Identification of a series of hair-cell MET channel blockers that protect against aminoglycoside-induced ototoxicity.

To identify small molecules that shield mammalian sensory hair cells from the ototoxic side effects of aminoglycoside antibiotics, 10,240 compounds were initially screened in zebrafish larvae, selecting for those that protected lateral-line hair cells against neomycin and gentamicin. When the 64 hits from this screen were retested in mouse cochlear cultures, 8 protected outer hair cells (OHCs) from gentamicin in vitro without causing hair-bundle damage. These 8 hits shared structural features and blocked, to varying degrees, the OHC's mechano-electrical transducer (MET) channel, a route of aminoglycoside entry into hair cells. Further characterization of one of the strongest MET channel blockers, UoS-7692, revealed it additionally protected against kanamycin and tobramycin and did not abrogate the bactericidal activity of gentamicin. UoS-7692 behaved, like the aminoglycosides, as a permeant blocker of the MET channel; significantly reduced gentamicin-Texas red loading into OHCs; and preserved lateral-line function in neomycin-treated zebrafish. Transtympanic injection of UoS-7692 protected mouse OHCs from furosemide/kanamycin exposure in vivo and partially preserved hearing. The results confirmed the hair-cell MET channel as a viable target for the identification of compounds that protect the cochlea from aminoglycosides and provide a series of hit compounds that will inform the design of future otoprotectants.

Spontaneous Otoacoustic Emissions in TectaY1870C/+ Mice Reflect Changes in Cochlear Amplification and How It Is Controlled by the Tectorial Membrane.

Spontaneous otoacoustic emissions (SOAEs) recorded from the ear canal in the absence of sound reflect cochlear amplification, an outer hair cell (OHC) process required for the extraordinary sensitivity and frequency selectivity of mammalian hearing. Although wild-type mice rarely emit, those with mutations that influence the tectorial membrane (TM) show an incidence of SOAEs similar to that in humans. In this report, we characterized mice with a missense mutation in Tecta, a gene required for the formation of the striated-sheet matrix within the core of the TM. Mice heterozygous for the Y1870C mutation (TectaY1870C/+ ) are prolific emitters, despite a moderate hearing loss. Additionally, Kimura's membrane, into which the OHC stereocilia insert, separates from the main body of the TM, except at apical cochlear locations. Multimodal SOAEs are also observed in TectaY1870C/+ mice where energy is present at frequencies that are integer multiples of a lower-frequency SOAE (the primary). Second-harmonic SOAEs, at twice the frequency of a lower-frequency primary, are the most frequently observed. These secondary SOAEs are found in spatial regions where stimulus-evoked OAEs are small or in the noise floor. Introduction of high-level suppressors just above the primary SOAE frequency reduce or eliminate both primary and second-harmonic SOAEs. In contrast, second-harmonic SOAEs are not affected by suppressors, either above or below the second-harmonic SOAE frequency, even when they are much larger in amplitude. Hence, second-harmonic SOAEs do not appear to be spatially separated from their primaries, a finding that has implications for cochlear mechanics and the consequences of changes to TM structure.

Identification of ion-channel modulators that protect against aminoglycoside-induced hair cell death.

Aminoglycoside antibiotics are used to treat life-threatening bacterial infections but can cause deafness due to hair cell death in the inner ear. Compounds have been described that protect zebrafish lateral line hair cells from aminoglycosides, but few are effective in the cochlea. As the aminoglycosides interact with several ion channels, including the mechanoelectrical transducer (MET) channels by which they can enter hair cells, we screened 160 ion-channel modulators, seeking compounds that protect cochlear outer hair cells (OHCs) from aminoglycoside-induced death in vitro. Using zebrafish, 72 compounds were identified that either reduced loading of the MET-channel blocker FM 1-43FX, decreased Texas red-conjugated neomycin labeling, or reduced neomycin-induced hair cell death. After testing these 72 compounds, and 6 structurally similar compounds that failed in zebrafish, 13 were found that protected against gentamicin-induced death of OHCs in mouse cochlear cultures, 6 of which are permeant blockers of the hair cell MET channel. None of these compounds abrogated aminoglycoside antibacterial efficacy. By selecting those without adverse effects at high concentrations, 5 emerged as leads for developing pharmaceutical otoprotectants to alleviate an increasing clinical problem.

The acquisition of mechano-electrical transducer current adaptation in auditory hair cells requires myosin VI.

KEY POINTS: The transduction of sound into electrical signals occurs at the hair bundles atop sensory hair cells in the cochlea, by means of mechanosensitive ion channels, the mechano-electrical transducer (MET) channels. The MET currents decline during steady stimuli; this is termed adaptation and ensures they always work within the most sensitive part of their operating range, responding best to rapidly changing (sound) stimuli. In this study we used a mouse model (Snell's waltzer) for hereditary deafness in humans that has a mutation in the gene encoding an unconventional myosin, myosin VI, which is present in the hair bundles. We found that in the absence of myosin VI the MET current fails to acquire its characteristic adaptation as the hair bundles develop. We propose that myosin VI supports the acquisition of adaptation by removing key molecules from the hair bundle that serve a temporary, developmental role. ABSTRACT: Mutations in Myo6, the gene encoding the (F-actin) minus end-directed unconventional myosin, myosin VI, cause hereditary deafness in mice (Snell's waltzer) and humans. In the sensory hair cells of the cochlea, myosin VI is expressed in the cell bodies and along the stereocilia that project from the cells' apical surface. It is required for maintaining the structural integrity of the mechanosensitive hair bundles formed by the stereocilia. In this study we investigate whether myosin VI contributes to mechano-electrical transduction. We report that Ca(2+) -dependent adaptation of the mechano-electrical transducer (MET) current, which serves to keep the transduction apparatus operating within its most sensitive range, is absent in outer and inner hair cells from homozygous Snell's waltzer mutant mice, which fail to express myosin VI. The operating range of the MET channels is also abnormal in the mutants, resulting in the absence of a resting MET current. We found that cadherin 23, a component of the hair bundle's transient lateral links, fails to be downregulated along the length of the stereocilia in maturing Myo6 mutant mice. MET currents of heterozygous littermates appear normal. We propose that myosin VI, by removing key molecules from developing hair bundles, is required for the development of the MET apparatus and its Ca(2+) -dependent adaptation.

Loss of the tectorial membrane protein CEACAM16 enhances spontaneous, stimulus-frequency, and transiently evoked otoacoustic emissions.

α-Tectorin (TECTA), ß-tectorin (TECTB), and carcinoembryonic antigen-related cell adhesion molecule 16 (CEACAM) are secreted glycoproteins that are present in the tectorial membrane (TM), an extracellular structure overlying the hearing organ of the inner ear, the organ of Corti. Previous studies have shown that TECTA and TECTB are both required for formation of the striated-sheet matrix within which collagen fibrils of the TM are imbedded and that CEACAM16 interacts with TECTA. To learn more about the structural and functional significance of CEACAM16, we created a Ceacam16-null mutant mouse. In the absence of CEACAM16, TECTB levels are reduced, a clearly defined striated-sheet matrix does not develop, and Hensen's stripe, a prominent feature in the basal two-thirds of the TM in WT mice, is absent. CEACAM16 is also shown to interact with TECTB, indicating that it may stabilize interactions between TECTA and TECTB. Although brain-stem evoked responses and distortion product otoacoustic emissions are, for most frequencies, normal in young mice lacking CEACAM16, stimulus-frequency and transiently evoked emissions are larger. We also observed spontaneous otoacoustic emissions (SOAEs) in 70% of the homozygous mice. This incidence is remarkable considering that <3% of WT controls have SOAEs. The predominance of SOAEs >15 kHz correlates with the loss of Hensen's stripe. Results from mice lacking CEACAM16 are consistent with the idea that the organ of Corti evolved to maximize the gain of the cochlear amplifier while preventing large oscillations. Changes in TM structure appear to influence the balance between energy generation and dissipation such that the system becomes unstable.

Transduction without tip links in cochlear hair cells is mediated by ion channels with permeation properties distinct from those of the mechano-electrical transducer channel.

Tip links between adjacent stereocilia are believed to gate mechano-electrical transducer (MET) channels and mediate the electrical responses of sensory hair cells. We found that mouse auditory hair cells that lack tip links due to genetic mutations or exposure to the Ca(2+) chelator BAPTA can, however, still respond to mechanical stimuli. These MET currents have unusual properties and are predominantly of the opposite polarity relative to those measured when tip links are present. There are other striking differences, for example, the channels are usually all closed when the hair cell is not stimulated and the currents in response to strong stimuli can be substantially larger than normal. These anomalous MET currents can also be elicited early in development, before the onset of mechano-electrical transduction with normal response polarity. Current-voltage curves of the anomalous MET currents are linear and do not show the rectification characteristic of normal MET currents. The permeant MET channel blocker dihydrostreptomycin is two orders of magnitude less effective in blocking the anomalous MET currents. The findings suggest the presence of a large population of MET channels with pore properties that are distinct from those of normal MET channels. These channels are not gated by hair-bundle links and can be activated under a variety of conditions in which normal tip-link-mediated transduction is not operational.

A mouse model for human deafness DFNB22 reveals that hearing impairment is due to a loss of inner hair cell stimulation.

The gene causative for the human nonsyndromic recessive form of deafness DFNB22 encodes otoancorin, a 120-kDa inner ear-specific protein that is expressed on the surface of the spiral limbus in the cochlea. Gene targeting in ES cells was used to create an EGFP knock-in, otoancorin KO (Otoa(EGFP/EGFP)) mouse. In the Otoa(EGFP/EGFP) mouse, the tectorial membrane (TM), a ribbon-like strip of ECM that is normally anchored by one edge to the spiral limbus and lies over the organ of Corti, retains its general form, and remains in close proximity to the organ of Corti, but is detached from the limbal surface. Measurements of cochlear microphonic potentials, distortion product otoacoustic emissions, and basilar membrane motion indicate that the TM remains functionally attached to the electromotile, sensorimotor outer hair cells of the organ of Corti, and that the amplification and frequency tuning of the basilar membrane responses to sounds are almost normal. The compound action potential masker tuning curves, a measure of the tuning of the sensory inner hair cells, are also sharply tuned, but the thresholds of the compound action potentials, a measure of inner hair cell sensitivity, are significantly elevated. These results indicate that the hearing loss in patients with Otoa mutations is caused by a defect in inner hair cell stimulation, and reveal the limbal attachment of the TM plays a critical role in this process.

Hair bundle defects and loss of function in the vestibular end organs of mice lacking the receptor-like inositol lipid phosphatase PTPRQ.

Recent studies have shown that mutations in PTPRQ, a gene encoding a receptor-like inositol lipid phosphatase, cause recessive, nonsyndromic, hereditary hearing loss with associated vestibular dysfunction. Although null mutations in Ptprq cause the loss of high-frequency auditory hair cells and deafness in mice, a loss of vestibular hair cells and overt behavioral defects characteristic of vestibular dysfunction have not been described. Hair bundle structure and vestibular function were therefore examined in Ptprq mutant mice. Between postnatal days 5 and 16, hair bundles in the extrastriolar regions of the utricle in Ptprq(-/-) mice become significantly longer than those in heterozygous controls. This increase in length (up to 50%) is accompanied by the loss and fusion of stereocilia. Loss and fusion of stereocilia also occurs in the striolar region of the utricle in Ptprq(-/-) mice, but is not accompanied by hair bundle elongation. These abnormalities persist until 12 months of age but are not accompanied by significant hair cell loss. Hair bundle defects are also observed in the saccule and ampullae of Ptprq(-/-) mice. At ∼3 months of age, vestibular evoked potentials were absent from the majority (12 of 15) of Ptprq(-/-) mice examined, and could only be detected at high stimulus levels in the other 3 mutants. Subtle but distinct defects in swimming behavior were detected in most (seven of eight) mutants tested. The results reveal a distinct phenotype in the vestibular system of Ptprq(-/-) mice and suggest similar hair bundle defects may underlie the vestibular dysfunction reported in humans with mutations in PTPRQ.

Stereocilin-deficient mice reveal the origin of cochlear waveform distortions.

Although the cochlea is an amplifier and a remarkably sensitive and finely tuned detector of sounds, it also produces conspicuous mechanical and electrical waveform distortions. These distortions reflect nonlinear mechanical interactions within the cochlea. By allowing one tone to suppress another (masking effect), they contribute to speech intelligibility. Tones can also combine to produce sounds with frequencies not present in the acoustic stimulus. These sounds compose the otoacoustic emissions that are extensively used to screen hearing in newborns. Because both cochlear amplification and distortion originate from the outer hair cells-one of the two types of sensory receptor cells-it has been speculated that they stem from a common mechanism. Here we show that the nonlinearity underlying cochlear waveform distortions relies on the presence of stereocilin, a protein defective in a recessive form of human deafness. Stereocilin was detected in association with horizontal top connectors, lateral links that join adjacent stereocilia within the outer hair cell's hair bundle. These links were absent in stereocilin-null mutant mice, which became progressively deaf. At the onset of hearing, however, their cochlear sensitivity and frequency tuning were almost normal, although masking was much reduced and both acoustic and electrical waveform distortions were completely lacking. From this unique functional situation, we conclude that the main source of cochlear waveform distortions is a deflection-dependent hair bundle stiffness resulting from constraints imposed by the horizontal top connectors, and not from the intrinsic nonlinear behaviour of the mechanoelectrical transducer channel.

Molecular identity and functional properties of a novel T-type Ca2+ channel cloned from the sensory epithelia of the mouse inner ear.

The molecular identity of non-Cav1.3 channels in auditory and vestibular hair cells has remained obscure, yet the evidence in support of their roles to promote diverse Ca2+-dependent functions is indisputable. Recently, a transient Cav3.1 current that serves as a functional signature for the development and regeneration of hair cells has been identified in the chicken basilar papilla. The Cav3.1 current promotes spontaneous activity of the developing hair cell, which may be essential for synapse formation. Here, we have isolated and sequenced the full-length complementary DNA of a distinct isoform of Cav3.1 in the mouse inner ear. The channel is derived from alternative splicing of exon14, exon25A, exon34, and exon35. Functional expression of the channel in Xenopus oocytes yielded Ca2+ currents, which have a permeation phenotype consistent with T-type channels. However, unlike most multiion channels, the T-type channel does not exhibit the anomalous mole fraction effect, possibly reflecting comparable permeation properties of divalent cations. The Cav3.1 channel was expressed in sensory and nonsensory epithelia of the inner ear. Moreover, there are profound changes in the expression levels during development. The differential expression of the channel during development and the pharmacology of the inner ear Cav3.1 channel may have contributed to the difficulties associated with identification of the non-Cav1.3 currents.

The very large G-protein-coupled receptor VLGR1: a component of the ankle link complex required for the normal development of auditory hair bundles.

Sensory hair bundles in the inner ear are composed of stereocilia that can be interconnected by a variety of different link types, including tip links, horizontal top connectors, shaft connectors, and ankle links. The ankle link antigen is an epitope specifically associated with ankle links and the calycal processes of photoreceptors in chicks. Mass spectrometry and immunoblotting were used to identify this antigen as the avian ortholog of the very large G-protein-coupled receptor VLGR1, the product of the Usher syndrome USH2C (Mass1) locus. Like ankle links, Vlgr1 is expressed transiently around the base of developing hair bundles in mice. Ankle links fail to form in the cochleae of mice carrying a targeted mutation in Vlgr1 (Vlgr1/del7TM), and the bundles become disorganized just after birth. FM1-43 [N-(3-triethylammonium)propyl)-4-(4-(dibutylamino)styryl) pyridinium dibromide] dye loading and whole-cell recordings indicate mechanotransduction is impaired in cochlear, but not vestibular, hair cells of early postnatal Vlgr1/del7TM mutant mice. Auditory brainstem recordings and distortion product measurements indicate that these mice are severely deaf by the third week of life. Hair cells from the basal half of the cochlea are lost in 2-month-old Vlgr1/del7TM mice, and retinal function is mildly abnormal in aged mutants. Our results indicate that Vlgr1 is required for formation of the ankle link complex and the normal development of cochlear hair bundles.