Stereocilia Bundles Research Articles

Molecular specializations underlying phenotypic differences in inner ear hair cells of zebrafish and mice

IntroductionHair cells (HCs) are the sensory receptors of the auditory and vestibular systems in the inner ears of vertebrates that selectively transduce mechanical stimuli into electrical activity. Although all HCs have the hallmark stereocilia bundle for mechanotransduction, HCs in non-mammals and mammals differ in their molecular specialization in the apical, basolateral, and synaptic membranes. HCs of non-mammals, such as zebrafish (zHCs), are electrically tuned to specific frequencies and possess an active process in the stereocilia bundle to amplify sound signals. Mammalian HCs, in contrast, are not electrically tuned and achieve amplification by somatic motility of outer HCs (OHCs).MethodsTo understand the genetic mechanisms underlying differences between adult zebrafish and mammalian HCs, we compared their RNA-seq-characterized transcriptomes, focusing on protein-coding orthologous genes related to HC specialization.ResultsThere was considerable shared expression of gene orthologs among the HCs, including those genes associated with mechanotransduction, ion transport/channels, and synaptic signaling. However, there were some notable differences in expression among zHCs, OHCs, and inner HCs (IHCs), which likely underlie the distinctive physiological properties of each cell type. For example, OHCs highly express Slc26a5 which encodes the motor protein prestin that contributes to OHC electromotility. However, zHCs have only weak expression of slc26a5, and subsequently showed no voltage-dependent electromotility when measured. Notably, the zHCs expressed more paralogous genes including those associated with HC-specific functions and transcriptional activity, though it is unknown whether they have functions similar to their mammalian counterparts. There was overlap in the expressed genes associated with a known hearing phenotype.DiscussionOur analyses unveil substantial differences in gene expression patterns that may explain phenotypic specialization of zebrafish and mouse HCs. This dataset also includes several protein-coding genes to further the functional characterization of HCs and study of HC evolution from non-mammals to mammals.

Proteins required for stereocilia elongation during mammalian hair cell development ensure precise and steady heights during adult life

The hair bundle, or stereocilia bundle, is the mechanosensory compartment of hair cells (HCs) in the inner ear. To date, most mechanistic studies have focused on stereocilia bundle morphogenesis, and it remains unclear how this organelle critical for hearing preserves its precise dimensions during life in mammals. The GPSM2-GNAI complex occupies the distal tip of stereocilia in the tallest row and is required for their elongation during development. Here, we ablate GPSM2-GNAI in adult mouse HCs after normal stereocilia elongation is completed. We observe a progressive height reduction of the tallest row stereocilia totaling ~600 nm after 12 wk in Gpsm2 mutant inner HCs. To measure GPSM2 longevity at tips, we generated a HaloTag-Gpsm2 mouse strain and performed pulse-chase experiments in vivo. Estimates using pulse-chase or tracking loss of GPSM2 immunolabeling following Gpsm2 inactivation suggest that GPSM2 is relatively long-lived at stereocilia tips with a half-life of 9 to 10 d. Height reduction coincides with dampened auditory brainstem responses evoked by low-frequency stimuli in particular. Finally, GPSM2 is required for normal tip enrichment of elongation complex (EC) partners MYO15A, WHRN, and EPS8, mirroring their established codependence during development. Taken together, our results show that the EC is also essential in mature HCs to ensure precise and stable stereocilia height and for sensitive detection of a full range of sound frequencies.

F‐actin in the cuticular plate and junctions of auditory hair cells is regulated by ADF and cofilin to allow for normal stereocilia bundle patterning and maintenance

AbstractAuditory hair cells, which convert sound‐induced vibrations in the inner ear into neural signals, depend on multiple actin populations for normal function. Stereocilia are mechanosensory protrusions formed around a core of linear, crosslinked F‐actin. They are anchored in the cuticular plate, which predominantly consists of randomly oriented actin filaments. A third actin population is found near hair cell junctions, consisting of both parallel and branched filaments. Actin depolymerizing factor (ADF) and cofilin‐1 (CFL1) proteins disassemble actin filaments and are required to regulate F‐actin in stereocilia, but their effect on cuticular plate and junctional actin populations is unclear. Here, we show that loss of ADF and CFL1 disrupts the patterning of stereocilia into orderly bundles and that this phenotype correlates with defective development of the cuticular plate and junctional actin populations. ADF/CFL1 continue to regulate these actin populations in mature cells, which is necessary for long‐term maintenance of hair cell morphology.

CIB2 function is distinct from Whirlin in the development of cochlear stereocilia staircase pattern.

Variations in genes coding for calcium and integrin binding protein 2 (CIB2) and whirlin cause deafness both in humans and mice. We previously reported that CIB2 binds to whirlin, and is essential for normal staircase architecture of auditory hair cells stereocilia. Here, we refine the interacting domains between these proteins and provide evidence that both proteins have distinct role in the development and organization of stereocilia bundles required for auditory transduction. Using a series of CIB2 and whirlin deletion constructs and nanoscale pulldown (NanoSPD) assays, we localized the regions of CIB2 that are critical for interaction with whirlin. AlphaFold 2 multimer, independently identified the same interacting regions between CIB2 and whirlin proteins, providing a detailed structural model of the interaction between the CIB2 EF2 domain and whirlin HHD2 domain. Next, we investigated genetic interaction between murine Cib2 and Whrn using genetic approaches. Hearing in mice double heterozygous for functionally null alleles (Cib2 KO/+ ;Whrn wi/+ ) was similar to age-matched wild type mice, indicating that partial deficiency for both Cib2 and Whrn does not impair hearing. Double homozygous mutant mice (Cib2 KO/KO ;Whrn wi/wi ) had profound hearing loss and cochlear stereocilia exhibited a predominant phenotype seen in single Whrn wi/wi mutants. Furthermore, over-expression of Whrn in Cib2 KO/KO mice did not rescue the stereocilia morphology. These data suggest that, CIB2 is multifunctional, with key independent functions in development and/or maintenance of stereocilia staircase pattern in auditory hair cells.

Stereocilia fusion pathology in the cochlear outer hair cells at the nanoscale level.

The hair bundle of cochlear hair cells comprises specialized microvilli, the stereocilia, which fulfil the role of mechanotransduction. Genetic defects and environmental noise challenge the maintenance of hair bundle structure, critically contributing to age-related hearing loss. Stereocilia fusion is a major component of the hair bundle pathology in mature hair cells, but its role in hearing loss and its molecular basis are poorly understood. Here, we utilized super-resolution expansion microscopy to examine the molecular anatomy of outer hair cell stereocilia fusion in mouse models of age-related hearing loss, heightened endoplasmic reticulum stress and prolonged noise exposure. Prominent stereocilia fusion in our model of heightened endoplasmic reticulum stress, Manf (Mesencephalic astrocyte-derived neurotrophic factor)-inactivated mice in a background with Cadherin 23missense mutation, impaired mechanotransduction and calcium balance in stereocilia. This was indicated by reduced FM1-43 dye uptake through the mechanotransduction channels, reduced neuroplastin/PMCA2 expression and increased expression of the calcium buffer oncomodulin inside stereocilia. Sparse BAIAP2L2 and myosin 7a expression was retained in the fused stereocilia but mislocalized away from their functional sites at the tips. These hair bundle abnormalities preceded cell soma degeneration, suggesting a sequela from stereociliary molecular perturbations to cell death signalling. In the age-related hearing loss and noise-exposure models, stereocilia fusion was more restricted within the bundles, yet both models exhibited oncomodulin upregulation at the fusion sites, implying perturbed calcium homeostasis. We conclude that stereocilia fusion is linked with the failure to maintain cellular proteostasis and with disturbances in stereociliary calcium balance. KEY POINTS: Stereocilia fusion is a hair cell pathology causing hearing loss. Inactivation of Manf, a component of the endoplasmic reticulum proteostasis machinery, has a cell-intrinsic mode of action in triggering outer hair cell stereocilia fusion and the death of these cells. The genetic background with Cadherin 23missense mutation contributes to the high susceptibility of outer hair cells to stereocilia fusion, evidenced in Manf-inactivated mice and in the mouse models of early-onset hearing loss and noise exposure. Endoplasmic reticulum stress feeds to outer hair cell stereocilia bundle pathology and impairs the molecular anatomy of calcium regulation. The maintenance of the outer hair cell stereocilia bundle cohesion is challenged by intrinsic and extrinsic stressors, and understanding the underlying mechanisms will probably benefit the development of interventions to promote hearing health.

VASCilia (Vision Analysis StereoCilia): A Napari Plugin for Deep Learning-Based 3D Analysis of Cochlear Hair Cell Stereocilia Bundles.

Cochlear hair cell stereocilia bundles are key organelles required for normal hearing. Often, deafness mutations cause aberrant stereocilia heights or morphology that are visually apparent but challenging to quantify. Actin-based structures, stereocilia are easily and most often labeled with phalloidin then imaged with 3D confocal microscopy. Unfortunately, phalloidin non-specifically labels all the actin in the tissue and cells and therefore results in a challenging segmentation task wherein the stereocilia phalloidin signal must be separated from the rest of the tissue. This can require many hours of manual human effort for each 3D confocal image stack. Currently, there are no existing software pipelines that provide an end-to-end automated solution for 3D stereocilia bundle instance segmentation. Here we introduce VASCilia, a Napari plugin designed to automatically generate 3D instance segmentation and analysis of 3D confocal images of cochlear hair cell stereocilia bundles stained with phalloidin. This plugin combines user-friendly manual controls with advanced deep learning-based features to streamline analyses. With VASCilia, users can begin their analysis by loading image stacks. The software automatically preprocesses these samples and displays them in Napari. At this stage, users can select their desired range of z-slices, adjust their orientation, and initiate 3D instance segmentation. After segmentation, users can remove any undesired regions and obtain measurements including volume, centroids, and surface area. VASCilia introduces unique features that measures bundle heights, determines their orientation with respect to planar polarity axis, and quantifies the fluorescence intensity within each bundle. The plugin is also equipped with trained deep learning models that differentiate between inner hair cells and outer hair cells and predicts their tonotopic position within the cochlea spiral. Additionally, the plugin includes a training section that allows other laboratories to fine-tune our model with their own data, provides responsive mechanisms for manual corrections through event-handlers that check user actions, and allows users to share their analyses by uploading a pickle file containing all intermediate results. We believe this software will become a valuable resource for the cochlea research community, which has traditionally lacked specialized deep learning-based tools for obtaining high-throughput image quantitation. Furthermore, we plan to release our code along with a manually annotated dataset that includes approximately 55 3D stacks featuring instance segmentation. This dataset comprises a total of 1,870 instances of hair cells, distributed between 410 inner hair cells and 1,460 outer hair cells, all annotated in 3D. As the first open-source dataset of its kind, we aim to establish a foundational resource for constructing a comprehensive atlas of cochlea hair cell images. Together, this open-source tool will greatly accelerate the analysis of stereocilia bundles and demonstrates the power of deep learning-based algorithms for challenging segmentation tasks in biological imaging research. Ultimately, this initiative will support the development of foundational models adaptable to various species, markers, and imaging scales to advance and accelerate research within the cochlea research community.

Molecular Specializations Underlying Phenotypic Differences in Inner Ear Hair Cells of Zebrafish and Mice.

Hair cells (HCs) are the sensory receptors of the auditory and vestibular systems in the inner ears of vertebrates that selectively transduce mechanical stimuli into electrical activity. Although all HCs have the hallmark stereocilia bundle for mechanotransduction, HCs in non-mammals and mammals differ in their molecular specialization in the apical, basolateral and synaptic membranes. HCs of non-mammals, such as zebrafish (zHCs), are electrically tuned to specific frequencies and possess an active process in the stereocilia bundle to amplify sound signals. Mammalian cochlear HCs, in contrast, are not electrically tuned and achieve amplification by somatic motility of outer HCs (OHCs). To understand the genetic mechanisms underlying differences among adult zebrafish and mammalian cochlear HCs, we compared their RNA-seq-characterized transcriptomes, focusing on protein-coding orthologous genes related to HC specialization. There was considerable shared expression of gene orthologs among the HCs, including those genes associated with mechanotransduction, ion transport/channels, and synaptic signaling. For example, both zebrafish and mouse HCs express Tmc1, Lhfpl5, Tmie, Cib2, Cacna1d, Cacnb2, Otof, Pclo and Slc17a8. However, there were some notable differences in expression among zHCs, OHCs, and inner HCs (IHCs), which likely underlie the distinctive physiological properties of each cell type. Tmc2 and Cib3 were not detected in adult mouse HCs but tmc2a and b and cib3 were highly expressed in zHCs. Mouse HCs express Kcna10, Kcnj13, Kcnj16, and Kcnq4, which were not detected in zHCs. Chrna9 and Chrna10 were expressed in mouse HCs. In contrast, chrna10 was not detected in zHCs. OHCs highly express Slc26a5 which encodes the motor protein prestin that contributes to OHC electromotility. However, zHCs have only weak expression of slc26a5, and subsequently showed no voltage dependent electromotility when measured. Notably, the zHCs expressed more paralogous genes including those associated with HC-specific functions and transcriptional activity, though it is unknown whether they have functions similar to their mammalian counterparts. There was overlap in the expressed genes associated with a known hearing phenotype. Our analyses unveil substantial differences in gene expression patterns that may explain phenotypic specialization of zebrafish and mouse HCs. This dataset also includes several protein-coding genes to further the functional characterization of HCs and study of HC evolution from non-mammals to mammals.

Expression of the human usherin c.2299delG mutation leads to early-onset auditory loss and stereocilia disorganization

Usher syndrome (USH) is the leading cause of combined deafness and blindness, with USH2A being the most prevalent form. The mechanisms responsible for this debilitating sensory impairment remain unclear. This study focuses on characterizing the auditory phenotype in a mouse model expressing the c.2290delG mutation in usherin equivalent to human frameshift mutation c.2299delG. Previously we described how this model reproduces patient’s retinal phenotypes. Here, we present the cochlear phenotype, showing that the mutant usherin, is expressed during early postnatal stages. The c.2290delG mutation results in a truncated protein that is mislocalized within the cell body of the hair cells. The knock-in model also exhibits congenital hearing loss that remains consistent throughout the animal’s lifespan. Structurally, the stereocilia bundles, particularly in regions associated with functional hearing loss, are disorganized. Our findings shed light on the role of usherin in maintaining structural support, specifically in longer inner hair cell stereocilia, during development, which is crucial for proper bundle organization and hair cell function. Overall, we present a genetic mouse model with cochlear defects associated with the c.2290delG mutation, providing insights into the etiology of hearing loss and offering potential avenues for the development of effective therapeutic treatments for USH2A patients.

Rab11a Is Essential for the Development and Integrity of the Stereocilia and Kinocilia in the Mammalian Organ of Corti.

The cochlea hair cells transform mechanic sounds to neural signals with a remarkable sensitivity and resolution. This is achieved via the precisely sculpted mechanotransduction apparatus of the hair cells and the supporting structure of the cochlea. The shaping of the mechanotransduction apparatus, the staircased stereocilia bundles on the apical surface of the hair cells, requires an intricate regulatory network including planar cell polarity (PCP) and primary cilia genes in orienting stereocilia bundles and building molecular machinery of the apical protrusions. The mechanism linking these regulatory components is unknown. Here, we show that a small GTPase known for its role in protein trafficking, Rab11a, is required for ciliogenesis in hair cells during development in mice. In addition, in the absence of Rab11a, stereocilia bundles lost their cohesion and integrity, and mice are deaf. These data indicate an essential role of protein trafficking in the formation of hair cell mechanotransduction apparatus, implicating a role of Rab11a or protein trafficking in linking the cilia and polarity regulatory components with the molecular machinery in building the cohesive and precisely shaped stereocilia bundles.

TMEM30A is essential for hair cell polarity maintenance in postnatal mouse cochlea

BackgroundPhosphatidylserine is translocated to the inner leaflet of the phospholipid bilayer membrane by the flippase function of type IV P-tape ATPase (P4-ATPase), which is critical to maintain cellular stability and homeostasis. Transmembrane protein 30A (TMEM30A) is the β-subunit of P4-ATPase. Loss of P4-ATPase function causes sensorineural hearing loss and visual dysfunction in human. However, the function of TMEM30A in the auditory system is unclear.MethodsP4-ATPase subtype expression in the cochlea was detected by immunofluorescence staining and quantitative real-time polymerase chain reaction (qRT-PCR) at different developmental stages. Hair cell specific TMEM30A knockout mice and wild-type littermates were used for the following functional and morphological analysis. Auditory function was evaluated by auditory brainstem response. We investigated hair cell and stereocilia morphological changes by immunofluorescence staining. Scanning electron microscopy was applied to observe the stereocilia ultrastructure. Differentially expressed transcriptomes were analyzed based on RNA-sequencing data from knockout and wild-type mouse cochleae. Differentially expressed genes were verified by qRT-PCR.ResultsTMEM30A and subtypes of P4-ATPase are expressed in the mouse cochlea in a temporal-dependent pattern. Deletion of TMEM30A in hair cells impaired hearing onset due to progressive hair cell loss. The disrupted kinocilia placement and irregular distribution of spectrin-α in cuticular plate indicated the hair cell planar polarity disruption in TMEM30A deletion hair cells. Hair cell degeneration begins at P7 and finishes around P14. Transcriptional analysis indicates that the focal adhesion pathway and stereocilium tip-related genes changed dramatically. Without the TMEM30A chaperone, excessive ATP8A2 accumulated in the cytoplasm, leading to overwhelming endoplasmic reticulum stress, which eventually contributed to hair cell death.ConclusionsDeletion of TMEM30A led to disrupted planar polarity and stereocilia bundles, and finally led to hair cell loss and auditory dysfunction. TMEM30A is essential for hair cell polarity maintenance and membrane homeostasis. Our study highlights a pivotal role of TMEM30A in the postnatal development of hair cells and reveals the possible mechanisms underlying P4-ATPase-related genetic hearing loss.

Hair cell morphological patterns and polarity organization in the sea lamprey vestibular cristae.

The inner ear of the sea lamprey was examined by scanning electron microscopy, antibody labeling with tubulin, Myo7a, Spectrin, and Phalloidin stain to elucidate the canal cristae organization and the morphology and polarity of the hair cells. We characterized the hair cell stereocilia bundles and their morphological polarity with respect to the kinocilia. We identified three types of hair cells. In Type 1 hair cells, the kinocilia were slightly longer than the tallest stereocilia. This type was located along the medial bank of the crista and their polarity, based on kinocilia location, was uniformly pointed ampullipetally. Type 2 hair cells that had kinocilia that were much longer than the stereocilia, were most abundant in the central region of the crista. This type of hair cell displayed variable polarity. Type 3 hair cells had extremely long kinocilia (~40-50 μm long) and with extremely short stereocilia. They were mostly located in the lateral zone crista and displayed ampullipetal polarity. Myo7a and tubulin antibodies revealed that hair cells and vestibular afferents are distributed across the canal cristae in the lamprey, covering the area of cruciate eminence; a feature that is absent in more derived vertebrates. Spectrin shows hair cells of varying polarities in the central zone. In this zone, some cells followed the main polarity vector (lateral) like those in medial and lateral zones, whereas other cells displayed polarities that carried up to 40° from the main polarity vector.

Regenerated hair cells in the neonatal cochlea are innervated and the majority co-express markers of both inner and outer hair cells.

After a damaging insult, hair cells can spontaneously regenerate from cochlear supporting cells within the first week of life. While the regenerated cells express several markers of immature hair cells and have stereocilia bundles, their capacity to differentiate into inner or outer hair cells, and ability to form new synaptic connections has not been well-described. In addition, while multiple supporting cell subtypes have been implicated as the source of the regenerated hair cells, it is unclear if certain subtypes have a greater propensity to form one hair cell type over another. To investigate this, we used two CreER mouse models to fate-map either the supporting cells located near the inner hair cells (inner phalangeal and border cells) or outer hair cells (Deiters’, inner pillar, and outer pillar cells) along with immunostaining for markers that specify the two hair cell types. We found that supporting cells fate-mapped by both CreER lines responded early to hair cell damage by expressing Atoh1, and are capable of producing regenerated hair cells that express terminal differentiation markers of both inner and outer hair cells. The majority of regenerated hair cells were innervated by neuronal fibers and contained synapses. Unexpectedly, we also found that the majority of the laterally positioned regenerated hair cells aberrantly expressed both the outer hair cell gene, oncomodulin, and the inner hair cell gene, vesicular glutamate transporter 3 (VGlut3). While this work demonstrates that regenerated cells can express markers of both inner and outer hair cells after damage, VGlut3 expression appears to lack the tight control present during embryogenesis, which leads to its inappropriate expression in regenerated cells.

Selective binding and transport of protocadherin 15 isoforms by stereocilia unconventional myosins in a heterologous expression system

During hair cell development, the mechanoelectrical transduction (MET) apparatus is assembled at the stereocilia tips, where it coexists with the stereocilia actin regulatory machinery. While the myosin-based tipward transport of actin regulatory proteins is well studied, isoform complexity and built-in redundancies in the MET apparatus have limited our understanding of how MET components are transported. We used a heterologous expression system to elucidate the myosin selective transport of isoforms of protocadherin 15 (PCDH15), the protein that mechanically gates the MET apparatus. We show that MYO7A selectively transports the CD3 isoform while MYO3A and MYO3B transports the CD2 isoform. Furthermore, MYO15A showed an insignificant role in the transport of PCDH15, and none of the myosins tested transport PCDH15-CD1. Our data suggest an important role for MYO3A, MYO3B, and MYO7A in the MET apparatus formation and highlight the intricate nature of MET and actin regulation during development and functional maturation of the stereocilia bundle.

Deciphering the Molecular Interaction Between the Adhesion G Protein-Coupled Receptor ADGRV1 and its PDZ-Containing Regulator PDZD7.

Hearing relies on the transduction of sound-evoked vibrations into electrical signals, occurring in the stereocilia bundle of inner ear hair cells. The G protein-coupled receptor (GPCR) ADGRV1 and the multi-PDZ protein PDZD7 play a critical role in the formation and function of stereocilia through their scaffolding and signaling properties. During hair cell development, the GPCR activity of ADGRV1 is specifically inhibited by PDZD7 through an unknown mechanism. Here, we describe the key interactions mediated by the two N-terminal PDZ domains of PDZD7 and the cytoplasmic domain of ADGRV1. Both PDZ domains can bind to the C-terminal PDZ binding motif (PBM) of ADGRV1 with the critical contribution of atypical C-terminal β extensions. The two PDZ domains form a supramodule in solution, stabilized upon PBM binding. Interestingly, we showed that the stability and binding properties of the PDZ tandem are affected by two deafness-causing mutations located in the binding grooves of PDZD7 PDZ domains.

Age-related stereocilia pathology in the human cochlea

Age-related hearing loss in humans is characterized by progressive loss of threshold sensitivity, especially at high frequencies. A multivariable regression of histopathological metrics from normal-aging human cochleae (Wu et al., 2020) showed that hair cell loss better predicts the audiometric shifts than either neural loss or strial atrophy, however considerable variability in age-related threshold elevation remained unexplained. Here, we develop and apply an algorithm to quantify stereocilia pathology in high-power confocal images of inner and outer hair cells in normal aging human cochleae, aged 21 – 71 yrs. Microdissected epithelial wholemounts of the cochleae were immunostained for myosin VIIa and espin to show cuticular plates and stereocilia, respectively, and each cochlea was imaged at 10 log-spaced locations along the cochlear spiral. An approach based on Fourier transforms was used to quantify the regularity of each stereocilia bundle, and the outcome was compared to a parallel analysis by a human observer. Results show a significant age-related decline in stereocilia regularity and increase in stereocilia loss and fusion. Stereocilia pathology was especially severe on the outer hair cells and in the basal half of the cochlea, and may represent a key contributor to age-related threshold elevations. For the one case with an associated pre-mortem audiogram, the threshold shifts are better predicted from the pattern of stereocilia damage than from the pattern of hair cell loss alone.

Two kinematic gains underlying the organ of Corti mechanotransduction

The organ of Corti (OoC) is the sensory epithelium in the mammalian hearing organ where vibrations are encoded into neural signals. The OoC vibrates due to external and internal agitations. Externally, differential fluid pressures across the OoC cause vibrations. Internally, actuator cells (outer hair cells) are known to boost the vibrations. These two agitations result in distinct vibration patterns. Eventually, these vibrations deflect the stereocilia bundle to activate mechanotransduction channels in them. How OoC vibrations result in the hair cell mechanotransduction is critical to hearing research, but a fundamental information (kinematic gain) has not been quantified clearly. Using the optical coherence tomography, we measured 2-D vibrations of the OoC in cochlear turns acutely excised from young Mongolian gerbils. The excised tissues were stimulated either mechanically or electrically, which are comparable to external or internal agitations, respectively. The stereocilia deflection was estimated from relative motion between overlying and underlying structures. Two kinematic gains were defined. Passive kinematic gain represents the amplitude ratio between the stereocilia deflection and the basilar membrane transverse vibration. Active kinematic gain represents the amplitude ratio between the stereocilia deflection and the outer hair cell length change. (Work supported by NIH NIDCD R01 DC014685.)

VGLUT3-p.A211V variant fuses stereocilia bundles and elongates synaptic ribbons.

DFNA25 is an autosomal‐dominant and progressive form of human deafness caused by mutations in the SLC17A8 gene, which encodes the vesicular glutamate transporter type 3 (VGLUT3). To resolve the mechanisms underlying DFNA25, we studied phenotypes of mice harbouring the p.A221V mutation in humans (corresponding to p.A224V in mice). Using auditory brainstem response and distortion product otoacoustic emissions, we showed progressive hearing loss with intact cochlear amplification in the VGLUT3A224V/A224V mouse. The summating potential was reduced, indicating the alteration of inner hair cell (IHC) receptor potential. Scanning electron microscopy examinations demonstrated the collapse of stereocilia bundles in IHCs, leaving those from outer hair cells unaffected. In addition, IHC ribbon synapses underwent structural and functional modifications at later stages. Using super‐resolution microscopy, we observed oversized synaptic ribbons and patch‐clamp membrane capacitance measurements showed an increase in the rate of the sustained releasable pool exocytosis. These results suggest that DFNA25 stems from a failure in the mechano‐transduction followed by a change in synaptic transfer. The VGLUT3A224V/A224V mouse model opens the way to a deeper understanding and to a potential treatment for DFNA25.Key points The vesicular glutamate transporter type 3 (VGLUT3) loads glutamate into the synaptic vesicles of auditory sensory cells, the inner hair cells (IHCs).The VGLUT3‐p.A211V variant is associated with human deafness DFNA25.Mutant mice carrying the VGLUT3‐p.A211V variant show progressive hearing loss.IHCs from mutant mice harbour distorted stereocilary bundles, which detect incoming sound stimulation, followed by oversized synaptic ribbons, which release glutamate onto the afferent nerve fibres.These results suggest that DFNA25 stems from the failure of auditory sensory cells to faithfully transduce acoustic cues into neural messages.

MANF supports the inner hair cell synapse and the outer hair cell stereocilia bundle in the cochlea.

Failure in the structural maintenance of the hair cell stereocilia bundle and ribbon synapse causes hearing loss. Here, we have studied how ER stress elicits hair cell pathology, using mouse models with inactivation of Manf (mesencephalic astrocyte-derived neurotrophic factor), encoding an ER-homeostasis-promoting protein. From hearing onset, Manf deficiency caused disarray of the outer hair cell stereocilia bundle and reduced cochlear sound amplification capability throughout the tonotopic axis. In high-frequency outer hair cells, the pathology ended in molecular changes in the stereocilia taper region and in strong stereocilia fusion. In high-frequency inner hair cells, Manf deficiency degraded ribbon synapses. The altered phenotype strongly depended on the mouse genetic background. Altogether, the failure in the ER homeostasis maintenance induced early-onset stereociliopathy and synaptopathy and accelerated the effect of genetic causes driving age-related hearing loss. Correspondingly, MANF mutation in a human patient induced severe sensorineural hearing loss from a young age onward. Thus, we present MANF as a novel protein and ER stress as a mechanism that regulate auditory hair cell maintenance in both mice and humans.

Protective effect of berberine chloride against cisplatin-induced ototoxicity

BackgroundCisplatin (CP) is an effective anticancer drug broadly used for various types of cancers, but it has shown ototoxicity that results from oxidative stress. Berberine has been reported for its anti-oxidative stress suggesting its therapeutic potential for many diseases such as colitis, diabetes, and vascular dementia.ObjectiveOrgan of Corti of postnatal day 3 mouse cochlear explants were used to compare hair cells after the treatment with cisplatin alone or with berberine chloride (BC) followed by CP.MethodsWe investigated the potential of the anti-oxidative effect of BC against the cisplatin-induced ototoxicity. We observed a reduced aberrant bundle of stereocilia in hair cells in CP with BC pre-treated group. Caspase-3 immunofluorescence and TUNEL assay supported the hypothesis that BC attenuates the apoptotic signals induced by CP. Reactive oxygen species level in the mitochondria were investigated by MitoSOX Red staining and the mitochondrial membrane potentials were compared by JC-1 assay.ResultsBC decreased ROS generation with preserved mitochondrial membrane potentials in mitochondria as well as reduced DNA fragmentation in hair cells. In summary, our data indicate that BC might act as antioxidant against CP by reducing the stress in mitochondria resulting in cell survival.ConclusionOur result suggests the therapeutic potential of BC for prevention of the detrimental effect of CP-induced ototoxicity.

Rab11a Regulates the Development of Cilia and Establishment of Planar Cell Polarity in Mammalian Vestibular Hair Cells.

Vestibular organs have unique planar cell polarity (Figure 1A), and their normal development and function are dependent on the regular polarity of cilia (Figure 1B) requires. Rab11a is a small G protein that participates in the transportation of intracellular and extracellular materials required for polarity formation; however, our understanding of the mechanisms of the actions of Rab11a in vestibular organs is limited. Here, we showed that the general shape of the utricle was abnormal in Rab11aCKO/CKO mice. These mice also showed abnormal morphology of the stereocilia bundles, which were reduced in both length and number, as well as disturbed tissue-level polarity. Rab11a affected the distribution of polarity proteins in the vestibular organs, indicating that the normal development of cilia requires Rab11a and intraflagellar transportation. Furthermore, small G protein migration works together with intraflagellar transportation in the normal development of cilia. FIGURE 1Morphological changes of stereocilia in the extrastriolar hair cells from Rab11a single or Rab11a/IFT88 double-mutant utricles. (A) Medial view of a mouse left inner ear with its five vestibular sensory organs (gray). Enlarged are the utricle showing their subdivisions, LPR (yellow line), and striola (blue). LES, lateral extrastriola; MES, medial extrastriola; LPR, line of polarity reversal. (B) Schematic view of vestibular hair cell. Kinocilium is marked with ace-tubulin. Basal body is marked with γ-tubulin. (C,C1,D,D1) Normal appearance of the stereocilia of extrastriolar hair cells of wild-type controls. (E,E1,F,F1) Altered morphology in Rab11aCKO/CKO animals. (G,G1,H,H1) The changes in the stereocilia morphology were more severe in Rab11aCKO/CKO/IFT 88CKO/+ mice. (I–L) Higher magnification of confocal images of hair cells. (M–P) Scanning electron microscopy images of hair cells from wild-type controls and Rab11a mutants. (I,M) Morphology of normal. hair cells of wild-type controls. (J,N) The number of stereocilia on a single hair cell was deceased in the Rab11a mutant. (K,O) Stereocilia were shorter in mutants compared to the wild-type controls. (L,P) The staircase-like hair bundle architecture of hair cells was lost in Rab11a mutant mice. (Q) The percentage of hair cells with abnormal development of static cilia bundles in the extrastriola region was counted as a percentage of the total (n = 5). The percentage of abnormal hair cells was higher in Rab11aCKO/CKO, IFT88CKO/+ mice compared to Rab11aCKO/CKO. The abnormal ratios of single and double knockout hair cells were 42.1 ± 5.7 and 71.5 ± 10.4, respectively. In (A–J), for all primary panels, hair cell stereociliary bundles were marked with phalloidin (green), the actin-rich cuticular plate of hair cells was labeled with β-spectrin (red), while the basal body of the hair cell was labeled with γ-tubulin (blue). Scale bars: 10 μm (C–H1), 5 μm (J–N). *P < 0.05.