ABR/DPOAE publications

Hearing loss is the most common sensory disorder, with a substantial proportion caused by genetic mutations. KCNQ4, a voltage-gated potassium channel highly expressed in cochlear outer hair cells, is a common genetic etiology implicated in autosomal dominant progressive hearing loss (DFNA2). The dominant-negative KCNQ4 p.W276S (c.827G>C) mutation represents a mutational hotspot in DFNA2, yet no effective treatments exist. Here, we developed allele-preferential antisense oligonucleotides (ASOs) targeting this dominant-negative KCNQ4 mutation. In a systemic in vitro screen, ASO-123 demonstrated a knockdown of mutant Kcnq4 while preserving wild-type transcripts. In a Kcnq4 p.W277S knockin mouse model mimicking DFNA2, ASO-123 preferentially suppressed mutant transcripts, attenuated progressive hearing loss, and improved outer hair cell survival while enhancing their electrophysiologic function. Comprehensive transcriptomic analyses further validated the efficacy of ASO-123. Thus, our findings establish ASO-based therapy as a promising strategy for treating hereditary hearing loss caused by dominant-negative KCNQ4 mutations.

Biallelic loss-of-function variants in the Otoferlin gene (OTOF) lead to congenital hearing loss in both humans and mice. We developed DB-OTO, a hair-cell-specific adeno-associated virus (AAV)-based dual-vector gene therapy designed to restore hearing in individuals with OTOF-related hearing loss. DB-OTO is composed of two AAV1 vectors that reconstitute a functional OTOF gene expression cassette to form the full-length human OTOF variant 5 (h OTOFv5). A synthetic promoter (mMyo15) based on the murine Myosin 15 a gene was engineered to restrict the transgene expression to hair cells. The promoter construct was chosen after comparison of multiple variants using a GFP reporter in ex vivo murine explant models, followed by in vivo studies in wild-type mice and cynomolgus macaques. Comparison between the 1.0 kb mMyo15 and a ubiquitous promoter driving expression of h OTOFv5 transgene in mice showed that hair-cell-specific expression is critical for safe expression of otoferlin. DB-OTO induced dose-dependent establishment of auditory brainstem response, and OTOF expression in inner hair cells over a 10-fold dose range sustained for at least 3 months in a mouse model of OTOF deficiency. These data supported the initiation of an ongoing phase I/II clinical trial of DB-OTO in pediatric patients with OTOF-related hearing loss.

Noise-induced hearing loss can lead to degeneration of spiral ganglion neurons (SGN), resulting in impairment of auditory signalling. However, the specific mechanism is unclear. In our study, we investigated the function of mitochondrial activity following noise exposure. Findings revealed a reduction in ATP synthesis and alterations in the levels of mitochondrial electron transport chain complexes II, Ⅲ, IV and complex I subunit ND3 reduction by the seventh day post-noise exposure. These findings indicate that SGN mitochondrial dysfunction can impair SGN function. This is further demonstrated by the reduced expression of the mitochondrial marker protein Tom20 and apoptosis-inducing factor (AIF) showed inner membrane but not outer membrane resulted in changes in mitochondrial ETC complexes. And the findings of calpain 2 but not calpain 1 high expression was accompanied in AIF reduction, suggesting AIF has not yet caused SGN deaths. Calpain 2 regulated decreased expression of AIF may relieve the low function of mitochondrion in SGN after noise.

Tinnitus, the perception of a phantom sound, often occurs as a clinical sequela of auditory traumas. However, the underlying mechanisms of tinnitus are largely unknown. In our previous studies, we found more gamma-aminobutyric acid A receptor alpha 1 subunit (GABAAR-α1)-positive cells in the dorsal cochlear nucleus (DCN) after noise exposure, however, we were not able to identify the specific types of DCN cells that up-regulated GABAAR-α1 after the insult. In the current study, we used Nissl staining, Purkinje cell protein 4 (PCP4) and glutamic acid decarboxylase 67 (GAD67) immunolabeling to identify GABAAR-α1-positive cells in the DCN. Each type of GABAAR-α1-positive cells was quantified and statistically analyzed using immunostaining and Nissl staining according to their morphology, size and location in the DCN. GABAAR-α1-positive cartwheel cells, Golgi cells, as well as ML-stellate and vertical cells were confirmed by dual immunolabeling. In the DCN, the most common GABAAR-α1-positive cells were Golgi cells followed by vertical cells and cartwheel cells while very few other cells were GABAAR-α1-positive in all conditions. We found significantly more GABAAR-α1-positive Golgi cells in the DCN of noise-exposed rats without behavioral evidence of tinnitus compared to normal controls and noise-exposed rats with behavioral evidence of tinnitus. This heightened "context-dependent" inhibition may help to maintain a balanced neuronal network, preventing the potential for tinnitus-related hyperactivity in the auditory pathways.

Outer hair cells (OHCs) in the cochlea contain specialized stereociliary structures essential for auditory function. These include horizontal top connectors (HTCs), linking adjacent stereocilia and tectorial membrane (TM)-attachment crowns (ACs), anchoring the tallest stereocilia to the TM. The known molecular components of these structures, such as stereocilin, otogelin, otogelin-like, and tubby, lack transmembrane domains, suggesting the existence of anchoring proteins. This study identifies TMEM145, a transmembrane protein with a Golgi dynamics domain, as a crucial OHC stereocilia component. TMEM145 is expressed in both OHCs and spiral ganglion neurons, with specific localization to TM-ACs and HTCs in OHCs. Tmem145 knockout (KO) mice exhibit profound hearing impairment at 3 weeks of age, with complete loss of distortion product otoacoustic emissions, indicating OHC dysfunction. Immunostaining and scanning electron microscopy reveal the absence of TM-ACs and HTCs in Tmem145 KO mice. In heterologous cell systems, TMEM145 interacts with stereocilin and tubby, facilitating their extracellular secretion. TMEM145 is undetectable in Stereocilin KO and tubby mutant mice, indicating interdependence among these proteins. These findings establish TMEM145 as an essential membrane protein for the structural integrity of OHC stereocilia, providing insights into the molecular architecture of cochlear hair cells and their role in auditory function.

Introduction Age-related hearing loss (presbycusis) is the most common sensory deficit in the elderly, yet it lacks effective pharmacological treatments. The decline of the cyclic AMP (cAMP) signaling pathway is implicated in aging, but its functional role in the auditory system remains largely unknown. Methods To investigate whether enhancing cAMP signaling could counteract presbycusis and elucidate the underlying molecular mechanism, we used a D-galactose-induced cellular senescence model and an in vivo aged mouse model. The effects of the cAMP analog dibutyryl-cAMP (dbcAMP) on cellular senescence, inflammation, mitochondrial function, and cochlear structure were evaluated. The role of Mitofusin-2 (MFN2) was further assessed using siRNA-mediated knockdown. Results dbcAMP potently suppressed cellular senescence and the associated inflammatory phenotype. In addition, cAMP treatment alleviated mitochondrial dysfunction, as indicated by improved mitochondrial bioenergetics and morphology. The anti-senescence effects of cAMP were significantly blunted upon siRNA-mediated knockdown of Mfn2, establishing MFN2 as a key, though perhaps not the sole, component of this protective pathway. Importantly, systemic administration of dbcAMP to aged mice significantly preserved hearing function and protected cochlear hair cells. This in vivo protection was accompanied by an upregulation of cochlear MFN2 and a coordinated suppression of senescence and inflammation markers. Discussion Our study reveals a critical protective role for the cAMP-MFN2 axis in auditory aging by suppressing cellular senescence. These findings identify this axis as a novel and promising therapeutic target for the treatment of age-related hearing loss.

Context‐dependent sensory processing within the predictive coding framework relies on detecting mismatches between incoming stimuli and internal predictive models. Sensory deviants elicit prediction errors, seen as enhanced neural responses, that update these models and influence attention and behaviour. Although prediction errors have been widely observed across brain regions, the downstream processes remain poorly understood. In this study, we recorded electrocorticography in five urethane‐anaesthetised rats and identified cortical slow oscillations, characterised by spontaneous transitions between ‘Up’ and ‘Down’ states. Deviant stimuli in an auditory oddball paradigm evoked an initial positive prediction error, followed by a prolonged, all‐or‐nothing response which spread in a travelling wave across the cortex. Identified as putative evoked cortical Up states, these responses were not evoked by standards, omissions or a many‐standards control. Up states following deviants occurred more recently after a previous Up state when compared to spontaneous Up states. In preliminary data from an awake rat, long‐latency Up states were not present spontaneously or evoked. In a different rat, anaesthetic depth was key to spontaneous and evoked Up states, with more robust Up/Down states and more reliable triggering of Up states under deeper anaesthesia. These results suggest that sensory deviants may cause shifts in cortical state under anaesthesia, explaining the long‐latency mismatch response under anaesthesia and sleep.

Purpose Measures of the human medial olivocochlear reflex (MOCR) typically rely on long duration (> 100 ms) or continuously presented broadband elicitors. MOCR gain reduction measured by otoacoustic emissions (OAE) exhibits multiple time constants, including in the hundreds of milliseconds, when elicited by broadband noise. Psychoacoustic studies of gain reduction have largely adopted these elicitor characteristics, but less is known about how broadband elicitor duration affects auditory perception. Additionally, the literature on the relationship between psychoacoustic and OAE measures of gain reduction has yielded mixed results, which is counterintuitive if both measures reflect the same mechanism. In this study, the effects of ipsilateral broadband elicitor duration were evaluated using forward masking psychoacoustic and transient-evoked OAE (TEOAE) paradigms in individuals with normal hearing (N = 19; m = 7, f = 12). Methods Ipsilateral pink broadband noise was used as the elicitor in both experiments, presented at 50 dB SPL (50–800 ms) for the psychoacoustic measures and 50 dB FPL (50–400 ms) for the TEOAE measures. Gain reduction was quantified as the change in signal threshold (2 kHz) and the change in TEOAE level (1/3rd-octave band centered at 2 kHz) with and without the presence of the elicitor. Results The average time constants for psychoacoustic and TEOAE gain reduction were similarly short (< 100 ms), with near-maximal effects observed for elicitor durations of 200 ms. However, individual comparisons of TEOAE and psychoacoustic gain reduction revealed mixed results. Potential factors contributing to this discrepancy are discussed. Conclusion The human MOCR reduces cochlear gain on relatively short time scales (< 100 ms) with ipsilateral broadband elicitors.

Objectives: Following deafness, a cochlear implant (CI) can be used for the restoration of hearing. CI effectiveness relies on the condition of the auditory nerve, which typically degenerates after deafness. The nerve’s condition can be assessed with the electrically evoked compound action potential (eCAP), the whole-nerve response to an electric pulse. Changes in the eCAP following an increase in the inter-phase gap (IPG) of a biphasic pulse have been reported to be informative of neural survival. This IPG effect can be explained by the temporal separation of the hyperpolarizing phase from the depolarizing phase. We hypothesize that increasing the phase duration (PD) has a similar effect. Design: We investigated the PD effect in normal hearing and in ototoxically deafened guinea pigs (total N = 40) with various conditions of the auditory nerve by recording eCAPs to biphasic current pulses with alternating polarity and with varying PD and IPG. The eCAP data were obtained from both chronically and acutely implanted guinea pigs by using CI stimulation paradigms with a fixed charge and varying PD (30, 50, or 100 µs) and IPG (2.1 or 30 µs). We evaluated six eCAP measures: five derived from the amplitude growth function and the N 1 latency. We examined the relationships of PD and IPG effects with the survival of the spiral ganglion cells. Results: The PD effects were stronger for latency than IPG effects, but weaker for the other five evaluated eCAP measures. The PD effect did not correlate as well with neural survival as the IPG effect. The IPG effect decreased with increasing PD, and accordingly, the stronger correlations between the IPG effect and neural survival were found for a short PD. Notably, the latency increase with increasing PD was greater than 1, which indicates the second phase of the pulse significantly contributes to the eCAP. This second-phase contribution was larger for lower neural survival. Conclusions: The PD effect of latency has predictive power in assessing neural survival. When using other eCAP measures than latency, the best approach to assess neural survival is using the IPG effect with a short PD (around 30 µs).

Noise-induced hearing loss (NIHL) affects approximately 5% of the global population, significantly impairing health-related quality of life, and is of particular interest in the aging population due to its association with an increased risk of cognitive decline. The pathophysiology of NIHL involves a complex interplay of damage to multiple cellular structures within the cochlea. While the pathophysiology of NIHL has been extensively studied in young mice, limited research exists on the molecular mechanisms underlying NIHL in older adult populations. This study provides the first insights into temporal differences in gene expression following noise exposure in older mice with an average age of 17 months. We exposed mice to broadband noise of 115 dB sound pressure level (SPL) for 2 h and conducted auditory testing before and after noise exposure to confirm the presence of NIHL. Cochleae were collected at 24 h, 72 h, and 1 week post exposure for RNA sequencing (RNA-seq). Pathway enrichment analysis using the Ingenuity Pathway Analysis (IPA) tool revealed distinct temporal patterns in the cochlear transcriptome. Acute inflammatory pathways were prominently activated at 24 h following noise exposure, while pathways associated with collagen degradation, hormone signaling, potassium channel activity, and GABA receptor activation peaked at 72 h or 1 week after noise exposure. These findings shed light on the age-specific molecular responses to acoustic trauma, largely consistent with previous experiments in younger mice, and highlight critical windows for cochlear damage and repair. Understanding these temporal patterns in older adults provides information for the development of targeted treatment strategies to mitigate NIHL and its associated risks.

Noise-induced hearing loss (NIHL) is a primary contributor to tinnitus, involving mechanisms such as inflammatory damage, central sensitization, and auditory cortex remodeling. However, not all cases of tinnitus are accompanied by NIHL, and the precise relationship between the two remains incompletely understood. Phosphorylation/dephosphorylation, as a core mechanism for fine cellular regulation, influences neuronal excitability, immune responses, and disease development by modulating protein activity, signal transduction, and gene expression. We hypothesized that aberrant phosphorylation levels may alter auditory cortex neuron function, leading to pathological changes at the protein level. Leveraging auditory cortex tissue from a noise-induced tinnitus model, we systematically investigated the pathogenesis of tinnitus and its distinction from NIHL through integrated proteomic and phosphoproteomic analyses. Compared to animals with NIHL alone, the tinnitus model exhibited enhanced neuronal excitability, synaptic dysfunction, hyperactive energy metabolism, and weakened neuroprotection, with disordered membrane receptor function playing a critical role. Multi-omics analysis further revealed that tinnitus development primarily depends on phosphorylation-mediated post-translational modifications reshaping cellular function, rather than changes in protein abundance caused by alterations in gene transcription levels. Collectively, this study elucidates the physiological and cellular structural alterations in noise-induced tinnitus from the dimensions of protein expression and phosphorylation modification. It confirms that tinnitus leads to neural dysfunction through abnormal membrane receptor activity, and the characteristic proteins and phosphorylation sites identified offer novel therapeutic targets for modulating central hyperexcitability in tinnitus.

Acquired sensorineural hearing loss (SNHL) is primarily caused by the damage or loss of hair cells (HCs), induced by factors such as noise exposure and ototoxic drugs. However, clinical treatments for SNHL remain limited. Here, the role of the apoptosis‐inducing gene Cidea in SNHL is investigated. It is initially observed that Cidea expression is specifically increased in neomycin‐damaged HCs at both the protein and mRNA levels. To further explore its role, Cidea knockout (Cidea‐/‐) mice are obtained, and it is found that the absence of Cidea effectively alleviates HC apoptosis caused by neomycin treatment and noise exposure in vivo. Moreover, a novel therapeutic strategy for SNHL has been developed by delivering CRISPR/SlugCas9‐HF via AAV to edit Cidea , and this approach significantly reduced HC loss induced by both neomycin and noise exposure. These findings suggest that Cidea may serve as a promising target for the prevention of neomycin‐ and noise‐induced SNHL in clinical settings.

Background Bmi1, a key component of the Polycomb repressive complex 1, plays a critical role in regulating gene expression by modulating chromatin structure. Its depletion is known to cause hair cell loss in the neonatal mouse cochlea. This study aimed to investigate the epigenetic mechanisms and transcriptional consequences of Bmi1 depletion in the neonatal auditory sensory epithelium. Results Analysis of neonatal Bmi1 knockout mice using H3K27me3 chromatin immunoprecipitation sequencing, assay for transposase-accessible chromatin sequencing, and RNA sequencing revealed significant transcriptional alterations, particularly in genes governing cell proliferation, senescence, and death. Bmi1 depletion resulted in widespread gene upregulation and increased chromatin accessibility, which correlated with reduced H3K27me3 enrichment. Notably, expression of Cdkn2c , a key cell cycle regulator, was significantly upregulated. Inhibition of Cdkn2c rescued the proliferative capacity of inner ear epithelial cells in Bmi1 knockout mice. Conclusions These findings demonstrate that Bmi1 maintains transcriptional repression and chromatin state in the developing cochlea, primarily through H3K27me3 deposition. Depletion disrupts this control, leading to Cdkn2c overexpression and impaired cell proliferation. This identifies Cdkn2c and its regulatory pathway as potential therapeutic targets for hearing loss associated with hair cell depletion.

Normally hearing humans can localize sound sources quite accurately, with minimum audible angles as small as 1°. To achieve this, these auditory pathways combine information from multiple acoustic cues, including interaural time and interaural level differences (ITDs and ILDs). Patients relying on cochlear implants (CIs) to hear the world do not match normal performance. These deficits are most pronounced in patients with little or no hearing experience early in life, and they appear to result from impaired sensitivity to ITDs, but not to ILDs. However, little is known about how ITD and ILD sensitivities develop and interact in an early deafened auditory system shortly after CI implantation. Neonatally deafened rats were fitted with bilateral CIs, and, providing informative ITDs and ILDs from stimulation onset, were trained to lateralize CI stimuli. These animals are exquisitely sensitive to both ILDs and ITDs of CI stimulus pulses, and combined information from both cues in a weighted sum. Importantly, ITDs are weighted heavily in the CI rats, such that only very modest ITDs pointing in one direction can confound quite large ILDs pointing in the opposite direction. This underlines the importance of informative ITDs for maximizing the potential for spatial hearing with CI devices.

Heavy metal ion exposure has become a global public health concern. Among them, lead is an extremely toxic heavy metal that poses serious health hazards to humans, particularly threatening vulnerable groups such as children and the elderly. Currently, the effects of chronic low‐dose lead exposure on the auditory system have yet to be reported. In this study, we established a chronic lead exposure mouse model and conducted comprehensive auditory function assessments. The results demonstrated that lead‐exposed mice developed high‐frequency hearing loss at early stages, which progressively worsened over time. Morphological examination of the inner ear revealed hair cell loss, reduced synaptic ribbon numbers, and disruption of gap junctions in lead‐exposed mice. Furthermore, immunofluorescence staining confirmed significantly decreased expression of the mitochondrial protease LONP1, along with markedly increased expression of its substrate HMGCS2, in the stria vascularis, sensory epithelium, and spiral ganglia of lead‐exposed mice. These findings indicate that chronic low‐level lead exposure causes inner ear damage and irreversible auditory dysfunction, while accelerating age‐related hearing loss in C57BL/6J mice. These preclinical results suggest that chronic lead exposure may represent a significant risk factor for age‐related hearing loss, deepen our understanding of lead‐induced auditory system impairment, and hold profound implications for preventing hearing damage in populations at high risk of lead exposure.

Background CLN3 disease, also known as juvenile Batten disease, is a recessively inherited neurodevelopmental disorder caused by mutations in the CLN3 gene. It represents the most common form of Neuronal Ceroid Lipofuscinoses (NCLs), a group of lysosomal storage disorders that impair brain function. Clinical features include progressive vision loss, language impairment, and cognitive decline. The early onset of visual deficits complicates the neurological assessment of cognitive dysfunction, while the rarity of CLN3 cases limits the study of sex-specific disease trajectories in humans. Therefore, there is a critical need for objective, translational biomarkers to monitor disease progression and support therapeutic development in preclinical animal models. Methods Building on our recent studies in individuals with CLN3 disease, we developed a parallel experimental paradigm using high-density electroencephalography (EEG) in Cln3 knockout ( Cln3-/-) mice to longitudinally assess auditory neurophysiological changes. We applied a duration-based mismatch negativity (MMN) paradigm, similar to that used in our human studies, to evaluate automatic detection of auditory pattern changes in male and female mice between 3 and 9 months of age. Results Wild-type (WT) mice of both sexes showed robust and stable duration MMN responses across this age range. In contrast, Cln3-/- mice showed marked sex- and age-dependent deficits: female mutants displayed persistent MMN deficits, whereas male mutants exhibited early MMN abnormalities that unexpectedly improved with age. Auditory brainstem responses confirmed intact peripheral hearing in Cln3-/- mice, indicating a central origin for the observed abnormalities. Further analyses revealed that MMN impairments were driven by age- and sex-specific alterations in auditory evoked potentials to both standard and deviant stimuli. Conclusions These findings demonstrate sex- and age-dependent disruptions in central auditory processing in Cln3-/- mice and support auditory duration MMN as a sensitive, translational biomarker of brain dysfunction in CLN3 disease. This approach offers a functional, cross-species measure for tracking disease progression and evaluating therapeutic interventions in Batten disease.

Noise-induced hearing loss (NIHL) involves a biphasic pathophysiology. Intense noise exposure causes immediate cochlear vasoconstriction and ischemia, leading to transient hypoxia. Subsequent reperfusion triggers excess reactive oxygen species (ROS) production, resulting in oxidative stress and hair cell injury. This study therefore developed two oxygenated albumin microbubble (OMB) formulations-ionic-bond metformin-coated (iMet-OMBs) and covalent-bond metformin-encapsulated (cMet-OMBs)-and combined them with transcranial ultrasound (US) to enhance targeted delivery to the cochlea. This approach aims to provide transient oxygen supplementation while simultaneously reducing ROS-mediated injury. Microbubbles were characterized for morphology, oxygen loading, and metformin content. Based on their superior stability and drug-loading profile, cMet-OMBs were selected for in vivo evaluation. In a mouse NIHL model, animals were administered cMet-OMBs systemically via retro-orbital injection, followed by US triggering over the temporal bone. Auditory brainstem response (ABR) thresholds, cochlear oxygen tension, and outer hair cell (OHC) survival were assessed. US-mediated cMet-OMBs rupture transiently increased intracochlear oxygen tension, counteracting early hypoxia after noise exposure. Metformin released from cMet-OMBs attenuated ROS production through mitochondrial complex I inhibition and antioxidant pathway activation. Mice treated with cMet-OMBs + US showed significantly lower ABR threshold shifts and better OHC preservation compared with controls. This dual-action strategy combines transient oxygen supplementation from OMBs with sustained antioxidant protection from metformin. While oxygen delivery raises intracochlear oxygen tension, metformin suppresses ROS generation through mitochondrial complex I inhibition and AMPK/Nrf2 activation. This controlled, US-triggered release achieves net cochlear protection against NIHL without excessive oxidative burden.

Cisplatin (CDDP), a widely used chemotherapeutic agent, is limited by severe ototoxicity side effects. Local drug delivery via the middle ear represents the most effective approach for treating inner ear disease. However, therapeutic efficacy is constrained by poor middle ear retention and limited permeability across the round window membrane (RWM). Quercetin (QU) exhibits potent activity against CDDP-induced cytotoxicity but suffers from delivery challenges. To address this, we developed amino-functionalized mesoporous silica nanoparticles (NH2-MSNs) loaded with QU (QU-N-MSNs), leveraging the permselective properties of the RWM. This system was noninvasively administered to the cochlea via trans-tympanic delivery. The synthesized QU-N-MSNs demonstrated a uniform particle size of approximately 116–124 nm, positive charge, and sustained drug release properties. Compared to free QU, QU-N-MSNs demonstrated significantly enhanced antiapoptotic and cytoprotective activities in vitro. In vivo studies confirmed nanoparticle retention within the inner ear for ≥14 days post administration and efficient RWM penetration. Pretreatment with QU-N-MSNs prior to CDDP exposure in murine models substantially mitigated ototoxicity, as evidenced by reduced hearing threshold shifts across multiple frequencies, preservation of hair cells (HCs) and spiral ganglion neurons (SGNs), and attenuation of mitochondrial-mediated SGN apoptosis. These findings establish QU-N-MSNs as an effective RWM-penetrating delivery platform, offering a promising strategy to enhance hydrophobic drug bioavailability in the inner ear and prevent CDDP-induced ototoxicity.

Background: Millions of older adults have age-related hearing loss (ARHL), a disorder where potassium-secreting cells in the cochlea's lateral wall often degenerate. The degeneration reduces the force that drives ions into the sensory cells during sound stimulation, which is traditionally thought to explain the loss of hearing. Here we describe previously unknown mechanisms underlying this metabolic form of ARHL. Methods: Fluorescence spectroscopy and live-cell imaging was used in Dunkin-Hartley guinea pigs of either sex to investigate the effects of lateral wall dysfunction on the hearing organ and its accessory structures. Critical findings were confirmed by studying samples of human temporal bones. Findings: Lateral wall dysfunction caused calcium levels in the inner ear to decline, a change that was most pronounced in the tectorial membrane, an accessory structure crucial for transmitting acoustic stimuli to sensory cell stereocilia. This calcium depletion deprived the sensory cells of an essential ion. Additionally, the tectorial membrane detached from stereocilia, significantly impairing their ability to respond to sound. Sound-evoked responses were further decreased by sustained contraction of the entire hearing organ. Interpretation: These findings establish the tectorial membrane as a key factor in metabolic ARHL, which needs to be considered when developing better diagnostic tools or treatments. Funding: Swedish Research Council grants 2017-06092 and 2022-00548, Swedish Brain foundation grant FO2023-0171 and US National Institutes of Health grant R01DC000141-44.

Objective: Screen for differential proteins in the cochlea of mice associated with Age-Related Hearing Loss (ARHL), analyze and validate the expression of specific differential proteins and genes in the cochlea and auditory cortex of ARHL mice, and preliminarily explore their potential mechanisms of action. Methods: ABR (Auditory Brainstem Response) hearing tests were conducted to select 15-month-old C57BL/6 mice with significantly decreased hearing as the experimental group and 2-month-old mice with normal hearing as the control group. Cochleae were dissected, and unlabeled quantitative proteomics was employed to identify and analyze differentially expressed proteins in the inner ear of the two groups of mice. Key node proteins were selected via the STRING database and Cytoscape analysis. The expression of two selected proteins, ApoE and Spp1, in the cochlea was detected using qRT-PCR, Western blot, and immunofluorescence techniques, and their expression in the auditory cortex of the brain was further explored. Results: Label-free quantitative proteomics identified 115 differentially expressed proteins in the cochlea of 15-month-old ARHL mice compared to 2-month-old hearing-normal mice, including 42 upregulated and 73 downregulated proteins. GO and KEGG enrichment analyses revealed significant enrichment of differentially expressed proteins in functions and signaling pathways associated with neurodegenerative diseases and neurotransmission. Protein-Protein Interaction (PPI) analysis using the STRING database and Cytoscape selection identified ApoE and Spp1 as key hub proteins. Validation via qRT-PCR, Western blot, and immunofluorescence demonstrated that both ApoE and Spp1 were highly expressed in the cochlea and auditory cortex of the ARHL mice compare to 2-month-old hearing-normal mice. Conclusion: ApoE and Spp1 are upregulated in the cochlea of ARHL mice, particularly in spiral ganglion neurons, and in the auditory cortex, suggesting their potential involvement in the pathogenesis and progression of ARHL through the modulation of auditory neural conduction systems. Level of evidence: Level 2.