Inner Hair Cell Stereocilia Research Articles

Live-cell single-molecule fluorescence microscopy for protruding organelles reveals regulatory mechanisms of MYO7A-driven cargo transport in stereocilia of inner ear hair cells.

Stereocilia are unidirectional F-actin-based cylindrical protrusions on the apical surface of inner ear hair cells and function as biological mechanosensors of sound and acceleration. Development of functional stereocilia requires motor activities of unconventional myosins to transport proteins necessary for elongating the F-actin cores and to assemble the mechanoelectrical transduction (MET) channel complex. However, how each myosin localizes in stereocilia using the energy from ATP hydrolysis is only partially understood. In this study, we develop a methodology for live-cell single-molecule fluorescence microscopy of organelles protruding from the apical surface using a dual-view light-sheet microscope, diSPIM. We demonstrate that MYO7A, a component of the MET machinery, traffics as a dimer in stereocilia. Movements of MYO7A are restricted when scaffolded by the plasma membrane and F-actin as mediated by MYO7A's interacting partners. Here, we discuss the technical details of our methodology and its future applications including analyses of cargo transportation in various organelles.

Generation and characterization of a human iPSC line (JUFMDOi007-A) from a patient with Usher syndrome due to mutation in USH2A

Usher syndrome type 2A (USH2A) gene mutations have been identified as the most frequent genetic causes of hereditary deafness in Usher syndrome, and an effective treatment has yet to be established. The encoded protein, Usherin, is essential for the ankle link associated with extracellular connections between the stereocilia of inner ear hair cells. We report the generation of a patient-derived USH2A iPSC line with compound mutations c.1907_1912ATGTTT > TCACAG (p.D636V + V637T + C638G) and c.8328_8329delAA (p.L2276fs*12). The iPSC showed the expression of pluripotency markers, the ability to differentiate into three germ layers in vitro, and USH2A mutations with normal karyotype.

Effect of perinatal and postnatal thiamine deficiency on auditory pathway of the Wistar-Albino rats.

In this study, we created an animal model to demonstrate the effects of thiamine on the hearing pathways of new-borns during pregnancy and lactation by inducing a dietary thiamine deficiency in the mother. The study included 16 female Wistar albino rats. The animals were separated into four groups and provided the appropriate amounts of dietary thiamine according to their groups during pre-pregnancy, pregnancy, and lactation periods. Three pups from each mother were included in the study, and 12 pups were selected from each group. On the fortieth day after birth, the auditory pathways of 48 pups in the 4 groups were examined electro physiologically and ultra-structurally. In Group N-N, morphology of hair cells stereocilia degeneration was not obtained in all turns of cochlea. In Group N-T, Inner Hair Cells (IHCs) and Outher Hair Cells (OHCs) stereocilia didn't show degeneration in all turns of cochlea but had rupture inrows of HCs stereocilia. In group T-N IHCs stereocilia less degeneration was observed in all turns of cochlea. OHC stereocilia partial loss was observed only in basal turn of cochlea. In Group T-T IHCs stereocilia was observed less degeneration and rupture in all turns of cochlea. Thiamine is vital for the development of cochlear hair cells during both prenatal and postnatal periods. Even partial deficiency of thiamine causes significant degeneration to the auditory pathway. The level of evidence of this article is 5. This article is an experimental animal and laboratory study.

Coupling between the Stereocilia of Rat Sensory Inner-Hair-Cell Hair Bundles Is Weak, Shaping Their Sensitivity to Stimulation.

The hair bundle is the universal mechanosensory organelle of auditory, vestibular, and lateral-line systems. A bundle comprises mechanically coupled stereocilia, whose displacements in response to stimulation activate a receptor current. The similarity of stereociliary displacements within a bundle regulates fundamental properties of the receptor current like its speed, magnitude, and sensitivity. However, the dynamics of individual stereocilia from the mammalian cochlea in response to a known bundle stimulus has not been quantified. We developed a novel high-speed system, which dynamically stimulates and tracks individual inner-hair-cell stereocilia from male and female rats. Stimulating two to three of the tallest stereocilia within a bundle (nonuniform stimulation) caused dissimilar stereociliary displacements. Stereocilia farther from the stimulator moved less, but with little delay, implying that there is little slack in the system. Along the axis of mechanical sensitivity, stereocilium displacements peaked and reversed direction in response to a step stimulus. A viscoelastic model explained the observed displacement dynamics, which implies that coupling between the tallest stereocilia is effectively viscoelastic. Coupling elements between the tallest inner-hair-cell stereocilia were two to three times stronger than elements anchoring stereocilia to the surface of the cell but were 100-10,000 times weaker than those of a well-studied noncochlear hair bundle. Coupling was too weak to ensure that stereocilia move similarly in response to nonuniform stimulation at auditory frequencies. Our results imply that more uniform stimulation across the tallest stereocilia of an inner-hair-cell bundle in vivo is required to ensure stereociliary displacement similarity, increasing the speed, sensitivity, and magnitude of the receptor current.SIGNIFICANCE STATEMENT Generation of the receptor current of the hair cell is the first step in electrically encoding auditory information in the hearing organs of all vertebrates. The receptor current is shaped by mechanical coupling between stereocilia in the hair bundle of each hair cell. Here, we provide foundational information on the mechanical coupling between stereocilia of cochlear inner-hair cells. In contrast to other types of hair cell, coupling between inner-hair-cell stereocilia is weak, causing slower, smaller, and less sensitive receptor currents in response to stimulation of few, rather than many, stereocilia. Our results imply that inner-hair cells need many stereocilia to be stimulated in vivo to ensure fast, large, and sensitive receptor currents.

Syntaxin 4 is essential for hearing in human and zebrafish.

Congenital hearing impairment (HI) is a genetically highly heterogeneous disorder in which prompt recognition and intervention are crucial to optimize outcomes. In this study, we used exome sequencing to investigate a large consanguineous Pakistani family with eight affected individuals showing bilateral severe-to-profound HI. This identified a homozygous splice region variant in STX4 (c.232 + 6T>C), which causes exon skipping and a frameshift, that segregated with HI (two-point logarithm of odds (LOD) score = 5.9). STX4, a member of the syntaxin family, is a component of the SNARE machinery involved in several vesicle transport and recycling pathways. In silico analysis showed that murine orthologue Stx4a is highly and widespread expressed in the developing and adult inner ear. Immunofluorescent imaging revealed localization of STX4A in the cell body, cell membrane and stereocilia of inner and outer hair cells. Furthermore, a morpholino-based knockdown of stx4 in zebrafish showed an abnormal startle response, morphological and developmental defects, and a disrupted mechanotransduction function in neuromast hair cells measured via FM1-43 uptake. Our findings indicate that STX4 dysfunction leads to HI in humans and zebrafish and supports the evolutionary conserved role of STX4 in inner ear development and hair cell functioning.

Inner hair cell stereocilia displacement in response to focal stimulation of the basilar membrane in the ex vivo gerbil cochlea

The inner hair cells in the mammalian cochlea transduce mechanical signals to electrical signals that provide input to the auditory nerve. The spatial-temporal displacement of the inner hair cell stereocilia (IHCsc) relative to basilar membrane (BM) displacement is central to characterizing the transduction process. This study specifically focuses on measuring displacement of the stereocilia hair bundles in the radial dimensions where they are most sensitive. To simplify the mechanical response of the cochlear partition, a mechanical probe was used to drive the BM. Optical imaging was used to measure radial displacement of the inner hair cell stereocilia local to the probe in ex vivo gerbil cochleae. The mechanical probe displaced the BM in the transverse direction using sinusoidal stimuli with frequencies ranging from 10 Hz to 42.5 kHz. IHCsc displacement measurements were made in the radial dimension as a function of their longitudinal location along the length of the BM. The results were used to quantify the frequency response, longitudinal space coupling, traveling wave velocity, and wavelength of the radial displacement of the stereocilia.The measurements were centered at two best frequency locations along the BM: Proximal to the round window (first turn), and in the second turn. At both locations, frequency tuning was seen that was consistent with published place maps. At both locations, traveling waves were observed simultaneously propagating basal and apical from the probe. The velocity of the traveling waves at the center frequency (CF) of the location was higher in the first turn than in the second. As the stimulus frequency increased and approached CF for a location, the traveling wavelength decreased. Differential motion of the BM and IHCsc was observed in the second turn as the stimulus frequency increased toward CF. The longitudinal coupling measured in this study was longer than observed in previous studies. In summary the results suggest that the shape of the wave patterns present on the BM are not sufficient to characterize the displacement of the IHCsc.

Inner hair cell stereocilia are embedded in the tectorial membrane

Mammalian hearing depends on sound-evoked displacements of the stereocilia of inner hair cells (IHCs), which cause the endogenous mechanoelectrical transducer channels to conduct inward currents of cations including Ca2+. Due to their presumed lack of contacts with the overlaying tectorial membrane (TM), the putative stimulation mechanism for these stereocilia is by means of the viscous drag of the surrounding endolymph. However, despite numerous efforts to characterize the TM by electron microscopy and other techniques, the exact IHC stereocilia-TM relationship remains elusive. Here we show that Ca2+-rich filamentous structures, that we call Ca2+ ducts, connect the TM to the IHC stereocilia to enable mechanical stimulation by the TM while also ensuring the stereocilia access to TM Ca2+. Our results call for a reassessment of the stimulation mechanism for the IHC stereocilia and the TM role in hearing.

Functional Role of Class III Myosins in Hair Cells.

Cytoskeletal motors produce force and motion using the energy from ATP hydrolysis and function in a variety of mechanical roles in cells including muscle contraction, cargo transport, and cell division. Actin-based myosin motors have been shown to play crucial roles in the development and function of the stereocilia of auditory and vestibular inner ear hair cells. Hair cells can contain hundreds of stereocilia, which rely on myosin motors to elongate, organize, and stabilize their structure. Mutations in many stereocilia-associated myosins have been shown to cause hearing loss in both humans and animal models suggesting that each myosin isoform has a specific function in these unique parallel actin bundle-based protrusions. Here we review what is known about the classes of myosins that function in the stereocilia, with a special focus on class III myosins that harbor point mutations associated with delayed onset hearing loss. Much has been learned about the role of the two class III myosin isoforms, MYO3A and MYO3B, in maintaining the precise stereocilia lengths required for normal hearing. We propose a model for how class III myosins play a key role in regulating stereocilia lengths and demonstrate how their motor and regulatory properties are particularly well suited for this function. We conclude that ongoing studies on class III myosins and other stereocilia-associated myosins are extremely important and may lead to novel therapeutic strategies for the treatment of hearing loss due to stereocilia degeneration.

Dexamethasone-loaded chitosan-based genipin-cross-linked hydrogel for prevention of cisplatin induced ototoxicity in Guinea pig model

ObjectivesThe aim of this study was to investigate the protective effects of a sustained release form of dexamethasone (dex) loaded chitosan-based genipin-cross-linked hydrogel (CBGCH) in a guinea pig model of cisplatin (CP) induced hearing loss. MethodsImplantation of CBGCH was made by intratympanic (IT) injection. Ototoxicity was produced by intraperitoneal (IP) single dose of 14 mg/kg CP. Animals were randomly divided into four groups with 6 guinea pigs in each. Group 1 received only IP CP; group 2 received only IT dex-loaded CBGCH injections. Group 3 and group 4 received IP CP, plus IT nondrug CBGCH and IT dex-loaded CBGCH respectively 24 h prior to IP CP injections. Distortion product otoacoustic emissions (DPOAEs) and auditory brainstem response (ABR) measurements were obtained before the treatments and solely ABR measurements were done after 3 and 10 days. The ultrastructural effects were investigated by scanning electron microscopy (SEM) analysis. ResultsThe postCP ABR thresholds at 4, 8, 12, 16, 32 kHz frequencies were significantly better in group 4 than groups 1 and 3 (p < 0.05). The comparison of time effective ABR thresholds between groups 1 and 4 and between groups 3 and 4 showed significantly lower ABR thresholds in group 4 (p < 0.05). The SEM analysis showed that stereocilia of inner and outer hair cells were preserved in group 4, almost like group 2, whereas cytotoxic degenerations were noted in groups 1 and 3. ConclusionsIntratympanic administration of dex-loaded CBGCH has been shown to provide functional and structural protection against CP-induced ototoxicity.

Mechanotransduction current is essential for stability of the transducing stereocilia in mammalian auditory hair cells.

Mechanotransducer channels at the tips of sensory stereocilia of inner ear hair cells are gated by the tension of 'tip links' interconnecting stereocilia. To ensure maximal sensitivity, tip links are tensioned at rest, resulting in a continuous influx of Ca2+ into the cell. Here, we show that this constitutive Ca2+ influx, usually considered as potentially deleterious for hair cells, is in fact essential for stereocilia stability. In the auditory hair cells of young postnatal mice and rats, a reduction in mechanotransducer current, via pharmacological channel blockers or disruption of tip links, leads to stereocilia shape changes and shortening. These effects occur only in stereocilia that harbor mechanotransducer channels, recover upon blocker washout or tip link regeneration and can be replicated by manipulations of extracellular Ca2+ or intracellular Ca2+ buffering. Thus, our data provide the first experimental evidence for the dynamic control of stereocilia morphology by the mechanotransduction current.

OSBPL2 encodes a protein of inner and outer hair cell stereocilia and is mutated in autosomal dominant hearing loss (DFNA67).

BackgroundEarly-onset hearing loss is mostly of genetic origin. The complexity of the hearing process is reflected by its extensive genetic heterogeneity, with probably many causative genes remaining to be identified. Here, we aimed at identifying the genetic basis for autosomal dominant non-syndromic hearing loss (ADNSHL) in a large German family.MethodsA panel of 66 known deafness genes was analyzed for mutations by next-generation sequencing (NGS) in the index patient. We then conducted genome-wide linkage analysis, and whole-exome sequencing was carried out with samples of two patients. Expression of Osbpl2 in the mouse cochlea was determined by immunohistochemistry. Because Osbpl2 has been proposed as a target of miR-96, we investigated homozygous Mir96 mutant mice for its upregulation.ResultsOnset of hearing loss in the investigated ADNSHL family is in childhood, initially affecting the high frequencies and progressing to profound deafness in adulthood. However, there is considerable intrafamilial variability. We mapped a novel ADNSHL locus, DFNA67, to chromosome 20q13.2-q13.33, and subsequently identified a co-segregating heterozygous frameshift mutation, c.141_142delTG (p.Arg50Alafs*103), in OSBPL2, encoding a protein known to interact with the DFNA1 protein, DIAPH1. In mice, Osbpl2 was prominently expressed in stereocilia of cochlear outer and inner hair cells. We found no significant Osbpl2 upregulation at the mRNA level in homozygous Mir96 mutant mice.ConclusionThe function of OSBPL2 in the hearing process remains to be determined. Our study and the recent description of another frameshift mutation in a Chinese ADNSHL family identify OSBPL2 as a novel gene for progressive deafness.Electronic supplementary materialThe online version of this article (doi:10.1186/s13023-015-0238-5) contains supplementary material, which is available to authorized users.

Power Dissipation in the Subtectorial Space of the Mammalian Cochlea Is Modulated by Inner Hair Cell Stereocilia

The stereocilia bundle is the mechano-transduction apparatus of the inner ear. In the mammalian cochlea, the stereocilia bundles are situated in the subtectorial space (STS)—a micrometer-thick space between two flat surfaces vibrating relative to each other. Because microstructures vibrating in fluid are subject to high-viscous friction, previous studies considered the STS as the primary place of energy dissipation in the cochlea. Although there have been extensive studies on how metabolic energy is used to compensate the dissipation, much less attention has been paid to the mechanism of energy dissipation. Using a computational model, we investigated the power dissipation in the STS. The model simulates fluid flow around the inner hair cell (IHC) stereocilia bundle. The power dissipation in the STS because of the presence IHC stereocilia increased as the stimulating frequency decreased. Along the axis of the stimulating frequency, there were two asymptotic values of power dissipation. At high frequencies, the power dissipation was determined by the shear friction between the two flat surfaces of the STS. At low frequencies, the power dissipation was dominated by the viscous friction around the IHC stereocilia bundle—the IHC stereocilia increased the STS power dissipation by 50- to 100-fold. There exists a characteristic frequency for STS power dissipation, CFSTS, defined as the frequency where power dissipation drops to one-half of the low frequency value. The IHC stereocilia stiffness and the gap size between the IHC stereocilia and the tectorial membrane determine the characteristic frequency. In addition to the generally assumed shear flow, nonshear STS flow patterns were simulated. Different flow patterns have little effect on the CFSTS. When the mechano-transduction of the IHC was tuned near the vibrating frequency, the active motility of the IHC stereocilia bundle reduced the power dissipation in the STS.

The collagen receptor DDR1 co-localizes with the non-muscle myosin IIA in mice inner ear and contributes to the cytoarchitecture and stability of motile cells.

Discoidin domain receptor 1 (DDR1) is a tyrosine kinase receptor activated by native collagen. DDRs regulate cell adhesion, migration and various other cell functions. Deletion of the DDR1 gene in mice is associated with a severe decrease in auditory function and substantial structural alterations in a heterogeneous group of cells, including cells containing actin/myosin contractile elements, e.g., outer hair cells (OHCs) (Meyer zum Gottesberge et al. Lab Invest, 88: 27-37, 2008). The non-muscle myosin heavy chain isoform IIA (NM-IIA), encoded by MYH9, is implicated in the regulation of cell spreading, cellular reshaping and movement and cell migration and adhesion. In this study, we identify DDR1 and NM-IIA co-localization in the type III fibrocytes (tension fibrocytes) of the spiral ligament, the OHCs and the stereocilia of both OHCs and inner hair cells. We show for the first time that DDR1 malfunction causes OHC deformation and the separation of the lateral wall, the location of the cellular motor responsible for the electromotile property, explicitly in those regions showing DDR1 and NM-IIA co-localization. On the basis of our results, we propose that DDR1 acts in concert with proteins of the actin/myosin complex to maintain mechanical forces in the inner ear and to stabilize OHC cellular shape for proper auditory signal transduction.

Absence of plastin 1 causes abnormal maintenance of hair cell stereocilia and a moderate form of hearing loss in mice.

Hearing relies on the mechanosensory inner and outer hair cells (OHCs) of the organ of Corti, which convert mechanical deflections of their actin-rich stereociliary bundles into electrochemical signals. Several actin-associated proteins are essential for stereocilia formation and maintenance, and their absence leads to deafness. One of the most abundant actin-bundling proteins of stereocilia is plastin 1, but its function has never been directly assessed. Here, we found that plastin 1 knock-out (Pls1 KO) mice have a moderate and progressive form of hearing loss across all frequencies. Auditory hair cells developed normally in Pls1 KO, but in young adult animals, the stereocilia of inner hair cells were reduced in width and length. The stereocilia of OHCs were comparatively less affected; however, they also showed signs of degeneration in ageing mice. The hair bundle stiffness and the acquisition of the electrophysiological properties of hair cells were unaffected by the absence of plastin 1, except for a significant change in the adaptation properties, but not the size of the mechanoelectrical transducer currents. These results show that in contrast to other actin-bundling proteins such as espin, harmonin or Eps8, plastin 1 is dispensable for the initial formation of stereocilia. However, the progressive hearing loss and morphological defects of hair cells in adult Pls1 KO mice point at a specific role for plastin 1 in the preservation of adult stereocilia and optimal hearing. Hence, mutations in the human PLS1 gene may be associated with relatively mild and progressive forms of hearing loss.

A computational study on different flow patterns around the inner hair cell stereocilia

The mechano-transduction of the mammalian cochlea occurs in the micro-fluid domain between the tectorial membrane and the reticular lamina called the subtectorial space. The subtectorial fluid bathes the bundled stereocilia of the inner hair cells (IHCs). These cells are responsible for the onset of neural impulses in the auditory nerve fibers. Despite the generally accepted postulation that the IHC stereocilia are deflected by shear flow between the two layers, there have been suggestions that other flow modes exist besides the shear flow. We developed a computational model of fluid dynamics in the subtectorial space. The model simulates IHC mechano-transduction excited by different flow patterns. In order to compare different modes of fluid dynamical stimulation, the power efficiency of IHC mechano-transduction was introduced (dissipated power normalized by IHC mechano-transduction current). Besides different flow patterns, the effect of mechanical parameters (such as the gap size between the stereociliary tip and the tectorial membrane, stereociliar bundle stiffness) were investigated. The results demonstrate that the power efficiency for the IHC mechano-transduction depends on the flow pattern in the subtectorial space.

Caprice/MISP is a novel F‐actin bundling protein critical for actin‐based cytoskeletal reorganizations

Caprice [C19orf21 actin-bundling protein in characteristic epithelial cells, also called mitotic interactor and substrate of Plk1 (MISP)] is a novel actin-related protein identified in the highly-insoluble subcellular scaffold proteins. This protein contains multiple actin-binding sites, forms characteristic mesh-like F-actin bundles in vitro, and exhibits capricious localization and expression patterns in vivo. Overexpression or knock-down of Caprice resulted in a dramatic effect on cellular morphology by inducing stress fiber-like thick filaments or filopodial formations, respectively. Caprice is expressed and localized in distinct cells and tissues with specialized actin-based structures, such as growth cones of migrating neurons and stereocilia of inner ear hair cells. However, Caprice gene expression is varied among different cell types; especially enriched in several epithelial cells whereas relatively suppressed in a subset of epithelial cells, fibroblasts, and neuroblastoma cells at the transcriptional level. Thus, this protein is expected to be an effector for cell type-specific actin reorganization with its direct actin-binding properties and provides a novel model of cell morphology regulation by a non-ubiquitous single actin-bundling protein.

Different uptake of gentamicin through TRPV1 and TRPV4 channels determines cochlear hair cell vulnerability

Hair cells at the base of the cochlea appear to be more susceptible to damage by the aminoglycoside gentamicin than those at the apex. However, the mechanism of base-to-apex gradient ototoxicity by gentamicin remains to be elucidated. We report here that gentamicin caused rodent cochlear hair cell damages in a time- and dose-dependent manner. Hair cells at the basal turn were more vulnerable to gentamicin than those at the apical turn. Gentamicin-conjugated Texas Red (GTTR) uptake was predominant in basal turn hair cells in neonatal rats. Transient receptor potential vanilloid 1 (TRPV1) and 4 (TRPV4) expression was confirmed in the cuticular plate, stereocilia and hair cell body of inner hair cells and outer hair cells. The involvement of TRPV1 and TRPV4 in gentamicin trafficking of hair cells was confirmed by exogenous calcium treatment and TRPV inhibitors, including gadolinium and ruthenium red, which resulted in markedly inhibited GTTR uptake and gentamicin-induced hair cell damage in rodent and zebrafish ototoxic model systems. These results indicate that the cytotoxic vulnerability of cochlear hair cells in the basal turn to gentamicin may depend on effective uptake of the drug, which was, in part, mediated by the TRPV1 and TRPV4 proteins.

Diaphanous homolog 3 (Diap3) Overexpression Causes Progressive Hearing Loss and Inner Hair Cell Defects in a Transgenic Mouse Model of Human Deafness

We previously demonstrated that a mutation in the 5′ untranslated region of Diaphanous homolog 3 (DIAPH3) results in 2 to 3-fold overexpression of the gene, leading to a form of delayed onset, progressive human deafness known as AUNA1 (auditory neuropathy, nonsyndromic, autosomal dominant, 1). To investigate the mechanism of deafness, we generated two lines of transgenic mice overexpressing Diap3, the murine ortholog of DIAPH3, on an FVB/NJ background. Line 771 exhibits a relatively mild 20 dB hearing loss at 12 kHz at 4 and 8 weeks of age, progressing to 40 dB and 60 dB losses at 16 and 24 weeks, respectively, at 12 and 24 kHz. Line 924 shows no hearing loss at 4 or 8 weeks, but manifests 35 and 50 dB threshold shifts at 16 and 24 weeks, respectively, at both 12 and 24 kHz. Notably, mice from the two transgenic lines retain distortion product otoacoustic emissions, indicative of normal cochlear outer hair cell (OHC) function despite elevation of auditory thresholds. Scanning electron microscopy of the organ of Corti demonstrates striking anomalies of the inner hair cell (IHC) stereocilia, while OHCs are essentially intact. Over time, IHCs of both lines develop elongated stereocilia that appear fused with neighboring stereocilia, in parallel to the time course of hearing loss in each line. Furthermore, we observe significant reduction in the number of IHC ribbon synapses over 24 weeks in both lines, although this reduction does not correlate temporally with onset and progression of hearing loss or stereociliary anomalies. In summary, overexpression of wild-type Diap3 in two lines of transgenic mice results in hearing loss that recapitulates human AUNA1 deafness. These findings suggest an essential role of Diap3 in regulating assembly and/or maintenance of actin filaments in IHC stereocilia, as well as a potential role at the IHC ribbon synapse.

Alterations of the CIB2 calcium- and integrin-binding protein cause Usher syndrome type 1J and nonsyndromic deafness DFNB48

Sensorineural hearing loss is genetically heterogeneous. Here we report that mutations in CIB2, encoding a Ca2+- and integrin-binding protein, are associated with nonsyndromic deafness (DFNB48) and Usher syndrome type 1J (USH1J). There is one mutation of CIB2 that is a prevalent cause of DFNB48 deafness in Pakistan; other CIB2 mutations contribute to deafness elsewhere in the world. In rodents, CIB2 is localized in the mechanosensory stereocilia of inner ear hair cells and in retinal photoreceptor and pigmented epithelium cells. Consistent with molecular modeling predictions of Ca2+ binding, CIB2 significantly decreased the ATP-induced Ca2+ responses in heterologous cells, while DFNB48 mutations altered CIB2 effects on Ca2+ responses. Furthermore, in zebrafish and Drosophila, CIB2 is essential for the function and proper development of hair cells and retinal photoreceptor cells. We show that CIB2 is a new member of the vertebrate Usher interactome.

How are inner hair cells stimulated? Evidence for multiple mechanical drives

Recent studies indicate that the gap over outer hair cells (OHCs) between the reticular lamina (RL) and the tectorial membrane (TM) varies cyclically during low-frequency sounds. Variation in the RL-TM gap produces radial fluid flow in the gap that can drive inner hair cell (IHC) stereocilia. Analysis of RL-TM gap changes reveals three IHC drives in addition to classic SHEAR. For upward basilar-membrane (BM) motion, IHC stereocilia are deflected in the excitatory direction by SHEAR and OHC-MOTILITY, but in the inhibitory direction by TM-PUSH and CILIA-SLANT. Upward BM motion causes OHC somatic contraction which tilts the RL, compresses the RL-TM gap over IHCs and expands the RL-TM gap over OHCs, thereby producing an outward (away from the IHCs) radial fluid flow which is the OHC-MOTILITY drive. For upward BM motion, the force that moves the TM upward also compresses the RL-TM gap over OHCs causing inward radial flow past IHCs which is the TM-PUSH drive. Motions that produce large tilting of OHC stereocilia squeeze the supra-OHC RL-TM gap and caused inward radial flow past IHCs which is the CILIA-SLANT drive. Combinations of these drives explain: (1) the reversal at high sound levels of auditory nerve (AN) initial peak (ANIP) responses to clicks, and medial olivocochlear (MOC) inhibition of ANIP responses below, but not above, the ANIP reversal, (2) dips and phase reversals in AN responses to tones in cats and chinchillas, (3) hypersensitivity and phase reversals in tuning-curve tails after OHC ablation, and (4) MOC inhibition of tail-frequency AN responses. The OHC-MOTILITY drive provides another mechanism, in addition to BM motion amplification, that uses active processes to enhance the output of the cochlea. The ability of these IHC drives to explain previously anomalous data provides strong, although indirect, evidence that these drives are significant and presents a new view of how the cochlea works at frequencies below 3 kHz.