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Key Points: The aim was to determine whether detachment of the tectorial membrane (TM) from the organ of Corti in Tecta/Tectb -/- mice affects the biophysical properties of cochlear outer hair cells (OHCs). Tecta/Tectb -/- mice have highly elevated hearing thresholds, but OHCs mature normally. Mechanoelectrical transducer (MET) channel resting open probability (P o ) in mature OHC is ∼50% in endolymphatic [Ca 2+ ], resulting in a large standing depolarizing MET current that would allow OHCs to act optimally as electromotile cochlear amplifiers. MET channel resting P o in vivo is also high in Tecta/Tectb -/- mice, indicating that the TM is unlikely to statically bias the hair bundles of OHCs. Distortion product otoacoustic emissions (DPOAEs), a readout of active, MET-dependent, non-linear cochlear amplification in OHCs, fail to exhibit long-lasting adaptation to repetitive stimulation in Tecta/Tectb -/- mice. We conclude that during prolonged, sound-induced stimulation of the cochlea the TM may determine the extracellular Ca 2+ concentration near the OHC's MET channels., Abstract: The tectorial membrane (TM) is an acellular structure of the cochlea that is attached to the stereociliary bundles of the outer hair cells (OHCs), electromotile cells that amplify motion of the cochlear partition and sharpen its frequency selectivity. Although the TM is essential for hearing, its role is still not fully understood. In Tecta/Tectb -/- double knockout mice, in which the TM is not coupled to the OHC stereocilia, hearing sensitivity is considerably reduced compared with that of wild-type animals. In vivo, the OHC receptor potentials, assessed using cochlear microphonics, are symmetrical in both wild-type and Tecta/Tectb -/- mice, indicating that the TM does not bias the hair bundle resting position. The functional maturation of hair cells is also unaffected in Tecta/Tectb -/- mice, and the resting open probability of the mechanoelectrical transducer (MET) channel reaches values of ∼50% when the hair bundles of mature OHCs are bathed in an endolymphatic-like Ca 2+ concentration (40 μM) in vitro. The resultant large MET current depolarizes OHCs to near -40 mV, a value that would allow optimal activation of the motor protein prestin and normal cochlear amplification. Although the set point of the OHC receptor potential transfer function in vivo may therefore be determined primarily by endolymphatic Ca 2+ concentration, repetitive acoustic stimulation fails to produce adaptation of MET-dependent otoacoustic emissions in vivo in the Tecta/Tectb -/- mice. Therefore, the TM is likely to contribute to the regulation of Ca 2+ levels around the stereocilia, and thus adaptation of the OHC MET channel during prolonged sound stimulation., (© 2021 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

The aging cochlea is subjected to a number of pathological changes to play a role in the onset of age-related hearing loss (ARHL). Although ARHL has often been thought of as the result of the loss of hair cells, it is in fact a disorder with a complex etiology, arising from the changes to both the organ of Corti and its supporting structures. In this study, we examine two aging pathologies that have not been studied in detail despite their apparent prevalence; the fusion, elongation, and engulfment of cochlear inner hair cell stereocilia, and the changes that occur to the tectorial membrane (TM), a structure overlying the organ of Corti that modulates its physical properties in response to sound. Our work demonstrates that similar pathological changes occur in these two structures in the aging cochleae of both mice and humans, examines the ultrastructural changes that underlie stereocilial fusion, and identifies the lost TM components that lead to changes in membrane structure. We place these changes into the context of the wider pathology of the aging cochlea, and identify how they may be important in particular for understanding the more subtle hearing pathologies that precede auditory threshold loss in ARHL., (© 2019 Wiley Periodicals, Inc.)

The transduction of acoustic information by hair cells depends upon mechanosensitive stereociliary bundles that project from their apical surface. Mutations or absence of the stereociliary protein EPS8 cause deafness in humans and mice, respectively. Eps8 knockout mice (Eps8 -/- ) have hair cells with immature stereocilia and fail to become sensory receptors. Here, we show that exogenous delivery of Eps8 using Anc80L65 in P1-P2 Eps8 -/- mice in vivo rescued the hair bundle structure of apical-coil hair cells. Rescued hair bundles correctly localize EPS8, WHIRLIN, MYO15, and BAIAP2L2, and generate normal mechanoelectrical transducer currents. Inner hair cells with normal-looking stereocilia re-expressed adult-like basolateral ion channels (BK and KCNQ4) and have normal exocytosis. The number of hair cells undergoing full recovery was not sufficient to rescue hearing in Eps8 -/- mice. Adeno-associated virus (AAV)-transduction of P3 apical-coil and P1-P2 basal-coil hair cells does not rescue hair cells, nor does Anc80L65-Eps8 delivery in adult Eps8 -/- mice. We propose that AAV-induced gene-base therapy is an efficient strategy to recover the complex hair-cell defects in Eps8 -/- mice. However, this therapeutic approach may need to be performed in utero since, at postnatal ages, Eps8 -/- hair cells appear to have matured or accumulated damage beyond the point of repair.

ABSTRACT Early postnatal mouse cochlear cultures were treated with a small panel of kinase inhibitors to elucidate the mechanisms underlying the maintenance of hair-bundle structure in the developing inner ear. At low concentrations (1-10 nM), staurosporine causes the collapse and loss of hair bundles without provoking hair-cell death, as judged by lack of terminal transferase dUTP nick end labeling (TUNEL) labeling or reactivity to anti-activated caspase-3. Staurosporine exposure results in the fusion of the hair bundle's stereocilia, a resorption of the parallel actin bundles of the stereocilia into the cytoplasm of the hair cell, a detachment of the apical, non-stereociliary membrane of the hair cell from the underlying cuticular plate, and a severing of the hair-bundle's rootlets from the actin cores of the stereocilia. It does not block membrane retrieval at the apical pole of the hair cells, nor does it elicit the externalization of phosphatidylserine. Staurosporine treatment causes a reduction in levels of the phosphorylated forms of ezrin, radixin, and moesin in cochlear cultures during the period of hair-bundle loss, indicating the integrity of the hair bundle may be actively maintained by the phosphorylation status of these proteins. J. Comp. Neurol. 522:3281-3294, 2014. © 2014 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Protocadherin-15 (Pcdh15) is a component of the tip-links, the extracellular filaments that gate hair cell mechano-electrical transduction channels in the inner ear. There are three Pcdh15 splice isoforms (CD1, CD2 and CD3), which only differ by their cytoplasmic domains; they are thought to function redundantly in mechano-electrical transduction during hair-bundle development, but whether any of these isoforms composes the tip-link in mature hair cells remains unknown. By immunolabelling and both morphological and electrophysiological analyses of post-natal hair cell-specific conditional knockout mice ( Pcdh15ex38-fl/ex38-fl Myo15- cre+/−) that lose only this isoform after normal hair-bundle development, we show that Pcdh15-CD2 is an essential component of tip-links in mature auditory hair cells. The finding, in the homozygous or compound heterozygous state, of a PCDH15 frameshift mutation (p.P1515Tfs*4) that affects only Pcdh15-CD2, in profoundly deaf children from two unrelated families, extends this conclusion to humans. These results provide key information for identification of new components of the mature auditory mechano-electrical transduction machinery. This will also serve as a basis for the development of gene therapy for deafness caused by PCDH15 defects. [ABSTRACT FROM AUTHOR]

This review focuses on the cellular and molecular mechanisms underlying the development of the sensory hair bundle, an apical specialisation of the hair cell that is essential for mechanotransduction. The structure, function and development of the hair bundle is described, with an emphasis on the properties and possible roles played by the different link types that interconnect the individual elements of the hair bundle - the multiple stereocilia and the single kinocilium. Studies of mouse and zebrafish mutants have revealed that several classes of molecule are required for the genesis and maintenance of hair-bundle structure. These include cell surface molecules that are associated with the different hair-bundle links, along with myosin motors, scaffolding proteins and an actin cross-linker. Finally we consider how differences in the form and shape of hair bundles within and between different sensory organs are generated. [ABSTRACT FROM AUTHOR]

Sound and acceleration are detected by hair bundles, mechanosensory structures located at the apical pole of hair cells in the inner ear. The different elements of the hair bundle, the stereocilia and a kinocilium, are interconnected by a variety of link types. One of these links, the tip link, connects the top of a shorter stereocilium with the lateral membrane of an adjacent taller stereocilium and may gate the mechanotransducer channel of the hair cell. Mass spectrometric and Western blot analyses identify the tip-link antigen, a hitherto unidentified antigen specifically associated with the tip and kinocilial links of sensory hair bundles in the inner ear and the ciliary calyx of photoreceptors in the eye, as an avian ortholog of human protocadherin-15, a product of the gene for the deaf/blindness Usher syndrome type 1F/DFNB23 locus. Multiple protocadherin-15 transcripts are shown to be expressed in the mouse inner ear, and these define four major isoform classes, two with entirely novel, previously unidentified cytoplasmic domains. Antibodies to the three cytoplasmic domain-containing isoform classes reveal that each has a different spatiotemporal expression pattern in the developing and mature inner ear. Two isoforms are distributed in a manner compatible for association with the tip-link complex. An isoform located at the tips of stereocilia is sensitive to calcium chelation and proteolysis with subtilisin and reappears at the tips of stereocilia as transduction recovers after the removal of calcium chelators. Protocadherin-15 is therefore associated with the tip-link complex and may be an integral component of this structure and/or required for its formation. [ABSTRACT FROM AUTHOR]

Abstract: Cadherin 23 is required for normal development of the sensory hair bundle, and recent evidence suggests it is a component of the tip links, filamentous structures thought to gate the hair cells'' mechano-electrical transducer channels. Antibodies against unique peptide epitopes were used to study the properties of cadherin 23 and its spatio-temporal expression patterns in developing cochlear hair cells. In the rat, intra- and extracellular domain epitopes are readily detected in the developing hair bundle between E18 and P5, and become progressively restricted to the distal tip of the hair bundle. From P13 onwards, these epitopes are no longer detected in hair bundles, but immunoreactivity is observed in the apical, vesicle-rich, pericuticular region of the hair cell. In the P2¿P3 mouse cochlea, immunogold labeling reveals cadherin 23 is associated with kinocilial links and transient lateral links located between and within stereociliary rows. At this stage, the cadherin 23 ectodomain epitope remains on the hair bundle following BAPTA or La3+ treatment, but is lost following exposure to the protease subtilisin. In contrast, mechano-electrical transduction is abolished by BAPTA but unaffected by subtilisin. These results suggest cadherin 23 is associated with transient lateral links that have properties distinct from those of the tip-link. [Copyright &y& Elsevier]

Abstract: A subtracted library prepared from vestibular sensory areas [Nat. Genet. 26 (2000) 51] was used to identify a 960bp murine transcript preferentially expressed in the inner ear and testis. The cDNA predicts a basic 124aa protein that does not share any significant sequence homology with known proteins. Immunofluorescence and immunoelectron microscopy revealed that the protein is located mainly in the kinocilium of sensory cells in the inner ear. The protein was thus named kinocilin. In the mouse, kinocilin is first detected in the kinocilia of vestibular and auditory hair cells at embryonic days 14.5, and 18.5, respectively. In the mature vestibular hair cells, kinocilin is still present in the kinocilium. As the auditory hair cells begin to lose the kinocilium during postnatal development, kinocilin becomes distributed in an annular pattern at the apex of these cells, where it co-localizes with the tubulin belt [Hear. Res. 42 (1989) 1]. In mature auditory hair cells, kinocilin is also present at the level of the cuticular plate, at the base of each stereocilium. In addition, as the kinocilium regresses from developing auditory hair cells, kinocilin begins to be expressed by the pillar cells and Deiters cells, that both contain prominent transcellular and apical bundles of microtubules. By contrast, kinocilin was not detected in the supporting cells in the vestibular end organs. The protein is also present in the manchette of the spermatids, a transient structure enriched in interconnected microtubules. We propose that kinocilin has a role in stabilizing dense microtubular networks or in vesicular trafficking. [Copyright &y& Elsevier]