Hair Cells Research Articles

The Glutamine Synthetases Are Required for Sensory Hair Cell Formation and Auditory Function in Zebrafish

The development of sensory hair cells (HCs) is closely linked to hearing loss. There are still many unidentified genes that may play a crucial role in HC development and function. Glutamine synthetase, Glul, is expressed in sensory hair cells and auditory organs. However, the role of the Glul gene family in the auditory system remains largely unexplored. This study aims to investigate the function of the Glul gene family in the auditory system. The expression patterns of the glul gene family were examined via in situ hybridization in zebrafish embryos. It was revealed that the expression of glula occurred in the otic vesicle, while glulb was expressed in the neuromast. In contrast, glulc did not exhibit any discernible signal. glula loss of function caused abnormal otolith formation and reduced hair cell number in otic vesicles, while glulb knockdown caused a decrease in HC number in both neuromasts and otic vesicles and impaired auditory function. Furthermore, we found that the knockdown of glulb induces apoptosis of hair cells. Transcriptomic analysis of zebrafish with glula and glulb knockdown revealed significant alterations in the expression of many genes associated with auditory organs. The current study sheds light on the requirement of glula and glulb in zebrafish hair cell formation and auditory function.

Endolymphatic hydrops and cochlear synaptopathy after noise exposure are distinct sequelae of hair cell stereociliary bundle trauma

Endolymphatic hydrops, increased endolymphatic fluid within the cochlea, is the key pathologic finding in patients with Meniere’s disease, a disease of episodic vertigo, fluctuating hearing loss, tinnitus, and aural fullness. Endolymphatic hydrops also can occur after noise trauma and its presence correlates with cochlear synaptopathy, a form of hearing loss caused by reduced numbers of synapses between hair cells and auditory nerve fibers. Here we tested whether there is a mechanistic link between these two phenomena by using multimodal imaging techniques to analyze the cochleae of transgenic mice exposed to blast and osmotic challenge. In vivo cochlear imaging after blast exposure revealed dynamic increases in endolymph that involved hair cell mechanoelectrical transduction channel block but not the synaptic release of glutamate at the hair cell–auditory nerve synapse. In contrast, ex vivo and in vivo auditory nerve imaging revealed that synaptopathy requires glutamate release from hair cells but not endolymphatic hydrops. Thus, although endolymphatic hydrops and cochlear synaptopathy are both observed after noise exposure, one does not cause the other. They are simply co-existent sequelae that derive from the traumatic stimulation of hair cell stereociliary bundles. Importantly, these data argue that Meniere’s disease derives from hair cell transduction channel blockade.

Relationship between hearing loss and Glasgow prognostic score in patients with cancer

ObjectiveOxidative stress damages cochlear hair cells in vitro. However, the effect of systemic inflammation on hearing loss remains unclear. Growing evidence suggests that malnutrition influences the development of hearing loss. In this study, we aimed to investigate the influence of the Glasgow prognostic score (GPS), which is calculated based on systemic inflammatory responses and malnutrition, on auditory threshold increases in patients with cancer. MethodsThis single-center retrospective cohort study included patients with cancer who underwent standard pure-tone audiometry (PTA) between November 2014 and May 2023. Patients with complete data in their electronic medical records within 90 days before undergoing standard PTA were included. Multivariate analysis was performed using auditory threshold as the response variable. Covariates, including GPS, were obtained from blood data and physical data before standard PTA. The GPS was classified into three levels based on serum albumin and C-reactive protein levels. ResultsStandard PTA was performed 14,868 times in 5,462 patients. Of these, 742 had cancer and 384 met the inclusion criteria. Multivariate analysis revealed that older age, creatinine clearance <60 mL/min, and high GPS significantly increased the auditory threshold at frequencies of 500–8,000 Hz. A history of platinum drug use and male sex increased the auditory threshold at frequencies >4,000 and >2,000 Hz, respectively. ConclusionThe GPS was independently associated with elevated standard PTA thresholds in patients with cancer. These results suggest an association between malnutrition/chronic inflammation and hearing loss and provide new information for planning clinical research on hearing loss prevention.

Precise genetic control of ATOH1 enhances maturation of regenerated hair cells in the mature mouse utricle

Vestibular hair cells are mechanoreceptors critical for detecting head position and motion. In mammals, hair cell loss causes vestibular dysfunction as spontaneous regeneration is nearly absent. Constitutive expression of exogenous ATOH1, a hair cell transcription factor, increases hair cell regeneration, however, these cells fail to fully mature. Here, we profiled mouse utricles at 14 time points, and defined transcriptomes of developing and mature vestibular hair cells. To mimic native hair cells which downregulate endogenous ATOH1 as they mature, we engineered viral vectors carrying the supporting cell promoters GFAP and RLBP1. In utricles damaged ex vivo, both CMV-ATOH1 and GFAP-ATOH1 increased regeneration more effectively than RLBP1-ATOH1, while GFAP-ATOH1 and RLBP1-ATOH1 induced hair cells with more mature transcriptomes. In utricles damaged in vivo, GFAP-ATOH1 induced regeneration of hair cells expressing genes indicative of maturing type II hair cells, and more hair cells with bundles and synapses than untreated organs. Together our results demonstrate the efficacy of spatiotemporal control of ATOH1 overexpression in inner ear hair cell regeneration.

Notoginsenoside R1 Attenuates Cisplatin-Induced Ototoxicity by Inducing Heme Oxygenase-1 Expression and Suppressing Oxidative Stress

Cisplatin-induced ototoxicity occurs in approximately half of patients treated with cisplatin, and pediatric patients are more likely to be affected than adults. The oxidative stress elicited by cisplatin is a key contributor to the pathogenesis of ototoxicity. Notoginsenoside R1 (NGR1), the main bioactive compound of Panax notoginseng saponins, has antioxidant and antiapoptotic effects. This study investigated the ability of NGR1 to protect against cisplatin-induced damage in auditory HEI-OC1 cells and neonatal murine cochlear explants. The viability of HEI-OC1 cells treated with NGR1 and cisplatin was greater than that of cells treated with cisplatin alone. The results of Western blots and immunostaining for cleaved caspase-3 revealed that the level of cleaved caspase-3 in the cells treated with cisplatin was repressed by NGR1. NGR1 attenuated cisplatin-induced cytotoxicity in HEI-OC1 cells. Intracellular reactive oxygen species (ROS) were detected with a DCFDA assay and immunostaining for 4-HNE. The result revealed that its expression was induced by cisplatin and was significantly reduced by NGR1. Moreover, NGR1 can promote heme oxygenase-1 (HO-1) expression at both the mRNA and protein levels. ZNPPIX, an HO-1 inhibitor, was administered to cisplatin-treated cells to investigate the role of HO-1 in the protective effect of NGR1. The suppression of HO-1 activity by ZNPPIX markedly abolished the protective effect of NGR1 on cisplatin-treated cells. Therefore, NGR1 protects cells from cisplatin-induced damage by activating HO-1 and its antioxidative activity. In cochlear explants, NGR1 protects cochlear hair cells and attenuates cisplatin-induced ototoxicity by inhibiting ROS generation. In the group treated with cisplatin alone, prominent loss of outer hair cells and severe damage to the structure of the stereociliary bundles of inner and outer hair cells were observed. Compared with the group treated with cisplatin alone, less loss of outer hair cells (p = 0.009) and better preservation of the stereociliary bundles of hair cells were observed in the group treated with cisplatin and NGR1. In conclusion, these findings indicate that NGR1 can protect against cisplatin-induced ototoxicity by inducing HO-1 expression and suppressing oxidative stress.

Absence of oncomodulin increases susceptibility to noise-induced outer hair cell death and alters mitochondrial morphology

Cochlear outer hair cells (OHCs) play a fundamental role in the hearing sensitivity and frequency selectivity of mammalian hearing and are especially vulnerable to noise-induced damage. The OHCs depend on Ca2+ homeostasis, which is a balance between Ca2+ influx and extrusion, as well as Ca2+ buffering by proteins and organelles. Alterations in OHC Ca2+ homeostasis is not only an immediate response to noise, but also associated with impaired auditory function. However, there is little known about the contribution of Ca2+ buffering proteins and organelles to the vulnerability of OHCs to noise. In this study, we used a knockout (KO) mouse model where oncomodulin (Ocm), the major Ca2+ binding protein preferentially expressed in OHCs, is deleted. We show that Ocm KO mice were more susceptible to noise induced hearing loss compared to wildtype (WT) mice. Following noise exposure (106 dB SPL, 2 h), Ocm KO mice had higher threshold shifts and increased OHC loss and TUNEL staining, compared to age-matched WT mice. Mitochondrial morphology was significantly altered in Ocm KO OHCs compared to WT OHCs. Before noise exposure, Ocm KO OHCs showed decreased mitochondrial abundance, volume, and branching compared to WT OHCs, as measured by immunocytochemical staining of outer mitochondrial membrane protein, TOM20. Following noise exposure, mitochondrial proteins were barely visible in Ocm KO OHCs. Using a mammalian cell culture model of prolonged cytosolic Ca2+ overload, we show that OCM has protective effects against changes in mitochondrial morphology and apoptosis. These experiments suggest that disruption of Ca2+ buffering leads to an increase in noise vulnerability and mitochondrial-associated changes in OHCs.

Vital Dye Uptake of YO-PRO-1 and DASPEI Depends Upon Mechanoelectrical Transduction Function in Zebrafish Hair Cells.

Vital dyes allow the visualization of cells in vivo without causing tissue damage, making them a useful tool for studying lateral line and inner ear hair cells in living zebrafish and other vertebrates. FM1-43, YO-PRO-1, and DASPEI are three vital dyes commonly used for hair cell visualization. While it has been established that FM1-43 enters hair cells of zebrafish and other organisms through the mechanoelectrical transduction (MET) channel, the mechanism of entry into hair cells for YO-PRO-1 and DASPEI has not been established despite widespread use. We hypothesize that YO-PRO-1 and DASPEI entry into zebrafish hair cells is MET channel uptake dependent similar to FM1-43. To test this hypothesis, we used both genetic and pharmacologic means to block MET channel function. Genetic based MET channel assays were conducted with two different mechanotransduction defective zebrafish lines, specifically the myo7aa-/- loss of function mutant tc320b (p.Y846X) and cdh23-/- loss of function mutant (c.570-571del). Pharmacologic assays were performed with Gadolinium(III) Chloride (Gad(III)), a compound that can temporarily block mechanotransduction activity. Five-day post fertilization (5dpf) myo7aa-/- and cdh23-/- larvae incubated with FM1-43, YO-PRO-1, and DASPEI all showed nearly absent uptake of each vital dye. Treatment of wildtype zebrafish larvae with Gad(III) significantly reduces uptake of FM1-43, YO-PRO-1, and DASPEI vital dyes. These results indicate that YO-PRO-1 and DASPEI entry into zebrafish hair cells is MET channel dependent similar to FM1-43. This knowledge expands the repertoire of vital dyes that can be used to assess mechanotransduction and MET channel function in zebrafish and other vertebrate models of hair cell function.

Mitochondrial-derived peptides, HNG and SHLP3, protect cochlear hair cells against gentamicin.

Preservation of hair cells is critical for maintaining hearing function, as damage to sensory cells potentially leads to irreparable sensorineural hearing loss. Hair cell loss is often associated with inflammation and oxidative stress. One promising class of bioactive peptides is mitochondrial-derived peptides (MDPs), which have already been proven to protect various tissues from cellular stresses and delay aging processes. Humanin (HN) is one of the best-known members of this family, and recently, we have shown its protective effect in hair cells. The synthetic derivate HN S14G (HNG) has a more potent protective effect than natural HN making it a more useful peptide candidate to promote cytoprotection. A less-known MDP is small humanin-like peptide 3 (SHLP3), which has cytoprotective effects similar to HN, but likely acts through different signaling pathways. Therefore, we examined the effect of exogenous HNG and SHLP3 in auditory hair cells and investigated the molecular mechanisms involved. For this purpose, explants of the organ of Corti (OC) were treated with gentamicin in the presence and absence of HNG or SHLP3. Administration of HNG and SHLP3 reduced gentamicin-induced hair cell loss. The protective mechanisms of HNG and SHLP3 in OC explants included, in part, modulation of AKT and AMPKα. In addition, treatment with HNG and SHLP3 reduced gentamicin-induced oxidative stress and inflammatory gene overexpression. Overall, our data show that HNG and SHLP3 protect hair cells from gentamicin-induced toxicity. This offers new perspectives for the development of therapeutic strategies with MDPs against hearing loss.

Pathological mechanisms and candidate therapeutic approaches in the hearing loss of mice carrying human MIR96 mutations.

Progressive hearing loss is a common problem in the human population with no effective therapeutics currently available. However, it has a strong genetic contribution, and investigating the genes and regulatory interactions underlying hearing loss offers the possibility of identifying therapeutic candidates. Mutations in regulatory genes are particularly useful for this, and an example is the microRNA miR-96, a post-transcriptional regulator which controls hair cell maturation. Mice and humans carrying mutations in miR-96 all exhibit hearing impairment, in homozygosis if not in heterozygosis, but different mutations result in different physiological, structural and transcriptional phenotypes. Here we present our characterisation of two lines of mice carrying different human mutations knocked-in to Mir96. We have carried out auditory brainstem response tests to examine their hearing with age and after noise exposure and have used confocal and scanning electron microscopy to examine the ultrastructure of the organ of Corti and hair cell synapses. Bulk RNA-seq was carried out on the organs of Corti of postnatal mice, followed by bioinformatic analyses to identify candidate targets. While mice homozygous for either mutation are profoundly deaf from 2weeks old, the heterozygous phenotypes differ markedly, with only one mutation resulting in hearing impairment in heterozygosis. Investigations of the structural phenotype showed that one mutation appears to lead to synaptic defects, while the other has a much more severe effect on the hair cell stereociliary bundles. Transcriptome analyses revealed a wide range of misregulated genes in both mutants which were notably dissimilar. We used the transcriptome analyses to investigate candidate therapeutics, and tested one, finding that it delayed the progression of hearing loss in heterozygous mice. Our work adds further support for the importance of the gain of novel targets in microRNA mutants and offers a proof of concept for the identification of pharmacological interventions to maintain hearing.

A Novel PTPRQ c.3697del Variant Causes Autosomal Dominant Progressive Hearing Loss in Both Humans and Mice.

PTPRQ plays an important role in the development of inner ear hair cell stereocilia. While many autosomal recessive variants in PTPRQ have been identified as the pathogenic cause for nonsyndromic hearing loss (DFNB84A), so far only one autosomal dominant PTPRQ variant, c.6881G>A (p.Trp2294*), has been reported for late-onset, mild-to-severe hearing loss (DFNA73). By using targeted next-generation sequencing, this study identified a novel PTPRQ truncating pathogenic variant, c.3697del (p.Leu1233Phefs*11), from a Chinese Han family that co-segregated with autosomal dominant, postlingual, progressive hearing loss. A Ptprq-3700del knock-in mouse model was generated by CRISPR-Cas9 and characterized for its hearing function and inner ear morphology. While the homozygous knock-in mice exhibit profound hearing loss at all frequencies at the age of 3 weeks, the heterozygous mutant mice resemble the human patients in mild, progressive hearing loss from age 3 to 12 weeks, primarily affecting high frequencies. At this stage, the homozygous knock-in mice have a normal hair cell count but disorganized stereocilia. Cochlear proteosome analysis of the homozygous mutant mice revealed differentially expressed genes and pathways involved in oxidative phosphorylation, regulation of angiogenesis and synaptic vesicle cycling. Our study provides a valuable animal model for further functional studies of the pathogenic mechanisms underlying DFNA73.

A Frameshift Variant in ANKRD24 Implicates Its Role in Human Non-Syndromic Hearing Loss.

Hearing loss (HL) is the most prevalent sensorineural disorders, affecting about one in 1000 newborns. Over half of the cases are attributed to genetic factors; however, due to the extensive clinical and genetic heterogeneity, many cases remain without a conclusive genetic diagnosis. The advent of next-generation sequencing methodologies in recent years has greatly helped unravel the genetic etiology of HL by identifying numerous genes and causative variants. Despite this, much remains to be uncovered about the genetic basis of sensorineural hearing loss (SNHL). Here, we report an Iranian consanguineous family with postlingual, moderate-to-severe autosomal recessive SNHL. After first excluding plausible variants in known deafness-causing genes using whole exome sequencing, we reanalyzed the data, using a gene/variant prioritization pipeline established for novel gene discovery for HL. This approach identified a novel homozygous frameshift variant c.1934_1937del; (p.Thr645Lysfs*52) in ANKRD24, which segregated with the HL phenotype in the family. Recently, ANKRD24 has been shown to be a pivotal constituent of the stereocilia rootlet in cochlea hair cells and interacts with TRIOBP, a protein already implicated in human deafness. Our data implicate for the first time, ANKRD24 in human nonsyndromic HL (NSHL) and expands the genetic spectrum of HL.

"Pathological Insights into Lassa Virus-Induced Vestibular Dysfunction in Mice: Histopathological Analysis of the Inner Ear Vestibular Apparatus".

Lassa fever (LF), caused by Lassa virus (LASV) infection, typically leads to mild symptoms in humans, yet some survivors experience audiovestibular problems. Here we present vestibular histopathological insights in our LF model mice. We observed: 1) hemorrhage within the vestibular ganglion and stroma beneath the sensory epithelium, 2) preserved hair cells and supporting cells, 3) LASV antigen presence in the vestibular ganglion cells and the stroma beneath the sensory epithelium, and 4) CD3-positive T lymphocyte infiltration in the vestibular ganglion and the stroma underlying the sensory epithelium. LASV and/or its immune response likely contributes to the pathogenesis of vestibular dysfunction.

Photodynamic therapy‐induced precise attenuation of light‐targeted semicircular canals for treating intractable vertigo

AbstractVertigo is a common symptom of various diseases that affects a large number of people worldwide. Current leading treatments for intractable peripheral vertigo are to intratympanically inject ototoxic drugs such as gentamicin to attenuate the semicircular canal function but inevitably cause hearing injury. Photodynamic therapy (PDT) is a noninvasive therapeutic approach by precisely targeting the diseased tissue. Here, we developed a PDT‐based method for treating intractable peripheral vertigo in a mouse model using a polymer‐coated photosensitizer chlorin e6 excited by red light. We found that a high dose of PDT attenuated the function of both semicircular canals and otolith organs and damaged their hair cells. Conversely, the PDT exerted no effect on hearing function or cochlear hair‐cell viability. These results suggest the therapeutic potential of PDT for treating intractable peripheral vertigo without hurting hearing. Besides, the attenuation level and affected area can be precisely controlled by adjusting the light exposure time. Furthermore, we demonstrated the potential of this therapeutic approach to be minimally invasive with light irradiation through bone results. Thus, our PDT‐based approach for attenuating the function of the semicircular canals offers a basis for developing a less‐invasive and targeted therapeutic option for treating vertigo.

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.

ATP-gated P2x7 receptor is a major channel type at type II auditory nerves and required for hearing sensitivity efferent controlling and noise protection.

Hearing sensitivity and noise protection are mediated and determined by negative feedback of the cochlear efferent system. Type II auditory nerves (ANs) innervate outer hair cells (OHCs) in the cochlea and provide an input to this efferent control. However, little is known about underlying channel information. Here, we report that ATP-gated P2x7 receptor had a predominant expression at type II ANs and the synaptic areas under inner hair cells and OHCs with lateral and medial olivocochlear efferent nerves. Knockout (KO) of P2x7 increased hearing sensitivity with enhanced acoustic startle response (ASR), auditory brainstem response (ABR), and cochlear microphonics (CM) by increasing OHC electromotility, an active cochlear amplifier in mammals. P2x7 KO also increased susceptibility to noise. Middle level noise exposure could impair active cochlear mechanics resulting in permanent hearing loss in P2x7 KO mice. These data demonstrate that P2x7 receptors have a critical role in type II AN function and the cochlear efferent system to control hearing sensitivity; deficiency of P2x7 receptors can impair the cochlear efferent suppression leading to hearing oversensitivity and susceptibility to noise.

CRISPR-Mediated Gene Editing Tool Development for Patients with Usher Syndrome Type II

Usher syndrome, a rare genetic disease affecting hearing and vision, is an autosomal recessive condition affecting 4 to 17 per 100,000 people and accounting for about 50 percent of hereditary deaf-blindness cases. Usher syndrome type II is caused by mutations of the USH2A gene, a 15.7 kb gene encoding the protein usherin, which localizes to photoreceptor cilium and cochlear hair cells. This subtype of Usher syndrome includes hearing loss from birth and progressive loss of vision, prompting retinitis pigmentosa (RP). Prime editing is a viable option for addressing USH2A mutations and restoring usherin expression and function. To begin, a HEK293T cell line needs to be established for subsequent testing of prime editors. Since prime editing to make the cell line was unsuccessful, homology-directed repair was done and successfully created a cell population with the mutation of interest (single base-pair deletion). Now that this cell population has been created, prime editors will be tested and optimized to address the mutation in a HEK293T cell line and potentially patient-derived induced pluripotent stem cells that are differentiated into photoreceptor precursor cells.

Risk factors contributing to outer hair cell damage in children with chronic kidney disease

Risk factors contributing to outer hair cell damage in children with chronic kidney disease

State-of-the-Art on the Impact of Bimodal Acoustic Stimulation on Speech Perception in Noise in Adults: A Systematic Review.

Bimodal stimulation (BS), which combines the use of a cochlear implant (CI) in one ear and a hearing aid (HA) in the opposite ear, is an established strategy to treat hearing loss by exploiting the unique capabilities of each device. CIs stimulate the auditory nerve by bypassing damaged hair cells, while HAs amplify sounds by requiring a functional hearing residual. The aim of this systematic review is to investigate the advantages and disadvantages of BS such as speech perception in noise. We examined clinical studies published from October 2020 to July 2024, following the PRISMA guidelines, focusing on the advantages and disadvantages of BS on speech perception in noise in adulthood. BS in adult patients significantly improves speech perception in quiet and noisy environments, especially for those with increased residual hearing. Unilateral CIs and BS perform similarly in quiet conditions, but BS significantly improves speech discrimination in noisy environments if loudness between the two devices is appropriately balanced. Directional microphones and programming software are new technologies that succeed in reducing environmental noise and improving verbal perception outcomes, although their features in the literature are controversial. In addition, the individuals using BS may face temporal mismatches mainly due to differing device latencies, affecting sound localization. Compensating for these mismatches can enhance localization accuracy. However, modulated noise remains a significant obstacle to verbal perception in noise. Valuable assessment tools such as music tests provide further information on hearing performance and quality of life. More research is needed to define certain selection criteria.

Inactivity of Stat3 in sensory and non-sensory cells of the mature cochlea.

Signal transducer and activator of transcription 3 (Stat3) plays a role in various cellular processes such as differentiation, inflammation, cell survival and microtubule dynamics, depending on the cell type and the activated signaling pathway. Stat3 is highly expressed in the hair cells and supporting cells of the cochlea and is essential for the differentiation of mouse hair cells in the early embryonic stage. However, it is unclear how Stat3 contributes to the correct function of cells in the organ of Corti postnatally. To investigate this, an inducible Cre/loxp system was used to knock out Stat3 in either the outer hair cells or the supporting cells. The results showed that the absence of Stat3 in either the outer hair cells or the supporting cells resulted in hearing loss without altering the morphology of the organ of Corti. Molecular analysis of the outer hair cells revealed an inflammatory process with increased cytokine production and upregulation of the NF-kB pathway. However, the absence of Stat3 in the supporting cells resulted in reduced microtubule stability. In conclusion, Stat3 is a critical protein for the sensory epithelium of the cochlea and hearing and functions in a cell and function-specific manner.

The p. S178L mutation in Tbc1d24 disrupts endosome-mediated synaptic vesicle trafficking of cochlear hair cells and leads to hearing impairment in mice.

The ribbon synapses of cochlear inner hair cells (IHCs) employ efficient vesicle resupply to enable fast and sustained release rates. However, the molecular mechanisms of these physiological activities remain unelucidated. Previous studies showed that the RAB-specific GTPase-activating protein TBC1D24 controls the endosomal trafficking of the synaptic vesicles (SVs) in Drosophila and mammalian neurons, and mutations in TBC1D24 may lead to non-syndromic hearing loss or hearing loss associated with the DOORS syndrome in humans. In this study, we generated a knock-in mouse model for the p. S178L mutation in TBC1D24, which leads to autosomal dominant non-syndromic hearing loss (DFNA65). The p.S178L mutant mice show mild hearing loss and progressively declined wave I amplitude of the auditory brainstem responses. Despite the normal gross and cellular morphology of the cochlea, transmission electron microscopy reveals accumulation of endosome-like vacuoles and a lower-than-normal number of SVs directly associated with the ribbons in the IHCs. Consistently, patch clamp of the IHCs shows reduced exocytosis under prolonged stimulus. ARF6, a TBC1D24-interacting protein also involved in endosomal membrane trafficking, was underexpressed in the cochleae of the mutant mouse and has weakened in vitro interaction with the p.S178L mutant TBC1D24. Our results suggest an important role of TBC1D24 in maintaining endosomal-mediated vesicle recycling and sustained exocytosis of hair cell ribbon synapses.