ABR/DPOAE publications

Loss of auditory-nerve (AN) afferent cochlear innervation is a prevalent human condition that does not affect audiometric thresholds and therefore remains largely undetectable with standard clinical tests. AN loss is widely expected to cause hearing difficulties in noise, known as “hidden hearing loss,” but support for this hypothesis is controversial. Here, we used operant conditioning procedures to examine the perceptual impact of AN loss on behavioral tone-in-noise (TIN) sensitivity in the budgerigar ( Melopsittacus undulatus ; of either sex), an avian animal model with complex hearing abilities similar to humans. Bilateral kainic acid (KA) infusions depressed compound AN responses by 40–70% without impacting otoacoustic emissions or behavioral tone sensitivity in quiet. Surprisingly, animals with AN damage showed normal thresholds for tone detection in noise (0.1 ± 1.0 dB compared to control animals; mean difference ± SE), even under a challenging roving-level condition with random stimulus variation across trials. Furthermore, decision-variable correlations (DVCs) showed no difference for AN-damaged animals in their use of energy and envelope cues to perform the task. These results show that AN damage has less impact on TIN detection than generally expected, even under a difficult roving-level condition known to impact TIN detection in individuals with sensorineural hearing loss (SNHL). Perceptual deficits could emerge for different perceptual tasks or with greater AN loss but are potentially minor compared with those caused by SNHL. SIGNIFICANCE STATEMENT Loss of auditory-nerve (AN) cochlear innervation is a common problem in humans that does not affect audiometric thresholds on a clinical hearing test. AN loss is widely expected to cause hearing problems in noise, known as “hidden hearing loss,” but existing studies are controversial. Here, using an avian animal model with complex hearing abilities similar to humans, we examined for the first time the impact of an experimentally induced AN lesion on behavioral tone sensitivity in noise. Surprisingly, AN-lesioned animals showed no difference in hearing performance in noise or detection strategy compared with controls. These results show that perceptual deficits from AN damage are smaller than generally expected, and potentially minor compared with those caused by sensorineural hearing loss (SNHL).

Background This study aimed to investigate the changes in the expression of hippocampal genes upon acute noise exposure. Methods Three-week-old Sprague–Dawley rats were assigned to control (n = 15) and noise (n = 15) groups. White noise (2–20 kHz, 115 dB sound pressure level [SPL]) was delivered for 4 h per day for 3 days to the noise group. All rats were sacrificed on the last day of noise exposure, and gene expression in the hippocampus was analyzed using a microarray. Pathway analyses were conducted for genes that showed differential expression ≥ 1.5-fold and P ≤ 0.05 compared to the control group. The genes included in the putative pathways were measured using quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Results Thirty-eight upregulated genes and 81 downregulated genes were identified. The pathway analyses revealed that upregulated genes were involved in the cellular responses to external stimuli and immune system pathways. qRT-PCR confirmed the upregulation of the involved genes. The downregulated genes were involved in neuronal systems and synapse-related pathways, and qRT-PCR confirmed the downregulation of the involved genes. Conclusions Acute noise exposure upregulated the expression of immune-related genes and downregulated the expression of neurotransmission-related genes in the hippocampus.

Thioredoxin 2 (TXN2) is a small redox protein found in nearly all organisms. As a mitochondrial member of the thioredoxin antioxidant defense system, TXN2 interacts with peroxiredoxin 3 (PRDX3) to remove hydrogen peroxide. Accordingly, TXN2 is thought to play an important role in maintaining the appropriate mitochondrial redox environment and protecting the mitochondrial components against oxidative stress. In the current study, we investigated the effects of Txn2 haplodeficiency on cochlear antioxidant defenses, auditory function, and cochlear cell loss across the lifespan in wild-type (WT) and Txn2 heterozygous knockout (Txn2+/-) mice backcrossed onto CBA/CaJ mice, a well-established model of age-related hearing loss. Txn2+/- mice displayed a 58% decrease in TXN2 protein levels in the mitochondria of the inner ears compared to WT mice. However, Txn2 haplodeficiency did not affect the thioredoxin or glutathione antioxidant defense in both the mitochondria and cytosol of the inner ears of young mice. There were no differences in the levels of mitochondrial biogenesis markers, mitochondrial DNA content, or oxidative DNA and protein damage markers in the inner ears between young WT and Txn2+/- mice. In a mouse inner ear cell line, knockdown of Txn2 did not affect cell viability under hydrogen peroxide treatment. Consistent with the tissue and cell line results, there were no differences in hair cell loss or spiral ganglion neuron density between WT and Txn2+/- mice at 3-5 or 23-25 months of age. Furthermore, Txn2 haplodeficiency did not affect auditory brainstem response threshold, wave I latency, or wave I amplitude at 3-5, 15-16, or 23-25 months of age. Therefore, Txn2 haplodeficiency does not affect cochlear antioxidant defenses, accelerate degeneration of cochlear cells, or affect auditory function in mice across the lifespan.

Background and objectives: Animal results have suggested that auditory brainstem responses (ABRs) to transient sounds presented at supra-threshold levels may be useful for measuring hearing damage that is hidden to current audiometric tests. Evaluating such ABRs requires extracting the latencies and amplitudes of relevant deflections, or "waves". Currently, this is mostly done by human observers manually picking the waves' peaks and troughs in each individual response - a process that is both time-consuming and requiring of expert experience. Here, we propose a highly automated procedure for extracting individual ABR wave latencies and amplitudes based on the well-established methodology of non-linear curve registration. Methods: First, the to-be-analysed individual ABRs are temporally aligned - either with one another or, if available, with a pre-existing template - by locally compressing or stretching their time axes with smooth and invertible time warping functions. Then, the individual latencies and amplitudes of relevant ABR waves are obtained by picking the latencies of the waves' peaks and troughs on the common (aligned) time axis and combining these with the individual aligned responses and inverse time warping functions. Results: Using an example ABR data set with a wide range of response latencies and signal-to-noise ratios (SNRs), we test different choices for fitting the time warping functions. We cross-validate the warping results using independent response replicates and compare automatically and manually extracted latencies and amplitudes for ABR waves I and V. Using a Bayesian approach, we show that, for the best registration condition, automatic and manual data were statistically similar. Conclusions: Non-linear curve registration can be used to temporally align individual ABRs and extract their wave latencies and amplitudes in a way that closely matches results from manual picking.

Exposure to excessive sound causes noise-induced hearing loss through complex mechanisms and represents a common and unmet neurological condition. We investigate how noise insults affect the cochlea with proteomics and functional assays. Quantitative proteomics reveals that exposure to loud noise causes proteotoxicity. We identify and confirm hundreds of proteins that accumulate, including cytoskeletal proteins, and several nodes of the proteostasis network. Transcriptomic analysis reveals that a subset of the genes encoding these proteins also increases acutely after noise exposure, including numerous proteasome subunits. Global cochlear protein ubiquitylation levels build up after exposure to excess noise, and we map numerous posttranslationally modified lysines residues. Several collagen proteins decrease in abundance, and Col9a1 specifically localizes to pillar cell heads. After two weeks of recovery, the cochlea selectively elevates the abundance of the protein synthesis machinery. We report that overstimulation of the auditory system drives a robust cochlear proteotoxic stress response.

The mammalian hearing organ, the cochlea, contains an active amplifier to boost the vibrational response to low level sounds. Hallmarks of this active process are sharp location-dependent frequency tuning and compressive nonlinearity over a wide stimulus range. The amplifier relies on outer hair cell (OHC)-generated forces driven in part by the endocochlear potential, the ∼+80 mV potential maintained in scala media, generated by the stria vascularis. We transiently eliminated the endocochlear potential in vivo by an intravenous injection of furosemide and measured the vibrations of different layers in the cochlea's organ of Corti using optical coherence tomography. Distortion product otoacoustic emissions were also monitored. After furosemide injection, the vibrations of the basilar membrane lost the best frequency (BF) peak and showed broad tuning similar to a passive cochlea. The intra-organ of Corti vibrations measured in the region of the OHCs lost the BF peak and showed low-pass responses but retained nonlinearity. This strongly suggests that OHC electromotility was operating and being driven by nonlinear OHC current. Thus, although electromotility is presumably necessary to produce a healthy BF peak, the mere presence of electromotility is not sufficient. The BF peak recovered nearly fully within 2 h, along with the recovery of odd-order distortion product otoacoustic emissions. The recovery pattern suggests that physical shifts in operating condition are a critical step in the recovery process.

During cochlear development, the Notch ligand JAGGED 1 (JAG1) plays an important role in the specification of the prosensory region, which gives rise to sound-sensing hair cells and neighboring supporting cells (SCs). While JAG1's expression is maintained in SCs through adulthood, the function of JAG1 in SC development is unknown. Here, we demonstrate that JAG1 is essential for the formation and maintenance of Hensen's cells, a highly specialized SC subtype located at the edge of the auditory epithelium. Using Sox2 CreERT2 /+ :: Jag1 loxP/loxP mice of both genders, we show that Jag1 deletion at the onset of differentiation, at embryonic day 14.5, disrupted Hensen's cell formation. Similar loss of Hensen's cells was observed when Jag1 was deleted after Hensen's cell formation at postnatal day (P) 0/P1 and fate-mapping analysis revealed that in the absence of Jag1 , some Hensen's cells die, but others convert into neighboring Claudius cells. In support of a role for JAG1 in cell survival, genes involved in mitochondrial function and protein synthesis were downregulated in the sensory epithelium of P0 cochlea lacking Jag1 . Finally, using Fgfr3-iCreER T2 :: Jag1 loxP/loxP mice to delete Jag1 at P0, we observed a similar loss of Hensen's cells and found that adult Jag1 mutant mice have hearing deficits at the low-frequency range. SIGNIFICANCE STATEMENT Hensen's cells play an essential role in the development and homeostasis of the cochlea. Defects in the biophysical or functional properties of Hensen's cells have been linked to auditory dysfunction and hearing loss. Despite their importance, surprisingly little is known about the molecular mechanisms that guide their development. Morphologic and fate-mapping analyses in our study revealed that, in the absence of the Notch ligand JAGGED1, Hensen's cells died or converted into Claudius cells, which are specialized epithelium-like cells outside the sensory epithelium. Confirming a link between JAGGED1 and cell survival, transcriptional profiling showed that JAGGED1 maintains genes critical for mitochondrial function and tissue homeostasis. Finally, auditory phenotyping revealed that JAGGED1's function in supporting cells is necessary for low-frequency hearing.

In deaf subjects using a cochlear implant (CI) for hearing restoration, the auditory nerve is subject to degeneration, which may negatively impact CI effectiveness. This nerve degeneration can be reduced by neurotrophic treatment. Here, we compare the preservative effects of the naturally occurring tyrosine receptor kinase B (TrkB) agonist brain-derived neurotrophic factor (BDNF) and the small-molecule TrkB agonist 7,8,3′-trihydroxyflavone (THF) on the auditory nerve in deafened guinea pigs. THF may be more effective than BDNF throughout the cochlea because of better pharmacokinetic properties. The neurotrophic compounds were delivered by placement of a gelatin sponge on the perforated round window membrane. To complement the histology of spiral ganglion cells (SGCs), electrically evoked compound action potential (eCAP) recordings were performed four weeks after treatment initiation. We analyzed the eCAP inter-phase gap (IPG) effect and measures derived from pulse-train evoked eCAPs, both indicative of SGC healthiness. BDNF but not THF yielded a significantly higher survival of SGCs in the basal cochlear turn than untreated controls. Regarding IPG effect and pulse-train responses, the BDNF-treated animals exhibited more normal responses than both untreated and THF-treated animals. We have thus confirmed the protective effect of BDNF, but we have not confirmed previously reported protective effects of THF with our clinically applicable delivery method.

Atg7 is an indispensable factor that plays a role in canonical nonselective autophagy. Here we show that genetic ablation of Atg7 in outer hair cells (OHCs) in mice caused stereocilium damage, somatic electromotility disturbances, and presynaptic ribbon degeneration over time, which led to the gradual wholesale loss of OHCs and subsequent early-onset profound hearing loss. Impaired autophagy disrupted OHC mitochondrial function and triggered the accumulation of dysfunctional mitochondria that would otherwise be eliminated in a timely manner. Atg7-independent autophagy/mitophagy processes could not compensate for Atg7 deficiency and failed to rescue the terminally differentiated, non-proliferating OHCs. Our results show that OHCs orchestrate intricate nonselective and selective autophagic/mitophagy pathways working in concert to maintain cellular homeostasis. Overall, our results demonstrate that Atg7-dependent autophagy plays a pivotal cytoprotective role in preserving OHCs and maintaining hearing function.

Noise-induced hearing loss (NIHL) is a common health concern with significant social, psychological, and cognitive implications. Moderate levels of acoustic overstimulation associated with tinnitus and impaired speech perception cause cochlear synaptopathy, characterized physiologically by reduction in wave I of the suprathreshold auditory brainstem response (ABR) and reduced number of synapses between sensory hair cells and auditory neurons. The unfolded protein response (UPR), an endoplasmic reticulum stress response pathway, has been implicated in the pathogenesis and treatment of NIHL as well as neurodegeneration and synaptic damage in the brain. In this study, we used the small molecule UPR modulator Integrated Stress Response InhiBitor (ISRIB) to treat noise-induced cochlear synaptopathy in a mouse model. Mice pretreated with ISRIB prior to noise-exposure were protected against noise-induced synapse loss. Male, but not female, mice also exhibited ISRIB-mediated protection against noise-induced suprathreshold ABR wave-I amplitude reduction. Female mice had higher baseline wave-I amplitudes but greater sensitivity to noise-induced wave-I reduction. Our results suggest that the UPR is implicated in noise-induced cochlear synaptopathy, and can be targeted for treatment.

Sensorineural hearing loss is a prevalent problem that adversely impacts quality of life by compromising interpersonal communication. While hair cell damage is readily detectable with the clinical audiogram, this traditional diagnostic tool appears inadequate to detect lost afferent connections between inner hair cells and auditory nerve (AN) fibers, known as cochlear synaptopathy. The envelope-following response (EFR) is a scalp-recorded response to amplitude modulation, a critical acoustic feature of speech. Because EFRs can have greater amplitude than wave I of the auditory brainstem response (ABR; i.e., the AN-generated component) in humans, the EFR may provide a more sensitive way to detect cochlear synaptopathy. We explored the effects of kainate- (kainic acid) induced excitotoxic AN injury on EFRs and ABRs in the budgerigar (Melopsittacus undulatus), a parakeet species used in studies of complex sound discrimination. Kainate reduced ABR wave I by 65-75 % across animals while leaving otoacoustic emissions unaffected or mildly enhanced, consistent with substantial and selective AN synaptic loss. Compared to wave I loss, EFRs showed similar or greater percent reduction following kainate for amplitude-modulation frequencies from 380 to 940 Hz and slightly less reduction from 80 to 120 Hz. In contrast, forebrain-generated middle latency responses showed no consistent change post-kainate, potentially due to elevated "central gain" in the time period following AN damage. EFR reduction in all modulation frequency ranges was highly correlated with wave I reduction, though within-animal effect sizes were greater for higher modulation frequencies. These results suggest that even low-frequency EFRs generated primarily by central auditory nuclei might provide a useful noninvasive tool for detecting synaptic injury clinically.

The mechanical and electrical responses of the mammalian cochlea to acoustic stimuli are nonlinear and highly tuned in frequency. This is due to the electromechanical properties of cochlear outer hair cells (OHCs). At each location along the cochlear spiral, the OHCs mediate an active process in which the sensory tissue motion is enhanced at frequencies close to the most sensitive frequency (called the characteristic frequency, CF). Previous experimental results showed an approximate 0.3 cycle phase shift in the OHC-generated extracellular voltage relative the basilar membrane displacement, which was initiated at a frequency approximately one-half octave lower than the CF. Findings in the present paper reinforce that result. This shift is significant because it brings the phase of the OHC-derived electromotile force near to that of the basilar membrane velocity at frequencies above the shift, thereby enabling the transfer of electrical to mechanical power at the basilar membrane. In order to seek a candidate physical mechanism for this phenomenon, we used a comprehensive electromechanical mathematical model of the cochlear response to sound. The model predicts the phase shift in the extracellular voltage referenced to the basilar membrane at a frequency approximately one-half octave below CF, in accordance with the experimental data. In the model, this feature arises from a minimum in the radial impedance of the tectorial membrane and its limbal attachment. These experimental and theoretical results are consistent with the hypothesis that a tectorial membrane resonance introduces the correct phasing between mechanical and electrical responses for power generation, effectively turning on the cochlear amplifier.

Neuroinflammation has been implicated in noise-induced auditory processing disorder and tinnitus. Certain non-auditory neurological disorders can also increase the levels of proinflammatory cytokines in the brain. To investigate the impact of increased brain proinflammatory cytokine levels on the central auditory pathway, we infused recombinant TNF-α into the right lateral cerebral ventricle, and examined auditory processing and cytoarchitecture of the auditory cortex. Microglial deramification was observed in the auditory cortex of mice that had received both TNF-α infusion and exposure to an 86-dB noise, but not in mice that had received either TNF-α infusion or noise exposure alone. In addition, we observed reduced cortical PV+ neuron density and impaired performances in gap detection and prepulse inhibition (PPI) only in mice that received both TNF-α infusion and the noise exposure. These results suggest that disease-related increase in brain proinflammatory cytokine release could be a risk factor for noise-induced auditory processing disorder and tinnitus.

Large-scale epidemiological surveys suggest that hearing loss (HL) is a significant risk factor for dementia. We previously showed that noise-induced HL (NIHL) impairs hippocampal cognitive function and decreases hippocampal neurogenesis and neuronal complexity, suggesting a causal role of HL in dementia. To further investigate the influence of acquired peripheral HL on hippocampal neurogenesis with the aging process as well as the underlying mechanism, we produced NIHL in male CBA/J mice and assessed hippocampal neurogenesis and microglial morphology in the auditory brain and hippocampus at 4 days post-noise exposure (DPN) or 1, 3, 6, or 12 months post-noise exposure (MPN) by immunofluorescence labeling. We found that the age-related decline in hippocampal neurogenesis was accelerated in mice with NIHL. Furthermore, in mice with NIHL, prolonged microglial activation occurred from 1 MPN to 12 MPN across multiple auditory nuclei, while aggravated microglial deterioration occurred in the hippocampus and correlated with the age-related decline in hippocampal neurogenesis. These results suggest that acquired peripheral HL accelerates the age-related decline in hippocampal neurogenesis and that hippocampal microglial degeneration may contribute to the development of neurodegeneration following acquired peripheral HL.

Currently, there are limited reports available regarding the treatment and prevention of progressive age-related hearing loss. This is because age-related hearing loss is not a critical disease with direct fatalities and has several well-established countermeasures such as hearing aids and cochlear implants. This study evaluated the efficacy of Ninjinyoeito (NYT) in the treatment of age-related hearing loss. C57BL/6J mice were divided into three groups: baseline group, untreated group, and NYT-treated group, with the latter receiving NYT treatment for 2 months. The mice were fed with NYT extract mixed with 4% mouse normal chow. Hearing loss was confirmed by a reduction in intact cell density of the auditory nerve from the age of 5-7 months. The suppression of hearing loss with aging and decrease in the intact cell density of the auditory nerve were significant in mice fed with NYT for 2 months. NYT has been reported to improve blood flow and enhance mitochondrial activity and may exert its protective effects on spiral neurons through these mechanisms. There was no decrease in the size of the stria vascularis from the age of 5-7 months in C57BL/6J mice. The present model failed to reveal the effect of NYT on atrophy of the stria vascularis of the cochlear duct. In conclusion, NYT appears to have a protective effect on the auditory nerve and suppress the progression of age-related hearing loss by reducing age-related auditory nerve degeneration.

The round window of the cochlea provides an ideal route for delivering medicines and gene therapy reagents that can cross the round window membrane (RWM) into the inner ear. Recombinant adeno‐associated viruses (rAAVs) have several advantages and are recommended as viral vectors for gene transfection. However, rAAVs cannot cross an intact RWM. Consequently, ultrasound‐mediated microbubble (USMB) cavitation is potentially useful, because it can sonoporate the cell membranes, and increase their permeability to large molecules. The use of USMB cavitation for drug delivery across the RWM has been tested in a few animal studies but has not been used in the context of AAV‐mediated gene transfection. The currently available large size of the ultrasound probe appears to be a limiting factor in the application of this method to the RWM. In this study, we used home‐made ultrasound probe with a decreased diameter to 1.5 mm, which enabled the easy positioning of the probe close to the RWM. In guinea pigs, we used this probe to determine that (1) USMB cavitation caused limited damage to the outer surface layer or the RWM, (2) an eGFP‐gene carrying rAAV could effectively pass the USMB‐treated RWM and reliably transfect cochlear cells, and (3) the hearing function of the cochlea remained unchanged. Our results suggest that USMB cavitation of the RWM is a good method for rAAV‐mediated cochlear gene transfection with clear potential for clinical translation. We additionally discuss several advantages of the small probe size.

Signal transducer and activator of transcription 1 (STAT1) is known to be an important player in inflammatory responses. STAT1 as a transcription factor regulates the expression of multiple proinflammatory genes. Inflammatory response is one of the common effects of ototoxicity. Our group reported that hair cells of STAT1 knockout (STAT1-KO) mice are less sensitive to ototoxic agents in-vitro. The effect of inflammatory responses in STAT1-KO mice has primarily been studied challenging them with several pathogens and analyzing different organs of those mice. However, the effect of STAT1 ablation in the mouse inner ear has not been reported. Therefore, we evaluated the cochlear function of wild type and STAT1-KO mice via auditory brain stem response (ABR) and performed histopathologic analysis of their temporal bones. We found ABR responses were affected in STAT1-KO mice with cases of bilateral and unilateral hearing impairment. Histopathologic examination of the middle and inner ears showed bilateral and unilateral otitis media. Otitis media was characterized by effusion of middle and inner ear that varied between the mice in volume and inflammatory cell content. In addition, the thickness of the middle ear mucosae in STAT1-KO mice were more pronounced than those in wild type mice. The degree of middle and inner ear inflammation correlated with ABR threshold elevation in STAT1-KO mice. It appears that a number of mice with inflammation underwent spontaneous resolution. The ABR thresholds were variable and showed a tendency to increase in homozygous and heterozygous STAT1-KO mice. These findings suggest that STAT1 ablation confers an increased susceptibility to otitis media leading to hearing impairment. Thus, the study supports the new role of STAT1 as otitis media predisposition gene.

Background In addition to the direct effects of irradiation, the induced inflammatory response may play an important role in the damage to the inner ear caused by radiotherapy for the treatment of head and neck cancers. Resolvin E1 (RvE1) has anti-inflammatory activity, acting by reducing neutrophil infiltration and proinflammatory cytokine expression. Therefore, in this study we sought to confirm whether the inflammation induced by irradiation was involved in damage to the inner ear after radiotherapy and to investigate the protective effect and underlying mechanism of RvE1 using mouse models. Methods A dose of RvE1 was delivered by intraperitoneal injection to mice before irradiation. Changes in the auditory brainstem response (ABR), relative balance ability, inner ear morphology and the expression levels of inflammatory factors in the inner ear were analyzed on days 7 and 14 after irradiation and compared among different experimental groups. Results Changes of ABR and relative balance ability showed the inner functions of experimental mice presented severe damage after irradiation, but the damage was significantly alleviated after RvE1 pretreatment compared to irradiation alone. Morphological analysis of the inner ear showed severe damage to the cochlea and vestibule after irradiation. In contrast, damage to the cochlea and vestibule was significantly reduced in the RvE1-pretreated group compared to that in the irradiation alone group. Along with these functional and morphological changes, the mRNA expression level of anti-inflammatory factors interleukin-2 was significantly increased, while those of proinflammatory factors interleukin-6 and tumor necrosis factor-α were significantly decreased in the inner ear of mice after RvE1 pretreatment compared to irradiation alone. Conclusions We believe that inflammation induced by irradiation is involved in the damage to the inner ear caused by radiotherapy, and that RvE1 reduces the damage caused by irradiation to the inner ear by regulating the induced inflammatory response.

Age-related hearing loss (ARHL) is the most common sensory disorder among the elderly, associated with aging and auditory hair cell death due to oxidative-stress-induced mitochondrial dysfunction. Although transgenic mice and long-term aging induction cultures have been used to study ARHL, there are currently no ARHL animal models that can be stimulated by intermittent environmental changes. In this study, an ARHL animal model was established by inducing continuous oxidative stress to promote short-term aging of cells, determined on the basis of expression of hearing-loss-induced phenotypes and aging-related factors. The incidence of hearing loss was significantly higher in dual- and triple-exposure conditions than in intermittent hypoxic conditions, high-fat diet (HFD), or d-galactose injection alone. Continuous oxidative stress and HFD accelerated cellular aging. An increase in Ucp2, usually expressed during mitochondrial dysfunction, was observed. Expression of Cdh23, Slc26a4, Kcnq4, Myo7a, and Myo6, which are ARHL-related factors, were modified by oxidative stress in the cells of the hearing organ. We found that intermittent hypoxia, HFD, and galactose injection accelerated cellular aging in the short term. Thus, we anticipate that the development of this hearing loss animal model, which reflects the effects of intermittent environmental changes, will benefit future research on ARHL.

Human pathogenic variants of TBC1D24 are associated with clinically heterogeneous phenotypes, including recessive nonsyndromic deafness DFNB86, dominant nonsyndromic deafness DFNA65, seizure accompanied by deafness, a variety of isolated seizure phenotypes and DOORS syndrome, characterized by deafness, onychodystrophy, osteodystrophy, intellectual disability and seizures. Thirty-five pathogenic variants of human TBC1D24 associated with deafness have been reported. However, functions of TBC1D24 in the inner ear and the pathophysiology of TBC1D24-related deafness are unknown. In this study, a novel splice-site variant of TBC1D24 c.965 + 1G > A in compound heterozygosity with c.641G > A p.(Arg214His) was found to be segregating in a Pakistani family. Affected individuals exhibited, either a deafness-seizure syndrome or nonsyndromic deafness. In human temporal bones, TBC1D24 immunolocalized in hair cells and spiral ganglion neurons, whereas in mouse cochlea, Tbc1d24 expression was detected only in spiral ganglion neurons. We engineered mouse models of DFNB86 p.(Asp70Tyr) and DFNA65 p.(Ser178Leu) nonsyndromic deafness and syndromic forms of deafness p.(His336Glnfs*12) that have the same pathogenic variants that were reported for human TBC1D24. Unexpectedly, no auditory dysfunction was detected in Tbc1d24 mutant mice, although homozygosity for some of the variants caused seizures or lethality. We provide some insightful supporting data to explain the phenotypic differences resulting from equivalent pathogenic variants of mouse Tbc1d24 and human TBC1D24.