ABR/DPOAE publications

Background DFNB16, the second most common genetic cause of hearing loss, is caused by mutations of the STRC gene encoding stereocilin, a protein essential for the effective functioning of outer hair cells (OHCs) as cochlear amplifiers. Strc −/− mice, which lack stereocilin, display severe to profound deafness and constitute a relevant preclinical model for DFNB16. Methods Using Strc − / − mice, we developed a gene therapy strategy based on the use of dual AAV9‐PHP.eB vectors to deliver the full‐length Strc cDNA. Therapeutic efficacy was assessed by evaluating stereocilin expression, OHC bundle architecture, and their attachment to the tectorial membrane, together with functional recovery using distortion product otoacoustic emissions (DPOAEs), auditory brainstem responses (ABR) measurements and Go/No‐Go behavioral testing with psychometric analysis. Results Dual‐AAV–mediated Strc gene delivery restored stereocilin expression, OHC bundle architecture and their attachment to the tectorial membrane, leading to the recovery of cochlear amplification and hearing to near normal thresholds, as confirmed by distortion product otoacoustic emission (DPOAE) and auditory brainstem response measurements. Behavioural assessment showed that treated Strc −/− mice regained normal frequency discrimination, indicating a restoration of higher‐order auditory processing, up to 100 days post‐treatment. Conclusion These findings provide the first proof‐of‐principle that peripheral gene therapy can restore OHC function, cochlear amplification and central auditory perception in a DFNB16 model. Key points Dual AAV‐mediated gene delivery restored peripheral hearing in a DFNB16 preclinical mouse model. The same treatment also restored central auditory processing. AAV‐mediated gene therapy represents a promising curative strategy for DFNB16. These results reinforce the translational potential for treating human genetic deafness.

It is widely known that users of cochlear implants (CIs) often show deficits in binaural hearing. In particular, interaural time difference (ITD) discrimination tends to be substantially worse in CI users. However, only very few studies have investigated how CI stimulation affects the “precedence effect” (PE), and how this might be affected by abnormal hearing experience during development. We performed behavioral experiments to measure temporal weighting functions (TWFs) to quantify the PE in three different cohorts of rats: normally hearing (NH) acoustically stimulated, neonatally deafened (ND) CI-stimulated, and adult deafened (AD) CI-stimulated animals. Lateralization responses were recorded to bursts of eight stimulus pulses in which the ITD of each pulse varied independently, and probit analysis revealed the perceptual weight of each pulse. The NH and ND-CI cohorts were tested at 50, 300, and 900 pps; the AD-CI animals were tested only at 300 pps. The NH animals show a pronounced “onset dominance” at higher pulse rates, that is, the weighting of the first pulse was much larger than for subsequent pulses, exactly as one would expect given previous studies on human listeners. In contrast, the TWFs for the ND-CI and AD-CI animals were much flatter, with a reduction of onset dominance by ∼75%, especially for higher pulse rates. Since neither of our CI cohorts had any experience of reverberation in their electric hearing, we interpret these results to indicate that the PE may require domain-specific experience to develop.

Background: Cisplatin is a widely used chemotherapeutic agent, but its clinical application is limited by serious ototoxic side effects, including hearing loss, tinnitus, and vertigo. Inflammation is now recognized as a significant contributor to cisplatin-induced hearing loss, but the upstream regulators remain largely undefined. This study aimed to identify novel regulators of cisplatin-induced ototoxicity through CRISPR-Cas9 screening and to determine the role of growth arrest and DNA damage-inducible alpha (GADD45A) in cochlear inflammation and hearing loss. Methods: To identify upstream regulators of cisplatin-induced ototoxicity, a CRISPR-Cas9-based loss-of-function screen was first performed in OC1 cells. Following the identification of Gadd45a as a candidate gene, its effects on cisplatin-induced cytotoxicity were evaluated using cell viability assays, flow cytometry, and TUNEL staining. Integrated transcriptomic and proteomic analyses were subsequently conducted to elucidate the downstream molecular mechanisms. Finally, the protective role of Gadd45a inhibition was validated in vivo through siRNA delivery into the posterior semicircular canal and in Gadd45a conditional knockout (cKO) mice. Results: Gadd45a knockout significantly inhibited cisplatin-induced cell death in the OC1 cell line while simultaneously enhancing autophagy signaling. Transcriptomic profiling revealed a marked downregulation of the expression of C-X-C motif chemokine ligand (CXCL) family cytokines following Gadd45a loss, which was further supported by proteomic analysis of culture supernatants. Mechanistically, Gadd45a knockout specifically inhibited nuclear factor κB subunit 1 (NF-κB1) but not RELA proto-oncogene, NF-κB subunit (RELA). Immunofluorescence staining demonstrated that Gadd45a knockout inhibited the nuclear translocation of the NF-κB1 protein, and subsequent studies revealed that the NF-κB1 protein was degraded through the lysosomal pathway. In vivo, both si-Gadd45a injection into the semicircular canal and the knockout of Gadd45a effectively attenuated cisplatin-induced ototoxicity. Conclusions: This study identifies GADD45A as a key regulator of cisplatin ototoxicity and links the balance between apoptosis and autophagy with NF-κB1-CXCL-mediated inflammation. Targeted inhibition of GADD45A signaling may represent a promising therapeutic strategy to prevent hearing loss in patients receiving cisplatin chemotherapy.

Background Usher syndrome (USH) is an autosomal recessive disorder characterized by hearing loss, retinitis pigmentosa, and variable vestibular dysfunction. USH2A is one of the causative genes of USH. This study is aimed at exploring the mechanism of hearing loss induced by USH2A gene knockout. Method USH2A knockout (Ush2a −/− ) mice were used, and auditory brainstem response testing was performed on WT, Ush2a −/− , and Ush2a +/− mice. Then, the cochlea tissues were used to carry out immunofluorescence staining, hematoxylin and eosin (H&E) staining, and scanning electron microscopy (SEM). The mRNA expressions were detected by RT‐qPCR. Finally, the differentially expressed genes (DEGs) in cochlear tissues of Ush2a −/− and WT mice were identified by transcriptome sequencing. Results Compared to WT mice, Ush2a −/− and Ush2a +/− mice exhibited moderate‐to‐severe nonprogressive hearing loss, with more pronounced deficits at low (4 kHz) and high (32/24 kHz) frequencies. HE staining and immunofluorescence staining showed that the modiolus, stria vascularis, basilar membrane, and the number of inner hair cells and outer hair cells (OHCs) in USH2A knockout mice have not changed. However, SEM results showed that severe stereociliary collapse was evident in OHCs of the Ush2a −/− group. In addition, through transcriptomic analysis, 3632 upregulated genes and 2921 downregulated genes were obtained in the Ush2a −/− mice. Among these DEGs, the most DEGs associated with hearing loss were Scn2a, Shank2, Bsn, Fcer1g, Prkce, Tgfb1, and Irf7. Conclusion This study demonstrates that USH2A deficiency disrupts auditory function through stereociliary instability and dysregulation of genes critical for synaptic transmission and cytoskeletal dynamics.

Hearing impairments, as a prevalent and debilitating non-motor symptom of Parkinson's disease (PD), remain unclear in mechanisms. In this work, we established PD mouse and rat models by using 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and 6-hydroxydopamine (6-OHDA), respectively, and investigated their hearing functions and potential mechanisms through auditory brainstem response (ABR), distortion product otoacoustic Emissions (DPOAE), noise exposure, immunofluorescence labeling, volumetric measurement, and colocalization analysis. In MPTP-induced PD mice, we observed significant cholinergic fibers decompensation, heterogeneous dopaminergic fibers damage of cochlear efferent fibers, and adrenergic sympathetic fibers marked loss in the osseous spiral lamina (OSL), corresponding to insignificant cochlear hair cells, ribbon synapse alteration, and auditory sensitivity injury. While in 6-OHDA-induced PD rats, asymmetric alterations in cochlear cholinergic, dopaminergic fibers were found, accompanied by inconsistent adrenergic changes in the OSL, which matched unilateral hair cells, ribbon synapse damage, and hearing loss. Overall, findings from this work indicate that pathological alterations in the cochlea of PD mice and rats, particularly in efferent fibers, may be closely relevant to peripheral hearing alterations.

Sensorineural hearing loss (SNHL) is significantly more prevalent in individuals with chronic kidney disease (CKD) than in the general population. Although a strong independent association has been observed between kidney dysfunction and the risk of hearing loss, the underlying mechanisms linking these conditions remain poorly understood. This study investigated the pathophysiology of hearing impairment using adriamycin nephropathy (AN), a well-established animal model of CKD. AN was induced in male BALB/c mice using 10 or 12 mg/kg of adriamycin (ADR), resulting in severe kidney injury and concurrent hearing loss. ADR-treated mice exhibited significant glomerular injury, podocyte damage, and elevated renal neutrophil gelatinase-associated lipocalin (NGAL), along with increased serum creatinine and blood urea nitrogen levels. Hearing impairment was evident after 4-8 weeks of ADR treatment, as assessed by auditory brainstem response and distortion-product otoacoustic emissions, and was accompanied by cochlear hair cell loss and ribbon synapse degeneration. AN affected cochlear function by altering ion channel expression in the stria vascularis and inducing blood-labyrinth barrier (BLB) hyperpermeability, along with changes in endothelial cells, pericytes, and perivascular-resident macrophage-like melanocytes. AN significantly increased cochlear NGAL and NLRP3 levels at 4 and 8 weeks following ADR administration. NGAL was highly expressed in the tectorial membrane, cochlear neurons, and organ of Corti, while its receptor, 24p3R, was co-localized with NGAL. These findings demonstrated the role of NGAL and BLB disruption in ADR-induced SNHL, providing novel insights into the mechanistic link between CKD and hearing loss.

The hippocampus is classically linked to memory, yet increasing evidence points to a broader role in perceptual inference and deviance detection. Predictive coding theories propose that perception minimizes mismatches between expected and actual sensory input, expressed in neural signatures such as mismatch negativity (MMN) and P300. Although MMN arises mainly from sensory and prefrontal cortices, the hippocampus is anatomically interconnected with both and may also contribute to prediction error processing. We recorded single- and multi-unit activity and local field potentials (LFPs) from DG and CA1 in urethane-anesthetized rats during an auditory oddball paradigm and a no-repetition control sequence to dissociate prediction error from repetition suppression. Approximately 20% of hippocampal neurons were sound responsive, and a subset showed deviant selectivity. Spiking activity predominantly reflected prediction errors, while LFPs revealed complementary contributions from repetition suppression and prediction error. Early LFP components were enhanced for randomly presented deviants, whereas later components within the P300 latency range were stronger for predictable deviants, indicating temporally distinct phases of error signaling and top-down modulation. These findings identify the hippocampus as an active contributor to auditory deviance detection and support a hierarchical model in which hippocampal circuits participate in predictive sensory processing beyond memory.

Although age-related hearing loss (ARHL) is the most common sensory loss in older adults, underlying mechanisms remain unclear. Recent genome-wide association studies (GWAS) linked variation in several genes with ARHL risk, including KLHDC7B , a gene of unknown function not previously linked to hearing. We demonstrate Klhdc7b is expressed exclusively in sensory hair cells in mouse cochlea. Utilizing two independent mouse knockout models ( Klhdc7b IMPC-/- and Klhdc7b Regn Δ/Δ ) we find that absence of Klhdc7b leads to severe early-onset, progressive hearing loss. Hair cells appear to develop normally, but outer hair cells are progressively lost from base-to-apex of the cochlea, a common pathology in ARHL. These results suggest KLHDC7B is required for maintenance rather than development, of auditory function. The validation in mouse of a human ARHL GWAS association suggests other novel candidates should be investigated. Our work provides two mouse models to study KLHDC7B function, and for development of therapeutic tools for ARHL.

Auditory hypersensitivity is a prominent symptom in Fragile X syndrome (FXS), the most prevalent monogenic cause of autism and intellectual disability. FXS arises through the loss of the protein encoded by the FMR1 (Fragile X Messenger Ribonucleoprotein 1) gene, FMRP, required for normal neural circuit excitability. In the brainstem, FMRP is necessary for normal development of acoustic reactivity, and its loss has been implicated in audiogenic seizures (AGS) in Fmr1 knockout (KO) mice, modeling auditory hypersensitivity and seizures in FXS patients. The present study investigated the correlation between auditory brainstem function and behavioral expression of AGS at the early (postnatal day P20, infancy) and late (P32, juvenile) stages of auditory development in Fmr1 KO mice compared with wildtype (WT) mice, and in both females and males. We tested responsiveness to pure tones of select auditory pathway elements through auditory brainstem responses, and neural synchronization to amplitude envelopes of modulated acoustic stimuli through auditory steady‐state responses. AGS behavior was categorized for severity during 5‐min exposure to loud sound. Expression of the immediate early gene cFos was quantified as a marker for neuronal activity in the inferior colliculus. During infancy, more severe AGS expression in Fmr1 KO mice compared with WT mice was accompanied by increased responsiveness to acoustic stimuli at the level of the superior olivary complex and inferior colliculus, and stronger neural synchronicity in subcortical auditory neurons. Fmr1 KO mice also had higher cFos positive cell counts in the inferior colliculus after exposure to loud sound. With age, both AGS susceptibility and exaggerated acoustic stimulus‐evoked activity in the Fmr1 KO mice subsided. Intriguingly, Fmr1 KO mice displayed an altered developmental profile in both the threshold and amplitude of auditory brainstem response. Our findings support evidence that AGS activity relies upon hyperexcitability in the auditory system, including in the lower brainstem, possibly due to disturbed auditory maturation. Hyper‐synchronization to modulated sounds in subcortical auditory neurons seemed to predict AGS severity. The developmental trajectory of the auditory hyperresponsiveness and hypersynchrony suggests a transient processing alteration underlying heightened AGS susceptibility in Fmr1 KO mice. A better understanding of FXS‐related circuit and behavioral symptoms of auditory processing across development provides the potential to identify therapeutic strategies to achieve auditory function recovery in FXS.

Purpose Although human temporal bones suggest that cochlear synapse numbers decline with age and noise exposure, no validated diagnostic method exists. In animal models, cochlear synaptopathy is associated with reduced auditory brainstem response (ABR) wave 1 amplitude and envelope following response (EFR) magnitude for a sinusoidally amplitude modulated (SAM) tone. However, measuring SAM EFR at the optimal modulation frequency (1000 Hz) is difficult in humans. A rectangular amplitude modulated (RAM) tone may be more sensitive to synaptopathy, but this has not been validated in animals. In addition, because synaptopathy likely co-occurs with outer hair cell dysfunction (OHC), a diagnostic assay needs to be robust to abnormal auditory thresholds. The objective of this study was to evaluate the relative ability of ABR and EFR measures to predict synapse numbers in mice with varying degrees of synaptopathy and OHC dysfunction. Methods Distortion product otoacoustic emissions (DPOAEs), ABR, SAM EFR, and RAM EFR were recorded from 57 mice with a range of auditory thresholds and degrees of synaptopathy. Cross-validation was used to compare the relative ability of linear regression models incorporating different measures to predict synapse numbers. Predictions were confirmed histologically. Results RAM EFR modulated at 1000 Hz was the single best predictor of synapse numbers for broad synapse loss across frequency, while combining RAM EFR with ABR further improved predictions. In contrast, ABR best predicted focal synaptopathy. Incorporating DPOAEs improved predictions for EFR, but not ABR. Conclusion RAM EFR, ABR, and DPOAEs should be used in the future when predicting synapse numbers.

The cochlear implant is a widely available hearing restoration technology that can provide speech understanding in quiet environments. This technology struggles however, in noisy settings or situations involving multiple speakers. The primary cause of these performance limitations is a poor neural interface, in which the bony wall of the cochlea separates the electrode surface from the auditory nerve fibers, thus causing unwanted current spread and non-specific frequency activation. This study utilizes an alternative auditory prosthetic technology (auditory nerve implant, ANI) that enables direct auditory nerve stimulation, which provides a potentially superior neural interface and enables more precise targeting of auditory nerve fibers than traditional cochlear implants. As auditory nerve implants progress towards clinical translation, new implant designs and stimulation strategies will be created. Animal models to efficiently test and iterate through these new designs will be useful for the continued development of ANI technology. We present a viable surgical approach in the non-human primate (rhesus macaque) along with electrophysiological results that demonstrate robust activation of the auditory system at low current levels via intraneural stimulation. Our findings indicate that the rhesus macaque, which possesses an inner ear anatomy more similar to the human compared to other animal models used in the hearing field (e.g., rodents, felines and ferrets), has strong potential as a useful preclinical testbed involving an upright head model for future ANI prototypes and stimulation strategy development.

No pharmacological interventions exist that can restore or preserve auditory function in the mammalian cochlea. Auditory hair cells (HCs) do not spontaneously regenerate, leading to permanent hearing loss. In non-mammalian vertebrates, HC regeneration happens through proliferation and differentiation of their clonally related supporting cells (SCs). The present study supports the potential of quinoxaline (Qx), a nonsteroidal anti-inflammatory compound, to stimulate SC proliferation in the auditory sensory epithelium, a process that may prime the tissue for future HC regeneration. We synthesized a series of Qx derivatives by introducing various substitutions, ranging from hydrophilic to lipophilic. Seventy analogs were generated and tested in vitro and in vivo. Among those, only one (Qx-100) exhibited the best medicinal chemistry profile and was further modified to expand the structure–activity relationship of the chemotype, develop additional analogs, and optimize potency, bioavailability, and in vivo efficacy. Ten new lead variants were generated. Of those, Qx-294 and Qx-301 demonstrated promising in vitro Absorption, Distribution, Metabolism, and Excretion (ADME) profiles and were selected for further testing. Overall, both compounds were rapidly absorbed in zebrafish and mice and promoted cell proliferation in vitro and in vivo without signs of apoptosis, supporting their potential for sensory HC regeneration.

The gene therapy achieved by AAV-mediated otoferlin-overexpression is an effective therapeutic strategy for congenital deafness. However, achieving its physiological and endogenous patterns of expression remains challenging. Here, we generate the homologous mutation Otof c.1315 C > T (p.R439*), equivalent to OTOF c.1273 C > T (p.R425*) found in humans with profound deafness, to create a nonsense mutation-induced deaf mouse model. We then deliver the ‘RESTART v3’ system, which is a CRISPR-free RNA base editor for nonsense mutation suppression, into the cochlea of the mice. We achieve physiological otoferlin expression, and the edited premature termination codon is reverse-mutated to the original amino acid. We observe significant hearing restoration and enhancement of the behavioral auditory startle reflex. Thus, our study presents a successful RNA editing strategy to significantly restore hereditary deafness in mice carrying the specific Otof nonsense mutation, which holds great promise for future clinical translation.

Ciliogenesis requires an orchestrated interaction between the Golgi apparatus and centrioles via vesicle trafficking, yet this process is still poorly understood. Phosphatidylinositol 4-kinase β (PI4KB) is a conserved kinase that localizes to the Golgi for generating phosphatidylinositol 4-phosphate, an important lipid component related to cilium formation. Here, we demonstrate a previously uncharacterized mechanism of PI4KB in regulating Rab11a to enable proper ciliogenesis. PI4KB kinase activity maintains the normal vesicle density around the Golgi and Rab11a localization to centrioles in ciliogenesis. Inhibition of PI4KB activity leads to the reduced centriole localization but accumulation of Rab11a on the Golgi. We identified that the activation of Rab11a relies on PI4KB activity, while inactive Rab11a–guanosine diphosphate stably associates with Golgi and fails to undergo outward delivery. Autosomal-dominant nonsyndromic sensorineural hearing loss mutations in PI4KB abnormally intensify PI4KB interaction with Rab11a, leading to the aberrant subcellular Rab11a localization and defective ciliogenesis. Collectively, our study delineates a critical role for PI4KB in post–Golgi vesicle formation and ciliogenesis.

Objective: To investigate the delivery efficiency of human umbilical cord mesenchymal stem cell-derived exosomes (hucMSC-EXOs) via intratympanic injection into vestibular end organs, evaluate their protective effects against gentamicin-induced vestibular dysfunction and hearing loss on gentamicin-induced vestibular dysfunction and hearing loss, and explore their regulatory mechanisms on hair cell apoptosis and autophagy. Methods: Exosome characteristics were identified by transmission electron microscopy, nanoparticle tracking analysis, and Western blot. PKH26 labeling was used to trace their distribution in the vestibule. SD rats were randomly divided into four groups: control group, gentamicin group (GEN group), gentamicin + exosome group (GEN + EXO group), and gentamicin + dexamethasone group (GEN + DEX group). On day 6 after administration, vestibular function was assessed via open-field test and beam balance test. On day 7, high-frequency hearing (32 kHz) was detected by auditory brainstem response (ABR). The quantity and structural changes of hair cells were analyzed by immunofluorescence staining and scanning electron microscopy. Proteomics was used to analyze differentially expressed proteins in vestibular tissues treated with dexamethasone or hucMSC-EXOs. The regulatory effects on Caspase-3 (apoptosis) and LC3 (autophagy) were validated by immunofluorescence. Results: hucMSC-EXOs administered via intratympanic injection were found to target the utricle, saccule, and crista ampullaris. Behavioral studies showed that the GEN + EXO group exhibited significant suppression of gentamicin-induced reduction in total movement distance (p < 0.05) and movement speed (p < 0.05, superior to the GEN + DEX group), with a 60.5% reduction in beam balance test passage time (p < 0.05). ABR results revealed that the auditory threshold at 32 kHz in the GEN + EXO group was 18.3 dB SPL lower than that in the injury group (p < 0.01), with no statistical difference compared to the GEN + DEX group. Hair cell counting showed significant protective effects of exosomes in reducing hair cell loss in the utricular striola (+ 25%), saccular striola (+ 44%), and central crista ampullaris (+ 44%), with partial regions outperforming the GEN + DEX group (e.g., 109% repair rate in the saccular striola). Scanning electron microscopy confirmed that exosomes reduced hair cell cilia loss and structural vacuolization. Proteomics showed significant activation of the complement and coagulation cascades in the GEN group, while the GEN + EXO group enhanced autophagy via enriching the vesicle transport-related SNARE interaction pathway and improved energy metabolism and oxidative stress by regulating metabolic pathways. Immunofluorescence demonstrated that exosomes significantly reduced the expression of the apoptosis marker Caspase-3 (p < 0.01) and enhanced the activity of the autophagy marker LC3 (p < 0.05). Conclusions: hucMSC-EXOs delivered via intratympanic injection effectively target vestibular sensory epithelia, significantly suppressing gentamicin-induced balance dysfunction and high-frequency hearing loss, with superior efficacy in partial indicators (e.g., movement speed, saccular hair cell repair) compared to dexamethasone. hucMSC-EXOs exert protective effects through a dual mechanism of "anti-apoptosis and pro-autophagy," providing a novel therapeutic strategy for vestibular hair cell injury.

Sensorineural hearing loss (SNHL) caused by human cytomegalovirus (HCMV) infection involves alterations in both the central auditory pathways and cochlear structures. Immediate early (IE) proteins are critical for HCMV pathogenicity and have been associated with neurodevelopmental disorders; however, their contribution to HCMV-associated SNHL remains unclear. Here, we generated transgenic mouse models expressing HCMV IE1 and IE2 protein to investigate their effects on auditory function and cochlear pathology. Auditory brainstem response (ABR) measurements revealed that expression of IE2, but not IE1, led to significantly elevated ABR thresholds and impaired auditory processing. IE2-transgenic mice exhibited synaptic loss, hair cell degeneration, and neuronal atrophy in auditory regions. scRNA-seq analysis indicated broad activation of inflammatory pathways and cytokines within the cochlea, along with disruptions in mitochondrial and metabolic pathways, suggesting that IE2 may contribute to hearing loss through mitochondrial impairment and inflammation. Transmission electron microscopy of cochlear tissues showed severe morphological abnormalities and a marked reduction in mitochondrial number in spiral ganglion neurons (SGNs). Further mechanistic investigation demonstrated that IE2 interacts with TSC2, leading to hyperactivation of mTOR signaling, metabolic dysregulation, and mitochondrial dysfunction. Importantly, administration of mTOR inhibitors substantially alleviated IE2-induced auditory deficits, hair cell degeneration, and neuronal atrophy. These findings identify IE2 through TSC2-mTOR-mediated mitochondrial dysfunction, metabolic reprogramming, and neuroinflammation leading to SNHL. Our study reveals a previously unrecognized mechanism linking IE2 protein expression to auditory neurodegeneration and suggests mTOR modulation as a potential therapeutic strategy for congenital HCMV infection and associated hearing loss.

Damage to inner hair cells (IHCs) is a leading cause of hearing loss, typically initiating at the base region of the basilar membrane. However, the mechanisms and preventative strategies for IHC damage remain to be elucidated. This study revealed that IHCs in the low-frequency region exhibit a significantly faster calcium clearance rate than high-frequency IHCs. This difference is associated with different PMCA1 expression. We then generated an IHC-specific Pmca1 knockout mouse model ( Pmca1 CKO) exhibiting profound hearing loss and IHC death. Using single-cell RNA-seq analysis, we found that the differentially expressed genes (DEGs) were related to tetrahydrofolate biosynthesis, DNA damage, and DNA repair dysfunction. We therefore treated Pmca1 CKO mice with folic acid and found that it protected IHCs by reducing γ-H2A.X levels. In addition, we found that folic acid protected IHCs from noise-induced damage. Overall, our findings suggest that disrupted calcium homeostasis plays a role in IHC damage and that folic acid may be a promising therapeutic agent for protecting hair cells.

Age-related hearing loss (ARHL) represents a progressive auditory disorder. Growing evidence indicates that degradation of connexin 26 (Cx26) in cochlear may constitute one of the pathogenic mechanisms in ARHL. However, the molecular mechanisms underlying age-related Cx26 degradation remain unclear. We systematically analyzed Cx26 expression patterns in C57BL/6J mice across different age groups and confirmed an age-dependent downregulation of Cx26 in the mouse cochlea. Notably, we observed concurrent upregulation of endoplasmic reticulum (ER) stress markers Glucose-regulated protein 78 (GRP78) and protein disulfide isomerase (PDI) in aged cochlear tissues. Furthermore, in vitro experiments demonstrated that ER stress significantly reduced Cx26 protein levels in cochlear explants. Mechanistically, we observed enhanced expression of ubiquitination-related proteins Nedd4 and Eps15 with evident colocalization with Cx26 in an ER stress mouse model. In summary, our findings demonstrate that Cx26 ubiquitination mediated by cochlear ER stress is the main cause of Cx26 degradation in the cochlea of aged mice.