Auditory Research publications

The lateral superior olive (LSO) is a key structure in the central auditory system of mammals that exerts efferent control on cochlear sensitivity and is involved in the processing of binaural level differences for sound localization. Understanding how the LSO contributes to these processes requires knowledge about the resident cells and their connections with other auditory structures. We used standard histological stains and retrograde tracer injections into the inferior colliculus (IC) and cochlea in order to characterize two basic groups of neurons: (1) Principal and periolivary (PO) neurons have projections to the IC as part of the ascending auditory pathway; and (2) lateral olivocochlear (LOC) intrinsic and shell efferents have descending projections to the cochlea. Principal and intrinsic neurons are intermixed within the LSO, exhibit fusiform somata, and have disk-shaped dendritic arborizations. The principal neurons have bilateral, symmetric, and tonotopic projections to the IC. The intrinsic efferents have strictly ipsilateral projections, known to be tonotopic from previous publications. PO and shell neurons represent much smaller populations (<10% of principal and intrinsic neurons, respectively), have multipolar somata, reside outside the LSO, and have non-topographic, bilateral projections. PO and shell neurons appear to have widespread projections to their targets that imply a more diffuse modulatory function. The somata and dendrites of principal and intrinsic neurons form a laminar matrix within the LSO and share quantifiably similar alignment to the tonotopic axis. Their restricted projections emphasize the importance of frequency in binaural processing and efferent control for auditory perception. This study addressed and expanded on previous findings of cell types, circuit laterality, and projection tonotopy in the LSO of the mouse.

Centrosomal protein dysfunction might cause ciliopathies. However, the role of centrosomal proteins in male infertility remains poorly defined. Here, we identified a pathogenic splicing mutation in CEP78 in male infertile patients with severely reduced sperm number and motility, and the typical multiple morphological abnormalities of the sperm flagella phenotype. We further created Cep78 knockout mice, which showed an extremely low sperm count, completely aberrant sperm morphology, and approximately null sperm motility. The infertility of the patients and knockout mice could not be rescued by an intracytoplasmic sperm injection treatment. Mechanistically, CEP78 might regulate USP16 expression, which further stabilizes Tektin levels via the ubiquitination pathway. Cep78 knockout mice also exhibited impairments in retina and outer hair cells of the cochlea. Collectively, our findings identified nonfunctional CEP78 as an indispensable factor contributing to male infertility and revealed a role for this gene in regulating retinal and outer hair cell function in mice.

Nuclear positioning is important for the functionality of many cell types and is mediated by interactions of cytoskeletal elements and nucleoskeleton proteins. Nesprin proteins, part of the linker of nucleoskeleton and cytoskeleton (LINC) complex, have been shown to participate in nuclear positioning in multiple cell types. Outer hair cells (OHCs) in the inner ear are specialized sensory epithelial cells that utilize somatic electromotility to amplify auditory signals in the cochlea. Recently, Nesprin-4 (encoded by Syne4 ) was shown to play a crucial role in nuclear positioning in OHCs. Syne4 deficiency in humans and mice leads to mislocalization of the OHC nuclei and cell death resulting in deafness. However, it is unknown how Nesprin-4 mediates the position of the nucleus, and which other molecular components are involved in this process. Here, we show that the interaction of Nesprin-4 and the microtubule motor kinesin-1 is mediated by a conserved 4 amino-acid motif. Using in vivo AAV gene delivery, we show that this interaction is critical for nuclear positioning and hearing in mice. Nuclear mislocalization and cell death of OHCs coincide with the onset of hearing and electromotility and are solely restricted to outer, but not inner, hair cells. Likewise, the C. elegans functional homolog of Nesprin-4, UNC-83, uses a similar motif to mediate interactions between migrating nuclei and kinesin-1. Overall, our results suggest that OHCs require unique cellular machinery for proper nuclear positioning at the onset of electromotility. This machinery relies on the interaction between Nesprin-4 and kinesin-1 motors supporting a microtubule cargo model for nuclear positioning.

Foraging on nocturnally migrating birds is one of the most challenging foraging tasks in the animal kingdom. Only three bat species (e.g., Ia io) known to date can prey on migratory birds. However, how these bats have exploited this challenging dietary niche remains unknown. Here, we demonstrate that I. io hunts at the altitude of migrating birds during the bird migration season. The foraging I. io exhibited high flight altitudes (up to 4945 m above sea level) and high flight speeds (up to 143.7 km h-1). I. io in flight can actively prey on birds in the night sky via echolocation cues. Genes associated with DNA damage repair, hypoxia adaptation, biting and mastication, and digestion and metabolism have evolved to adapt to this species' avivorous habits. Our results suggest that the evolution of behavioral innovation and genomic novelty are associated with the exploitation of challenging dietary opportunities.

The transduction of acoustic information by hair cells depends upon mechanosensitive stereociliary bundles that project from their apical surface. Mutations or absence of the stereociliary protein EPS8 cause deafness in humans and mice, respectively. Eps8 knockout mice (Eps8 -/- ) have hair cells with immature stereocilia and fail to become sensory receptors. Here, we show that exogenous delivery of Eps8 using Anc80L65 in P1-P2 Eps8 -/- mice in vivo rescued the hair bundle structure of apical-coil hair cells. Rescued hair bundles correctly localize EPS8, WHIRLIN, MYO15, and BAIAP2L2, and generate normal mechanoelectrical transducer currents. Inner hair cells with normal-looking stereocilia re-expressed adult-like basolateral ion channels (BK and KCNQ4) and have normal exocytosis. The number of hair cells undergoing full recovery was not sufficient to rescue hearing in Eps8 -/- mice. Adeno-associated virus (AAV)-transduction of P3 apical-coil and P1-P2 basal-coil hair cells does not rescue hair cells, nor does Anc80L65-Eps8 delivery in adult Eps8 -/- mice. We propose that AAV-induced gene-base therapy is an efficient strategy to recover the complex hair-cell defects in Eps8 -/- mice. However, this therapeutic approach may need to be performed in utero since, at postnatal ages, Eps8 -/- hair cells appear to have matured or accumulated damage beyond the point of repair.

Noise-induced hearing impairment can mediate delayed injury of spiral neurons (SGNs), resulting in degeneration of nerve fibers, synaptic degeneration and even death of SGNs. We believe that delayed injury is related to mitochondrial energy metabolism disorders. Therefore, we investigated ATP and the electron transport chain (ETC) in rat SGNs after noise injury and found that with prolonged injury time, ATP synthesis and the expression of complexes II and IV decreased, indicating the functional decline of the ETC. The maintenance of ETC function is related to subunit import and assembly of the complex. The disulfide relay mechanism controlled by the apoptosis inducing factor/coiled-coil-helix-coiled-coil-helix domain-containing protein 4(AIF/CHCHD4) pathway can regulate mitochondrial protein import. The results showed that AIF expression in SGNs decreased after noise exposure, indicating that noise damage to SGNs can restore intramitochondrial protein input by downregulating the AIF/CHCHD4 pathway, hindering ETC function; insufficient ETC function is a possible reason for the delayed injury of SGNs.

Background: Cryopyrin-associated periodic syndrome (CAPS) is an inherited autoinflammatory disease caused by a gain-of-function mutation in NLRP3. Although CAPS patients frequently suffer from sensorineural hearing loss, it remains unclear whether CAPS-associated mutation in NLRP3 is associated with the progression of hearing loss. Methods: We generated a mice with conditional expression of CAPS-associated NLRP3 mutant (D301N) in cochlea-resident CX3CR1 macrophages and examined the susceptibility of CAPS mice to inflammation-mediated hearing loss in a local and systemic inflammation context. Findings: Upon lipopolysaccharide (LPS) injection into middle ear cavity, NLRP3 mutant mice exhibited severe cochlear inflammation, inflammasome activation and hearing loss. However, this middle ear injection model induced a considerable hearing loss in control mice and inevitably caused an inflammation-independent hearing loss possibly due to ear tissue damages by injection procedure. Subsequently, we optimized a systemic LPS injection model, which induced a significant hearing loss in NLRP3 mutant mice but not in control mice. Peripheral inflammation induced by a repetitive low dose of LPS injection caused a blood-labyrinth barrier disruption, macrophage infiltration into cochlea and cochlear inflammasome activation in an NLRP3-dependent manner. Interestingly, both cochlea-infiltrating and -resident macrophages contribute to peripheral inflammation-mediated hearing loss of CAPS mice. Furthermore, NLRP3-specific inhibitor, MCC950, as well as an interleukin-1 receptor antagonist significantly alleviated systemic LPS-induced hearing loss and inflammatory phenotypes in NLRP3 mutant mice. Interpretation: Our findings reveal that CAPS-associated NLRP3 mutation is critical for peripheral inflammation-induced hearing loss in our CAPS mice model, and an NLRP3-specific inhibitor can be used to treat inflammation-mediated sensorineural hearing loss. Funding: National Research Foundation of Korea Grant funded by the Korean Government and the Team Science Award of Yonsei University College of Medicine.

Clinical exome sequencing has yielded extensive disease-related missense single-nucleotide variants (SNVs) of uncertain significance, leading to diagnostic uncertainty. KCNQ4 is one of the most commonly responsible genes for autosomal dominant nonsyndromic hearing loss. According to the gnomAD cohort, approximately one in 100 people harbors missense variants in KCNQ4 (missense variants with minor allele frequency > 0.1% were excluded), but most are of unknown consequence. To prospectively characterize the function of all 4085 possible missense SNVs of human KCNQ4 , we recorded the whole-cell currents using the patch-clamp technique and categorized 1068 missense SNVs as loss of function, as well as 728 loss-of-function SNVs located in the transmembrane domains. Further, to mimic the heterozygous condition in Deafness nonsyndromic autosomal dominant 2 (DFNA2) patients caused by KCNQ4 variants, we coexpressed loss-of-function variants with wild-type KCNQ4 and found 516 variants showed impaired or only partially rescued heterogeneous channel function. Overall, our functional classification is highly concordant with the auditory phenotypes in Kcnq4 mutant mice and the assessments of pathogenicity in clinical variant interpretations. Taken together, our results provide strong functional evidence to support the pathogenicity classification of newly discovered KCNQ4 missense variants in clinical genetic testing.

Over 27 million people worldwide currently receive daily antiretroviral therapy for the management of HIV/AIDS. In order to prevent the continued spread of HIV, the World Health Organization (WHO) recommends the use of highly active antiretroviral therapy by pregnant and nursing women. There is currently little research into the auditory effects of this therapy on children exposed during pregnancy and breastfeeding, and research to date on the direct effects of antiretroviral exposure on the auditory system is inconclusive. The current study examined the effects of WHO-recommended first-line antiretrovirals in a well-controlled animal model to evaluate the potential for auditory damage and dysfunction following these exposures. Female breeding mice were each exposed to one of four antiretroviral cocktails or a vehicle control once daily during pregnancy and breastfeeding. Offspring of these mice had their auditory status evaluated after weaning using auditory brainstem responses and distortion-product otoacoustic emissions (DPOAEs). Auditory brainstem response thresholds following antiretroviral exposure during gestation and breastfeeding showed elevated thresholds and increased wave latencies in offspring of exposed mice when compared to unexposed controls, but no corresponding decrease in DPOAE amplitude. These differences in threshold were small and so may explain the lack of identified hearing loss in antiretroviral-exposed children during hearing screenings at birth. Minimal degrees of hearing impairment in children have been correlated with decreased academic performance and impaired auditory processing, and so these findings, if also seen in human children, suggest significant implications for children exposed to antiretrovirals during development despite passing hearing screenings at birth.

The progressive motor neuropathy (PMN) mouse is a model of an inherited motor neuropathy disease with progressive neurodegeneration. Axon degeneration associates with homozygous mutations of the TBCE gene encoding the tubulin chaperone E protein. TBCE is responsible for the correct dimerization of alpha and beta-tubulin. Strikingly, the PMN mouse also develops a progressive hearing loss after normal hearing onset, characterized by degeneration of the auditory nerve and outer hair cell (OHC) loss. However, the development of this neuronal and cochlear pathology is not fully understood yet. Previous studies with pegylated insulin-like growth factor 1 (peg-IGF-1) treatment in this mouse model have been shown to expand lifespan, weight, muscle strength, and motor coordination. Accordingly, peg-IGF-1 was evaluated for an otoprotective effect. We investigated the effect of peg-IGF-1 on the auditory system by treatment starting at postnatal day 15 (p15). Histological analysis revealed positive effects on OHC synapses of medial olivocochlear (MOC) neuronal fibers and a short-term attenuation of OHC loss. Peg-IGF-1 was able to conditionally restore the disorganization of OHC synapses and maintain the provision of cholinergic acetyltransferase in presynapses. To assess auditory function, frequency-specific auditory brainstem responses and distortion product otoacoustic emissions were recorded in animals on p21 and p28. However, despite the positive effect on MOC fibers and OHC, no restoration of hearing could be achieved. The present work demonstrates that the synaptic pathology of efferent MOC fibers in PMN mice represents a particular form of “efferent auditory neuropathy.” Peg-IGF-1 showed an otoprotective effect by preventing the degeneration of OHCs and efferent synapses. However, enhanced efforts are needed to optimize the treatment to obtain detectable improvements in hearing performances.

Anxiety is characteristic comorbidity of noise-induced hearing loss (NIHL), which causes physiological changes within the dentate gyrus (DG), a subfield of the hippocampus that modulates anxiety. However, which DG circuit underlies hearing loss-induced anxiety remains unknown. We utilize an NIHL mouse model to investigate short- and long-term synaptic plasticity in DG networks. The recently discovered longitudinal DG-DG network is a collateral of DG neurons synaptically connected with neighboring DG neurons and displays robust synaptic efficacy and plasticity. Furthermore, animals with NIHL demonstrate increased anxiety-like behaviors similar to a response to chronic restraint stress. These behaviors are concurrent with enhanced synaptic responsiveness and suppressed short- and long-term synaptic plasticity in the longitudinal DG-DG network but not in the transverse DG-CA3 connection. These findings suggest that DG-related anxiety is typified by synaptic alteration in the longitudinal DG-DG network.

Objectives To systematically explore the differences in acoustic changes and healing outcomes of tympanic membranes (TMs) with pars flaccida perforation (PFP) and pars tensa perforation (PTP). Methods We created PFPs and PTPs of various sizes in Sprague–Dawley rats, and evaluated TM umbo velocity and hearing function using laser Doppler vibrometry and auditory brainstem response (ABR) measurement before and immediately after perforation. Two weeks later, hearing was reevaluated and TMs were investigated by immunohistochemical staining. Results Small PFPs and PTPs did not significantly affect umbo velocity and hearing function. Large PFPs increased umbo velocity loss at low frequency (1.5 kHz) and elevated ABR thresholds within 1–2 kHz. Large PTP caused significant velocity loss at low frequencies from 1.5 to 3.5 kHz and threshold elevations at full frequencies (1–2 kHz). Two weeks after the perforation, the hearing function of rats with healed PFPs recovered completely. However, high‐frequency hearing loss (16–32 kHz) persisted in rats with healed PTPs. Morphological staining revealed that no increase in the thickness and obvious increase in collagen I level of regenerated par flaccida; regenerated pars tensa exhibited obvious increase in thickness and increased collagen I, while the collagen II regeneration was limited with discontinuous and disordered structure in regenerated pars tensa. Conclusion The hearing loss caused by large PFP limits at low frequencies while large PTP can lead to hearing loss at wide range frequencies. PFP and PTP have different functional outcomes after spontaneous healing, which is determined by the discrepant structure reconstruction and collagen regeneration.

The cochlea consists of multiple types of cells, including hair cells, supporting cells and spiral ganglion neurons, and is responsible for converting mechanical forces into electric signals that enable hearing. Genetic and environmental factors can result in dysfunctions of cochlear and auditory systems. In recent years, gene therapy has emerged as a promising treatment in animal deafness models. One major challenge of the gene therapy for deafness is to effectively deliver genes to specific cells of cochleae. Here, we screened and identified an AAV-ie mutant, AAV-ie-K558R, that transduces hair cells and supporting cells in the cochleae of neonatal mice with high efficiency. AAV-ie-K558R is a safe vector with no obvious deficits in the hearing system. We found that AAV-ie-K558R can partially restore the hearing loss in Prestin KO mice and, importantly, deliver Atoh1 into cochlear supporting cells to generate hair cell-like cells. Our results demonstrate the clinical potential of AAV-ie-K558R for treating the hearing loss caused by hair cell death.

Age-related hearing loss (AHL) is the most common sensory disorder amongst the elderly population. Although the degeneration of spiral ganglion neurons (SGNs) and hair cells (HCs) is considered to play a critical role in AHL, the mechanism has not been fully outlined. The repressor element 1-silencing transcription factor (REST) has recently been associated with mediating cell death in neurodegenerative diseases. However, whether REST induces degeneration of cochlear HCs and SGNs to contribute to AHL remains unknown. Here, we report that REST expression was decreased in HCs and SGNs in AHL mice. Conditional deletion of Rest in HCs and SGNs of 2-month-old mice resulted in hearing loss accompanied by the upregulation of p53, TNFR1(tumor necrosis factor receptor-1), and cleaved caspase-3. The p53 inhibitor pifithrin-α significantly attenuated SGN and HC damage and rescued hearing impairment in Rest cKO mice. Furthermore, downregulation of REST by H 2 O 2 treatment induced apoptosis in the House Ear Institute Organ of Corti 1 cell, through the upregulation of p53. In contrast, overexpression of REST reversed the changes in p53 expression. In addition, REST was further shown to bind directly to the p53 promoter site, thereby inhibiting the effect of p53. Finally, in aged mice, the p53 inhibitor significantly reduced loss of HCs and SGNs, and subsequently improved hearing. In summary, our findings indicate that REST has a protective role in AHL, and that its deficiency upregulates p53 and induces cochlear cell apoptosis, which that leads to deafness.

Background Endolymphatic hydrops (EH) is considered as the pathological correlate of Menière’s disease (MD) and cause of hearing loss. The mechanism of EH, remaining unrevealed, poses challenges for formalized clinical trials. Objective This study aims to investigate the development of hearing loss, as well as the effect of dehydration treatment on EH animal models. Methods In this study, different severity EH animal models were created. The laser Doppler vibrometer (LDV) and auditory brainstem responses (ABR) were used to study the effects of EH and the dehydration effects of mannitol. The LDV was used to measure the vibration of the round window membrane (RWM) reflecting the changes in inner ear impedance. ABR was used to evaluate the hearing changes. Furthermore, tissue section and scanning electron microscopy (SEM) observations were used to analyze the anatomical change to the cochlea and outer hair cells. Results The RWM vibrations decreased with the severity of EH, indicating an increase in the cochlear impedance. The dehydration therapy lowered the impedance to restore acoustic transduction in EH 10- and 20-day animal models. Simultaneously, the ABR thresholds increased in EH models and were restored after dehydration. Moreover, a difference in the hearing was found between ABR and LDV results in severe EH animal models, and the dehydration therapy was less effective, indicating a sensorineural hearing loss (SNHL). Conclusion Endolymphatic hydrops causes hearing loss by increasing the cochlear impedance in all tested groups, and mannitol dehydration is an effective therapy to restore hearing. However, SNHL occurs for the EH 30-day animal models, limiting the effectiveness of dehydration. Our results suggest the use of dehydrating agents in the early stage of EH.

The laryngeal echolocation is regarded as one of the conspicuous traits that play major roles in flourishing bats. Whether the laryngeal echolocation in bats originated once, however, is still controversial. We here address this question by performing molecular convergence analyses between ancestral branches of bats and toothed whales. Compared with controls, the molecular convergences were enriched in hearing-related genes for the last common ancestor of bats (LCAB) and extant echolocating bats, but not for the LCA of Old World fruit bats (LCAP). And the convergent hearing gene prestin of the LCAB and the extant echolocating bats functionally converged. More importantly, the high-frequency hearing of the LCAP-prestin knock-in mice decreased with lower cochlear outer hair cell function compared with the LCAB-prestin knock-in mice. Together, our findings provide multiple lines of evidence suggesting a single origin of laryngeal echolocation in the LCAB and the subsequent loss in the LCAP.

Voltage-gated Ca 2+ channels are critical for the development and mature function of the nervous system. Variants in the CACNA2D4 gene encoding the α 2 δ-4 auxiliary subunit of these channels are associated with neuropsychiatric and neurodevelopmental disorders. α 2 δ-4 is prominently expressed in the retina and is crucial for vision, but extra-retinal functions of α 2 δ-4 have not been investigated. Here, we sought to fill this gap by analyzing the behavioral phenotypes of α 2 δ-4 knockout (KO) mice. α 2 δ-4 KO mice (both males and females) exhibited significant impairments in prepulse inhibition that were unlikely to result from the modestly elevated auditory brainstem response thresholds. Whereas α 2 δ-4 KO mice of both sexes were hyperactive in various assays, only females showed impaired motor coordination in the rotarod assay. α 2 δ-4 KO mice exhibited anxiolytic and anti-depressive behaviors in the elevated plus maze and tail suspension tests, respectively. Our results reveal an unexpected role for α 2 δ-4 in sensorimotor gating and motor function and identify α 2 δ-4 KO mice as a novel model for studying the pathophysiology associated with CACNA2D4 variants.

Age-related hearing loss (ARHL) is a major hearing impairment characterized by pathological changes in both the peripheral and central auditory systems. Low-grade inflammation was observed in the cochlea of deceased human subjects with ARHL and animal models of early onset ARHL, which suggests that inflammation contributes to the development of ARHL. However, it remains elusive how chronic inflammation progresses during normal aging in the cochlea, and especially the accompanying changes of neuroinflammation in the central auditory system. To address this, we investigated chronic inflammation in both the cochlea and the cochlear nucleus (CN) of CBA/CaJ mice, an inbred mouse strain that undergoes normal aging and develops human, like-late-onset ARHL. Using immunohistochemistry, confocal microscopy, and quantitative image processing, we measured the accumulation and activation of macrophages in the cochlea and microglia in the CN using their shared markers: ionized calcium binding adaptor molecule 1 (Iba1) and CD68—a marker of phagocytic activity. We found progressive increases in the area covered by Iba1-labeled macrophages and enhanced CD68 staining in the osseous spiral lamina of the cochlea that correlated with elevated ABR threshold across the lifespan. During the process, we further identified significant increases in microglial activation and C1q deposition in the CN, indicating increased neuroinflammation and complement activation in the central auditory system. Our study suggests that during normal aging, chronic inflammation occurs in both the peripheral and the central auditory system, which may contribute in coordination to the development of ARHL.

CRISPR/RfxCas13d (CasRx) editing system can specifically and precisely cleave single-strand RNAs, which is a promising treatment for various disorders by downregulation of related gene expression. Here, we tested this RNA-editing approach on Beethoven ( Bth ) mice, an animal model for human DFNA36 due to a point mutation in Tmc1 . We first screened 30 sgRNAs in cell cultures and found that CasRx with sgRNA3 reduced the Tmc1 Bth transcript by 90.8%, and the Tmc1 wild type transcript ( Tmc1 + ) by 44.3%. We then injected a newly developed AAV vector (AAV-PHP.eB) based CasRx into the inner ears of neonatal Bth mice, and we found that Tmc1 Bth was reduced by 70.2% in 2 weeks with few off-target effects in the whole transcriptome. Consistently, we found improved hair cell survival, rescued hair bundle degeneration, and reduced mechanoelectrical transduction current. Importantly, the hearing performance, measured in both ABR and DPOAE thresholds, was improved significantly in all ages over 8 weeks. We, therefore, have validated the CRISPR/CasRx-based RNA editing strategy in treating autosomal-dominant hearing loss, paving way for its further application in many other hereditary diseases in hearing and beyond.

The sound-evoked electrical compound potential known as auditory brainstem response (ABR) represents the firing of a heterogenous population of auditory neurons in response to sound stimuli, and is often used for clinical diagnosis based on wave amplitude and latency. However, recent ABR applications to detect human cochlear synaptopathy have led to inconsistent results, mainly due to the high variability of ABR wave-1 amplitude. Here, rather than focusing on the amplitude of ABR wave 1, we evaluated the use of ABR wave curvature to detect cochlear synaptic loss. We first compared four curvature quantification methods using simulated ABR waves, and identified that the cubic spline method using five data points produced the most accurate quantification. We next evaluated this quantification method with ABR data from an established mouse model with cochlear synaptopathy. The data clearly demonstrated that curvature measurement is more sensitive and consistent in identifying cochlear synaptic loss in mice compared to the amplitude and latency measurements. We further tested this curvature method in a different mouse model presenting with otitis media. The change in curvature profile due to middle ear infection in otitis media is different from the profile of mice with cochlear synaptopathy. Thus, our study suggests that curvature quantification can be used to address the current ABR variability issue, and may lead to additional applications in the clinic diagnosis of hearing disorders.