Auditory Research publications

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.

Oncomodulin (OCM) is the most abundant Ca2+ buffering protein found in mature outer hair cells (OHCs). Cadherin 23 (CDH23) is a crucial component of the tip-links in hair cell stereocilia. The absence or dysfunction of these two proteins contributes to the early onset of age-related hearing loss (AHL). In this study, we investigated the effects of the Cdh23753G→A mutation on OHC function using new Ocm-knockout (KO) mouse models (Ocmtm1a/tm1a) with or without the Cdh23753G→A mutation. Despite having the same genetic background, Ocm-KO mice carrying the Cdh23753G→A mutation displayed a notable decline in OHC function across all measured frequencies as early as three months of age. In contrast, Ocm-KO mice without the Cdh23753G→A mutation did not exhibit comparable hearing loss until they reached twelve months of age. Additionally, we examined the role of OCM in preserving OHC function under ototoxic stress induced by HPβCD (2-hydroxypropyl-β-cyclodextrin). The distortion product otoacoustic emission data show that the administration of HPβCD resulted in a more pronounced decline in OHC function in Ocm-KO mice compared to wild-type (WT) mice. Time-lapse recording also shows that HPβCD treatment led to greater structural deterioration and more rapid rupture events in OHCs from Ocm-KO mice than in those from WT mice. These findings suggest that the Cdh23753G→A mutation, rather than other potential strain-specific genetic factors associated with AHL, significantly exacerbates the early onset of AHL phenotypes in Ocm-KO mice. Furthermore, our data indicates that the OCM protein in OHCs enhances their ability to withstand ototoxic stimuli.

The CUGBP Elav-like family 4 (CELF4), an RNA-binding protein, is dynamically expressed in spiral ganglion neurons (SGNs) in the cochlea over development, but how Celf4 is involved in regulating hearing functions is poorly understood. In this study, we generated a Celf4 ± mouse line and examined changes in the first two synapses along the auditory pathways. Firstly, we found that hearing functions were largely intact in Celf4 ± mice, with exception of reduced amplitude for Wave II of auditory brainstem responses (ABRs) and increased delays for Wave II and IV, both in case of 4 kHz only. Secondly, we found that counts of inner and outer hair cells (IHCs and OHCs) and SGNs remained unchanged in Celf4 ± mice, and that the number and function of ribbon synapses between IHCs and SGNs were comparable between WT and Celf4 ± mice. Lastly, function of the endbulb of Held synapse, formed between auditory nerve fibers (ANFs) of SGNs and bushy cells in the cochlear nucleus, was significantly altered in Celf4 ± mice. Specifically, synaptic vesicle release was subtly reduced, and excitability of bushy cells was significantly dampened, likely due to a hyperpolarized resting membrane potential. Furthermore, we found that spike kinetics was significantly faster in Celf4 ± bushy cells, likely caused by a larger fast-inactivating A-type potassium current (I A ) found in these cells. In conclusion, we found that Celf4 haploinsufficiency altered transmission at the endbulb of Held synapses in the cochlear nucleus in a significant and multifaceted manner, revealing the roles of Celf4 in regulating hearing functions. Graphical Abstract Title: Schematic representation of the experimental approach and major findings. Legend: In mice with Celf4 haploinsufficiency, we found that while function at hair cell ribbon synapses was largely preserved, transmission at the endbulb of Held synapse was significantly altered, including subtly reduced release of synaptic vesicles from the endbulb, dampened excitability in bushy cells, and accelerated spike kinetics caused by enhanced voltage-gated K + current.

Activity-dependent modulation of presynaptic voltage-gated Ca 2+ channels (Ca V 2) regulates Ca 2+ influx to control neuronal circuit output. Although Ca V 2.1 can undergo robust Ca 2+ dependent facilitation (CDF), whether it occurs in native central nervous system neuronal circuits is disputed. Accurate auditory information processing requires precise and reliable synaptic transmission at high activity rates in the auditory brainstem. To determine if Ca V 2.1 CDF is a key regulator of high-fidelity synaptic transmission, we expressed Ca V 2.1 splice variants capable (Ca V 2.1 37a) or incapable (Ca V 2.1 37b) of CDF at the calyx of Held presynaptic terminal in the auditory brainstem. We found no difference in basal Ca V 2.1 currents or synaptic transmission. However, Ca V 2.1 37b terminals lacked CDF, synaptic facilitation and had a decreased reliability and precision of postsynaptic action-potential firing. Additionally, the Wave III amplitude of the auditory brainstem responses was reduced. We propose that Ca V 2.1 CDF is essential for accurate auditory information processing.

Cochlear implants (CIs) have successfully restored hearing in more than one million patients with severe to profound hearing loss worldwide. Although CIs effectively restore speech perception in quiet environments, sound localization remains challenging for bilateral CI users, particularly their ability to utilize interaural time differences (ITDs). The majority of clinical CI processors use a coding strategy that encodes ITD information only in the envelope of electrical pulse trains rather than their pulse timing, which may contribute to the poorer spatial hearing perception of CI users. We recently demonstrated in a behavioural study on early deafened, bilaterally CI‐implanted rats that pulse timing ITDs completely dominate ITD perception, whereas sensitivity to envelope ITDs is almost negligible in comparison. Building on this, we here investigated the neurophysiological sensitivity of the inferior colliculus (IC) to envelope and pulse timing ITDs at two different pulse rates (900 and 4500 pulses/s) and three different stimulation modulations (5, 20 and 100 Hz) in CI rats with different hearing experiences. Our results indicate that IC neurons exhibit far greater sensitivity to pulse timing ITD than envelope ITD independent of pulse rate, modulation rate or hearing experience. These findings suggest that to improve binaural hearing outcomes in bilateral CI users, clinical stimulation strategies should provide informative pulse timing ITDs. image Key points Current bilateral cochlear implants mostly provide time cues of arriving sound in the envelope of stimulation rather than in the timing of pulses. A behavioural study on cochlear implanted rats has shown that pulse timing dominates binaural hearing. Here we examined the neural sensitivity to independent changes in time cues presented on the envelope or pulse timing, respectively, in rats with bilateral cochlear implants and different hearing experiences. Auditory midbrain neurons were consistently more sensitive to changes in pulse timing rather than envelope, irrespective of stimulus pulse rate, modulation rate or an animal's hearing experience.

Oxidative stress and inflammation are interconnected drivers of cellular damage in pathologies ranging from neurodegenerative disorders to sensorineural hearing loss. We aimed to develop a chitosan-Prussian blue nanozyme (CS-PB) to target these processes in sensorineural hearing loss. CS-PB (35 μg/mL) pretreatment of H 2 O 2 -injured HEI-OC1 cochlear cells for 4 h markedly decreased the levels of reactive oxygen species (ROS) inside cells (from 3.8 to 2.4 relative fluorescence intensity) by approximately 37% ( P < 0.001)-and concomitantly significantly decreased expression of the oxidative damage markers 4-HNE and 3-NT (both P < 0.001). The rate of apoptosis decreased from 27.5% in the H 2 O 2 -treated group to 14.1% following CS-PB treatment (P < 0.01). This reduction was accompanied by a significant downregulation of the pro-apoptotic proteins Bax (P < 0.05) and Cleaved-caspase-3 (P < 0.001), as well as an upregulation of the anti-apoptotic protein Bcl-XL (P < 0.01).Western blot analysis confirmed that CS–PB significantly downregulated TLR4 expression and inhibited downstream phosphorylation of p-P65/P65, while upregulating p-IκBα/IκBα protein (all P < 0.01) associated with reduction in the production of inflammatory cytokines like TNF-α, IL-1β, and IL-6. Treatment with the specific TLR4 antagonist TAK-242 (2 μM) mimicked the effect of CS-PB, with coadministration showing no additional ant-inflammatory effect (although HEI-OC1 viability was additionally increased), indicating that the anti-inflammatory properties of CS-PB were facilitated via the TLR4/NF-κB signaling pathway. In vivo , CS-PB pretreatment (2 mg/mL, 2 μL) was administered by carefully applying the solution to the round window membrane (RWM) using a microsyringe in a noise-induced hearing loss rat model, which significantly curtailed the noise-induced activation of the TLR4/NF-κB pathway in the cochlea. After 24 hours, the levels of p-P65/P65 and TLR4 decreased by 54% and 50%, respectively. ( P < 0.001), and levels of the inflammatory cytokines IL-6, TNF-α and IL-1β were decreased by 53%, 48%, and 51% ( P < 0.001). Furthermore, the degree of pathway inhibition showed a strong correlation with cytokine reduction (r = 0.87–0.89, all P < 0.001). CS-PB showed no significant cytotoxicity in vitro or in vivo , suggesting that its combined ROS-scavenging and TLR4/NF-κB–modulating properties may represent a potential therapeutic strategy, pending further mechanistic and translational validation.

Many neurological and psychiatric disorders stem from maladaptive conditioning and resist treatment. Tinnitus, as one example, is the distressing perception of sound in the absence of an external source. One of the network systems implicated in multiple disorders is the 'salience network', associated with positive/negative reward, habit formation, motivation, valuation and mood. However, it is not known whether directly modulating the 'salience network' at its anatomical and physiological core, in the ventral striatum, more specifically the nucleus accumbens, could be beneficial. Here we show that a novel treatment approach, targeting the salience network, can reduce tinnitus perception in a rat model. We found that bilateral rewarding stimulation of the nucleus accumbens, when paired with non-tinnitus tones, was most effective. A combined approach using rewarding accumbens stimulation and dysrewarding lateral habenula stimulation - paired with non-tinnitus and tinnitus tones, respectively - also suppressed tinnitus but caused negative effects. Our results demonstrate how the rewarding stimulation can recondition brain networks to effectively remove the salience of tinnitus frequencies. We anticipate that our unique reconditioning strategy has broader therapeutic applications for other brain disorders rooted in maladaptive learning.

Cisplatin is widely used in oncology yet causes dose-limiting, irreversible hearing loss through cochlear hair-cell injury. Although mitochondrial dysfunction is central to this ototoxicity, how the outer mitochondrial membrane (OMM) fails in hair cells remains unclear. We identify BAX-associated mitochondrial membrane rupture and abnormal permeabilization as key drivers. In HEI-OC1 cells and murine cochlear explants, super-resolution and transmission electron microscopy revealed cisplatin-induced BAX translocation and oligomerization on the OMM, forming ring-like assemblies with ultrastructural damage (rupture, herniation, fragmentation). Subcellular fractionation and functional assays demonstrated consequent mitochondrial outer membrane permeabilization, cytochrome c release, loss of membrane potential, and accumulation of reactive oxygen species (ROS). Pharmacologic inhibition of BAX oligomerization with BAX inhibitor peptide V5 (BipV5) preserved mitochondrial integrity, reduced ROS, and limited hair-cell loss in vitro. In vivo, repeated transtympanic delivery of BipV5 conferred sustained functional protection and reduced outer hair cell loss. These findings establish a structural mechanism linking mitochondrial membrane rupture to redox imbalance and apoptosis in cochlear hair cells and nominate BAX oligomerization as a drug-targetable node for preventing cisplatin ototoxicity without compromising anticancer therapy.

Sensorineural hearing loss (SNHL) induced by noise or aminoglycoside antibiotics is a significant public health concern without any FDA‐approved pharmaceutical therapies. Dysregulation of iron homeostasis and its subsequently induced ferroptosis has increasingly been identified as a key mechanism underlying cochlear hair cell (HC) damage. Nevertheless, the therapeutic targets for restoring iron balance for hearing protection remain poorly investigated. In this study, we uncover a previously unrecognized role of the SMO pathway in regulating iron homeostasis. SMO expression was rapidly upregulated and activated in HCs following injury. Both genetic ablation of Smo and pharmacological inhibition of SMO reduced iron accumulation and lipid peroxidation, promoting HC survival and preserving auditory function in mouse models of ototoxic‐ and noise‐induced hearing loss. Mechanistically, SMO inhibition suppressed ATF2 phosphorylation, resulting in downregulation of IRP1, which decreased iron accumulation via downregulation of Tfrc and upregulation of Fpn , ultimately protecting HCs from ferroptosis. Notably, treatment with the SMO inhibitor SANT‐1 nearly restored auditory thresholds to baseline levels in mice subjected to ototoxic injury. Our findings identify the SMO–ATF2–IRP1–FPN/TFRC axis as a central regulator of cochlear iron homeostasis and propose SMO inhibition as a promising therapeutic strategy for SNHL through precise modulation of iron metabolism.

Lipid metabolism is an important biological process that maintains the dynamic balance of several key functions, such as intracellular energy metabolism, signal transduction, and membrane remodeling. However, the role of lipid metabolism in auditory function and the underlying mechanisms remain unclear. Our results reveal that the neomycin exposure disrupts lipid metabolism in auditory system. We find that neomycin-induced hair cells (HC) damage leads to abnormal lipid droplets (LD) accumulation. Further research reveals that decreased YAP expression is a key factor that contributes to abnormal LD accumulation. In both in vivo and in vitro studies, Yap overexpression reduces abnormal LD accumulation and mitigated HC damage. To further investigate its downstream mechanisms, we perform a cross-analysis of Yap -related and lipid metabolism–related genes, identifying that Cox2 is a key downstream target of Yap that contributes to LD accumulation and HC damage. Our work provides clear evidence for the role of lipid metabolism in neomycin-induced hearing loss and elucidates underlying mechanism of Yap/Cox2 pathway. These findings provide new perspectives and avenues for the clinical treatment of sensorineural hearing loss.

Cisplatin (CDDP) is an effective chemotherapeutic agent used to treat solid tumors, but it can cause irreversible hearing loss. Currently, there are no specific preventive measures available for this side effect. G protein-coupled receptor 55 (GPR55) exhibits antioxidant, anti-inflammatory, and anti-apoptotic properties and is implicated in various disease processes. Nevertheless, whether GPR55 plays a role in CDDP-induced hearing loss remains unclear. We explored the effects and mechanisms of O-1602, a GPR55 agonist, on CDDP-induced ototoxicity. Our results showed that GPR55 is present in cochlear hair cells and HEI-OC1 cells, with increased expression following CDDP exposure. Moreover, O-1602-induced activation of GPR55 markedly mitigated the ototoxic effects of CDDP in HEI-OC1 cells, cochlear explants, and mouse models by preventing oxidative stress and apoptosis. In addition, GPR55 protected against CDDP-induced damage via inhibiting the MAPK pathway. Therefore, GPR55 is a potential therapeutic target for preventing CDDP-induced ototoxicity.

Background Ménière’s disease (MD) is a disabling inner-ear disorder. Although the key pathologic alteration of MD is characterized as endolymphatic hydrops (EH), the underlying pathogenic mechanism remains unclear, and no definitive curative treatments are available. Emerging evidence implicates oxidative stress and innate immunity as key contributors to MD pathogenesis. Methods Human vestibular end organs were collected to characterize the reactive oxygen species (ROS) levels and the STING signaling activation, findings that were further corroborated in an LPS-induced EH mouse model. Transfection and transwell co-culture were performed to investigate the feedback loop between sensory epithelial cells and macrophages in vitro. Molecular biology experiments were performed to elucidate the underlying regulatory mechanisms. Additionally, STING and CUL4B deficiency mice were used to explore potential therapeutic targets in vivo. Results The oxidative stress, cytoplasmic dsDNA leakage, and STING activation were elevated in both MD patients and LPS-induced EH mouse model. The dsDNA released from ROS-damaged hair cells activates STING in vestibular macrophages. STING-specific knockout in macrophages alleviates EH, audio-vestibular dysfunction, and interferon-stimulated genes expression in mouse model. The CUL4B-DDB1-ROC1 complex ubiquitinates STING at K370, promoting its degradation and inhibiting activation. Notably, CUL4B deficiency exacerbates LPS-induced EH, audio-vestibular dysfunction, and interferon-stimulated genes (ISG) upregulation, while double conditional knockout reverses these effects. Conclusion Our study demonstrates that STING activation in vestibular macrophages contributes to audio-vestibular dysfunction and modulating this pathway has promising beneficial effects on audio-vestibular function in EH mice model, which is a potential therapeutic strategy for MD.

Ménière's disease (MD), a chronic inflammatory disorder with age‐related increased incidence, exhibits poorly understood pathogenesis and limited therapeutic options. Here, we demonstrate that cellular senescence, marked by mitochondrial damage, reactive oxygen species accumulation, and senescence‐associated secretory phenotype (SASP), is prevalent in the vestibular tissue of MD patients and an endolymphatic hydrops mouse model. The transcription factor GATA4 is upregulated in MD and mice, and its genetic deletion in hair cells alleviates LPS‐induced audio‐vestibular dysfunction and cellular senescence in mice and HEI‐OC1 cells. Mechanistically, HDAC6 interacts with GATA4 and restrains its nuclear transport, while RNA‐seq and ChIP‐seq identify HtrA1, a serine protease, as a direct transcriptional target of GATA4. Inhibition of HDAC6 or AAV‐mediated HtrA1 overexpression exacerbates MD‐like symptoms, whereas inhibition of HtrA1 by Galegenimab ameliorates these phenotypes in mice. In aged mice, GATA4 deletion reduces age‐related audio‐vestibular deficits and senescence markers. Collectively, our findings establish GATA4 as a critical regulator of cellular senescence and inflammaging in inner ear pathologies, providing promising therapeutic targets for MD and age‐related audio‐vestibular disorders.

Ultrasonic vocalizations (USVs) are widely used in rodent social communication, yet the functional significance of male–male vocal interactions in mice remains unclear. Here, we investigated whether USVs produced during specific social behaviors influence the behavior of conspecifics. Using playback experiments, we compared responses to vocalizations recorded during chasing and being chased in male–male interactions. We found that USVs emitted by chased intruders consistently elicited approach behavior in receiver mice, whereas those emitted by chasing individuals did not. Acoustic analyses revealed that these vocalizations differed in syllable composition, with intruder calls containing a higher proportion of upward frequency-modulated syllables and exhibiting higher mean frequencies. In addition, the temporal organization of syllables appeared to contribute to the behavioral response. Together, these results suggest that male mice respond selectively to certain USV patterns associated with specific social contexts, indicating that acoustic features and temporal structure may jointly influence social approach behavior in mice. Highlights Behavioral context (chased vs. chasing) shapes the composition of USV syllable types Male mice selectively approach USVs from chased intruders, but not chasing residents The approach response exhibits high temporal synchrony across individual receivers Temporal organization of syllables modulates approach behavior based on acoustic features

The molecular and cellular mechanisms underlying the function of the cochlear nucleus (CN) remain to be fully elucidated. Using single-nucleus RNA sequencing and single-cell spatial transcriptome analyses, we generated a comprehensive cell type atlas of the mouse CN, identified molecularly defined CN subregions, and quantified changes in gene expression and the spatial organization of CN cells in normal mice during postnatal development and in mutant mice with congenital hearing loss. We further identified a subtype of bushy cells expressing the osteopontin-encoding gene Spp1 as the primary CN cell type that exhibited hearing loss-induced alteration of gene expression. Among the highly affected genes in bushy cells, deletion of the auditory input-regulated gene Spp1 affected CN processing of auditory signals in mice. These results provide the most comprehensive cellular and molecular database to date for understanding auditory processing within the CN and identifying potential therapeutic targets for hearing restoration at the CN level.

Across sensory modalities, peripheral deafferentation can induce maladaptive central plasticity that amplifies sensory responses while distorting affective processing, rendering innocuous stimuli unpleasant, intrusive, or even painful. Here, we examined the affective dimension of compensatory plasticity in a mouse model of hyperacusis by introducing noise-induced focal cochlear lesions and tracking maladaptive plasticity in the lateral amygdala (LA), a key limbic hub for sensory-affective valuation. Whereas control mice habituated to neutral sounds, mice with noise-induced hearing loss (NIHL) showed sustained LA hyperresponsivity and abnormally strong temporal coupling between calcium transients and pupil dilations, an autonomic index of arousal. NIHL also disrupted discriminative auditory threat learning, producing poorly selective, non-extinguishing enhancement of LA responses and freezing to both threatening and non-threatening sounds. We posited that potentiating inhibition in the higher-order auditory cortex (HO-AC), a prominent source of auditory sensory input to the LA, could reinstate normal affective sound processing after NIHL. Brief bouts of 40-Hz optogenetic activation of HO-AC parvalbumin-expressing inhibitory neurons (PVNs) durably reversed LA sensitization, normalized pupil-indexed arousal dynamics, and reinstated discriminative auditory threat memory. These findings identify cortical inhibitory potentiation as a strategy for reversing neural, autonomic, and behavioral signatures of distorted affective sound processing after peripheral sensory injury.

Background: Kölliker’s organ (KO) support cells undergo orderly, time-dependent degeneration that is essential for auditory development and is accompanied by precisely regulated autophagic activity; however, the molecular hierarchy linking autophagy to this remodeling remains obscure. This study aimed to elucidate the regulatory mechanisms connecting autophagic flux to lysosomal biogenesis and auditory function during cochlear development. Method: We established an Atg5flox/flox; Sox2Cre+ mouse model with deletion of the autophagy gene Atg5 in cochlear-supporting cells. Auditory function was assessed via Auditory Brainstem Response (ABR) testing. Transcriptomic profiling of the neonatal basilar membrane was performed to screen for downstream targets. Mechanistic validation included spatiotemporal immunofluorescence mapping (E18–P30) and in vitro functional assays using siRNA-mediated knockdown and lysosomal tracking. Results: At 2 months of age, Atg5flox/flox; Sox2Cre+ mice exhibited moderate-to-severe sensorineural hearing loss accompanied by significant outer hair cell loss. Bulk RNA-seq of the basilar membrane identified fork-head box A3 (Foxa3) as a significantly downregulated transcription factor within the lysosomal–autophagy network. Spatiotemporal immunolabelling from embryonic day 18 to postnatal day 30 revealed that FOXA3 expression becomes progressively restricted to KO cells during postnatal development, with ATG5 loss reducing FOXA3 protein levels by 62.4%. In vitro, deficiency of either Atg5 or Foxa3 in primary KO cells resulted in comparable reductions in LAMP1-positive puncta. Conclusions: These findings support a model wherein the ATG5-FOXA3 axis contributes to lysosomal biogenesis in developing KO cells, with implications for understanding mechanisms of congenital sensorineural hearing loss.

Noise exposure is a known cause of hearing loss, and only a few effective preventive drugs are available. Therefore, in this study, we aimed to investigate the protective effects of brimonidine on noise‐induced inner ear hearing impairment in mice and explore its underlying mechanisms and long‐term outcomes. Mice were randomly divided into control, noise exposure, and brimonidine groups. A 62‐week follow‐up was conducted after noise exposure. Brimonidine inhibited the noise‐induced increase in inner ear glutamate concentration and downregulated inflammatory factors and immunoglobulins. Brimonidine decreased glutaminase and VGLUT2/3 expression and reduced glutamate synthesis and vesicle transport without affecting its clearance, thereby decreasing glutamate excitotoxicity and protecting synapses and spiral ganglion neurons long term. Mice exposed to noise could temporarily restore their hearing thresholds; however, their auditory function in old age remained significantly worse than those that received brimonidine‐mediated cochlear protection in youth. These findings highlight the importance of enhancing noise protection from an early age.

Approximately 50% of hereditary deafness cases and most age-related hearing loss result from cochlear hair cell dysfunction. Adeno-associated virus (AAV)-mediated gene therapy offers a promising strategy for hearing restoration. However, the cochlea's complex cellular composition poses a significant challenge for targeted gene delivery into hair cells. Here, we establish a cross-species screening platform for cochlear AAV tools, applicable from rodents to large animals. Using this platform, we identify human promoters that specifically target inner hair cells, outer hair cells, or all hair cells in mice. The hair cell-specific coProB2 promoter is further validated in Bama miniature pigs and cynomolgus monkeys, achieving specific transgene expression in hair cells without auditory toxicity. Importantly, coProB2-driven gene therapies in DFNB79 and DFNB9 mouse models restore auditory function to near wild-type levels. Thus, this study establishes a cross-species-compatible, hair cell-specific AAV system, providing a versatile toolbox for cochlear hair cell gene therapy.

Background: Genetic diagnosis plays a critical role in enhancing the identification and clinical management of pediatric hearing loss, ultimately improving both physiological outcomes and social-emotional development. As one of the most prevalent sensory disorders characterized by substantial genetic heterogeneity, hearing loss requires the implementation of efficient diagnostic approaches to inform targeted clinical interventions and personalized care strategies. Methods: Here, we recruited 3353 Han Chinese children with nonsyndromic hearing loss, including 598 multiplex cases with family history. We employed SNPscan, targeted panel sequencing (TPS), and whole-exome sequencing (WES) to delineate the mutation landscape. Findings: Utilizing a tiered genomic testing strategy, we performed SNPscan for all 3353 participants, TPS for 675 cases, and WES for 204 multiplex cases. We achieved an overall diagnostic yield of 48.3% (1620/3353) in this cohort. Our analysis identified 87 previously unreported variants across 24 genes, including a de novo FOXI1 mutation (c.479_481del) linked to Mondini malformation and enlarged vestibular aqueduct, validated through knock-in mouse models. Additionally, we discovered NEU4 as a candidate gene related to hearing loss, with knockout mice and cellular models demonstrating its role in auditory dysfunction via disrupted neuraminidase activity and axonal development. These findings enrich the spectrum of deafness-related genes and provide a new theoretical foundation for diagnosing hearing loss. Interpretation: This study not only expands the genetic spectrum of hearing loss but also underscores the clinical utility of advanced sequencing in guiding precision therapies and early syndromic surveillance. This study offers a detailed characterization of the genetic landscape of pathogenic variants associated with hearing loss in the Han Chinese population, which will contribute to the design of a cost-effective genetic screening scheme, and also provides valuable insights that contribute to the global understanding of the genetics of hearing loss. Funding: This work was supported by the Key Program of National Natural Science Foundation of China, the National Key Research and Development Program of China, the National Natural Science Foundation of China and the Major Fundamental Research Program of the Natural Science Foundation of Shandong Province, China.