the IHC response to sound stimulation. Altogether, our results demonstrate that the Cx26-containing epithelial gap junction network is essential for cochlear function and cell survival. We conclude that prevention of cell death in the sensory epithelium is essential for any attempt to restore the auditory function in DFNB1 patients.

Vezatin, a novel transmembrane protein, bridges myosin VIIA to the cadherin–catenins complex

Defects in myosin VIIA are responsible for deafness in the human and mouse. The role of this unconventional myosin in the sensory hair cells of the inner ear is not yet understood. Here we show that the C‐terminal FERM domain of myosin VIIA binds to a novel transmembrane protein, vezatin, which we identified by a yeast two‐hybrid screen. Vezatin is a ubiquitous protein of adherens cell–cell junctions, where it interacts with both myosin VIIA and the cadherin–catenins complex. Its recruitment to adherens junctions implicates the C‐terminal region of α‐catenin. Taken together, these data suggest that myosin VIIA, anchored by vezatin to the cadherin–catenins complex, creates a tension force between adherens junctions and the actin cytoskeleton that is expected to strengthen cell–cell adhesion. In the inner ear sensory hair cells vezatin is, in addition, concentrated at another membrane–membrane interaction site, namely at the fibrillar links interconnecting the bases of adjacent stereocilia. In myosin VIIA‐defective mutants, inactivity of the vezatin–myosin VIIA complex at both sites could account for splaying out of the hair cell stereocilia.

Localization of Usher 1 proteins to the photoreceptor calyceal processes, which are absent from mice.

Mice are a poor model for retinal defects caused by type I Usher syndrome (USH1) because their photoreceptors have almost no calyceal processes, the structures in which all USH1 proteins are detected in other vertebrates.

Usher type 1G protein sans is a critical component of the tip-link complex, a structure controlling actin polymerization in stereocilia

The mechanotransducer channels of auditory hair cells are gated by tip-links, oblique filaments that interconnect the stereocilia of the hair bundle. Tip-links stretch from the tips of stereocilia in the short and middle rows to the sides of neighboring, taller stereocilia. They are made of cadherin-23 and protocadherin-15, products of the Usher syndrome type 1 genes USH1D and USH1F, respectively. In this study we address the role of sans, a putative scaffold protein and product of the USH1G gene. In Ush1g−/− mice, the cohesion of stereocilia is disrupted, and both the amplitude and the sensitivity of the transduction currents are reduced. In Ush1gfl/flMyo15-cre+/− mice, the loss of sans occurs postnatally and the stereocilia remain cohesive. In these mice, there is a decrease in the amplitude of the total transducer current with no loss in sensitivity, and the tips of the stereocilia in the short and middle rows lose their prolate shape, features that can be attributed to the loss of tip-links. Furthermore, stereocilia from these rows undergo a dramatic reduction in length, suggesting that the mechanotransduction machinery has a positive effect on F-actin polymerization. Sans interacts with the cytoplasmic domains of cadherin-23 and protocadherin-15 in vitro and is absent from the hair bundle in mice defective for either of the two cadherins. Because sans localizes mainly to the tips of short- and middle-row stereocilia in vivo, we conclude that it belongs to a molecular complex at the lower end of the tip-link and plays a critical role in the maintenance of this link.

Shroom2, a myosin-VIIa- and actin-binding protein, directly interacts with ZO-1 at tight junctions

Defects in myosin VIIa lead to developmental anomalies of the auditory and visual sensory cells. We sought proteins interacting with the myosin VIIa tail by using the yeast two-hybrid system. Here, we report on shroom2, a submembranous PDZ domain-containing protein that is associated with the tight junctions in multiple embryonic and adult epithelia. Shroom2 directly interacts with the C-terminal MyTH4-FERM domain of myosin VIIa and with F-actin. In addition, a shroom2 fragment containing the region of interaction with F-actin was able to protect actin filaments from cytochalasin-D-induced disruption in MDCK cells. Transfection experiments in MDCK and LE (L fibroblasts that express E-cadherin) cells led us to conclude that shroom2 is targeted to the cell-cell junctions in the presence of tight junctions only. In Ca2+-switch experiments on MDCK cells, ZO-1 (also known as TJP1) preceded GFP-tagged shroom2 at the differentiating tight junctions. ZO-1 directly interacts with the serine- and proline-rich region of shroom2 in vitro. Moreover, the two proteins colocalize in vivo at mature tight junctions, and could be coimmunoprecipitated from brain and cochlear extracts. We suggest that shroom2 and ZO-1 form a tight-junction-associated scaffolding complex, possibly linked to myosin VIIa, that bridges the junctional membrane to the underlying cytoskeleton, thereby contributing to the stabilization of these junctions.

An unusually powerful mode of low-frequency sound interference due to defective hair bundles of the auditory outer hair cells

Significance We show that the submembrane scaffold protein Nherf1 is necessary for correct shaping of outer hair cells stereocilia bundles in the basal cochlea. The mild elevation of hearing thresholds (22–35 dB) of Nherf1−/− mice at high frequencies was inconsistent with the loss of outer hair cell functionality in the basal cochlea. Responses of Nherf1−/− mice to high-frequency test tones were masked by tones displaying inordinate characteristics in frequency, level, and growth response. We suggest that in Nherf1−/− mice, high-frequency vibrations are detected in the unaffected apical cochlea, thus accounting for the powerful masking effect of low-frequency sound. This source of misleading evaluation of high-frequency hearing thresholds and hypervulnerability to low-frequency sound interference should be systematically sought in hearing-impaired patients. A detrimental perceptive consequence of damaged auditory sensory hair cells consists in a pronounced masking effect exerted by low-frequency sounds, thought to occur when auditory threshold elevation substantially exceeds 40 dB. Here, we identified the submembrane scaffold protein Nherf1 as a hair-bundle component of the differentiating outer hair cells (OHCs). Nherf1−/− mice displayed OHC hair-bundle shape anomalies in the mid and basal cochlea, normally tuned to mid- and high-frequency tones, and mild (22–35 dB) hearing-threshold elevations restricted to midhigh sound frequencies. This mild decrease in hearing sensitivity was, however, discordant with almost nonresponding OHCs at the cochlear base as assessed by distortion-product otoacoustic emissions and cochlear microphonic potentials. Moreover, unlike wild-type mice, responses of Nherf1−/− mice to high-frequency (20–40 kHz) test tones were not masked by tones of neighboring frequencies. Instead, efficient maskers were characterized by their frequencies up to two octaves below the probe-tone frequency, unusually low intensities up to 25 dB below probe-tone level, and growth-of-masker slope (2.2 dB/dB) reflecting their compressive amplification. Together, these properties do not fit the current acknowledged features of a hypersensitivity of the basal cochlea to lower frequencies, but rather suggest a previously unidentified mechanism. Low-frequency maskers, we propose, may interact within the unaffected cochlear apical region with midhigh frequency sounds propagated there via a mode possibly using the persistent contact of misshaped OHC hair bundles with the tectorial membrane. Our findings thus reveal a source of misleading interpretations of hearing thresholds and of hypervulnerability to low-frequency sound interference.

Class III myosins shape the auditory hair bundles by limiting microvilli and stereocilia growth.

Analysis of mice deficient for myosin IIIa and myosin IIIb shows that class III myosins limit the elongation of stereocilia and of subsequently regressing microvilli, thus contributing to the early hair bundle shaping.