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Sound-induced mechanical stimuli are detected by elaborate mechanosensory transduction (MT) machinery in highly specialized hair cells of the inner ear. Genetic studies of inherited deafness in the past decades have uncovered several molecular constituents of the MT complex, and intense debate has surrounded the molecular identity of the pore-forming subunits. How the MT components function in concert in response to physical stimulation is not fully understood. In this review, we summarize and discuss multiple lines of evidence supporting the hypothesis that transmembrane channel-like 1 is a long-sought MT channel subunit. We also review specific roles of other components of the MT complex, including protocadherin 15, cadherin 23, lipoma HMGIC fusion partner-like 5, transmembrane inner ear, calcium and integrin-binding family member 2, and ankyrins. Based on these recent advances, we propose a unifying theory of hair cell MT that may reconcile most of the functional discoveries obtained to date. Finally, we discuss key questions that need to be addressed for a comprehensive understanding of hair cell MT at molecular and atomic levels.

The present study aimed to observe the changes in the cochlea ribbon synapses after repeated exposure to moderate-to-high intensity noise. Guinea pigs received 95 dB SPL white noise exposure 4 h a day for consecutive 7 days (we regarded it a medium-term and moderate-intensity noise, or MTMI noise). Animals were divided into four groups: Control, 1DPN (1-day post noise), 1WPN (1-week post noise), and 1MPN (1-month post noise). Auditory function analysis by auditory brainstem response (ABR) and compound action potential (CAP) recordings, as well as ribbon synapse morphological analyses by immunohistochemistry (Ctbp2 and PSD95 staining) were performed 1 day, 1 week, and 1 month after noise exposure. After MTMI noise exposure, the amplitudes of ABR I and III waves were suppressed. The CAP threshold was elevated, and CAP amplitude was reduced in the 1DPN group. No apparent changes in hair cell shape, arrangement, or number were observed, but the number of ribbon synapse was reduced. The 1WPN and 1MPN groups showed that part of ABR and CAP changes recovered, as well as the synapse number. The defects in cochlea auditory function and synapse changes were observed mainly in the high-frequency region. Together, repeated exposure in MTMI noise can cause hidden hearing loss (HHL), which is partially reversible after leaving the noise environment; and MTMI noise-induced HHL is associated with inner hair cell ribbon synapses., (© 2021 The Author(s).)

Background: The SCN11A gene, encoded Nav1.9 TTX resistant sodium channels, is a main effector in peripheral inflammation related pain in nociceptive neurons. The role of SCN11A gene in the auditory system has not been well characterized. We therefore examined the expression of SCN11A in the murine cochlea, the morphological and physiological features of Nav1.9 knockout (KO) ICR mice., Results: Nav1.9 expression was found in the primary afferent endings beneath the inner hair cells (IHCs). The relative quantitative expression of Nav1.9 mRNA in modiolus of wild-type (WT) mice remains unchanged from P0 to P60. The number of presynaptic CtBP2 puncta in Nav1.9 KO mice was significantly lower than WT. In addition, the number of SGNs in Nav1.9 KO mice was also less than WT in the basal turn, but not in the apical and middle turns. There was no lesion in the somas and stereocilia of hair cells in Nav1.9 KO mice. Furthermore, Nav1.9 KO mice showed higher and progressive elevated ABR threshold at 16 kHz, and a significant increase in CAP thresholds., Conclusions: These data suggest a role of Nav1.9 in regulating the function of ribbon synapses and the auditory nerves. The impairment induced by Nav1.9 gene deletion mimics the characters of cochlear synaptopathy.

The characteristic feature of noise-induced hearing loss (NIHL) is the loss or malfunction of the outer hair cells (OHC) and the inner hair cells (IHC) of the cochlea. 90-95% of the spiral ganglion neurons, forming the cell bodies of cochlear nerve, synapse with the IHCs. Glutamate is the most potent excitatory neurotransmitter for IHC-auditory nerve synapses. Excessive release of glutamate in response to acoustic trauma (AT), may cause excitotoxicity by causing damage to the spiral ganglion neurons (SGN) or loss of the spiral ganglion dendrites, post-synaptic to the IHCs. Another neurotransmitter, GABA, plays an important role in the processing of acoustic stimuli and central regulation after peripheral injury, so it is potentially related to the regulation of hearing function and sensitivity after noise. The aim of this study is to evaluate the effect of AT on the expressions of glutamate excitotoxicity, GABA inhibition and neurosteroid synthesis genes.We exposed 24 BALB/c mice to AT. Controls were sacrificed without exposure to noise, Post-AT(1) and Post-AT(15) were sacrificed on the 1st and 15th day, respectively, after noise exposure. The expressions of various genes playing roles in glutamate, GABA and neurosteroid pathways were compared between groups by real-time PCR.Expressions of Cyp11a1 , Gls , Gabra1 , Grin2b , Sult1a1 , Gad1, and Slc1a2 genes in Post-AT(15) mice were significantly decreased in comparison to control and Post-AT(1) mice. No significant differences in the expression of Slc6a1 and Slc17a8 genes was detected.These findings support the possible role of balance between glutamate excitotoxicity and GABA inhibition is disturbed during the post AT days and also the synthesis of some neurosteroids such as pregnenolone sulfate may be important in this balance.

Tinnitus, the phantom perception of sound, often occurs as a clinical sequela of auditory traumas. In an effort to develop an objective test and therapeutic approach for tinnitus, the present study was performed in blast-exposed rats and focused on measurements of auditory brainstem responses (ABRs), prepulse inhibition of the acoustic startle response, and presynaptic ribbon densities on cochlear inner hair cells (IHCs). Although the exact mechanism is unknown, the "central gain theory" posits that tinnitus is a perceptual indicator of abnormal increases in the gain (or neural amplification) of the central auditory system to compensate for peripheral loss of sensory input from the cochlea. Our data from vehicle-treated rats supports this rationale; namely, blast-induced cochlear synaptopathy correlated with imbalanced elevations in the ratio of centrally-derived ABR wave V amplitudes to peripherally-derived wave I amplitudes, resulting in behavioral evidence of tinnitus. Logistic regression modeling demonstrated that the ABR wave V/I amplitude ratio served as a reliable metric for objectively identifying tinnitus. Furthermore, histopathological examinations in blast-exposed rats revealed tinnitus-related changes in the expression patterns of key plasticity factors in the central auditory pathway, including chronic loss of Arc/Arg3.1 mobilization. Using a formulation of N-acetylcysteine (NAC) and disodium 2,4-disulfophenyl-N-tert-butylnitrone (HPN-07) as a therapeutic for addressing blast-induced neurodegeneration, we measured a significant treatment effect on preservation or restoration of IHC ribbon synapses, normalization of ABR wave V/I amplitude ratios, and reduced behavioral evidence of tinnitus in blast-exposed rats, all of which accorded with mitigated histopathological evidence of tinnitus-related neuropathy and maladaptive neuroplasticity., Competing Interests: The authors have read the journal’s policy, and the authors of this manuscript have the following competing interests to declare: RDK is a minor stockholder in Otologic Pharmaceutics Inc. The authors would like to declare the following patents/patent applications associated with this research: Patent families 10,555,915; 9,289,404; 10,111,843. The authors also declare the following product in development associated with this research: an oral combination drug (NAC plus HPN-07) being developed for prevention and/or treatment of acute sensorineural hearing loss. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Inner hair cell (IHC) ribbon synapses are the first synapse in the auditory system and can be degenerated by noise and aging, thereby leading to hidden hearing loss (HHL) and other hearing disorders. However, the mechanism underlying this cochlear synaptopathy remains unclear. Here, we report that elevation of extracellular K + , which is a consequence of noise exposure, could cause IHC ribbon synapse degeneration and swelling. Like intensity dependence in noise-induced cochlear synaptopathy, the K + -induced degeneration was dose-dependent, and could be attenuated by BK channel blockers. However, application of glutamate receptor (GluR) agonists caused ribbon swelling but not degeneration. In addition, consistent with synaptopathy in HHL, both K + and noise exposure only caused IHC but not outer hair cell ribbon synapse degeneration. These data reveal that K + excitotoxicity can degenerate IHC ribbon synapses in HHL, and suggest that BK channel may be a potential target for prevention and treatment of HHL.

Spag6 encodes an axoneme central apparatus protein that is required for normal flagellar and cilia motility. Recent findings suggest that Spag6 also plays a role in ciliogenesis, orientation of cilia basal feet, and planar polarity. Sensory cells of the inner ear display unique structural features that underlie their mechanosensitivity. They represent a distinctive form of cellular polarity, known as planar cell polarity (PCP). However, a role for Spag6 in the inner ear has not yet been explored. In the present study, the function of Spag6 in the inner ear was examined using Spag6 -deficient mice. Our results demonstrate hearing loss in the Spag6 mutants, associated with abnormalities in cellular patterning, cell shape, stereocilia bundles, and basal bodies, as well as abnormally distributed Frizzled class receptor 6 (FZD6), suggesting that Spag6 participates in PCP regulation. Moreover, we found that the subapical microtubule meshwork was disrupted. Our observations suggest new functions for Spag6 in hearing and PCP in the inner ear.

Key Points: Age-related hearing loss is a progressive hearing loss involving environmental and genetic factors, leading to a decrease in hearing sensitivity, threshold and speech discrimination. We compared age-related changes in inner hair cells (IHCs) between four mouse strains with different levels of progressive hearing loss. The surface area of apical coil IHCs (9-12 kHz cochlear region) decreases by about 30-40% with age. The number of BK channels progressively decreases with age in the IHCs from most mouse strains, but the basolateral membrane current profile remains unchanged. The mechanoelectrical transducer current is smaller in mice harbouring the hypomorphic Cdh23 allele Cdh23 ahl (C57BL/6J; C57BL/6NTac), but not in Cdh23-repaired mice (C57BL/6NTac Cdh23+ ), indicating that it could contribute to the different progression of hearing loss among mouse strains. The degree of efferent rewiring onto aged IHCs, most likely coming from the lateral olivocochlea fibres, was correlated with hearing loss in the different mouse strains., Abstract: Inner hair cells (IHCs) are the primary sensory receptors of the mammalian cochlea, transducing acoustic information into electrical signals that are relayed to the afferent neurons. Functional changes in IHCs are a potential cause of age-related hearing loss. Here, we have investigated the functional characteristics of IHCs from early-onset hearing loss mice harbouring the allele Cdh23 ahl (C57BL/6J and C57BL/6NTac), from late-onset hearing loss mice (C3H/HeJ), and from mice corrected for the Cdh23 ahl mutation (C57BL/6NTac Cdh23+ ) with an intermediate hearing phenotype. There was no significant loss of IHCs in the 9-12 kHz cochlear region up to at least 15 months of age, but their surface area decreased progressively by 30-40% starting from ∼6 months of age. Although the size of the BK current decreased with age, IHCs retained a normal KCNQ4 current and resting membrane potential. These basolateral membrane changes were most severe for C57BL/6J and C57BL/6NTac, less so for C57BL/6NTac Cdh23+ and minimal or absent in C3H/HeJ mice. We also found that lateral olivocochlear (LOC) efferent fibres re-form functional axon-somatic connections with aged IHCs, but this was seen only sporadically in C3H/HeJ mice. The efferent post-synaptic SK2 channels appear prior to the establishment of the efferent contacts, suggesting that IHCs may play a direct role in re-establishing the LOC-IHC synapses. Finally, we showed that the size of the mechanoelectrical transducer (MET) current from IHCs decreased significantly with age in mice harbouring the Cdh23 ahl allele but not in C57BL/6NTac Cdh23+ mice, indicating that the MET apparatus directly contributes to the progression of age-related hearing loss., (© 2020 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

Transmembrane channel-like protein 1 (TMC1) and lipoma HMGIC fusion partner-like 5 (LHFPL5) are recognized as two critical components of the mechanotransduction complex in inner-ear hair cells. However, the physical and functional interactions of TMC1 and LHFPL5 remain largely unexplored. We examined the interaction between TMC1 and LHFPL5 by using multiple approaches, including our recently developed ultrasensitive microbead-based single-molecule pulldown (SiMPull) assay. We demonstrate that LHFPL5 physically interacts with and stabilizes TMC1 in both heterologous expression systems and in the soma and hair bundle of hair cells. Moreover, the semidominant deafness mutation D572N in human TMC1 (D569N in mouse TMC1) severely disrupted LHFPL5 binding and destabilized TMC1 expression. Thus, our findings reveal previously unrecognized physical and functional interactions of TMC1 and LHFPL5 and provide insights into the molecular mechanism by which the D572N mutation causes deafness. Notably, these findings identify a missing link in the currently known physical organization of the mechanotransduction macromolecular complex. Furthermore, this study has demonstrated the power of the microbead-based SiMPull assay for biochemical investigation of rare cells such as hair cells., Competing Interests: The authors declare no competing interest.

In the mammalian cochlea, specialized ribbon-type synapses between sensory inner hair cells (IHCs) and postsynaptic spiral ganglion neurons ensure the temporal precision and indefatigability of synaptic sound encoding. These high-through-put synapses are presynaptically characterized by an electron-dense projection-the synaptic ribbon-which provides structural scaffolding and tethers a large pool of synaptic vesicles. While advances have been made in recent years in deciphering the molecular anatomy and function of these specialized active zones, the developmental assembly of this presynaptic interaction hub remains largely elusive. In this review, we discuss the dynamic nature of IHC (pre-) synaptogenesis and highlight molecular key players as well as the transport pathways underlying this process. Since developmental assembly appears to be a highly dynamic process, we further ask if this structural plasticity might be maintained into adulthood, how this may influence the functional properties of a given IHC synapse and how such plasticity could be regulated on the molecular level. To do so, we take a closer look at other ribbon-bearing systems, such as retinal photoreceptors and pinealocytes and aim to infer conserved mechanisms that may mediate these phenomena.

Sensorineural hearing loss and vertigo, resulting from lesions in the sensory epithelium of the inner ear, have a high incidence worldwide. The sensory epithelium of the inner ear may exhibit extreme degeneration and is transformed to flat epithelium (FE) in humans and mice with profound sensorineural hearing loss and/or vertigo. Various factors, including ototoxic drugs, noise exposure, aging, and genetic defects, can induce FE. Both hair cells and supporting cells are severely damaged in FE, and the normal cytoarchitecture of the sensory epithelium is replaced by a monolayer of very thin, flat cells of irregular contour. The pathophysiologic mechanism of FE is unclear but involves robust cell division. The cellular origin of flat cells in FE is heterogeneous; they may be transformed from supporting cells that have lost some features of supporting cells (dedifferentiation) or may have migrated from the flanking region. The epithelial-mesenchymal transition may play an important role in this process. The treatment of FE is challenging given the severe degeneration and loss of both hair cells and supporting cells. Cochlear implant or vestibular prosthesis implantation, gene therapy, and stem cell therapy show promise for the treatment of FE, although many challenges remain to be overcome.

Objective: To explore the connections between hair cells and spiral ganglion neurons (SGNs) during the development of the C57BL/6 mouse inner ear., Materials and Methods: The specimens of C57BL/6 mouse inner ear, from E15 (embryo day 15) to adult mouse, were collected; immunohistochemistry was employed to explore the frozen sections of specimens., Results: The development of cochlea starts sequentially from the basal turn to the apex turn. Morphological development of SGNs occurs mainly from E16 to P12 (postnatal day 12). Hair cells appear from E18 to P12, and inner hair cells (IHCs) develop earlier than outer hair cells (OHCs). The connections between hair cells and SGNs begin to develop during E18-P1, morphologically resemble mature synapses during P8-P12, and completely mature in adult mice., Conclusions: The genesis of auditory ribbon synapse occurs from E18 to P1. Synchronized with the development of SGNs and hair cells, the functional filaments remain connected to hair cells, while the spare ones get disconnected from the surface of hair cells. Connections between SGN nerve filaments and IHCs occur earlier than those between SGN nerve filaments and OHCs.

Inner ear hair cells are mechanosensitive cells responsible for sensing and transmitting signals to the brain to be interpreted as sound or head position/movement. The zinc-finger protein, gfi1, is expressed in differentiating neurons and inner ear hair cells. Gfi1 deficiency leads to a massive loss of cochlear hair cells in mice. However, the mechanism remains unclear. To develop an effective molecular therapy for hearing loss, it is critical to first understand the relationship between gfi1 and hair cell development. We demonstrated in the zebrafish model that gfi1.2 was initially expressed in the inner ear at 14 h post-fertilization (hpf), preceding the expression of gfi1.1 at 19 hpf. In the morpholino-mediated gfi1.2 knockdown mutants, hair cells reduced in number without altering the expression of pax2a, dlx3b, atho1a and pou4f3, the markers for otic patterning and specification. There was a down-regulation of the pro-neuronal genes, ngn1 and atoh1b in the context of gfi1.2 knockdown, which was rescued by the exogenous gfi1.2. We also found that gfi1.2 may regulate ngn1 expression by suppressing id2a. Our results suggested that knockdown of gfi1.2 may lead to deafness through promoting cells proliferation in the pro-sensory region and interrupting cell differentiation., (Copyright © 2020. Published by Elsevier B.V.)

Despite the known importance of the transcription factors ATOH1, POU4F3 and GFI1 in hair cell development and regeneration, their downstream transcriptional cascades in the inner ear remain largely unknown. Here, we have used Gfi1 cre ;RiboTag mice to evaluate changes to the hair cell translatome in the absence of GFI1. We identify a systematic downregulation of hair cell differentiation genes, concomitant with robust upregulation of neuronal genes in the GFI1-deficient hair cells. This includes increased expression of neuronal-associated transcription factors (e.g. Pou4f1 ) as well as transcription factors that serve dual roles in hair cell and neuronal development (e.g. Neurod1 , Atoh1 and Insm1 ). We further show that the upregulated genes are consistent with the NEUROD1 regulon and are normally expressed in hair cells prior to GFI1 onset. Additionally, minimal overlap of differentially expressed genes in auditory and vestibular hair cells suggests that GFI1 serves different roles in these systems. From these data, we propose a dual mechanism for GFI1 in promoting hair cell development, consisting of repression of neuronal-associated genes as well as activation of hair cell-specific genes required for normal functional maturation., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

Apoptosis of cochlear hair cells is a key step towards age-related hearing loss. Although numerous genes have been implicated in the genetic causes of late-onset, progressive hearing loss, few show direct links to the proapoptotic process. By genome-wide linkage analysis and whole exome sequencing, we identified a heterozygous p.L183V variant in THOC1 as the probable cause of the late-onset, progressive, non-syndromic hearing loss in a large family with autosomal dominant inheritance. Thoc1, a member of the conserved multisubunit THO/TREX ribonucleoprotein complex, is highly expressed in mouse and zebrafish hair cells. The thoc1 knockout (thoc1 mutant) zebrafish generated by gRNA-Cas9 system lacks the C-startle response, indicative of the hearing dysfunction. Both Thoc1 mutant and knockdown zebrafish have greatly reduced hair cell numbers, while the latter can be rescued by embryonic microinjection of human wild-type THOC1 mRNA but to significantly lesser degree by the c.547C>G mutant mRNA. The Thoc1 deficiency resulted in marked apoptosis in zebrafish hair cells. Consistently, transcriptome sequencing of the mutants showed significantly increased gene expression in the p53-associated signaling pathway. Depletion of p53 or applying the p53 inhibitor Pifithrin-α significantly rescued the hair cell loss in the Thoc1 knockdown zebrafish. Our results suggested that THOC1 deficiency lead to late-onset, progressive hearing loss through p53-mediated hair cell apoptosis. This is to our knowledge the first human disease associated with THOC1 mutations and may shed light on the molecular mechanism underlying the age-related hearing loss., Competing Interests: The authors have declared that no competing interests exist.

Perception of sound is initiated by mechanically gated ion channels at the tips of stereocilia. Mature mammalian auditory hair cells require transmembrane channel-like 1 (TMC1) for mechanotransduction, and mutations of the cognate genetic sequences result in dominant or recessive heritable deafness forms in humans and mice. In contrast, zebrafish lateral line hair cells, which detect water motion, require Tmc2a and Tmc2b. Here, we use standard and multiplex genome editing in conjunction with functional and behavioral assays to determine the reliance of zebrafish hearing and vestibular organs on Tmc proteins. Surprisingly, our approach using multiple mutant alleles demonstrates that hearing in zebrafish is not dependent on Tmc1, nor is it fully dependent on Tmc2a and Tmc2b. Hearing however is absent in triple-mutant zebrafish that lack Tmc1, Tmc2a and Tmc2b. These outcomes reveal a striking resemblance of Tmc protein reliance in the vestibular sensory epithelia of mammals to the maculae of zebrafish. Moreover, our findings disclose a logic of Tmc use where hearing depends on a complement of Tmc proteins beyond those employed to sense water motion., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Auditory inner hair cells (IHCs) convert sound vibrations into receptor potentials that drive synaptic transmission. For the precise encoding of sound qualities, receptor potentials are shaped by K + conductances tuning the properties of the IHC membrane. Using patch-clamp and computational modeling, we unravel this membrane specialization showing that IHCs express an exclusive repertoire of six voltage-dependent K + conductances mediated by K v 1.8, K v 7.4, K v 11.1, K v 12.1, and BK Ca channels. All channels are active at rest but are triggered differentially during sound stimulation. This enables non-saturating tuning over a far larger potential range than in IHCs expressing fewer current entities. Each conductance contributes to optimizing responses, but the combined activity of all channels synergistically improves phase locking and the dynamic range of intensities that IHCs can encode. Conversely, hypothetical simpler IHCs appear limited to encode only certain aspects (frequency or intensity). The exclusive channel repertoire of IHCs thus constitutes an evolutionary adaptation to encode complex sound through multifaceted receptor potentials., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Mature hair cells transduce information over a wide range of stimulus intensities and frequencies for prolonged periods of time. The efficiency of such a demanding task is reflected in the characteristics of exocytosis at their specialized presynaptic ribbons. Ribbons are electron-dense structures able to tether a large number of releasable vesicles allowing them to maintain high rates of vesicle release. Calcium entry through rapidly activating, non-inactivating Ca V 1.3 (L-type) Ca 2+ channels in response to cell depolarization causes a local increase in Ca 2+ at the ribbon synapses, which is detected by the exocytotic Ca 2+ sensors. The Ca 2+ dependence of vesicle exocytosis at mammalian vestibular hair cell (VHC) ribbon synapses is believed to be linear, similar to that observed in mature cochlear inner hair cells (IHCs). The linear relation has been shown to correlate with the presence of the Ca 2+ sensor synaptotagmin-4 (Syt-4). Therefore, we studied the exocytotic Ca 2+ dependence, and the release kinetics of different vesicle pool populations, in Type II VHCs of control and Syt-4 knockout mice using patch-clamp capacitance measurements, under physiological recording conditions. We found that exocytosis in mature control and knockout Type II VHCs displayed a high-order dependence on Ca 2+ entry, rather than the linear relation previously observed. Consistent with this finding, the Ca 2+ dependence and release kinetics of the ready releasable pool (RRP) of vesicles were not affected by an absence of Syt-4. However, we did find that Syt-4 could play a role in regulating the release of the secondary releasable pool (SRP) in these cells. Our findings show that the coupling between Ca 2+ influx and neurotransmitter release at mature Type II VHC ribbon synapses is faithfully described by a nonlinear relation that is likely to be more appropriate for the accurate encoding of low-frequency vestibular information, consistent with that observed at low-frequency mammalian auditory receptors., (© 2020 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.)

Proper functioning of the auditory nerve is of critical importance for auditory rehabilitation by cochlear implants. Here we used the Cldn14 -/- mouse to study in detail the effects of Claudin 14 loss on auditory synapses and the auditory nerve. Mutations in the tight junction protein Claudin 14 cause autosomal recessive non-syndromic hearing loss (DFNB29) in humans and mice, due to extensive degeneration of outer and inner hair cells. Here we show that massive inner hair cell loss in Cldn14 -/- mice starts after the third postnatal week. Immunohistochemical analysis, using presynaptic Ribeye and postsynaptic GluR2 or PSD 95 as markers, revealed the degeneration of full ribbon synapses in inner hair cells from apical cochlear regions already at postnatal day 12 (P12). At P20, significant reduction in number of ribbon synapses has been observed for all cochlear regions and the loss of synaptic ribbons becomes even more prominent in residual inner hair cells from middle and apical cochlear regions at P45, which by then lost more than 40% of all ribbon synapses. In contrast to excessive noise exposure, loss of Claudin 14 does not cause an increase in "orphan" ribbons with no postsynaptic counterpart due to a reduction of postsynaptic structures. Hair cell loss in Cldn14 -/- mice is associated with regression of peripheral auditory nerve processes, especially of outer radial fibers, which normally innervate the outer hair cells. The number of spiral ganglion neurons per area, however, was unchanged between the genotypes. Different effects were observed in the cochlear nucleus complex (CNC), the central projection area of the auditory nerve. While the dorsal cochlear nucleus (DCN) showed a significant 19.7% volume reduction, VGLUT-1 input was reduced by 34.4% in the ventral cochlear nucleus (VCN) but not in the DCN of Cldn14 -/- mice. Taken together, massive inner hair cell loss starts after the third postnatal week in Cldn14 -/- mice, but is preceded by the loss of ribbon synapses, which may be a first sign of an ongoing degeneration process in otherwise morphologically inconspicuously inner hair cells. In addition to the regression of peripheral nerve processes, reduced levels of VGLUT-1 in the VCN of Cldn14 -/- mice suggests that Claudin 14 loss does not only cause hair cell loss but also affects peripheral and central connectivity of the auditory nerve., Competing Interests: Declaration of competing interest The authors declare no competing financial interests., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Mammalian hearing requires the development of the organ of Corti, a sensory epithelium comprising unique cell types. The limited number of each of these cell types, combined with their close proximity, has prevented characterization of individual cell types and/or their developmental progression. To examine cochlear development more closely, we transcriptionally profile approximately 30,000 isolated mouse cochlear cells collected at four developmental time points. Here we report on the analysis of those cells including the identification of both known and unknown cell types. Trajectory analysis for OHCs indicates four phases of gene expression while fate mapping of progenitor cells suggests that OHCs and their surrounding supporting cells arise from a distinct (lateral) progenitor pool. Tgfβr1 is identified as being expressed in lateral progenitor cells and a Tgfβr1 antagonist inhibits OHC development. These results provide insights regarding cochlear development and demonstrate the potential value and application of this data set.

In different animal models, auditory nerve fibers display variation in spontaneous activity and response threshold. Functional and structural differences among inner hair cell ribbon synapses are believed to contribute to this variation. The relative volumes of synaptic proteins at individual synapses might be one such difference. This idea is based on the observation of opposing volume gradients of the presynaptic ribbons and associated postsynaptic glutamate receptor patches in mice along the pillar modiolar axis of the inner hair cell, the same axis along which fibers were shown to vary in their physiological properties. However, it is unclear whether these opposing gradients are expressed consistently across animal models. In addition, such volume gradients observed for separate populations of presynaptic ribbons and postsynaptic glutamate receptor patches suggest different relative volumes of these synaptic structures at individual synapses; however, these differences have not been examined in mice. Furthermore, it is unclear whether such gradients are limited to these synaptic proteins. Therefore, we analyzed organs of Corti isolated from CBA/CaJ, C57BL/6, and FVB/NJ mice using immunofluorescence, confocal microscopy, and quantitative image analysis. We find consistent expression of presynaptic volume gradients across strains of mice and inconsistent expression of postsynaptic volume gradients. We find differences in the relative volume of synaptic proteins, but these are different between CBA/CaJ mice, and C57BL/6 and FVB/NJ mice. We find similar results in C57BL/6 and FVB/NJ mice when using other postsynaptic density proteins (Shank1, Homer, and PSD95). These results have implications for the mechanisms by which volumes of synaptic proteins contribute to variations in the physiology of individual auditory nerve fibers and their vulnerability to excitotoxicity., Competing Interests: Declaration of competing interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

The retinoblastoma family of pocket proteins (pRBs), composed of Rb1, p107, and p130 are negative regulators of cell-cycle progression. The deletion of any individual pRB in the auditory system triggers hair cells' (HCs) and supporting cells' (SCs) proliferation to different extents. Nevertheless, accessing their combined role in the inner ear through conditional or complete knockout methods is limited by the early mortality of the triple knockout. In quiescent cells, hyperphosphorylation and inactivation of the pRBs are maintained through the activity of the Cyclin-D1-cdk4/6 complex. Cyclin D1 (CycD1) is expressed in the embryonic and neonatal inner ear. In the mature organ of Corti (OC), CycD1 expression is significantly downregulated, paralleling the OC mitotic quiescence. Earlier studies showed that CycD1 overexpression leads to cell-cycle reactivation in cultures of inner ear explants. Here, we characterize a Cre-activated, Doxycycline (Dox)-controlled, conditional CycD1 overexpression model, which when bred to a tetracycline-controlled transcriptional activator and the Atoh1-cre mouse lines, allow for transient CycD1 overexpression and pRBs' downregulation in the inner ear in a reversible fashion. Analyses of postnatal mice's inner ears at various time points revealed the presence of supernumerary cells throughout the length of the cochlea and in the vestibular end-organs. Notably, most supernumerary cells were observed in the inner hair cells' (IHCs) region, expressed myosin VIIa (M7a), and showed no signs of apoptosis at any of the time points analyzed. Auditory and vestibular phenotypes were similar between the different genotypes and treatment groups. The fact that no significant differences were observed in auditory and vestibular function supports the notion that the supernumerary cells detected in the adult mice cochlea and macular end-organs may not impair auditory functions., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Mutations in myosin-VIIa (MYO7A) cause Usher syndrome type 1, characterized by combined deafness and blindness. MYO7A is proposed to function as a motor that tensions the hair cell mechanotransduction (MET) complex, but conclusive evidence is lacking. Here we report that multiple MYO7A isoforms are expressed in the mouse cochlea. In mice with a specific deletion of the canonical isoform (Myo7a-ΔC mouse), MYO7A is severely diminished in inner hair cells (IHCs), while expression in outer hair cells is affected tonotopically. IHCs of Myo7a-ΔC mice undergo normal development, but exhibit reduced resting open probability and slowed onset of MET currents, consistent with MYO7A's proposed role in tensioning the tip link. Mature IHCs of Myo7a-ΔC mice degenerate over time, giving rise to progressive hearing loss. Taken together, our study reveals an unexpected isoform diversity of MYO7A expression in the cochlea and highlights MYO7A's essential role in tensioning the hair cell MET complex.

Cochlear ribbon synapses play a pivotal role in the prompt and precise acoustic signal transmission from inner hair cells (IHCs) to the spiral ganglion neurons, while noise and aging can damage ribbon synapses, resulting in sensorineural hearing loss. Recently, we described reduced fibroblast growth factor 22 (FGF22) and augmented myocyte enhancer factor 2D (MEF2D) in an ototoxicity mouse model with impaired ribbon synapses. Here, we investigated the mechanisms that underlie the FGF22/MEF2D- regulated impairment of ribbon synapses. We generated adeno-associated virus (AAV) carrying FGF22, shFGF22, MEF2D, shMEF2D, calcineurin (CalN), shCalN or corresponding scramble controls for transduction of cultured mouse hair cells. We found that FGF22 was a suppressor for MEF2D, but not vice versa. Moreover, FGF22 likely induced increases in the calcium influx into IHCs to activate CalN, which subsequently inhibited MEF2D. Cochlear infusion of AAV-shFGF22 activated MEF2D, reduced ribbon synapse number and impaired hearing function, which were all abolished by co-infusion of AAV-shMEF2D. Hence, our data suggest that the ribbon synapses may be regulated by FGF22/calcium/CalN/MEF2D signaling, which implied novel therapeutic targets for hearing loss.

Tsukushi (TSK)-a small, secreted, leucine-rich-repeat proteoglycan-interacts with and regulates essential cellular signaling cascades. However, its functions in the mouse inner ear are unknown. In this study, measurement of auditory brainstem responses, fluorescence microscopy, and scanning electron microscopy revealed that TSK deficiency in mice resulted in the formation of abnormal stereocilia in the inner hair cells and hearing loss but not in the loss of these cells. TSK accumulated in nonprosensory regions during early embryonic stages and in both nonprosensory and prosensory regions in late embryonic stages. In adult mice, TSK was localized in the organ of Corti, spiral ganglion cells, and the stria vascularis. Moreover, loss of TSK caused dynamic changes in the expression of key genes that drive the differentiation of the inner hair cells in prosensory regions. Finally, our results revealed that TSK interacted with Sox2 and BMP4 to control stereocilia formation in the inner hair cells. Hence, TSK appears to be an essential component of the molecular pathways that regulate inner ear development.

The mammalian cochlea detects sound and transmits this information to the brain. A cross section through the cochlea reveals functionally distinct epithelial domains arrayed around the circumference of a fluid-filled duct. Six major domains include two on the roof of the duct (Reissner's membrane medially and the stria vascularis laterally) and four across the floor of the duct, including the medial and lateral halves of the sensory domain, the organ of Corti. These radial domains are distinguishable in the embryonic cochlea by differential expression of transcription factors, and we focus here on a subset of the factors that can influence cochlear fates. We then move upstream of these genes to identify which of five signaling pathways (Notch, Fgf, Wnt, Bmp, and Shh) controls their spatial patterns of expression. We link the signaling pathways to their downstream genes, separating them by their radial position, to create putative gene regulatory networks (GRNs) from two time points, before and during the time when six radial compartments arise. These GRNs offer a framework for understanding the acquisition of positional information across the radial axis of the cochlea, and to guide therapeutic approaches to repair or regenerate distinct cochlear components that may contribute to hearing loss., (© 2019 Wiley Periodicals, Inc.)

To examine whether exposure to sodium salicylate disrupts expression of vesicular glutamate transporter 3 (VGLUT3) and whether the alteration in expression corresponds to increased risk for tinnitus. Rats were treated with saline (control) or sodium salicylate (treated) Rats were examined for tinnitus by monitoring gap-pre-pulse inhibition of the acoustic startle reflex (GPIAS). Auditory brainstem response (ABR) was applied to evaluate hearing function after treatment. Rats were sacrificed after injection to obtain the cochlea, cochlear nucleus (CN), and inferior colliculus (IC) for examination of VGLUT3 expression. No significant differences in hearing thresholds between groups were identified (p>0.05). Tinnitus in sodium salicylate-treated rats was confirmed by GPIAS. VGLUT3 encoded by solute carrier family 17 members 8 (SLC17a8) expression was significantly increased in inner hair cells (IHCs) of the cochlea in treated animals, compared with controls (p<0.01). No significant differences in VGLUT3 expression between groups were found for the cochlear nucleus (CN) or IC (p>0.05). Exposure to sodium salicylate may disrupt SLC17a8 expression in IHCs, leading to alterations that correspond to tinnitus in rats. However, the CN and IC are unaffected by exposure to sodium salicylate, suggesting that enhancement of VGLUT3 expression in IHCs may contribute to the pathogenesis of tinnitus.

Lateral olivocochlear (LOC) efferent neurons modulate auditory nerve fiber (ANF) activity using a large repertoire of neurotransmitters, including dopamine (DA) and acetylcholine (ACh). Little is known about how individual neurotransmitter systems are differentially utilized in response to the ever-changing acoustic environment. Here we present quantitative evidence in rodents that the dopaminergic LOC input to ANFs is dynamically regulated according to the animal's recent acoustic experience. Sound exposure upregulates tyrosine hydroxylase, an enzyme responsible for dopamine synthesis, in cholinergic LOC intrinsic neurons, suggesting that individual LOC neurons might at times co-release ACh and DA. We further demonstrate that dopamine down-regulates ANF firing rates by reducing both the hair cell release rate and the size of synaptic events. Collectively, our results suggest that LOC intrinsic neurons can undergo on-demand neurotransmitter re-specification to re-calibrate ANF activity, adjust the gain at hair cell/ANF synapses, and possibly to protect these synapses from noise damage., Competing Interests: JW, EY, MM, HJ, AL, EG No competing interests declared, (© 2020, Wu et al.)

High-throughput neurotransmission at ribbon synapses of cochlear inner hair cells (IHCs) requires tight coupling of neurotransmitter release and balanced recycling of synaptic vesicles (SVs) as well as rapid restoration of release sites. Here, we examined the role of the adaptor protein AP180 (also known as SNAP91) for IHC synaptic transmission by comparing AP180-knockout (KO) and wild-type mice using high-pressure freezing and electron tomography, confocal microscopy, patch-clamp membrane capacitance measurements and systems physiology. AP180 was found predominantly at the synaptic pole of IHCs. AP180-deficient IHCs had severely reduced SV numbers, slowed endocytic membrane retrieval and accumulated endocytic intermediates near ribbon synapses, indicating that AP180 is required for clathrin-dependent endocytosis and SV reformation in IHCs. Moreover, AP180 deletion led to a high prevalence of SVs in a multi-tethered or docked state after stimulation, a reduced rate of SV replenishment and a hearing impairment. We conclude that, in addition to its role in clathrin recruitment, AP180 contributes to release site clearance in IHCs.This article has an associated First Person interview with the first author of the paper., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

Cav1.3 L-type Ca 2+ channels (LTCCs) in cochlear inner hair cells (IHCs) are essential for hearing as they convert sound-induced graded receptor potentials into tonic postsynaptic glutamate release. To enable fast and indefatigable presynaptic Ca 2+ signaling, IHC Cav1.3 channels exhibit a negative activation voltage range and uniquely slow inactivation kinetics. Interaction with CaM-like Ca 2+ -binding proteins inhibits Ca 2+ -dependent inactivation, while the mechanisms underlying slow voltage-dependent inactivation (VDI) are not completely understood. Here we studied if the complex formation of Cav1.3 LTCCs with the presynaptic active zone proteins RIM2α and RIM-binding protein 2 (RBP2) can stabilize slow VDI. We detected both RIM2α and RBP isoforms in adult mouse IHCs, where they co-localized with Cav1.3 and synaptic ribbons. Using whole-cell patch-clamp recordings (tsA-201 cells), we assessed their effect on the VDI of the C-terminal full-length Cav1.3 (Cav1.3 L ) and a short splice variant (Cav1.3 42A ) that lacks the C-terminal RBP2 interaction site. When co-expressed with the auxiliary β3 subunit, RIM2α alone (Cav1.3 42A ) or RIM2α/RBP2 (Cav1.3 L ) reduced Cav1.3 VDI to a similar extent as observed in IHCs. Membrane-anchored β2 variants (β2a, β2e) that inhibit inactivation on their own allowed no further modulation of inactivation kinetics by RIM2α/RBP2. Moreover, association with RIM2α and/or RBP2 consolidated the negative Cav1.3 voltage operating range by shifting the channel's activation threshold toward more hyperpolarized potentials. Taken together, the association with "slow" β subunits (β2a, β2e) or presynaptic scaffolding proteins such as RIM2α and RBP2 stabilizes physiological gating properties of IHC Cav1.3 LTCCs in a splice variant-dependent manner ensuring proper IHC function.

Hair cells in the auditory organ of the vertebrate inner ear are the sensory receptors that convert acoustic stimuli into electrical signals that are conveyed along the auditory nerve to the brainstem. Hair cells are highly susceptible to ototoxic drugs, infection, and acoustic trauma, which can cause cellular degeneration. In mammals, hair cells that are lost after damage are not replaced, leading to permanent hearing impairments. By contrast, supporting cells in birds and other non-mammalian vertebrates regenerate hair cells after damage, which restores hearing function. The cellular mechanisms that regulate hair cell regeneration are not well understood. We investigated the role of vascular endothelial growth factor (VEGF) during regeneration of auditory hair cells in chickens after ototoxic injury. Using RNA-Seq, immunolabeling, and in situ hybridization, we found that VEGFA, VEGFC, VEGFR1, VEGFR2, and VEGFR3 were expressed in the auditory epithelium, with VEGFA expressed in hair cells and VEGFR1 and VEGFR2 expressed in supporting cells. Using organotypic cultures of the chicken cochlear duct, we found that blocking VEGF receptor activity during hair cell injury reduced supporting cell proliferation as well as the numbers of regenerated hair cells. By contrast, addition of recombinant human VEGFA to organ cultures caused an increase in both supporting cell division and hair cell regeneration. VEGF's effects on supporting cells were preserved in isolated supporting cell cultures, indicating that VEGF can act directly upon supporting cells. These observations demonstrate a heretofore uncharacterized function for VEGF signaling as a critical positive regulator of hair cell regeneration in the avian inner ear., (Copyright © 2019 Elsevier B.V. All rights reserved.)

ELMOD3, an ARL2 GTPase-activating protein, is implicated in causing hearing impairment in humans. However, the specific role of ELMOD3 in auditory function is still far from being elucidated. In the present study, we used the CRISPR/Cas9 technology to establish an Elmod3 knockout mice line in the C57BL/6 background (hereinafter referred to as Elmod3-/- mice) and investigated the role of Elmod3 in the cochlea and auditory function. Elmod3-/- mice started to exhibit hearing loss from 2 months of age, and the deafness progressed with aging, while the vestibular function of Elmod3-/- mice was normal. We also observed that Elmod3-/- mice showed thinning and receding hair cells in the organ of Corti and much lower expression of F-actin cytoskeleton in the cochlea compared with wild-type mice. The deafness associated with the mutation may be caused by cochlear hair cells dysfunction, which manifests with shortening and fusion of inner hair cells stereocilia and progressive degeneration of outer hair cells stereocilia. Our finding associates Elmod3 deficiencies with stereocilia dysmorphologies and reveals that they might play roles in the actin cytoskeleton dynamics in cochlear hair cells, and thus relate to hearing impairment., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

The adult mammalian inner ear lacks the capacity to divide or regenerate. Damage to inner ear generally leads to permanent hearing loss in humans. Here, we present that reprogramming of the adult inner ear induces renewed proliferation and regeneration of inner ear cell types. Co-activation of cell cycle activator Myc and inner ear progenitor gene Notch1 induces robust proliferation of diverse adult cochlear sensory epithelial cell types. Transient MYC and NOTCH activities enable adult supporting cells to respond to transcription factor Atoh1 and efficiently transdifferentiate into hair cell-like cells. Furthermore, we uncover that mTOR pathway participates in MYC/NOTCH-mediated proliferation and regeneration. These regenerated hair cell-like cells take up the styryl dye FM1-43 and are likely to form connections with adult spiral ganglion neurons, supporting that Myc and Notch1 co-activation is sufficient to reprogram fully mature supporting cells to proliferate and regenerate hair cell-like cells in adult mammalian auditory organs.

Glutamate is the major excitatory neurotransmitter in the CNS binding to a variety of glutamate receptors. Metabotropic glutamate receptors (mGluR1 to mGluR8) can act excitatory or inhibitory, depending on associated signal cascades. Expression and localization of inhibitory acting mGluRs at inner hair cells (IHCs) in the cochlea are largely unknown. Here, we analyzed expression of mGluR2, mGluR3, mGluR4, mGluR6, mGluR7, and mGluR8 and investigated their localization with respect to the presynaptic ribbon of IHC synapses. We detected transcripts for mGluR2, mGluR3, and mGluR4 as well as for mGluR7a, mGluR7b, mGluR8a, and mGluR8b splice variants. Using receptor-specific antibodies in cochlear wholemounts, we found expression of mGluR2, mGluR4, and mGluR8b close to presynaptic ribbons. Super resolution and confocal microscopy in combination with 3-dimensional reconstructions indicated a postsynaptic localization of mGluR2 that overlaps with postsynaptic density protein 95 on dendrites of afferent type I spiral ganglion neurons. In contrast, mGluR4 and mGluR8b were expressed at the presynapse close to IHC ribbons. In summary, we localized in detail 3 mGluR types at IHC ribbon synapses, providing a fundament for new therapeutical strategies that could protect the cochlea against noxious stimuli and excitotoxicity.-Klotz, L., Wendler, O., Frischknecht, R., Shigemoto, R., Schulze, H., Enz, R. Localization of group II and III metabotropic glutamate receptors at pre- and postsynaptic sites of inner hair cell ribbon synapses.

Mammalian inner hair cells (IHCs) transduce sound into depolarization and transmitter release. Big conductance and voltage- and Ca 2+ -activated K + (BK) channels are responsible for fast membrane repolarization and small time constants of mature IHCs. For unknown reasons, they activate at around -75 mV with a voltage of half-maximum activation ( V half ) of -50 mV although being largely insensitive to Ca 2+ influx. Ca 2+ -independent activation of BK channels was observed by others in heterologous expression systems if γ subunits leucine-rich repeat-containing protein (LRRC)26 (γ1) and LRRC52 (γ2) were coexpressed with the pore-forming BKα subunit, which shifted V half by -140 and -100 mV, respectively. Using nested PCR, we consistently detected transcripts for LRRC52 but not for LRRC26 in IHCs of 3-wk-old mice. Confocal immunohistochemistry showed synchronous up-regulation of LRRC52 protein with BKα at the onset of hearing. Colocalization of LRRC52 protein and BKα at the IHC neck within ≤40 nm was specified using an in situ proximity ligation assay. Mice deficient for the voltage-gated Ca v 1.3 Ca 2+ channel encoded by Cacna1d do not express BKα protein. LRRC52 protein was neither expressed in IHCs of BKα nor in IHCs of Ca v 1.3 knockout mice. Together, LRRC52 is a γ2 subunit of BK channel complexes and is a strong candidate for causing the Ca 2+ -independent activation of BK currents at negative membrane potentials in mouse IHCs.-Lang, I., Jung, M., Niemeyer, B. A., Ruth, P., Engel, J. Expression of the LRRC52 γ subunit (γ2) may provide Ca 2+ -independent activation of BK currents in mouse inner hair cells.

Noise exposures causing only transient threshold shifts can destroy auditory-nerve synapses without damaging hair cells. Here, we asked whether virally mediated neurotrophin3 (NT3) overexpression can repair this damage. CBA/CaJ mice at 6 wks were injected unilaterally with adeno-associated virus (AAV) containing either NT3 or GFP genes, via the posterior semicircular canal, 3 wks prior to, or 5 hrs after, noise exposure. Controls included exposed animals receiving vehicle only, and unexposed animals receiving virus. Thresholds were measured 2 wks post-exposure, just before cochleas were harvested for histological analysis. In separate virus-injected animals, unexposed cochleas were extracted for qRT-PCR. The GFP reporter showed that inner hair cells (IHCs) were transfected throughout the cochlea, and outer hair cells mainly in the apex. qRT-PCR showed 4- to 10-fold overexpression of NT3 from 1-21 days post-injection, and 1.7-fold overexpression at 40 days. AAV-NT3 delivered prior to noise exposure produced a dose-dependent reduction of synaptopathy, with nearly complete rescue at some cochlear locations. In unexposed ears, NT3 overexpression did not affect thresholds, however GFP overexpression caused IHC loss. In exposed ears, NT3 overexpression increased permanent threshold shifts. Thus, although NT3 overexpression can minimize noise-induced synaptic damage, the forced overexpression may be harmful to hair cells themselves during cochlear overstimulation.

Development of multicellular organs requires the coordination of cell differentiation and patterning. Critical for sound detection, the mammalian organ of Corti contains functional units arranged tonotopically along the cochlear turns. Each unit consists of sensory hair cells intercalated by nonsensory supporting cells, both specified and radially patterned with exquisite precision during embryonic development. However, how cell identity and radial patterning are jointly controlled is poorly understood. Here we show that β-catenin is required for specification of hair cell and supporting cell subtypes and radial patterning of the cochlea in vivo. In 2 mouse models of conditional β-catenin deletion, early specification of Myosin7-expressing hair cells and Prox1-positive supporting cells was preserved. While β-catenin-deficient cochleae expressed FGF8 and FGFR3, both of which are essential for pillar cell specification, the radial patterning of organ of Corti was disrupted, revealed by aberrant expression of cadherins and the pillar cell markers P75 and Lgr6. Moreover, β-catenin ablation caused duplication of FGF8-positive inner hair cells and reduction of outer hair cells without affecting the overall hair cell density. In contrast, in another transgenic model with suppressed transcriptional activity of β-catenin but preserved cell adhesion function, both specification and radial patterning of the organ of Corti were intact. Our study reveals specific functions of β-catenin in governing cell identity and patterning mediated through cell adhesion in the developing cochlea., Competing Interests: The authors declare no competing interest.

The TMC1 channel was identified as a protein essential for hearing in mouse and human, and recognized as one of a family of eight such proteins in mammals. The TMC family is part of a superfamily of seven branches, which includes the TMEM16s. Vertebrate hair cells express both TMC1 and TMC2. They are located at the tips of stereocilia and are required for hair cell mechanotransduction. TMC1 assembles as a dimer and its similarity to the TMEM16s has enabled a predicted tertiary structure with an ion conduction pore in each subunit of the dimer. Cysteine mutagenesis of the pore supports the role of TMC1 and TMC2 as the core channel proteins of a larger mechanotransduction complex that includes PCDH15 and LHFPL5, and perhaps TMIE, CIB2 and others., (Copyright © 2019 Cold Spring Harbor Laboratory Press; all rights reserved.)

The perception of sound relies on sensory hair cells in the cochlea that convert the mechanical energy of sound into release of glutamate onto postsynaptic auditory nerve fibers. The hair cell receptor potential regulates the strength of synaptic transmission and is shaped by a variety of voltage-dependent conductances. Among these conductances, the Ca 2+ - and voltage-activated large conductance Ca 2+ -activated K + channel (BK) current is prominent, and in mammalian inner hair cells (IHCs) displays unusual properties. First, BK currents activate at unprecedentedly negative membrane potentials (-60 mV) even in the absence of intracellular Ca 2+ elevations. Second, BK channels are positioned in clusters away from the voltage-dependent Ca 2+ channels that mediate glutamate release from IHCs. Here, we test the contributions of two recently identified leucine-rich-repeat-containing (LRRC) regulatory γ subunits, LRRC26 and LRRC52, to BK channel function and localization in mouse IHCs. Whereas BK currents and channel localization were unaltered in IHCs from Lrrc26 knockout (KO) mice, BK current activation was shifted more than +200 mV in IHCs from Lrrc52 KO mice. Furthermore, the absence of LRRC52 disrupted BK channel localization in the IHCs. Given that heterologous coexpression of LRRC52 with BK α subunits shifts BK current gating about -90 mV, to account for the profound change in BK activation range caused by removal of LRRC52, we suggest that additional factors may help define the IHC BK gating range. LRRC52, through stabilization of a macromolecular complex, may help retain some other components essential both for activation of BK currents at negative membrane potentials and for appropriate BK channel positioning., Competing Interests: The authors declare no conflict of interest.

The development of therapeutic interventions for hearing loss requires fundamental knowledge about the signaling pathways controlling tissue development as well as the establishment of human cell-based assays to validate therapeutic strategies ex vivo Recent advances in the field of stem cell biology and organoid culture systems allow the expansion and differentiation of tissue-specific progenitors and pluripotent stem cells in vitro into functional hair cells and otic-like neurons. We discuss how inner ear organoids have been developed and how they offer for the first time the opportunity to validate drug-based therapies, gene-targeting approaches and cell replacement strategies., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

Tissue-specific inducible Cre recombinase mouse lines allow precise genetic manipulations in spatiotemporal manners and are pivotal for functional studies of genes during development and in adults. Growth factor independence 1 (GFI1) is an essential transcription factor expressed in the hair cells of mouse inner ear and Gfi1 locus serves as an excellent anchor site to drive the expression of inducible Cre recombinase in mouse inner hair cells. In this study, we have generated Gfi1-P2A-GFP-CreERT2 (Gfi1-GCE) knock-in mouse line by in-frame fusion of a self-cleaving GCE to the C-terminus of GFI1. We have shown that as predicted, the expression of GCE and GFI1 was detected specifically in the cytosol and nuclei of hair cells, respectively, of uninduced Gfi1-GCE mice, suggesting the successful cleavage and simultaneous expression of GFI1 and GCE. In addition, the in-frame fusion of the self-cleaving GCE does not interrupt the function of Gfi1 in the inner ear. Administration of tamoxifen leads to nuclear translocation of GCE and results in an efficient activation of tdTomato reporter gene expression specifically in most hair cells throughout development and in adults. Thus, this inducible Gfi1-GCE mouse line is a highly efficient Cre deleter and is suitable for gene manipulation in developing and adult inner ear hair cells., (© 2019 Wiley Periodicals, Inc.)

Precise and efficient endocytosis is critical for sustained neurotransmission during continuous neuronal activity. Endocytosis is a prerequisite for maintaining the auditory function. However, the differences between the patterns of endocytosis in cochlear inner hair cells (IHCs) and outer hair cells (OHCs) remain unclear. Both IHCs and OHCs were obtained from adult C57 mice. Patterns of endocytosis in cells were estimated by analyzing the uptake of FM1-43, a fluorescent. The observations were made using live confocal imaging, fluorescence intensities were calculated statistically. Results revealed the details about following phenomenon, i) sites of entry: the FM1-43 dye was found to enter IHC at the apical area initially, the additional sites of entry were then found at basolateral membrane of the cells, The entry of the dye into OHCs initially appeared to be occurring around whole apical membranes area, which then diffused towards the other membrane surface of the cells, ii) capacity of endocytosis: fluorescence intensity in IHCs showed significantly higher than that of OHCs (P<0.01). We have found different patterns of endocytosis between IHCs and OHCs, this indicated functional distinctions between them. Moreover, FM1-43 dye can be potentially used as an indicator of the functional loss or repair of cochlear hair cells.

The bundle of stereocilia on inner ear hair cells responds to subnanometer deflections produced by sound or head movement. Stereocilia are interconnected by a variety of links and also carry an electron-dense surface coat. The coat may contribute to stereocilia adhesion or protect from stereocilia fusion, but its molecular identity remains unknown. From a database of hair-cell-enriched translated proteins, we identify Polycystic Kidney and Hepatic Disease 1-Like 1 (PKHD1L1), a large, mostly extracellular protein of 4249 amino acids with a single transmembrane domain. Using serial immunogold scanning electron microscopy, we show that PKHD1L1 is expressed at the tips of stereocilia, especially in the high-frequency regions of the cochlea. PKHD1L1-deficient mice lack the surface coat at the upper but not lower regions of stereocilia, and they develop progressive hearing loss. We conclude that PKHD1L1 is a component of the surface coat and is required for normal hearing in mice.

Manganese (Mn) is an essential cofactor for many enzymes and thus plays an important role in normal growth and development. However, persistent exposure to high Mn concentrations can result in deleterious effects on not only the central nervous system but also peripheral nerves, including nerves associated with the auditory system. Our initial research on cochlear organotypic cultures in vitro showed that N-acetylcysteine (NAC) clearly decreases Mn-induced losses in hair cells (HCs), auditory nerve fibers (ANFs) and spiral ganglion neurons (SGNs) in a concentration-dependent manner. Salidroside (SAL) (p-hydroxyphenethyl-b-d-glucoside; C14H20O7), which is extracted from Rhodiola rosea L, has many pharmacological actions and antioxidative, antiaging, neuroprotective and anticancer effects. We hypothesized that SAL could also protect HCs, ANFs and SGNs from Mn injury. Cochlear organotypic cultures were treated with 1 mM Mn alone or combined with SAL (1-1000 μM). The neurofilament staining results showed that HCs, ANFs and SGNs were seriously damaged at high concentrations (100-1000 μM) but less damaged at low concentrations (1-10 μM). SAL may protect against 1 mM Mn-induced HC loss and axonal degeneration, suggesting that SAL could be a promising drug for clinical applications., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Transmembrane channel-like protein isoform 1 (TMC1) is essential for the generation of mechano-electrical transducer currents in hair cells of the inner ear. TMC1 disruption causes hair cell degeneration and deafness in mice and humans. Although thought to be expressed at the cell surface in vivo, TMC1 remains in the endoplasmic reticulum when heterologously expressed in standard cell lines, precluding determination of its roles in mechanosensing and pore formation. Here, we report that the KCNQ1 Kv channel forms complexes with TMC1 and rescues its surface expression when coexpressed in Chinese Hamster Ovary cells. TMC1 rescue is specific for KCNQ1 within the KCNQ family, is prevented by a KCNQ1 trafficking-deficient mutation, and is influenced by KCNE β subunits and inhibition of KCNQ1 endocytosis. TMC1 lowers KCNQ1 and KCNQ1-KCNE1 K + currents, and despite the surface expression, it does not detectably respond to mechanical stimulation or high salt. We conclude that TMC1 is not intrinsically mechano- or osmosensitive but has the capacity for cell surface expression, and requires partner protein(s) for surface expression and mechanosensitivity. We suggest that KCNQ1, expression of which is not thought to overlap with TMC1 in hair cells, is a proxy partner bearing structural elements or a sequence motif reminiscent of a true in vivo TMC1 hair cell partner. Discovery of the first reported strategy to rescue TMC1 surface expression should aid future studies of the TMC1 function and native partners., (© 2019 Wiley Periodicals, Inc.)

DFNA2 is a progressive deafness caused by mutations in the voltage-activated potassium channel KCNQ4. Hearing loss develops with age from a mild increase in the hearing threshold to profound deafness. Studies using transgenic mice for Kcnq4 expressed in a mixed background demonstrated the implication of outer hair cells at the initial phase. However, it could not explain the last phase mechanisms of the disease. Genetic backgrounds are known to influence disease expressivity. To unmask the cause of profound deafness phenotype, we backcrossed the Kcnq4 knock-out allele to the inbred strain C3H/HeJ and investigated inner and outer hair cell and spiral ganglion neuron degeneration across the lifespan. In addition to the already reported outer hair cell death, the C3H/HeJ strain also exhibited inner hair cell and spiral ganglion neuron death. We tracked the spatiotemporal survival of cochlear cells by plotting cytocochleograms and neuronal counts at different ages. Cell loss progressed from basal to apical turns with age. Interestingly, the time-course of cell degeneration was different for each cell-type. While for outer hair cells it was already present by week 3, inner hair cell and neuronal loss started 30 weeks later. We also established that outer hair cell loss kinetics slowed down from basal to apical regions correlating with KCNQ4 expression pattern determined in wild-type mice. Our findings indicate that KCNQ4 plays differential roles in each cochlear cell-type impacting in their survival ability. Inner hair cell and spiral ganglion neuron death generates severe hearing loss that could be associated with the last phase of DFNA2., (Copyright © 2019 IBRO. Published by Elsevier Ltd. All rights reserved.)

Key Points: The physiological maturation of auditory hair cells and their innervation requires precise temporal and spatial control of cell differentiation. The transcription factor gata3 is essential for the earliest stages of auditory system development and for survival and synaptogenesis in auditory sensory afferent neurons. We show that during postnatal development in the mouse inner ear gata3 is required for the biophysical maturation, growth and innervation of inner hair cells; in contrast, it is required only for the survival of outer hair cells. Loss of gata3 in inner hair cells causes progressive hearing loss and accounts for at least some of the deafness associated with the human hypoparathyroidism, deafness and renal anomaly (HDR) syndrome. The results show that gata3 is critical for later stages of mammalian auditory system development where it plays distinct, complementary roles in the coordinated maturation of sensory hair cells and their innervation., Abstract: The zinc finger transcription factor gata3 regulates inner ear development from the formation of the embryonic otic placode. Throughout development, gata3 is expressed dynamically in all the major cochlear cell types. Its role in afferent formation is well established but its possible involvement in hair cell maturation remains unknown. Here, we find that in heterozygous gata3 null mice (gata3 +/- ) outer hair cells (OHCs) differentiate normally but their numbers are significantly lower. In contrast, inner hair cells (IHCs) survive normally but they fail to acquire adult basolateral membrane currents, retain pre-hearing current and efferent innervation profiles and have fewer ribbon synapses. Targeted deletion of gata3 driven by otoferlin-cre recombinase (gata3 fl/fl otof-cre +/- ) in IHCs does not affect OHCs or the number of IHC afferent synapses but it leads to a failure in IHC maturation comparable to that observed in gata3 +/- mice. Auditory brainstem responses in gata3 fl/fl otof-cre +/- mice reveal progressive hearing loss that becomes profound by 6-7 months, whilst distortion product otoacoustic emissions are no different to control animals up to this age. Our results, alongside existing data, indicate that gata3 has specific, complementary functions in different cell types during inner ear development and that its continued expression in the sensory epithelium orchestrates critical aspects of physiological development and neural connectivity. Furthermore, our work indicates that hearing loss in human hypoparathyroidism, deafness and renal anomaly (HDR) syndrome arises from functional deficits in IHCs as well as loss of function from OHCs and both afferent and efferent neurons., (© 2019 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

The channel that governs mechanotransduction (MT) by hair cells in the inner ear has been investigated intensively for 4 decades, but its precise molecular composition remains enigmatic. Transmembrane channel-like protein 1 (TMC1) was recently identified as a component of the MT channel, and lipoma HMGIC fusion partner-like 5 (LHFPL5) is considered to be part of the MT complex and may functionally couple the tip link to the MT channel. As components of the MT complex, TMC1 and LHFPL5 are expected to localize at the lower end of the tip link in hair cells, a notion generally supported by previous studies on neonatal mice. However, the localization of these 2 proteins, particularly in the hair cells of adult mice, remains incompletely elucidated. Because determination of TMC1 and LHFPL5 localization at distinct developmental stages is essential for understanding their function and regulation, we used several approaches to examine the localization of these proteins in neonatal and adult hair cells in the mouse. We report several notable findings: 1 ) TMC1 and LHFPL5 predominantly localize at the tip of the shorter rows of stereocilia in neonatal hair cells, which largely verifies the previously published findings in neonatal hair cells; 2 ) LHFPL5 persists in the hair bundle of hair cells after postnatal day (P)7, which clarifies the previously reported unexpected absence of LHFPL5 after P7 and supports the view that LHFPL5 is a permanent component in the MT complex; and 3 ) TMC1 and LHFPL5 remain at the tip of the shorter rows of stereocilia in adult outer hair cells, but in adult inner hair cells, TMC1 is uniformly distributed in both the tallest row and the shorter rows of stereocilia, whereas LHFPL5 is uniformly distributed in the shorter rows of stereocilia. These findings raise intriguing questions regarding the turnover rate, regulation, additional functions, and functional interaction of TMC1 and LHFPL5. Our study confirms the previous findings in neonatal hair cells and reveals several previously unidentified aspects of TMC1 and LHFPL5 localization in more mature hair cells.-Li, X., Yu, X., Chen, X., Liu, Z., Wang, G., Li, C., Wong, E. Y. M., Sham, M. H., Tang, J., He, J., Xiong, W., Liu, Z., Huang, P. Localization of TMC1 and LHFPL5 in auditory hair cells in neonatal and adult mice.

Barhl1 , a mouse homologous gene of Drosophila BarH class homeobox genes, is highly expressed within the inner ear and crucial for the long-term maintenance of auditory hair cells that mediate hearing and balance, yet little is known about the molecular events underlying Barhl1 regulation and function in hair cells. In this study, through data mining and in vitro report assay, we firstly identified Barhl1 as a direct target gene of Atoh1 and one E-box (E3) in Barhl1 3' enhancer is crucial for Atoh1-mediated Barhl1 activation. Then we generated a mouse embryonic stem cell (mESC) line carrying disruptions on this E3 site E-box (CAGCTG) using CRISPR/Cas9 technology and this E3 mutated mESC line is further subjected to an efficient stepwise hair cell differentiation strategy in vitro . Disruptions on this E3 site caused dramatic loss of Barhl1 expression and significantly reduced the number of induced hair cell-like cells, while no affections on the differentiation toward early primitive ectoderm-like cells and otic progenitors. Finally, through RNA-seq profiling and gene ontology (GO) enrichment analysis, we found that this E3 box was indispensable for Barhl1 expression to maintain hair cell development and normal functions. We also compared the transcriptional profiles of induced cells from CDS mutated and E3 mutated mESCs, respectively, and got very consistent results except the Barhl1 transcript itself. These observations indicated that Atoh1-mediated Barhl1 expression could have important roles during auditory hair cell development. In brief, our findings delineate the detail molecular mechanism of Barhl1 expression regulation in auditory hair cell differentiation.