Endogenous Sox2 expression is essential for Atoh1-induced hair cell addition and regeneration in the mouse utricle.

Pharmacological activation of GPX4 by selenomethionine attenuates cisplatin-induced ototoxicity and hearing loss.

USH2A knockdown induces apoptosis and reduces prestin expression in HEI-OC1 hair cell model

Engineering vascularized inner ear organoids: Challenges and advances in recapitulating the cochlear microenvironment.

Cisplatin is an effective chemotherapeutic agent, but its clinical use is limited by dose-dependent ototoxicity that leads to irreversible sensorineural hearing loss. Accumulating evidence implicates impaired autophagy-lysosomal homeostasis in cisplatin-induced ototoxicity. Transcription factor EB (TFEB), a master regulator of autophagy and lysosomal biogenesis, represents a promising therapeutic target. Clotrimazole, an FDA-approved antifungal drug with emerging cytoprotective properties, has not been investigated for its potential to mitigate cisplatin-induced ototoxicity. We evaluated the protective effects of clotrimazole using House Ear Institute-Organ of Corti 1 cells, cochlear explants, and an adult C57BL/6J mouse model of transtympanic cisplatin ototoxicity. Apoptosis, reactive oxygen species (ROS), and autophagy flux were assessed using biochemical assays and imaging. RNA-sequencing was performed to identify transcriptional pathways regulated by clotrimazole. TFEB dependence was verified using small interfering RNA knockdown and pharmacological inhibition of AMP-activated protein kinase (AMPK). Cochlear function was assessed using auditory brainstem responses (ABRs), and hair-cell and synapse survival were quantified by immunofluorescence. Clotrimazole significantly reduced cisplatin-induced apoptosis, ROS generation, and calcium overload. Transcriptomic profiling and functional assays revealed robust activation of autophagy. Clotrimazole promoted AMPK activation, suppressed mTORC1 signaling, and enhanced TFEB nuclear translocation. TFEB or AMPK inhibition abrogated these protective effects. In vivo, intratympanic clotrimazole preserved hair cell survival, maintained ribbon synapses, and significantly reduced ABR threshold shifts. Clotrimazole protects against cisplatin-induced ototoxicity by activating the AMPK-mTOR-TFEB axis and restoring autophagy lysosomal homeostasis. These findings support TFEB-targeted autophagy activation as a promising therapeutic strategy for preventing cisplatin-induced hearing loss. Antioxid. Redox Signal. 44, 928-950.

With orthologs for >70% of human genes, zebrafish represent a popular model for investigating development and disease, particularly when murine models are unsuitable. One caveat of zebrafish genetic models is the prevalence of paralogs, where compensation and transcriptional adaptation can complicate our understanding. Usher syndrome type 1 F (USH1F), caused by PCDH15 mutations, exemplifies the need for alternatives models; the primary site of injury, calyceal processes (CP), are uniquely lacking from photoreceptors of nocturnal rodents. Here we demonstrate that contrary to previous reports, zebrafish pcdh15a/b paralogs are not absolutely restricted to ear and eye tissues respectively. Instead, both paralogs are expressed and required in the mechanosensitive hair cells and retinal photoreceptors. Double mutants present the most severe phenotypes: loss of kinocilial and stereocilial links in the ear, and disorganization of CPs, inner/outer segment detachment, and photoreceptor cell death. We also demonstrate that ear and eye-specific phenotypes may be isolated via isoform-specific knockout. These data highlight the strengths of zebrafish to study pathomechanism in vivo. They should also caution researchers from defining paralogs as discrete, absolutely restricted functional units without exceptional evidence. When possible, double mutants may be preferable for studying gene function, and modelling diseases where humans have a single ortholog.

SLC26A4 is a major causative gene for hereditary hearing loss, its mutation spectrum shows pronounced population specificity. In Chinese populations, patients predominantly carry biallelic mutations, and compound heterozygous genotypes are prevalent, which results in a wide spectrum of auditory phenotypes. However, how different alleles interact within these contexts to shape phenotypic variability remains poorly understood. We employed cellular and mouse models to explore the allele-specific mechanisms associated with two novel mutations, a frameshift mutation and a missense mutation, in compound heterozygous that share the same splice-site pathogenic allele. In vitro, wild-type (WT) and mutant (c.574delC, c.1211C>A) SLC26A4 constructs were expressed in HeLa cells to assess pendrin localization. Both mutations reduced membrane enrichment and increased intracellular retention. In vivo, compound heterozygous knock-in mouse models (Slc26a4c.574delC/c.919-2A>G and Slc26a4c.1211C>A/c.919-2A>G) were generated using CRISPR/Cas9. The auditory function and cochlear pathology were investigated. Both compound mutants exhibited elevated ABR thresholds, with more severe hearing loss in Slc26a4c.574delC/c.919-2A>G mice. Correspondingly, these mice showed marked hair cell disruption, stereociliary loss, and cochlear structural abnormalities, whereas the Slc26a4c.1211C>A/c.919-2A>G mice displayed milder changes. Transcriptomic profiling examined by bulk RNA-sequencing revealed broader differential expression in Slc26a4c.574delC/c.919-2A>G mice, enriched in structural and developmental pathways, while the missense model showed predominantly immune-related signatures. Our findings demonstrate that allele-specific functional divergence in compound heterozygous SLC26A4 mutations leads to distinct auditory dysfunction, cochlear pathology, and transcriptional programs. These findings provide mechanistic insight into the phenotypic heterogeneity of hearing loss and may indicate future allele-specific interventions or therapeutic strategies.

Information about head motion and gravity is conveyed to the brain by vestibular nerve afferents which are subdivided by their spontaneous firing properties into regular and irregular subtypes, thought to be differentially responsible for vestibulo-ocular versus vestibulo-spinal reflexes. In the vestibular periphery, afferents make glutamatergic synapses with type II hair cells (HCs) in all vertebrates. During the evolutionary transition to land, however, amniotes (reptiles, birds, and mammals) additionally developed type I vestibular HCs in which unique calyceal afferent terminals cover the basolateral walls of one or more HCs, enabling a nonquantal (NQ) form of synaptic transmission. Most afferents receive inputs from both types of HCs, but the roles of type I versus type II HCs in generating vestibular afferent firing patterns and behaviors remains unclear. Using optogenetics in mice (both sexes), we confirm that stimulation of type II HCs drives conventional quantal glutamatergic transmission, whereas type I HC stimulation evokes NQ responses. In mice with disrupted glutamatergic quantal transmission, NQ transmission effectively drove afferent responses to a wide range of head movement frequencies, as assessed by both vestibular sensory evoked potentials and the vestibulo-ocular reflex. Although the distribution of afferent discharge regularity was unaffected, loss of glutamatergic transmission impaired detection of gravity as evidenced by abnormal contact righting reflex behavior. These results indicate that NQ glutamatergic transmission from type I HCs is sufficient to generate normal afferent firing patterns and dynamic vestibular behaviors and that glutamatergic release from type II HCs is required for the detection of gravity.

Although the effects of the auditory system on the balance system are known, we do not possess sufficient knowledge about the possible effects of the balance system on the auditory system. This study aims to investigate the impact of bithermal caloric stimulation on the outer hair cell functions of the contralateral ear. Distortion product otoacoustic emissions (DPOAEs) were recorded before, during, and after bithermal caloric stimulation. A repeated-measures multivariate analysis examined the effects of Condition (pre, cold, warm, post) and Frequency (13 f2 frequencies). DPOAE amplitudes decreased significantly during caloric-induced nystagmus. A significant effect was observed among DPOAE frequencies. The largest amplitude changes occurred in the midfrequency range, while smaller response changes were seen at both low and high frequencies. This significant frequency interaction indicates that caloric-induced suppression varies across frequencies. Furthermore, both cold and warm irrigation reduced DPOAE amplitudes with warm irrigation, in particular, generally provided greater suppression and showed that this effect was related to the rate of caloric stimulation. Posttest values showed that this reduction persisted, albeit to a lesser extent than during active irrigation. This study shows a significant decrease in DPOAE results for all frequencies during caloric stimulation, supporting the idea that vestibular inputs affect the auditory system through central mechanisms. These findings contribute to emerging evidence of bidirectional interactions between the vestibular and auditory systems.

Schizantherin B (SNB), a key bioactive ingredient of the Chinese traditional medicine Schisandra chinensis, possesses anti-inflammatory and antioxidant properties. Cisplatin (CDDP) is typically used to treat various malignant tumors, however, its clinical utility is often limited by significant off-target toxicities, most notably irreversible hearing loss. Various strategies have been explored to mitigate this side effect. In this study, we investigated the protective effects of SNB against cisplatin-induced hearing loss(CIHL) as a potential preclinical therapeutic strategy.Firstly, in the ex-vivo basilar membrane, we found that SNB alleviated CDDP-induced loss of hair cells, cochlear spiral ganglion cells, and ribbon synapses. Secondly, in vivo experiments showed that SNB protected animals against CHL, returning their response close to normal level. Thirdly, in multiple tumor cell lines, we found SNB did not interfere with CDDP's anti-tumor effects. Consistently, in a mouse model for breast cancer, we found that SNB did not obtrude CDDP's effects in reducing tumor mass. Finally, in examining the molecular mechanisms, we found that SNB reduced both oxidative stress and cell apoptosis in the auditory cell line of HEI-OC1 during CDDP treatment, likely through the Bcl-2/Bax/cleaved-Caspase-3 signal pathways. Collectively, our study demonstrated that SNB mitigates CIHL without interfering with its therapeutic effects in treating cancer, suggesting that SNB is a potential drug candidate for preventing CIHL in cancer patients.

Ketamine is widely used as an anesthetic agent, yet its cellular effects on the auditory system, particularly on cochlear outer hair cells (OHCs), remain incompletely understood. In this study, we investigated the electrophysiological effects of ketamine on isolated OHCs from young Sprague Dawley rats using whole-cell patch-clamp techniques. OHCs were acutely dissociated and exposed to ketamine at varying concentrations to evaluate its impact on membrane currents. Ketamine produced a dose- and voltage-dependent reduction in outward membrane currents, particularly at membrane potentials more positive than -36mV. Pharmacological blockade with iberiotoxin and ion substitution experiments using intracellular Cs+ support that the ketamine-sensitive current is predominantly mediated by BK-like Ca2+-activated K+ channels. Ketamine had minimal effects on resting membrane potential and on voltage-activated K+ currents at hyperpolarized potentials, indicating selective modulation of depolarization-activated conductances. Acetylcholine (ACh)-evoked outward currents recorded at a depolarized holding potential (+ 3mV) were not significantly altered by ketamine. Under these conditions, the measured current primarily reflects secondary Ca2+-activated K+ channel activity rather than direct α9α10 nicotinic receptor-mediated currents. Therefore, the present experimental design does not allow determination of whether ketamine directly affects α9α10 receptor function. These findings demonstrate that ketamine modulates BK-like potassium currents in OHCs and may influence cochlear electrophysiological function. However, the precise mechanism-whether through direct channel interaction or indirect modulation via calcium signaling-remains to be determined.

A Tumor-Bearing Mouse Model of Cisplatin-Induced Hearing Loss for Preclinical Otoprotectant Evaluation.

A multi-metric diagnostic approach was used with chinchilla models of inner-hair-cell (IHC) damage and cochlear synaptopathy (CS).IHC damage and synaptopathy both cause suprathreshold deficits "hidden" from the audiogram.IHC damage results in more severe temporal envelope coding degradation than does synaptopathy.A combination of EFR "peakiness", ABR Wave V/I ratio, and Wideband Middle Ear Muscle Reflex (WB-MEMR) appear to be useful measures for profiling IHC damage and CS.

Cycloastragenol protects cochlear hair cells against cisplatin-induced ototoxicity by preserving mitochondrial function, suppressing apoptosis, and activating the PI3K/Akt/mTOR signaling pathway. These findings highlight the potential of CAG as a therapeutic candidate for preventing cisplatin-induced hearing loss. CAG significantly improved cell viability and reduced cisplatin-induced apoptosis in cochlear hair cells, as demonstrated by decreased TUNEL positivity, reduced apoptotic rates, and modulation of apoptosis-related proteins. CAG also preserved hair-cell marker expression and maintained cochlear hair-cell morphology. Furthermore, CAG restored mitochondrial function by increasing ATP production, maintaining ΔΨm, reducing ROS accumulation, and enhancing respiratory chain complex activities, while preventing mitochondrial fragmentation. In vivo, CAG markedly attenuated cisplatin-induced hearing loss, as indicated by improved ABR thresholds. Mechanistically, CAG activated the PI3K/Akt/mTOR signaling pathway, and inhibition of this pathway abolished its protective effects. Cisplatin-induced ototoxicity, a major adverse effect of chemotherapy, results in irreversible sensorineural hearing loss primarily due to apoptotic loss of cochlear sensory hair cells. Cycloastragenol (CAG), a naturally occurring triterpenoid saponin derived from Astragalus, possesses antioxidant and anti-apoptotic properties. However, its potential protective effects against cisplatin-induced cochlear injury and the underlying mechanisms remain unclear. HEI-OC1 cells, cochlear explant cultures (including the ex vivo cochlear explant model), and a mouse model were used to evaluate the protective effects of CAG against cisplatin-induced ototoxicity. Cell viability was assessed using the CCK-8 assay, while apoptosis was evaluated by TUNEL staining, flow cytometry, and Western blotting of apoptosis-related proteins. Hair-cell markers (Myo7a and Prestin) and cochlear hair-cell morphology were examined by immunofluorescence staining. Mitochondrial function was assessed by measuring ATP levels, mitochondrial membrane potential (ΔΨm), reactive oxygen species (ROS), and respiratory chain complex activities, along with mitochondrial morphology analysis using immunofluorescence and transmission electron microscopy. Auditory function in mice was evaluated by auditory brainstem response (ABR) measurements. The involvement of the PI3K/Akt/mTOR pathway was analyzed by Western blotting and inhibition assays using the PI3K inhibitor LY294002.

During development, coordinated cell behaviors drive epithelial morphogenesis toward precise three-dimensional architectures essential for physiological function. How such coordination arises in epithelia composed of multiple cell types remains unclear. Here, we study development of the avian auditory epithelium comprising sensory hair cells (HCs) and nonsensory supporting cells (SCs). Initially, HCs and SCs are arranged into mosaics by Notch-Delta signaling. As development proceeds, HCs partially extrude from the epithelium, establish a tenfold gradient in apical surface area across the tissue, and rearrange with SCs to form near-hexagonal order. Using experiments combined with a three-dimensional vertex model, we show that increased contractility at apical junctions between SCs relative to HC-SC junctions drives spatial organization both within the epithelial plane and along the apical-basal axis. Consistent with experimental findings, our simulation shows systematic differences in HC apical area expansion generate opposing coordinated movements of HCs and SCs, establishing gradients in HC apical surface area and density while maintaining uniform hexagonal order. Together, these results demonstrate that spatial patterning of junctional contractility coordinates cell behavior across both the plane and depth of a mixed epithelium, producing quasi-stratified architecture and tissue-scale three-dimensional order.

Regeneration is triggered by cells dying due to various types of injuries. However, it remains unclear whether different types of cell death elicit distinct regeneration responses. Here, we systematically profile dynamic transcriptional responses of macrophages and lateral line cells to different cell death modalities during zebrafish sensory hair cell regeneration. We show that chemogenetically induced programmed hair cell death triggers a diminished inflammatory response compared to pharmacologically induced cell lysis, characterized by minimal neutrophil recruitment, distinct transcriptional profiles in phagocytosing macrophages and reduced expression of injury-responsive genes in lateral line cells. Nevertheless, regeneration ultimately converges on a shared set of regeneration-specific genes. Importantly, preventing immune cell recruitment enhances injury-induced support cell proliferation in response to programmed, but not unprogrammed cell death, highlighting cell death-dependent regenerative outcomes following immune cell inhibition. Our findings demonstrate that different forms of cell death trigger distinct molecular events, with implications for tailoring regenerative therapies to specific injury contexts.

Inner ear transplantation of cochlear progenitor cells restores acute sensorineural hearing loss.

Recent advances in novel drug carriers and advanced administration routes for inner ear drug delivery: challenges and future perspectives.

Ototoxic aminoglycosides collect inside the acellular tectorial membrane of the inner ear, likely due to electrostatic interactions, and the structural status of that membrane modulates the toxic effect of those aminoglycosides on sensory hair cells.

Root hairs are tubular tip-growing extensions of root epidermal cells that enhance root surface area for water and nutrient acquisition. While the mechanisms governing root hair fate, polarity and tip growth are well understood, the regulation of root hair longevity remains largely unknown. Here we show that root hair cells employ high levels of autophagy to maximize their lifespan in Arabidopsis thaliana. Loss-of-function mutations in the genes encoding the autophagy regulators ATG2, ATG5 or ATG7 induce a premature, cell-autonomous cell death program. This cell death is activated via a gene regulatory network promoted by the NAC transcription factors ANAC046 and ANAC087. Our findings uncover an antagonistic relationship between autophagy and senescence-controlled programmed cell death in root hair lifespan regulation, with potential implications for optimizing plant nutrient and water uptake in crop breeding.