Cochlear Ribbon Synapses in Aged Gerbils.

In mammalian hearing, type-I afferent auditory nerve fibers comprise the basis of the afferent auditory pathway. They are connected to inner hair cells of the cochlea via specialized ribbon synapses. Auditory nerve fibers of different physiological types differ subtly in their synaptic location and morphology. Low-spontaneous-rate auditory nerve fibers typically connect on the modiolar side of the inner hair cell, while high-spontaneous-rate fibers are typically found on the pillar side. In aging and noise-damaged ears, this fine-tuned balance between auditory nerve fiber populations can be disrupted and the functional consequences are currently unclear. Here, using immunofluorescent labeling of presynaptic ribbons and postsynaptic glutamate receptor patches, we investigated changes in synaptic morphology at three different tonotopic locations along the cochlea of aging gerbils compared to those of young adults. Quiet-aged gerbils showed about 20% loss of afferent ribbon synapses. While the loss was random at apical, low-frequency cochlear locations, at the basal, high-frequency location it almost exclusively affected the modiolar-located synapses. The subtle differences in volumes of pre- and postsynaptic elements located on the inner hair cell's modiolar versus pillar side were unaffected by age. This is consistent with known physiology and suggests a predominant, age-related loss in the low-spontaneous-rate auditory nerve population in the cochlear base, but not the apex.

Letting the calcium flow.

Two calcium-binding proteins, CaBP1 and CaBP2, cooperate to keep calcium channels in the hair cells of the inner ear open.

Disruption of Cdh23 exon 68 splicing leads to progressive hearing loss in mice by affecting tip-link stability.

Inner ear hair cells are characterized by the F-actin-based stereocilia that are arranged into a staircase-like pattern on the apical surface of each hair cell. The tips of shorter-row stereocilia are connected with the shafts of their neighboring taller-row stereocilia through extracellular links named tip links, which gate mechano-electrical transduction (MET) channels in hair cells. Cadherin 23 (CDH23) forms the upper part of tip links, and its cytoplasmic tail is inserted into the so-called upper tip-link density (UTLD) that contains other proteins such as harmonin. The Cdh23 gene is composed of 69 exons, and we show here that exon 68 is subjected to hair cell-specific alternative splicing. Tip-link formation is not affected in genetically modified mutant mice lacking Cdh23 exon 68. Instead, the stability of tip links is compromised in the mutants, which also suffer from progressive and noise-induced hearing loss. Moreover, we show that the cytoplasmic tail of CDH23(+68) but not CDH23(-68) cooperates with harmonin in phase separation-mediated condensate formation. In conclusion, our work provides evidence that inclusion of Cdh23 exon 68 is critical for the stability of tip links through regulating condensate formation of UTLD components.

The Piezo channel is a mechano-sensitive complex component in the mammalian inner ear hair cell.

The inner ear is the hub where hair cells (HCs) transduce sound, gravity, and head acceleration stimuli to the brain. Hearing and balance rely on mechanosensation, the fastest sensory signals transmitted to the brain. The mechanoelectrical transducer (MET) channel is the entryway for the sound-balance-brain interface, but the channel-complex composition is not entirely known. Here, we report that the mouse utilizes Piezo1 (Pz1) and Piezo2 (Pz2) isoforms as MET-complex components. The Pz channels, expressed in HC stereocilia, and cell lines are co-localized and co-assembled with MET complex partners. Mice expressing non-functional Pz1 and Pz2 at the ROSA26 locus have impaired auditory and vestibular traits that can only be explained if the Pzs are integral to the MET complex. We suggest that Pz subunits constitute part of the MET complex and that interactions with other MET complex components yield functional MET units to generate HC MET currents.

Zinc deficiency triggers hearing loss by reducing ribbon synapses of inner hair cells in CBA/N mice.

Zinc plays a vital role in our metabolism, encompassing antioxidant regulation, immune response, and auditory function. Several studies have reported that zinc levels correlate with hearing loss. We have previously demonstrated that the auditory brainstem response (ABR) threshold increased in mice fed a zinc-deficient diet. However, the effects of zinc deficiency on hearing were not fully elucidated. The present study investigated whether zinc deficiency affects hearing in association with neuronal components or cochlear structures. CBA/N mice were fed a normal or zinc-deficient diet for 8 weeks and assessed for ABR and distortion product otoacoustic emissions (DPOAE). The cochlear sections were stained with hematoxylin and eosin solution. Also, we observed the expression of synaptic ribbons, neurofilaments, and alpha-synuclein (α-Syn). The 8-week zinc-deficient diet mice had an elevated ABR threshold but no changed DPOAE threshold or cochlear structures. A reduced number of synaptic ribbons of inner hair cells (IHCs) and impaired efferent nerve fibers were observed in the zinc-deficient diet mice. The number of outer hair cells (OHCs) and expression of α-Syn remained unchanged. Our results suggest that zinc-mediated hearing loss is associated with the loss of neuronal components of IHCs.

Proteomic Analysis Reveals the Composition of Glutamatergic Organelles of Auditory Inner Hair Cells.

In the ear, inner hair cells (IHCs) employ sophisticated glutamatergic ribbon synapses with afferent neurons to transmit auditory information to the brain. The presynaptic machinery responsible for neurotransmitter release in IHC synapses includes proteins such as the multi-C2-domain protein otoferlin and the vesicular glutamate transporter 3 (VGluT3). Yet, much of this likely unique molecular machinery remains to be deciphered. The scarcity of material has so far hampered biochemical studies which require large amounts of purified samples. We developed a subcellular fractionation workflow combined with immunoisolation of VGluT3-containing membrane vesicles, allowing for the enrichment of glutamatergic organelles that are likely dominated by synaptic vesicles (SVs) of IHCs. We have characterized their protein composition in mice before and after hearing onset using mass spectrometry and confocal imaging and provide a fully annotated proteome with hitherto unidentified proteins. Despite the prevalence of IHC marker proteins across IHC maturation, the profiles of trafficking proteins differed markedly before and after hearing onset. Among the proteins enriched after hearing onset were VAMP-7, syntaxin-7, syntaxin-8, syntaxin-12/13, SCAMP1, V-ATPase, SV2, and PKCα. Our study provides an inventory of the machinery associated with synaptic vesicle-mediated trafficking and presynaptic activity at IHC ribbon synapses and serves as a foundation for future functional studies.

Ca2+ regulation of glutamate release from inner hair cells of hearing mice.

In our hearing organ, sound is encoded at ribbon synapses formed by inner hair cells (IHCs) and spiral ganglion neurons (SGNs). How the underlying synaptic vesicle (SV) release is controlled by Ca2+ in IHCs of hearing animals remained to be investigated. Here, we performed patch-clamp SGN recordings of the initial rate of release evoked by brief IHC Ca2+-influx in an ex vivo cochlear preparation from hearing mice. We aimed to closely mimic physiological conditions by perforated-patch recordings from IHCs kept at the physiological resting potential and at body temperature. We found release to relate supralinearly to Ca2+-influx (power, m: 4.3) when manipulating the [Ca2+] available for SV release by Zn2+-flicker-blocking of the single Ca2+-channel current. In contrast, a near linear Ca2+ dependence (m: 1.2 to 1.5) was observed when varying the number of open Ca2+-channels during deactivating Ca2+-currents and by dihydropyridine channel-inhibition. Concurrent changes of number and current of open Ca2+-channels over the range of physiological depolarizations revealed m: 1.8. These findings indicate that SV release requires ~4 Ca2+-ions to bind to their Ca2+-sensor of fusion. We interpret the near linear Ca2+-dependence of release during manipulations that change the number of open Ca2+-channels to reflect control of SV release by the high [Ca2+] in the Ca2+-nanodomain of one or few nearby Ca2+-channels. We propose that a combination of Ca2+ nanodomain control and supralinear intrinsic Ca2+-dependence of fusion optimally links SV release to the timing and amplitude of the IHC receptor potential and separates it from other IHC Ca2+-signals unrelated to afferent synaptic transmission.

The actin cytoskeleton in hair bundle development and hearing loss.

Inner ear hair cells assemble mechanosensitive hair bundles on their apical surface that transduce sounds and accelerations. Each hair bundle is comprised of ∼ 100 individual stereocilia that are arranged into rows of increasing height and width; their specific and precise architecture being necessary for mechanoelectrical transduction (MET). The actin cytoskeleton is fundamental to establishing this architecture, not only by forming the structural scaffold shaping each stereocilium, but also by composing rootlets and the cuticular plate that together provide a stable foundation supporting each stereocilium. In concert with the actin cytoskeleton, a large assortment of actin-binding proteins (ABPs) function to cross-link actin filaments into specific topologies, as well as control actin filament growth, severing, and capping. These processes are individually critical for sensory transduction and are all disrupted in hereditary forms of human hearing loss. In this review, we provide an overview of actin-based structures in the hair bundle and the molecules contributing to their assembly and functional properties. We also highlight recent advances in mechanisms driving stereocilia elongation and how these processes are tuned by MET.

The cytosolic N-terminal region of heterologously-expressed transmembrane channel-like protein 1 (TMC1) can be cleaved in HEK293 cells.

Transmembrane channel-like protein 1 (TMC1) is a transmembrane protein forming mechano-electrical transduction (MET) channel, which transduces mechanical stimuli into electrical signals at the top of stereocilia of hair cells in the inner ear. As an unexpected phenomenon, we found that the cytosolic N-terminal (Nt) region of heterologously-expressed mouse TMC1 (mTMC1) was localized in nuclei of a small population of the transfected HEK293 cells. This raised the possibility that the Nt region of heterologously-expressed mTMC1 was cleaved and transported into the nucleus. To confirm the cleavage, we performed western blot analyses. The results revealed that at least a fragment of the Nt region was produced from heterologously-expressed mTMC1. Site-directed mutagenesis experiments identified amino acid residues which were required to produce the fragment. The accumulation of the heterologously-expressed Nt fragment into the nuclei depended on nuclear localization signals within the Nt region. Furthermore, a structural comparison showed a similarity between the Nt region of mTMC1 and basic region leucine zipper (bZIP) transcription factors. However, transcriptome analyses using a next-generation sequencer showed that the heterologously-expression of the Nt fragment of mTMC1 hardly altered expression levels of genes. Although it is still unknown what is the precise mechanism and the physiological significance of this cleavage, these results showed that the cytosolic Nt region of heterologously-expressed mTMC1 could be cleaved in HEK293 cells. Therefore, it should be taken into account that the cleavage of Nt region might influence the functional analysis of TMC1 by the heterologous-expression system using HEK293 cells.

Repair of noise-induced damage to stereocilia F-actin cores is facilitated by XIRP2 and its novel mechanosensor domain.

Prolonged exposure to loud noise has been shown to affect inner ear sensory hair cells in a variety of deleterious manners, including damaging the stereocilia core. The damaged sites can be visualized as 'gaps' in phalloidin staining of F-actin, and the enrichment of monomeric actin at these sites, along with an actin nucleator and crosslinker, suggests that localized remodeling occurs to repair the broken filaments. Herein, we show that gaps in mouse auditory hair cells are largely repaired within 1 week of traumatic noise exposure through the incorporation of newly synthesized actin. We provide evidence that Xin actin binding repeat containing 2 (XIRP2) is required for the repair process and facilitates the enrichment of monomeric γ-actin at gaps. Recruitment of XIRP2 to stereocilia gaps and stress fiber strain sites in fibroblasts is force-dependent, mediated by a novel mechanosensor domain located in the C-terminus of XIRP2. Our study describes a novel process by which hair cells can recover from sublethal hair bundle damage and which may contribute to recovery from temporary hearing threshold shifts and the prevention of age-related hearing loss.

Epistatic genetic interactions between Insm1 and Ikzf2 during cochlear outer hair cell development.

The cochlea harbors two types of sound receptors, outer hair cells (OHCs) and inner hair cells (IHCs). OHCs transdifferentiate into IHCs in Insm1 mutants, and OHCs in Ikzf2-deficient mice are dysfunctional and maintain partial IHC gene expression. Insm1 potentially acts as a positive but indirect regulator of Ikzf2, considering that Insm1 is expressed earlier than Ikzf2 and primarily functions as a transcriptional repressor. However, direct evidence of this possibility is lacking. Here, we report the following results: first, Insm1 overexpression in IHCs leads to ectopic Ikzf2 expression. Second, Ikzf2 expression is repressed in Insm1-deficient OHCs, and forced expression of Ikzf2 mitigates the OHC abnormality in Insm1 mutants. Last, dual ablation of Insm1 and Ikzf2 generates a similar OHC phenotype as does Insm1 ablation alone. Collectively, our findings reveal the transcriptional cascade from Insm1 to Ikzf2, which should facilitate future investigation of the molecular mechanisms underlying OHC development and regeneration.

Cochlear transcript diversity and its role in auditory functions implied by an otoferlin short isoform.

Isoforms of a gene may contribute to diverse biological functions. In the cochlea, the repertoire of alternative isoforms remains unexplored. We integrated single-cell short-read and long-read RNA sequencing techniques and identified 236,012 transcripts, 126,612 of which were unannotated in the GENCODE database. Then we analyzed and verified the unannotated transcripts using RNA-seq, RT-PCR, Sanger sequencing, and MS-based proteomics approaches. To illustrate the importance of identifying spliced isoforms, we investigated otoferlin, a key protein involved in synaptic transmission in inner hair cells (IHCs). Upon deletion of the canonical otoferlin isoform, the identified short isoform is able to support normal hearing thresholds but with reduced sustained exocytosis of IHCs, and further revealed otoferlin functions in endocytic membrane retrieval that was not well-addressed previously. Furthermore, we found that otoferlin isoforms are associated with IHC functions and auditory phenotypes. This work expands our mechanistic understanding of auditory functions at the level of isoform resolution.

GIPC3 couples to MYO6 and PDZ domain proteins, and shapes the hair cell apical region.

GIPC3 has been implicated in auditory function. Here, we establish that GIPC3 is initially localized to the cytoplasm of inner and outer hair cells of the cochlea and then is increasingly concentrated in cuticular plates and at cell junctions during postnatal development. Early postnatal Gipc3KO/KO mice had mostly normal mechanotransduction currents, but had no auditory brainstem response at 1 month of age. Cuticular plates of Gipc3KO/KO hair cells did not flatten during development as did those of controls; moreover, hair bundles were squeezed along the cochlear axis in mutant hair cells. Junctions between inner hair cells and adjacent inner phalangeal cells were also severely disrupted in Gipc3KO/KO cochleas. GIPC3 bound directly to MYO6, and the loss of MYO6 led to altered distribution of GIPC3. Immunoaffinity purification of GIPC3 from chicken inner ear extracts identified co-precipitating proteins associated with adherens junctions, intermediate filament networks and the cuticular plate. Several of immunoprecipitated proteins contained GIPC family consensus PDZ-binding motifs (PBMs), including MYO18A, which bound directly to the PDZ domain of GIPC3. We propose that GIPC3 and MYO6 couple to PBMs of cytoskeletal and cell junction proteins to shape the cuticular plate.

Control of stereocilia length during development of hair bundles.

Assembly of the hair bundle, the sensory organelle of the inner ear, depends on differential growth of actin-based stereocilia. Separate rows of stereocilia, labeled 1 through 3 from tallest to shortest, lengthen or shorten during discrete time intervals during development. We used lattice structured illumination microscopy and surface rendering to measure dimensions of stereocilia from mouse apical inner hair cells during early postnatal development; these measurements revealed a sharp transition at postnatal day 8 between stage III (row 1 and 2 widening; row 2 shortening) and stage IV (final row 1 lengthening and widening). Tip proteins that determine row 1 lengthening did not accumulate simultaneously during stages III and IV; while the actin-bundling protein EPS8 peaked at the end of stage III, GNAI3 peaked several days later-in early stage IV-and GPSM2 peaked near the end of stage IV. To establish the contributions of key macromolecular assemblies to bundle structure, we examined mouse mutants that eliminated tip links (Cdh23v2J or Pcdh15av3J), transduction channels (TmieKO), or the row 1 tip complex (Myo15ash2). Cdh23v2J/v2J and Pcdh15av3J/av3J bundles had adjacent stereocilia in the same row that were not matched in length, revealing that a major role of these cadherins is to synchronize lengths of side-by-side stereocilia. Use of the tip-link mutants also allowed us to distinguish the role of transduction from effects of transduction proteins themselves. While levels of GNAI3 and GPSM2, which stimulate stereocilia elongation, were greatly attenuated at the tips of TmieKO/KO row 1 stereocilia, they accumulated normally in Cdh23v2J/v2J and Pcdh15av3J/av3J stereocilia. These results reinforced the suggestion that the transduction proteins themselves facilitate localization of proteins in the row 1 complex. By contrast, EPS8 concentrates at tips of all TmieKO/KO, Cdh23v2J/v2J, and Pcdh15av3J/av3J stereocilia, correlating with the less polarized distribution of stereocilia lengths in these bundles. These latter results indicated that in wild-type hair cells, the transduction complex prevents accumulation of EPS8 at the tips of shorter stereocilia, causing them to shrink (rows 2 and 3) or disappear (row 4 and microvilli). Reduced rhodamine-actin labeling at row 2 stereocilia tips of tip-link and transduction mutants suggests that transduction's role is to destabilize actin filaments there. These results suggest that regulation of stereocilia length occurs through EPS8 and that CDH23 and PCDH15 regulate stereocilia lengthening beyond their role in gating mechanotransduction channels.

Three-Dimensional Structure of Inner Ear Hair Cell Ribbon Synapses in a Zebrafish Model of Usher Syndrome Type 1B.

Our understanding of inner ear hair cell ultrastructure has heretofore relied upon two-dimensional imaging; however, serial block-face scanning electron microscopy (SBFSEM) changes this paradigm allowing for three-dimensional evaluation. We compared inner ear hair cells of the apical cristae in myo7aa-/- null zebrafish, a model of human Usher Syndrome type 1B, to hair cells in wild-type zebrafish by SBFSEM to investigate possible ribbon synapse ultrastructural differences. Previously, it has been shown that compared to wild type, myo7aa-/- zebrafish neuromast hair cells have fewer ribbon synapses yet similar ribbon areas. We expect the recapitulation of these results within the inner ear apical crista hair cells furthering the knowledge of three-dimensional ribbon synapse structure while resolving the feasibility of therapeutically targeting myo7aa-/- mutant ribbons. In this report, we evaluated ribbon synapse number, volume, surface area, and sphericity. Localization of ribbons and their distance from the nearest innervation were also evaluated. We determined that myo7aa-/- mutant ribbon synapses are smaller in volume and surface area; however, all other measurements were not significantly different from wild-type zebrafish. Because the ribbon synapses are nearly indistinguishable between the myo7aa-/- mutant and wild type, it suggests that the ribbons are structurally receptive, supporting that therapeutic intervention may be feasible.

The hair cell analysis toolbox is a precise and fully automated pipeline for whole cochlea hair cell quantification.

Our sense of hearing is mediated by sensory hair cells, precisely arranged and highly specialized cells subdivided into outer hair cells (OHCs) and inner hair cells (IHCs). Light microscopy tools allow for imaging of auditory hair cells along the full length of the cochlea, often yielding more data than feasible to manually analyze. Currently, there are no widely applicable tools for fast, unsupervised, unbiased, and comprehensive image analysis of auditory hair cells that work well either with imaging datasets containing an entire cochlea or smaller sampled regions. Here, we present a highly accurate machine learning-based hair cell analysis toolbox (HCAT) for the comprehensive analysis of whole cochleae (or smaller regions of interest) across light microscopy imaging modalities and species. The HCAT is a software that automates common image analysis tasks such as counting hair cells, classifying them by subtype (IHCs versus OHCs), determining their best frequency based on their location along the cochlea, and generating cochleograms. These automated tools remove a considerable barrier in cochlear image analysis, allowing for faster, unbiased, and more comprehensive data analysis practices. Furthermore, HCAT can serve as a template for deep learning-based detection tasks in other types of biological tissue: With some training data, HCAT's core codebase can be trained to develop a custom deep learning detection model for any object on an image.

Current development of patient-specific induced pluripotent stem cells harbouring mitochondrial gene mutations and their applications in the treatment of sensorineural hearing loss.

Of all the human body's sensory systems, the auditory system is perhaps its most intricate. Hearing loss can result from even modest damage or cell death in the inner ear, and is the most common form of sensory loss. Human hearing is made possible by the sensory epithelium, the lateral wall, and auditory nerves. The most prominent functional cells in the sensory epithelium are outer hair cells (OHCs), inner hair cells (IHCs), and supporting cells. Different sound frequencies are processed by OHCs and IHCs in different cochlear regions, with those in the apex responsible for low frequencies and those in the basal region responsible for high frequencies. Hair cells can be damaged or destroyed by loud noise, aging process, genetic mutations, ototoxicity, infection, and illness. As such, they are a primary target for treating sensorineural hearing loss. Other areas known to affect hearing include spiral ganglion neurons (SGNs) in the auditory nerve. Age-related degradation of HCs and SGNs can also cause hearing loss. The aim of this review is to introduce the roles of mitochondria in human auditory system and the inner ear's main cell types and cellular functions, before going on to detail the likely health benefits of iPSC technology. We posit that patient-specific iPSCs with mitochondrial gene mutations will be an important aspect of regenerative medicine and will lead to significant progress in the treatment of SNHL.

Culture of organoids with vestibular cell-derived factors promotes differentiation of embryonic stem cells into inner ear vestibular hair cells.

Vestibular hair cells (V-HCs) residing in the inner ear have important roles related to balance. Although differentiation of pluripotent stem cells into HCs has been shown, an effective method has yet to be established. We previously reported that use of vestibular cell-derived conditioned medium (V-CM) was helpful to induce embryonic stem (ES) cells to differentiate into V-HC-like cells in two-dimensional (2D) cultures of ES-derived embryoid bodies (EBs). In the present report, V-CM was used with three-dimensional (3D) cultures of EBs, which resulted in augmented expression of V-HC-related markers (Math1, Myosin6, Brn3c, Dnah5), but not of the cochlear HC-related marker Lmod3. Gene expression analyses of both 2D and 3D EBs cultured for two weeks revealed a greater level of augmented induction of HC-related markers in the 3D-cultured EBs. These results indicate that a 3D culture in combination with use of V-CM is an effective method for producing V-HCs.

TBX2 specifies and maintains inner hair and supporting cell fate in the Organ of Corti.

The auditory function of the mammalian cochlea relies on two types of mechanosensory hair cells and various non-sensory supporting cells. Recent studies identified the transcription factors INSM1 and IKZF2 as regulators of outer hair cell (OHC) fate. However, the transcriptional regulation of the differentiation of inner hair cells (IHCs) and their associated inner supporting cells (ISCs) has remained enigmatic. Here, we show that the expression of the transcription factor TBX2 is restricted to IHCs and ISCs from the onset of differentiation until adulthood and examine its function using conditional deletion and misexpression approaches in the mouse. We demonstrate that TBX2 acts in prosensory progenitors as a patterning factor by specifying the inner compartment of the sensory epithelium that subsequently gives rise to IHCs and ISCs. Hair cell-specific inactivation or misexpression causes transdifferentiation of hair cells indicating a cell-autonomous function of TBX2 in inducing and maintaining IHC fate.

Loss of Pex1 in Inner Ear Hair Cells Contributes to Cochlear Synaptopathy and Hearing Loss.

Peroxisome Biogenesis Disorders (PBD) and Zellweger syndrome spectrum disorders (ZSD) are rare genetic multisystem disorders that include hearing impairment and are associated with defects in peroxisome assembly, function, or both. Mutations in 13 peroxin (PEX) genes have been found to cause PBD-ZSD with ~70% of patients harboring mutations in PEX1. Limited research has focused on the impact of peroxisomal disorders on auditory function. As sensory hair cells are particularly vulnerable to metabolic changes, we hypothesize that mutations in PEX1 lead to oxidative stress affecting hair cells of the inner ear, subsequently resulting in hair cell degeneration and hearing loss. Global deletion of the Pex1 gene is neonatal lethal in mice, impairing any postnatal studies. To overcome this limitation, we created conditional knockout mice (cKO) using Gfi1Creor VGlut3Cre expressing mice crossed to floxed Pex1 mice to allow for selective deletion of Pex1 in the hair cells of the inner ear. We find that Pex1 excision in inner hair cells (IHCs) leads to progressive hearing loss associated with significant decrease in auditory brainstem responses (ABR), specifically ABR wave I amplitude, indicative of synaptic defects. Analysis of IHC synapses in cKO mice reveals a decrease in ribbon synapse volume and functional alterations in exocytosis. Concomitantly, we observe a decrease in peroxisomal number, indicative of oxidative stress imbalance. Taken together, these results suggest a critical function of Pex1 in development and maturation of IHC-spiral ganglion synapses and auditory function.