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Aging, a complex process marked by molecular and cellular changes, inevitably influences tissue and organ homeostasis and leads to an increased onset or progression of many chronic diseases and conditions, one of which is age-related hearing loss (ARHL). ARHL, known as presbycusis, is characterized by the gradual and irreversible decline in auditory sensitivity, accompanied by the loss of auditory sensory cells and neurons, and the decline in auditory processing abilities associated with aging. The extended human lifespan achieved by modern medicine simultaneously exposes a rising prevalence of age-related conditions, with ARHL being one of the most significant. While our understanding of the molecular basis for aging has increased over the past three decades, a further understanding of the interrelationship between the key pathways controlling the aging process and the development of ARHL is needed to identify novel targets for the treatment of AHRL. The dysregulation of molecular pathways (AMPK, mTOR, insulin/IGF-1, and sirtuins) and cellular pathways (senescence, autophagy, and oxidative stress) have been shown to contribute to ARHL. However, the mechanistic basis for these pathways in the initiation and progression of ARHL needs to be clarified. Therefore, understanding how longevity pathways are associated with ARHL will directly influence the development of therapeutic strategies to treat or prevent ARHL. This review explores our current understanding of the molecular and cellular mechanisms of aging and hearing loss and their potential to provide new approaches for early diagnosis, prevention, and treatment of ARHL.

Biomolecular condensates or membraneless organelles (MLOs) formed by liquid-liquid phase separation (LLPS) divide intracellular spaces into discrete compartments for specific functions. Dysregulation of LLPS or aberrant phase transition that disturbs the formation or material states of MLOs is closely correlated with neurodegeneration, tumorigenesis, and many other pathological processes. Herein, we summarize the recent progress in development of methods to monitor phase separation and we discuss the biogenesis and function of MLOs formed through phase separation. We then present emerging proof-of-concept examples regarding the disruption of phase separation homeostasis in a diverse array of clinical conditions including neurodegenerative disorders, hearing loss, cancers, and immunological diseases. Finally, we describe the emerging discovery of chemical modulators of phase separation., (© 2024. Science China Press.)

Hearing loss is a major health concern in our society, affecting more than 400 million people worldwide. Among the causes, aminoglycoside therapy can result in permanent hearing loss in 40% to 60% of patients receiving treatment, and despite these high numbers, no drug for preventing or treating this type of hearing loss has yet been approved by the US Food and Drug Administration. We have previously conducted high-throughput screenings of bioactive compounds, using zebrafish as our discovery platform, and identified piplartine as a potential therapeutic molecule. In the present study, we expanded this work and characterized piplartine's physicochemical and therapeutic properties. We showed that piplartine had a wide therapeutic window and neither induced nephrotoxicity in vivo in zebrafish nor interfered with aminoglycoside antibacterial activity. In addition, a fluorescence-based assay demonstrated that piplartine did not inhibit cytochrome C activity in microsomes. Coadministration of piplartine protected from kanamycin-induced hair cell loss in zebrafish and protected hearing function, outer hair cells, and presynaptic ribbons in a mouse model of kanamycin ototoxicity. Last, we investigated piplartine's mechanism of action by phospho-omics, immunoblotting, immunohistochemistry, and molecular dynamics experiments. We found an up-regulation of AKT1 signaling in the cochleas of mice cotreated with piplartine. Piplartine treatment normalized kanamycin-induced up-regulation of TRPV1 expression and modulated the gating properties of this receptor. Because aminoglycoside entrance to the inner ear is, in part, mediated by TRPV1, these results suggested that by regulating TRPV1 expression, piplartine blocked aminoglycoside's entrance, thereby preventing the long-term deleterious effects of aminoglycoside accumulation in the inner ear compartment.

As a broad-spectrum anticancer drug, cisplatin is widely used in the treatment of tumors in various systems. Unfortunately, several serious side effects of cisplatin limit its clinical application, the most common of which are nephrotoxicity and ototoxicity. Studies have shown that cochlear hair cell degeneration is the main cause of cisplatin-induced hearing loss. However, the mechanism of cisplatin-induced hair cell death remains unclear. The present study aimed to explore the potential role of activating transcription factor 6 (ATF6), an endoplasmic reticulum (ER)-localized protein, on cisplatin-induced ototoxicity in vivo and in vitro. In this study, we observed that cisplatin exposure induced apoptosis of mouse auditory OC-1 cells, accompanied by a significant increase in the expression of ATF6 and C/EBP homologous protein (CHOP). In cell or cochlear culture models, treatment with an ATF6 agonist, an ER homeostasis regulator, significantly ameliorated cisplatin-induced cytotoxicity. Further, our in vivo experiments showed that subcutaneous injection of an ATF6 agonist almost completely prevented outer hair cell loss and significantly alleviated cisplatin-induced auditory brainstem response (ABR) threshold elevation in mice. Collectively, our results revealed the underlying mechanism by which activation of ATF6 significantly improved cisplatin-induced hair cell apoptosis, at least in part by inhibiting apoptosis signal-regulating kinase 1 expression, and demonstrated that pharmacological activation of ATF6-mediated unfolded protein response is a potential treatment for cisplatin-induced ototoxicity., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

The hair bundle of cochlear hair cells comprises specialized microvilli, the stereocilia, which fulfil the role of mechanotransduction. Genetic defects and environmental noise challenge the maintenance of hair bundle structure, critically contributing to age-related hearing loss. Stereocilia fusion is a major component of the hair bundle pathology in mature hair cells, but its role in hearing loss and its molecular basis are poorly understood. Here, we utilized super-resolution expansion microscopy to examine the molecular anatomy of outer hair cell stereocilia fusion in mouse models of age-related hearing loss, heightened endoplasmic reticulum stress and prolonged noise exposure. Prominent stereocilia fusion in our model of heightened endoplasmic reticulum stress, Manf (Mesencephalic astrocyte-derived neurotrophic factor)-inactivated mice in a background with Cadherin 23 missense mutation, impaired mechanotransduction and calcium balance in stereocilia. This was indicated by reduced FM1-43 dye uptake through the mechanotransduction channels, reduced neuroplastin/PMCA2 expression and increased expression of the calcium buffer oncomodulin inside stereocilia. Sparse BAIAP2L2 and myosin 7a expression was retained in the fused stereocilia but mislocalized away from their functional sites at the tips. These hair bundle abnormalities preceded cell soma degeneration, suggesting a sequela from stereociliary molecular perturbations to cell death signalling. In the age-related hearing loss and noise-exposure models, stereocilia fusion was more restricted within the bundles, yet both models exhibited oncomodulin upregulation at the fusion sites, implying perturbed calcium homeostasis. We conclude that stereocilia fusion is linked with the failure to maintain cellular proteostasis and with disturbances in stereociliary calcium balance. KEY POINTS: Stereocilia fusion is a hair cell pathology causing hearing loss. Inactivation of Manf, a component of the endoplasmic reticulum proteostasis machinery, has a cell-intrinsic mode of action in triggering outer hair cell stereocilia fusion and the death of these cells. The genetic background with Cadherin 23 missense mutation contributes to the high susceptibility of outer hair cells to stereocilia fusion, evidenced in Manf-inactivated mice and in the mouse models of early-onset hearing loss and noise exposure. Endoplasmic reticulum stress feeds to outer hair cell stereocilia bundle pathology and impairs the molecular anatomy of calcium regulation. The maintenance of the outer hair cell stereocilia bundle cohesion is challenged by intrinsic and extrinsic stressors, and understanding the underlying mechanisms will probably benefit the development of interventions to promote hearing health., (© 2024 The Author(s). The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

Our study aimed to investigate the role of ferroptosis in sevoflurane-induced hearing impairment and explore the mechanism of the microRNA-182-5p (miR-182-5p)/Glutathione Peroxidase 4 (GPX4) pathway in sevoflurane-induced ototoxicity. Immunofluorescence staining was performed using myosin 7a and CtBP2. Cell viability was assessed using the CCK-8 kit. Fe 2+ concentration was measured using FerroOrange and Mi-to-FerroGreen fluorescent probes. The lipid peroxide level was assessed using BODIPY 581/591 C11 and MitoSOX fluorescent probes. The auditory brainstem response (ABR) test was conducted to evaluate the hearing status. Bioinformatics tools and dual luciferase gene reporter analysis were used to confirm the direct targeting of miR-182-5p on GPX4 mRNA. GPX4 and miR-182-5p expression in cells was assessed by qRT-PCR and Western blot. Ferrostatin-1 (Fer-1) pretreatment significantly improved hearing impairment and damage to ribbon synapses in mice caused by sevoflurane exposure. Immunofluorescence staining revealed that Fer-1 pretreatment reduced intracellular and mitochondrial iron overload, as well as lipid peroxide accumulation. Our findings indicated that miR-182-5p was upregulated in sevoflurane-exposed HEI-OC1 cells, and miR-182-5p regulated GPX4 expression by binding to the 3'UTR of GPX4 mRNA. The inhibition of miR-182-5p attenuated sevoflurane-induced iron overload and lipid peroxide accumulation. Our study elucidated that the miR-182-5p/GPX4 pathway was implicated in sevoflurane-induced ototoxicity by promoting ferroptosis.

The disruption of ribbon synapses in the cochlea impairs the transmission of auditory signals from the cochlear sensory receptor cells to the auditory cortex. Although cisplatin-induced loss of ribbon synapses is well-documented, and studies have reported nitration of cochlear proteins after cisplatin treatment, yet the underlying mechanism of cochlear synaptopathy is not fully understood. This study tests the hypothesis that cisplatin treatment alters the abundance of cochlear synaptosomal proteins, and selective targeting of nitrative stress prevents the associated synaptic dysfunction. Auditory brainstem responses of mice treated with cisplatin showed a reduction in amplitude and an increase in latency of wave I, indicating cisplatin-induced synaptic dysfunction. The mass spectrometry analysis of cochlear synaptosomal proteins identified 102 proteins that decreased in abundance and 249 that increased in abundance after cisplatin treatment. Pathway analysis suggested that the dysregulated proteins were involved in calcium binding, calcium ion regulation, synapses, and endocytosis pathways. Inhibition of nitrative stress by co-treatment with MnTBAP, a peroxynitrite scavenger, attenuated cisplatin-induced changes in the abundance of 27 proteins. Furthermore, MnTBAP co-treatment prevented the cisplatin-induced decrease in the amplitude and increase in the latency of wave I. Together, these findings suggest a potential role of oxidative/nitrative stress in cisplatin-induced cochlear synaptic dysfunction., Competing Interests: Declaration of competing interest On behalf of all authors, the corresponding author states that there is no conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Age-related hearing loss (HL), or presbycusis, is a complex and heterogeneous condition, affecting a significant portion of older adults and involving various interacting mechanisms. Metabolic presbycusis, a type of age-related HL, is characterized by the dysfunction of the stria vascularis, which is crucial for maintaining the endocochlear potential necessary for hearing. Although attention on metabolic presbycusis has waned in recent years, research continues to identify strial pathology as a key factor in age-related HL. This narrative review integrates past and recent research, bridging findings from animal models and human studies, to examine the contributions of the stria vascularis to age-related HL. It provides a brief overview of the structure and function of the stria vascularis and then examines mechanisms contributing to age-related strial dysfunction, including altered ion transport, changes in pigmentation, inflammatory responses, and vascular atrophy. Importantly, this review outlines the contribution of metabolic mechanisms to age-related HL, highlighting areas for future research. It emphasizes the complex interdependence of metabolic and sensorineural mechanisms in the pathology of age-related HL and highlights the importance of animal models in understanding the underlying mechanisms. The comprehensive and mechanistic investigation of all factors contributing to age-related HL, including cochlear metabolic dysfunction, remains crucial to identifying the underlying mechanisms and developing personalized, protective, and restorative treatments.

Hearing loss is a pivotal risk factor for dementia. It has recently emerged that a disruption in the intercommunication between the cochlea and brain is a key process in the initiation and progression of this disease. However, whether the cochlear properties can be influenced by pathological signals associated with dementia remains unclear. In this study, using a mouse model of Alzheimer's disease (AD), we investigated the impacts of the AD-like amyloid β (Aβ) pathology in the brain on the cochlea. Despite little detectable change in the age-related shift of the hearing threshold, we observed quantitative and qualitative alterations in the protein profile in perilymph, an extracellular fluid that fills the path of sound waves in the cochlea. Our findings highlight the potential contribution of Aβ pathology in the brain to the disturbance of cochlear homeostasis., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Fukuda et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Mutations in AIFM1, encoding for apoptosis-inducing factor (AIF), cause AUNX1, an X-linked neurologic disorder with late-onset auditory neuropathy (AN) and peripheral neuropathy. Despite significant research on AIF, there are limited animal models with the disrupted AIFM1 representing the corresponding phenotype of human AUNX1, characterized by late-onset hearing loss and impaired auditory pathways. Here, we generated an Aifm1 p.R450Q knock-in mouse model (KI) based on the human AIFM1 p.R451Q mutation. Hemizygote KI male mice exhibited progressive hearing loss from P30 onward, with greater severity at P60 and stabilization until P210. Additionally, muscle atrophy was observed at P210. These phenotypic changes were accompanied by a gradual reduction in the number of spiral ganglion neuron cells (SGNs) at P30 and ribbons at P60, which coincided with the translocation of AIF into the nucleus starting from P21 and P30, respectively. The SGNs of KI mice at P210 displayed loss of cytomembrane integrity, abnormal nuclear morphology, and dendritic and axonal demyelination. Furthermore, the inner hair cells and myelin sheath displayed abnormal mitochondrial morphology, while fibroblasts from KI mice showed impaired mitochondrial function. In conclusion, we successfully generated a mouse model recapitulating AUNX1. Our findings indicate that disruption of Aifm1 induced the nuclear translocation of AIF, resulting in the impairment in the auditory pathway., (© The Author(s) 2024. Published by Oxford University Press.)

Cisplatin-induced hearing loss is a common side effect of cancer chemotherapy in clinics; however, the mechanism of cisplatin-induced ototoxicity is still not completely clarified. Cisplatin-induced ototoxicity is mainly associated with the production of reactive oxygen species, activation of apoptosis, and accumulation of intracellular lipid peroxidation, which also is involved in ferroptosis induction. In this study, the expression of TfR1, a ferroptosis biomarker, was upregulated in the outer hair cells of cisplatin-treated mice. Moreover, several key ferroptosis regulator genes were altered in cisplatin-damaged cochlear explants based on RNA sequencing, implying the induction of ferroptosis. Ferroptosis-related Gpx4 and Fsp1 knockout mice were established to investigate the specific mechanisms associated with ferroptosis in cochleae. Severe outer hair cell loss and progressive damage of synapses in inner hair cells were observed in Atoh1-Gpx4 -/- mice. However, Fsp1 -/- mice showed no significant hearing phenotype, demonstrating that Gpx4, but not Fsp1, may play an important role in the functional maintenance of HCs. Moreover, findings showed that FDA-approved luteolin could specifically inhibit ferroptosis and alleviate cisplatin-induced ototoxicity through decreased expression of transferrin and intracellular concentration of ferrous ions. This study indicated that ferroptosis inhibition through the reduction of intracellular ferrous ions might be a potential strategy to prevent cisplatin-induced hearing loss., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024. Published by Elsevier Inc.)

Local drug delivery has been exploited recently to treat hearing loss, as this method can both bypass the blood-labyrinth barrier and provide sustained drug release. Combined drug microcrystals (MCs) offer additional advantages for sensorineural hearing loss treatment via intratympanic (IT) injection due to their shape effect and combination strategy. In this study, to endow viscous effects of hydrogels, nonspherical dexamethasone (DEX) and lipoic acid (LA) MCs were incorporated into silk fibroin (SF) hydrogels, which were subsequently administered to the tympanic cavity to investigate their pharmaceutical properties. First, we prepared DEX and LA MCs by a traditional precipitation technique followed by SF hydrogel incorporation (SF+DEX+LA). After characterization of the physicochemical features, including morphology, rheology, and dissolution, both a suspension of combined DEX and LA MCs (DEX+LA) and SF+DEX+LA were administered to guinea pigs by IT injection, after which the pharmacokinetics, biodegradation and biocompatibility were evaluated. To our surprise, compared to the DEX+LA group, the pharmacokinetics of the SF+DEX+LA hydrogel group did not improve significantly, which may be ascribed to their nonspherical shape and deposition effects of the drugs MCs. The cochlear tissue in each group displayed good morphology, with no obvious inflammatory reactions. This combined MC suspension has the clear advantages of no vehicle, easy scale-up preparation, and good biocompatibility and outcomes, which paves the way for practical treatment of hearing loss via local drug delivery., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Objectives: Biochemical alterations due to diabetes development and progress are complex and diabetes-associated injury to various tissues has been well reported. Nevertheless, a close investigation of the literature demonstrates limited coverage regarding these biochemical and molecular alterations within the inner ear and their impact on the vestibulocochlear environment. A closer look at these may reveal pharmacological targets that could alleviate the severity of disease in patients., Key Findings: Tight control of glucose levels within the highly metabolic inner ear structures is crucial for their physiology and function. Impaired glucose homeostasis is well known to occur in vestibulocochlear malfunctioning. Moreover, the involvement of insulin signalling, and glucose transporters were recently confirmed in vestibulocochlear structures and are believed to play a crucial role in auditory and vestibular functions., Conclusion: Oxidative overload, glucolipotoxicity, perturbed blood rheology, endothelial dysfunction, compromised microvascular supply, and neurotoxicity are reported in many diabetic complications such as nephropathy, retinopathy, and diabetic neuropathy and are incriminated in the disruption of blood labyrinth barrier as well as vestibulocochlear neuritis. Dysfunctional insulin signalling was recently reported in the Organ of Corti. Insulin resistance in the inner ear niche warrants further studies to verify and uncover new pharmacological targets to manage this debilitating condition better., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Royal Pharmaceutical Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

In recent years, extensive research has been conducted on the pathogenesis of sensorineural hearing loss (SNHL). Apoptosis and necrosis have been identified to play important roles in hearing loss, but they cannot account for all hearing loss. Autophagy, a cellular process responsible for cell self-degradation and reutilization, has emerged as a significant factor contributing to hearing loss, particularly in cases of autophagy deficiency. Autophagy plays a crucial role in maintaining cell health by exerting cytoprotective and metabolically homeostatic effects in organisms. Consequently, modulating autophagy levels can profoundly impact the survival, death, and regeneration of cells in the inner ear, including hair cells (HCs) and spiral ganglion neurons (SGNs). Abnormal mitochondrial autophagy has been demonstrated in animal models of SNHL. These findings indicate the profound significance of comprehending autophagy while suggesting that our perspective on this cellular process holds promise for advancing the treatment of SNHL. Thus, this review aims to clarify the pathogenic mechanisms of SNHL and the role of autophagy in the developmental processes of various cochlear structures, including the greater epithelial ridge (GER), SGNs, and the ribbon synapse. The pathogenic mechanisms of age-related hearing loss (ARHL), also known as presbycusis, and the latest research on autophagy are also discussed. Furthermore, we underscore recent findings on the modulation of autophagy in SNHL induced by ototoxic drugs. Additionally, we suggest further research that might illuminate the complete potential of autophagy in addressing SNHL, ultimately leading to the formulation of pioneering therapeutic strategies and approaches for the treatment of deafness., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Inner ear hair cells are characterized by the F-actin-based stereocilia that are arranged into a staircase-like pattern on the apical surface of each hair cell. The tips of shorter-row stereocilia are connected with the shafts of their neighboring taller-row stereocilia through extracellular links named tip links, which gate mechano-electrical transduction (MET) channels in hair cells. Cadherin 23 (CDH23) forms the upper part of tip links, and its cytoplasmic tail is inserted into the so-called upper tip-link density (UTLD) that contains other proteins such as harmonin. The Cdh23 gene is composed of 69 exons, and we show here that exon 68 is subjected to hair cell-specific alternative splicing. Tip-link formation is not affected in genetically modified mutant mice lacking Cdh23 exon 68. Instead, the stability of tip links is compromised in the mutants, which also suffer from progressive and noise-induced hearing loss. Moreover, we show that the cytoplasmic tail of CDH23(+68) but not CDH23(-68) cooperates with harmonin in phase separation-mediated condensate formation. In conclusion, our work provides evidence that inclusion of Cdh23 exon 68 is critical for the stability of tip links through regulating condensate formation of UTLD components., Competing Interests: Competing interests statement:Author U.M. and Editor J.N. are at the same institution but have not collaborated directly.

DFNB61 is a recessively inherited nonsyndromic hearing loss caused by mutations in SLC26A5, the gene that encodes the voltage-driven motor protein, prestin. Prestin is abundantly expressed in the auditory outer hair cells that mediate cochlear amplification. Two DFNB61-associated SLC26A5 variants, p.W70X and p.R130S, were identified in patients who are compound heterozygous for these nonsense and missense changes (SLC26A5 W70X/R130S ). Our recent study showed that mice homozygous for p.R130S (Slc26a5 R130S/R130S ) suffer from hearing loss that is ascribed to significantly reduced motor kinetics of prestin. Given that W70X-prestin is nonfunctional, compound heterozygous Slc26a5 R130S/- mice were used as a model for human SLC26A5 W70X/R130S . By examining the pathophysiological consequences of p.R130S prestin when it is the sole allele for prestin protein production, we determined that this missense change results in progressive outer hair cell loss in addition to its effects on prestin's motor action. Thus, this study defines the pathogenic roles of p.R130S prestin and identifies a limited time window for potential clinical intervention. KEY POINTS: The voltage-driven motor protein, prestin, is encoded by SLC26A5 and expressed abundantly in cochlear outer hair cells (OHCs). The importance of prestin for normal hearing was demonstrated in mice lacking prestin; however, none of the specific SLC26A5 variants identified to date in human patients has been experimentally demonstrated to be pathogenic. In this study we used both cell lines and a mouse model to define the pathogenic role of compound heterozygous p.W70X (c.209G>A) and p.R130S (c.390A>C) SLC26A5 variants identified in patients with moderate to profound hearing loss. As in patients, mice carrying one copy of p.R130S Slc26a5 showed OHC dysfunction and progressive degeneration, which results in congenital progressive hearing loss. This is the first functional study reporting pathogenic SLC26A5 variants and pointing to the presence of a therapeutic time window for potential clinical interventions targeting the affected OHCs before they are lost., (© 2024 The Authors. The Journal of Physiology © 2024 The Physiological Society.)

The incidence and prevalence of hearing loss is increasing globally at an accelerated pace. Hair cells represent the sensory receptors of auditory and vestibular systems. Hair cell absence, loss or degeneration due to congenital diseases, trauma, toxicity, infection or advancing age, results in disabling hearing loss. Regenerative medicine approaches consisting in stem cell-based hair cell rescue or regeneration, gene therapy, as well as cell and tissue engineering are expected to dramatically improve the therapeutic arsenal available for addressing hearing loss. Current strategies that are using different stem cell types to rescue or to induce hair cell proliferation and regeneration are presented. Gene and cell therapy methods that modulates transdifferentiation of surrounding cell types into hair cells are presented, together with their specific advantages and limitations. Several modalities for improving therapeutic targeting to the inner ear such as nanoparticle-mediated cell and gene delivery are introduced. Further steps in building more relevant high-throughput models for testing novel drugs and advanced therapies are proposed as a modality to accelerate translation to clinical settings.

Puerarin (PUE), a flavonoid derivative with vasodilatory effects found in the traditional Chinese medicine kudzu, has anti-sensorineural hearing loss properties. However, the mechanism of its protective effect against ototoxicity is not well understood. In this study, we used in vitro and in vivo methods to investigate the protective mechanism of puerarin against cisplatin (CDDP)-induced ototoxicity. We established an ototoxicity model of CDDP in BALB/c mice and assessed the degree of hearing loss and cochlear cell damage. We used bioinformatics analysis, molecular docking, histological analysis, and biochemical and molecular biology to detect the expression of relevant factors. Our results show that puerarin improved CDDP-induced hearing loss and reduced hair cell loss. It also blocked CDDP-induced activation of TRPV1 and inhibited activation of IP3R1 to prevent intracellular calcium overload. Additionally, puerarin blocked CDDP-stimulated p65 activation, reduced excessive ROS production, and alleviated cochlear cell apoptosis. Our study provides new evidence and potential targets for the protective effect of puerarin against drug-induced hearing loss. Puerarin ameliorates cisplatin-induced ototoxicity and blocks cellular apoptosis by inhibiting CDDP activated TRPV1/IP3R1/p65 pathway, blocking induction of calcium overload and excessive ROS expression., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Inc.)

Hearing loss is a chronic disease affecting millions of people worldwide, yet no restorative treatment options are available. Although non-mammalian species can regenerate their auditory sensory hair cells, mammals cannot. Birds retain facultative stem cells known as supporting cells that engage in proliferative regeneration when surrounding hair cells die. Here, we investigated gene expression changes in chicken supporting cells during auditory hair cell death. This identified a pathway involving the receptor F2RL1, HBEGF, EGFR, and ERK signaling. We propose a cascade starting with the proteolytic activation of F2RL1, followed by matrix-metalloprotease-mediated HBEGF shedding, and culminating in EGFR-mediated ERK signaling. Each component of this cascade is essential for supporting cell S-phase entry in vivo and is integral for hair cell regeneration. Furthermore, STAT3-phosphorylation converges with this signaling toward upregulation of transcription factors ATF3, FOSL2, and CREM. Our findings could provide a basis for designing treatments for hearing and balance disorders., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Introduction: Previous studies have reported that hearing loss (HL) is associated with dementia, although the mechanistic underpinnings remain elusive. This study aimed to evaluate the changes in brain metabolism in patients with HL and different types of dementia., Methods: Patients with cognitive impairment (CI) and HL treated at the university-based memory clinic from May 2016 to October 2021 were included. In total, 108 patients with CI and HL prospectively underwent audiometry, neuropsychological test, magnetic resonance imaging, and 18 F-fluorodeoxyglucose positron emission tomography. Twenty-seven individuals without cognitive impairment and hearing loss were enrolled as a control group. Multivariable regression was performed to evaluate brain regions correlated with each pathology type after adjusting for confounding factors., Results: Multivariable regression analyses revealed that Alzheimer's disease-related CI (ADCI) was associated with hypometabolic changes in the right superior temporal gyrus (STG), right middle temporal gyrus (MTG), and bilateral medial temporal lobe. Lewy body disease-related CI (LBDCI) and vascular CI were associated with hypermetabolic and hypometabolic changes in the ascending auditory pathway, respectively. In the pure ADCI group, the degree of HL was positively associated with abnormal increase of brain metabolism in the right MTG, whereas it was negatively associated with decreased brain metabolism in the right STG in the pure LBDCI group., Conclusion: Each dementia type is associated with distinct changes in brain metabolism in patients with HL., (© 2024 The Authors. Brain and Behavior published by Wiley Periodicals LLC.)

Class III myosins localize to inner ear hair cell stereocilia and are thought to be crucial for stereocilia length regulation. Mutations within the motor domain of MYO3A that disrupt its intrinsic motor properties have been associated with non-syndromic hearing loss, suggesting that the motor properties of MYO3A are critical for its function within stereocilia. In this study, we investigated the impact of a MYO3A hearing loss mutation, H442N, using both in vitro motor assays and cell biological studies. Our results demonstrate the mutation causes a dramatic increase in intrinsic motor properties, actin-activated ATPase and in vitro actin gliding velocity, as well as an increase in actin protrusion extension velocity. We propose that both "gain of function" and "loss of function" mutations in MYO3A can impair stereocilia length regulation, which is crucial for stereocilia formation during development and normal hearing. Furthermore, we generated chimeric MYO3A constructs that replace the MYO3A motor and neck domain with the motor and neck domain of other myosins. We found that duty ratio, fraction of ATPase cycle myosin is strongly bound to actin, is a critical motor property that dictates the ability to tip localize within filopodia. In addition, in vitro actin gliding velocities correlated extremely well with filopodial extension velocities over a wide range of gliding and extension velocities. Taken together, our data suggest a model in which tip-localized myosin motors exert force that slides the membrane tip-ward, which can combat membrane tension and enhance the actin polymerization rate that ultimately drives protrusion elongation., Competing Interests: Conflict of interest The authors declare no conflict of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Transmembrane and tetratricopeptide repeat 4 (Tmtc4) is a deafness gene in mice. Tmtc4-KO mice have rapidly progressive postnatal hearing loss due to overactivation of the unfolded protein response (UPR); however, the cellular basis and human relevance of Tmtc4-associated hearing loss in the cochlea was not heretofore appreciated. We created a hair cell-specific conditional KO mouse that phenocopies the constitutive KO with postnatal onset deafness, demonstrating that Tmtc4 is a hair cell-specific deafness gene. Furthermore, we identified a human family in which Tmtc4 variants segregate with adult-onset progressive hearing loss. Lymphoblastoid cells derived from multiple affected and unaffected family members, as well as human embryonic kidney cells engineered to harbor each of the variants, demonstrated that the human Tmtc4 variants confer hypersensitivity of the UPR toward apoptosis. These findings provide evidence that TMTC4 is a deafness gene in humans and further implicate the UPR in progressive hearing loss.

The mitochondrial matrix peptidase CLPP is crucial during cell stress. Its loss causes Perrault syndrome type 3 (PRLTS3) with infertility, neurodegeneration, and a growth deficit. Its target proteins are disaggregated by CLPX, which also regulates heme biosynthesis via unfolding ALAS enzymes, providing access for pyridoxal-5'-phosphate (PLP). Despite efforts in diverse organisms with multiple techniques, CLPXP substrates remain controversial. Here, avoiding recombinant overexpression, we employed complexomics in mitochondria from three mouse tissues to identify endogenous targets. A CLPP absence caused the accumulation and dispersion of CLPX-VWA8 as AAA+ unfoldases, and of PLPBP. Similar changes and CLPX-VWA8 co-migration were evident for mitoribosomal central protuberance clusters, translation factors like GFM1-HARS2, the RNA granule components LRPPRC-SLIRP, and enzymes OAT-ALDH18A1. Mitochondrially translated proteins in testes showed reductions to <30% for MTCO1-3, the mis-assembly of the complex IV supercomplex, and accumulated metal-binding assembly factors COX15-SFXN4. Indeed, heavy metal levels were increased for iron, molybdenum, cobalt, and manganese. RT-qPCR showed compensatory downregulation only for Clpx mRNA; most accumulated proteins appeared transcriptionally upregulated. Immunoblots validated VWA8, MRPL38, MRPL18, GFM1, and OAT accumulation. Co-immunoprecipitation confirmed CLPX binding to MRPL38, GFM1, and OAT, so excess CLPX and PLP may affect their activity. Our data mechanistically elucidate the mitochondrial translation fidelity deficits which underlie progressive hearing impairment in PRLTS3.

Cholesterol is a risk factor for age-related hearing loss (ARHL). However, the effect of cholesterol on the organ of Corti during the onset of ARHL is unclear. We established a mouse model for the ARHL group (24 months, n = 12) and a young group (6 months, n = 12). Auditory thresholds were measured in both groups using auditory brainstem response (ABR) at frequencies of 8, 16, and 32 kHz. Subsequently, mice were sacrificed and subjected to histological analyses, including transmission electron microscopy (TEM), H&E, Sudan Black B (SBB), and Filipin staining, as well as biochemical assays such as IHC, enzymatic analysis, and immunoblotting. Additionally, mRNA extracted from both young and aged cochlea underwent RNA sequencing. To identify the mechanism, in vitro studies utilizing HEI-OC1 cells were also performed. RNA sequencing showed a positive correlation with increased expression of genes related to metabolic diseases, cholesterol homeostasis, and target of rapamycin complex 1 (mTORC1) signaling in the ARHL group as compared to the younger group. In addition, ARHL tissues exhibited increased cholesterol and lipofuscin aggregates in the organ of Corti, lateral walls, and spiral ganglion neurons. Autophagic flux was inhibited by the accumulation of damaged lysosomes and autolysosomes. Subsequently, we observed a decrease in the level of transcription factor EB (TFEB) protein, which regulates lysosomal biosynthesis and autophagy, together with increased mTORC1 activity in ARHL tissues. These changes in TFEB and mTORC1 expression were observed in a cholesterol-dependent manner. Treatment of ARHL mice with atorvastatin, a cholesterol synthesis inhibitor, delayed hearing loss by reducing the cholesterol level and maintaining lysosomal function and autophagy by inhibiting mTORC1 and activating TFEB. The above findings were confirmed using stress-induced premature senescent House Ear Institute organ of Corti 1 (HEI-OC1) cells. The findings implicate cholesterol in the pathogenesis of ARHL. We propose that atorvastatin could prevent ARHL by maintaining lysosomal function and autophagy by inhibiting mTORC1 and activating TFEB during the aging process.

Cingulin (CGN) is a cytoskeleton-associated protein localized at the apical junctions of epithelial cells. CGN interacts with major cytoskeletal filaments and regulates RhoA activity. However, physiological roles of CGN in development and human diseases are currently unknown. Here, we report a multi-generation family presenting with autosomal dominant non-syndromic hearing loss (ADNSHL) that co-segregates with a CGN heterozygous truncating variant, c.3330delG (p.Leu1110Leufs*17). CGN is normally expressed at the apical cell junctions of the organ of Corti, with enriched localization at hair cell cuticular plates and circumferential belts. In mice, the putative disease-causing mutation results in reduced expression and abnormal subcellular localization of the CGN protein, abolishes its actin polymerization activity, and impairs the normal morphology of hair cell cuticular plates and hair bundles. Hair cell-specific Cgn knockout leads to high-frequency hearing loss. Importantly, Cgn mutation knockin mice display noise-sensitive, progressive hearing loss and outer hair cell degeneration. In summary, we identify CGN c.3330delG as a pathogenic variant for ADNSHL and reveal essential roles of CGN in the maintenance of cochlear hair cell structures and auditory function., (© 2023 The Authors. Published under the terms of the CC BY 4.0 license.)

Previous studies indicate changes in neurotransmission along the auditory pathway in subjective tinnitus. Most authors, however, investigated brain regions including the primary auditory cortex, whose physiology can be affected by concurrent hearing deficits. In the present MR spectroscopy study we assumed increased levels of glutamate and glutamine (Glx), and other Central Nervous System metabolites in the temporal lobe outside the primary auditory cortex, in a region involved in conscious auditory perception and memory. We studied 52 participants with unilateral (n = 24) and bilateral (n = 28) tinnitus, and a control group without tinnitus (n = 25), all with no severe hearing losses and a similar hearing profile. None of the metabolite levels in the temporal regions of interest were found related to tinnitus status or laterality. Unexpectedly, we found a tendency of increased concentration of Glx in the control left medial frontal region in bilateral vs unilateral tinnitus. Slightly elevated depressive and anxiety symptoms were also shown in participants with tinnitus, as compared to healthy individuals, with the bilateral tinnitus group marginally more affected. We discuss no apparent effect in the temporal lobes, as well as the role of frontal brain areas, with respect to hearing loss, attention and psychological well-being in chronic tinnitus. We furthermore elaborate on the design-related and technical obstacles of MR spectroscopy., (© 2023. The Author(s).)

The issue of hearing protection in the presence of noise pollution is of great importance in the fields of environmental science and clinical medicine. Currently, the clinical significance of Klotho in relation to hearing has not been revealed. The aim of this study was to examine the correlation between serum Klotho levels and Pure Tone Average (PTA) hearing thresholds among individuals in the U.S.. The analysis involved a sample of 1,781 individuals aged 20 to 69, obtained from the 2007-2012 National Health and Nutrition Examination Survey. Various methods were utilized for the analysis, including univariate and multivariate linear regression, stratified analysis, smooth curve fitting, a two-segment linear regression model, and log-likelihood ratio analysis. The results of the univariate analysis indicated that serum Klotho concentration, age, education level, hypertension, diabetes, and smoking all exhibited a significant influence on PTAs. After adjusting for potential confounding factors, it was observed that a decrease in serum Klotho was significantly associated with PTA thresholds at low frequency (β = -0.002; 95% CI: -0.003, -0.001; P = 0.004), speech frequency (β = -0.002; 95% CI: -0.003, -0.001; P = 0.007), and high frequency (β = -0.002; 95% CI: -0.003, -0.001; P = 0.045). Specifically, for every 1 pg/ml decrease in serum Klotho concentration, the PTAs increased by 0.002 dB. Moreover, age and gender-specific analyses revealed significant associations. For individuals aged 59-69, a significant association was found between serum Klotho concentration and high-frequency PTA (β = -4.153; 95% CI: -7.948, -0.358; P = 0.032). Additionally, among females, significant associations were observed between serum Klotho concentration and speech-frequency PTA (β = -1.648, 95% CI: -3.197, -0.099; P = 0.037) as well as high-frequency PTA (β = -3.046; 95% CI: -5.319, -0.772; P = 0.009). Finally, the results of smooth curve fitting and threshold effect analyses indicated a potential negative linear correlation between serum Klotho concentration and PTA thresholds. In conclusion, a lower level of serum Klotho was found to be associated with increased hearing thresholds, particularly among the elderly population. This finding has significant implications for the prevention and treatment of hearing damage., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

POU4F3, a member of the POU family of transcription factors, commonly causes autosomal dominant deafness. Exome sequencing was used to identify four novel variants in POU4F3 (NM&#95;002700.2), including c.564dupA: p.Ala189SerfsTer26, c.743T > C:p.Leu248Pro, c.879C > A:p.Phe293Leu, and c.952G > A:p.Val318Met, and diverse aspects of the molecular consequences of their protein expression, stability, subcellular localization, and transcriptional activity were investigated. The expression of three mutant proteins, encoded by missense variants, was reduced compared to the wild-type protein, demonstrating that the mutants were unstable and vulnerable to degradation. Additionally, all the mutant proteins had distinct subcellular localization patterns. A mutant protein carrying p.Ala189SerfsTer26, in which both mono- and bi-partite nuclear localization signals were disrupted, showed abnormal subcellular localization. Resultantly, all the mutant proteins significantly reduced the transcriptional activity required to regulate the downstream target gene expression. Furthermore, we identified the altered expression of 14 downstream target genes associated with inner ear development using patient-derived lymphoblastoid cell lines. There was a significant correlation of the expression profile between patient-derived cells and the cochlear hair cells, which provided a breakthrough for cases where the collection of human cochlear samples for transcriptome studies was unfeasible. This study expanded the genotypic spectrum of POU4F3 in DFNA15, and further refined the molecular mechanisms underlying POU4F3-associated DFNA15., (© 2023. Springer Nature Limited.)

Previous studies have revealed that age-related hearing loss (AHL) in Cdk5 regulatory subunit-associated protein 1 (Cdk5rap1)-knockout mice is associated with pathology in the cochlea. Here, we aimed to identify mitochondrial alterations in the cochlea of Cdk5rap1-knockout mice with AHL. Mitochondria in the spiral ganglion neurons (SGNs) and hair cells (HCs) were normal despite senescence; however, the mitochondria of types I, II, and IV spiral ligament fibrocytes were ballooned, damaged, and ballooned, respectively, in the stria vascularis. Our results suggest that the accumulation of dysfunctional mitochondria in the lateral wall, rather than the loss of HCs and SGNs, leads to the onset of AHL. Our results provide valuable information regarding the underlying mechanisms of AHL and the relationship between aberrant tRNA modification-induced hearing loss and mitochondrial dysfunction., (© 2023 The Authors. FEBS Open Bio published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

The connexin gene family is the most prevalent gene that contributes to hearing loss. Connexins 26 and 30, encoded by GJB2 and GJB6 , respectively, are the most abundantly expressed connexins in the inner ear. Connexin 43, which is encoded by GJA1 , appears to be widely expressed in various organs, including the heart, skin, the brain, and the inner ear. The mutations that arise in GJB2 , GJB6 , and GJA1 can all result in comprehensive or non-comprehensive genetic deafness in newborns. As it is predicted that connexins include at least 20 isoforms in humans, the biosynthesis, structural composition, and degradation of connexins must be precisely regulated so that the gap junctions can properly operate. Certain mutations result in connexins possessing a faulty subcellular localization, failing to transport to the cell membrane and preventing gap junction formation, ultimately leading to connexin dysfunction and hearing loss. In this review, we provide a discussion of the transport models for connexin 43, connexins 30 and 26, mutations affecting trafficking pathways of these connexins, the existing controversies in the trafficking pathways of connexins, and the molecules involved in connexin trafficking and their functions. This review can contribute to a new way of understanding the etiological principles of connexin mutations and finding therapeutic strategies for hereditary deafness.

As the auditory and balance receptor cells in the inner ear, hair cells are responsible for converting mechanical stimuli into electrical signals, a process referred to as mechano-electrical transduction. Hair cell development and function are tightly regulated, and hair cell deficits are the main reasons for hearing loss and balance disorders. TMCC2 is an endoplasmic reticulum (ER)-residing transmembrane protein whose physiological function largely remains unknown. In the present work, we show that Tmcc2 is specifically expressed in the auditory hair cells of mouse inner ear. Tmcc2 knockout mice were then established to investigate its physiological role in hearing. Auditory brainstem responses measurements show that Tmcc2 knockout mice suffer from congenital hearing loss. Further investigations reveal progressive auditory hair cell loss in the Tmcc2 knockout mice. The general morphology and function of ER are unaffected in Tmcc2 knockout hair cells. However, increased ER stress was observed in Tmcc2 knockout mice and knockdown cells, suggesting that loss of TMCC2 leads to auditory hair cell death through elevated ER stress., (© The Author(s) 2023. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Background: We have developed 3D-printed microneedle technology for diagnostic aspiration of perilymph and intracochlear delivery of therapeutic agents. Single microneedle-mediated round window membrane (RWM) perforation does not cause hearing loss, heals within 48-72 h, and yields sufficient perilymph for proteomic analysis. In this study, we investigate the anatomic, physiologic, and proteomic consequences of repeated microneedle-mediated perforations of the same RWM at different timepoints., Methods: 100-μm-diameter hollow microneedles were fabricated using two-photon polymerization (2PP) lithography. The tympanic bullae of Hartley guinea pigs (n = 8) were opened with adequate exposure of the RWM. Distortion product otoacoustic emissions (DPOAE) and compound action potential (CAP) were recorded to assess hearing. The hollow microneedle was introduced into the bulla and the RWM was perforated; 1 μL of perilymph was aspirated from the cochlea over the course of 45 s. 72 h later, the above procedure was repeated with aspiration of an additional 1 μL of perilymph. 72 h after the second perforation, RWMs were harvested for confocal imaging. Perilymph proteomic analysis was completed using liquid chromatography-tandem mass spectrometry (LC-MS/MS)., Results: Two perforations and aspirations were performed in 8 guinea pigs. In six, CAP, DPOAE, and proteomic analysis were obtained; in one, only CAP and DPOAE results were obtained; and in one, only proteomics results were obtained. Hearing tests demonstrated mild hearing loss at 1-4 kHz and 28 kHz, most consistent with conductive hearing loss. Confocal microscopy demonstrated complete healing of all perforations with full reconstitution of the RWM. Perilymph proteomic analysis identified 1855 proteins across 14 samples. The inner ear protein cochlin was observed in all samples, indicating successful aspiration of perilymph. Non-adjusted paired t-tests with p < 0.01 revealed significant changes in 13 of 1855 identified proteins (0.7%) between the first and second aspirations., Conclusions: We demonstrate that repeated microneedle perforation of the RWM is feasible, allows for complete healing of the RWM, and minimally changes the proteomic expression profile. Thus, microneedle-mediated repeated aspirations in a single animal can be used to monitor the response to inner ear treatments over time., (Copyright © 2023. Published by Elsevier B.V.)

Aminoglycosides (AGs) are widely used to treat severe infections. However, systemically administered AGs preferentially kill cochlear hair cells, resulting in irreversible hearing loss. Recently, we found that AGs bind to RIPOR2 and trigger its rapid translocation in cochlear hair cells. Reducing RIPOR2 expression entirely prevents AG-induced hair cell death and subsequent hearing loss in mice. Next using yeast two-hybrid screening, we found that RIPOR2 interacts with GABARAP, a key macroautophagy/autophagy pathway protein. Following AG treatment, RIPOR2 colocalizes with GABARAP and regulates the activation of autophagy. Remarkably, reducing the expression of GABARAP, or another key autophagy protein MAP1LC3B/LC3B, entirely prevents AG-induced hair cell death and subsequent hearing loss in mice. Furthermore, we found that AGs activate the autophagy pathway specific to mitochondria. Reducing the expression of PINK1 or PRKN/parkin, two key mitophagy proteins, protects hair cells against AG toxicity. Thus, our findings demonstrated that RIPOR2-mediated autophagic dysfunction is essential for AG-induced hearing loss and provided potential therapeutic strategies for preventing AG toxicity.

Stem cell replacement holds the potential for sensorineural hearing loss (SNHL) treatment. However, its translation into clinical practice requires strategies for improving stem cell survival following intracochlear transplantation. Considering recent findings showing that the inner ear contains a resident population of immune cells, we hypothesized that immune evasion would improve the survival and residence time of transplanted stem cells in the cochlea, potentially leading to better outcomes. To test this, we leveraged genetic engineering techniques to develop hypoimmunogenic human-induced pluripotent stem cells (hi-iPSC), which lack human leukocyte antigen expression. We found that gene editing does not affect the biological properties of hi-iPSCs, including their capacity to differentiate into otic neural progenitors (ONPs). Compared to wild-type ONPs, more hypoimmunogenic ONPs (derived from hi-iPSCs) were found in the inner ear of immunocompetent mice ten days following cochlear xenotransplantation. This approach may open a new avenue for experimental and clinical SNHL treatments., (© 2023. The Author(s).)

Cisplatin is chemotherapy used for solid tumor treatment like lung, bladder, head and neck, ovarian and testicular cancers. However, cisplatin-induced ototoxicity limits the utility of this agent in cancer patients, especially when dose escalations are needed. Ototoxicity is associated with cochlear cell death through DNA damage, the generation of reactive oxygen species (ROS) and the consequent activation of caspase, glutamate excitotoxicity, inflammation, apoptosis and/or necrosis. Previous studies have demonstrated a role of CXC chemokines in cisplatin ototoxicity. In this study, we investigated the role of CXCL1, a cytokine which increased in the serum and cochlea by 24 h following cisplatin administration. Adult male Wistar rats treated with cisplatin demonstrated significant hearing loss, assessed by auditory brainstem responses (ABRs), hair cell loss and loss of ribbon synapse. Immunohistochemical studies evaluated the levels of CXCL1 along with increased presence of CD68 and CD45-positive immune cells in cochlea. Increases in CXCL1 was time-dependent in the spiral ganglion neurons and organ of Corti and was associated with progressive increases in CD45, CD68 and IBA1-positive immune cells. Trans-tympanic administration of SB225002, a chemical inhibitor of CXCR2 (receptor target for CXCL1) reduced immune cell migration, protected against cisplatin-induced hearing loss and preserved hair cell integrity. We show that SB225002 reduced the expression of CXCL1 , NOX3 , iNOS , TNF-α , IL-6 and COX-2 . Similarly, knockdown of CXCR2 by trans-tympanic administration of CXCR2 siRNA protected against hearing loss and loss of outer hair cells and reduced ribbon synapses. In addition, SB225002 reduced the expression of inflammatory mediators induced by cisplatin. These results implicate the CXCL1 chemokine as an early player in cisplatin ototoxicity, possibly by initiating the immune cascade, and indicate that CXCR2 is a relevant target for treating cisplatin ototoxicity., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Al Aameri, Alanisi, Oluwatosin, Al Sallami, Sheth, Alberts, Patel, Rybak and Ramkumar.)

Mitochondrial dynamics is essential for maintaining the physiological function of the mitochondrial network, and its disorders lead to a variety of diseases. Our previous study identified mitochondrial dynamics controlled anti-tumor immune responses and anxiety symptoms. However, how mitochondrial dynamics affects auditory function in the inner ear remains unclear. Here, we show that the deficiency of FAM73a or FAM73b, two mitochondrial outer membrane proteins that mediate mitochondrial fusion, leads to outer hair cells (HCs) damage and progressive hearing loss in FVB/N mice. Abnormal mitochondrial fusion causes elevated oxidative stress and apoptosis of HCs in the early stage. Thereafter, the activation of macrophages and CD4+ T cell is found in the mutant mice with the increased expression of the inflammatory cytokines IL-12 and IFN-γ compared with control mice. Strikingly, a dramatically decreased number of macrophages by Clophosome®-A-Clodronate Liposomes treatment alleviates the hearing loss of mutant mice. Collectively, our finding highlights that FAM73a or FAM73b deficiency affects HCs survival by disturbing the mitochondrial function, and the subsequent immune response in the cochleae worsens the damage of HCs., (© The Author(s) 2022. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Cisplatin-induced ototoxicity leads to hearing impairment, possibly through reactive oxygen species (ROS) production and DNA damage in cochlear hair cells (HC), although the exact mechanism is unknown. Avenanthramide-C (AVN-C), a natural, potent antioxidant, was evaluated in three study groups of normal adult C57Bl/6 mice (control, cisplatin, and AVN-C+cisplatin) for the prevention of cisplatin-induced hearing loss. Auditory brainstem responses and immunohistochemistry of outer hair cells (OHCs) were ascertained. Cell survival, ROS production, Phospho-H2AX-enabled tracking of DNA damage-repair kinetics, and expression levels of inflammatory cytokines ( TNF-α , IL-1β , IL6 , iNOS , and COX2 ) were assessed using House Ear Institute-Organ of Corti 1 (HEI-OC1 Cells). In the in vivo mouse model, following cisplatin-induced damage, AVN-C decreased the hearing thresholds and sheltered all cochlear turns' OHCs. In HEI-OC1 cells, AVN-C preserved cell viability and decreased ROS production, whereas cisplatin enhanced both ROS levels and cell viability. In HEI-OC1 cells, AVN-C downregulated IL6 , IL-1β , TNF-α , iNOS , and COX2 production that was upregulated by cisplatin treatment. AVN-C attenuated the cisplatin-enhanced nuclear H2AX activation. AVN-C had a strong protective effect against cisplatin-induced ototoxicity through inhibition of ROS and inflammatory cytokine production and DNA damage and is thus a promising candidate for preventing cisplatin-induced sensorineural hearing loss.

Hearing loss impacts a large proportion of the global population. Damage to the inner ear, in particular the sensitive hair cells, can impact individuals for the rest of their lives. There are very limited options for interventions after damage to these cells has occurred. Targeted gene delivery may provide an effective means to trigger appropriate differentiation of progenitor cells for effective replacement of these sensitive hair cells. There are several hurdles that need to be overcome to effectively deliver these genes. Nanoencapsulation technology has previously been used for the delivery of pharmaceuticals, proteins and nucleic acids, and may provide an effective means of delivering genes to trigger appropriate differentiation. This review investigates the background of hearing loss, current advancements and pitfalls of gene delivery, and how nanoencapsulation may be useful., (© 2022 Wiley-VCH GmbH.)

Cytomegalovirus (CMV)-induced sensorineural hearing loss (SNHL) is a worldwide epidemic. Recent studies have shown that the degree of spiral ganglion neuron (SGN) loss is correlated with hearing loss after CMV infection. We aimed to better understand the pathological mechanisms of CMV-related SGN death and to search for intervention measures. We found that both apoptosis and pyroptosis are involved in CMV-induced SGN death, which may be caused by the simultaneous activation of the p53/JNK and NLRP3/caspase-1 signaling pathways, respectively. Moreover, considering that mixed lineage kinase family (MLK1/2/3) are host restriction factors against viral infection and upstream regulators of the p53/JNK and inflammatory (including NLRP3-caspase1) signaling pathways, we further demonstrated that the MLKs inhibitor URMC-099 exhibited a protective effect against CMV-induced SGN death and hearing loss. These results indicate that MLKs signaling may be a key regulator and promising novel target for preventing apoptosis and even pyroptosis during the CMV infection of SGN cells and for treating hearing loss., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Hearing loss is the third most prevalent chronic health condition affecting older adults. Age-related hearing loss affects one in three adults over 65 years of age and is caused by both extrinsic and intrinsic factors, including genetics, aging, and exposure to noise and toxins. All cells possess antioxidant defense systems that play an important role in protecting cells against these factors. Reduced nicotinamide adenine dinucleotide phosphate (NADPH) serves as a co-factor for antioxidant enzymes such as glutathione reductase and thioredoxin reductase and is produced by glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, isocitrate dehydrogenase 1 (IDH1) or malic enzyme 1 in the cytosol, while in the mitochondria, NADPH is generated from mitochondrial transhydrogenase, glutamate dehydrogenase, malic enzyme 3 or IDH2. There are three isoforms of IDH: cytosolic IDH1, and mitochondrial IDH2 and IDH3. Of these, IDH2 is thought to be the major supplier of NADPH to the mitochondrial antioxidant defense system. The NADP + /NADPH and NAD + /NADH couples are essential for maintaining a large array of biological processes, including cellular redox state, and energy metabolism, mitochondrial function. A growing body of evidence indicates that mitochondrial dysfunction contributes to age-related structural or functional changes of cochlear sensory hair cells and neurons, leading to hearing impairments. In this review, we describe the current understanding of the roles of NADPH and IDHs in cochlear mitochondrial antioxidant defense and aging., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Tight junctions act as a barrier between epithelial cells to limit the transport of the paracellular substance, which is a required function in various tissues to sequestrate diverse microenvironments and maintain a normal physiological state. Tight junctions are complexes that contain various proteins, like transmembrane proteins, scaffolding proteins, signaling proteins, etc. Defects in those tight junction- related proteins can lead to hearing loss in humans which is also recapitulated in many model organisms. The disruption of the barrier between the endolymph and perilymph caused by tight junction abnormalities will affect the microenvironment of hair cells; and this could be the reason for this type of hearing loss. Besides their functions as a typical barrier and channel, tight junctions are also involved in many signaling networks to regulate gene expression, cell proliferation, and differentiation. This review will summarize the structures, localization, and related signaling pathways of hearingrelated tight junction proteins and their potential contributions to the hearing disorder., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Hearing loss is one of the most important public health matters worldwide, severely affecting people's social, psychological, and cognitive development. The perception of sound, movement, and balance in vertebrates depends on a special sensory organ called the cochlea, which contains hair cells and supporting cells in the inner ear. Genetic factors, epigenetics, the use of ototoxic drugs (some antibiotics and chemotherapeutics), noise, infections, or even aging can cause loss of hair cells and their related primary neurons, leading to sensorineural hearing loss. Although a sensorineural hearing loss, also known as permanent hearing loss, is treated with hearing aids and cochlear implants, treatment methods are limited. Since even the best implant cannot exhibit the characteristics of the original ear, the permanent sensory deficit will be permanent. For this reason, it has become important to develop regenerative treatment methods to regenerate and replace lost or damaged hair cells and neurons. Developments in stem cell technology have led to promising studies in regenerating damaged/lost hair cells or neurons with endogenous or exogenous cell-based therapies. Epigenetic mechanisms can turn hearing-related genes on and off and determine which proteins to copy. In addition, due to gene silencing, gene replacement, and CRISPR/CAS9 technology, gene therapy methods have accelerated, and studies have been carried out to treat dominant and recessive mutations that cause genetic-induced hearing loss or increase hair cell regeneration. In this paper, potential gene therapy and stem cell applications in the acquisition of cochlear function, which causes sensorineural hearing loss, and the difficulties encountered in these applications are compiled from a bioengineering perspective., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Peroxisome Biogenesis Disorders (PBD) and Zellweger syndrome spectrum disorders (ZSD) are rare genetic multisystem disorders that include hearing impairment and are associated with defects in peroxisome assembly, function, or both. Mutations in 13 peroxin ( PEX ) genes have been found to cause PBD-ZSD with ~70% of patients harboring mutations in PEX1 . Limited research has focused on the impact of peroxisomal disorders on auditory function. As sensory hair cells are particularly vulnerable to metabolic changes, we hypothesize that mutations in PEX1 lead to oxidative stress affecting hair cells of the inner ear, subsequently resulting in hair cell degeneration and hearing loss. Global deletion of the Pex1 gene is neonatal lethal in mice, impairing any postnatal studies. To overcome this limitation, we created conditional knockout mice (cKO) using Gfi1 Cre or VGlut3 Cre expressing mice crossed to floxed Pex1 mice to allow for selective deletion of Pex1 in the hair cells of the inner ear. We find that Pex1 excision in inner hair cells (IHCs) leads to progressive hearing loss associated with significant decrease in auditory brainstem responses (ABR), specifically ABR wave I amplitude, indicative of synaptic defects. Analysis of IHC synapses in cKO mice reveals a decrease in ribbon synapse volume and functional alterations in exocytosis. Concomitantly, we observe a decrease in peroxisomal number, indicative of oxidative stress imbalance. Taken together, these results suggest a critical function of Pex1 in development and maturation of IHC-spiral ganglion synapses and auditory function.

Hearing loss is the most common sensory deficit, affecting 466 million people worldwide. The vast and diverse genes involved reflect the complexity of auditory physiology, which requires the use of animal models in order to gain a fuller understanding. Among the loci with a yet-to-be validated gene is the DFNA58, in which ~200 Kb genomic duplication, including three protein-coding genes ( PLEK , CNRIP1 , and PPP3R1 's exon1), was found to segregate with autosomal dominant hearing loss. Through whole genome sequencing, the duplication was found to be in tandem and inserted in an intergenic region, without the disruption of the topological domains. Reanalysis of transcriptomes data studies (zebrafish and mouse), and RT-qPCR analysis of adult zebrafish target organs, in order to access their orthologues expression, highlighted promising results with Cnrip1a, corroborated by zebrafish in situ hybridization and immunofluorescence. Mouse data also suggested Cnrip1 as the best candidate for a relevant role in auditory physiology, and its importance in hearing seems to have remained conserved but the cell type exerting its function might have changed, from hair cells to spiral ganglion neurons.

Measurement of the motor protein prestin offers a novel approach to assessing outer hair cell (OHC) status using serological techniques. Motivated by our prior work showing reduced serum prestin levels in healthy young adults at-risk for noise damage, the current study examined serum prestin levels, measured from circulating blood, across the age span from 18 to 82 years old. Results suggest that serum prestin levels negatively correlate with age, with young adults having higher levels of circulating serum in the blood than older adults. Group-level analyses showed minimal differences in prestin levels between 18 and 29, 30-39, and 40-49 year olds, but significant reductions in the 50+ years-old age group compared to the three younger groups, even though all groups significantly differed from each other in audiometric thresholds and distortion product otoacoustic emissions signal-to-noise ratio. Serum prestin levels declined with increasing levels of hearing loss, with a statistically significant relationship emerging between prestin and low-frequency hearing thresholds (0.25-2 kHz) but a weaker non-significant relationship for high-frequency hearing thresholds (3-8 kHz). This differential pattern between low- and high- frequency thresholds is consistent with the basal-to-apical progression of OHC loss with age. Findings support the idea that serum prestin is the product of residual OHCs in the less age-affected apical regions. Moreover, when entered in a regression model with audiometric thresholds, age was a stronger predictor than pure tone hearing threshold level for predicting serum prestin levels., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The mammalian cochlea is an exceptionally well-organized epithelium composed of hair cells, supporting cells, and innervating neurons. Loss or defects in any of these cell types, particularly the specialized sensory hair cells, leads to deafness. The Notch pathway is known to play a critical role in the decision to become either a hair cell or a supporting cell during embryogenesis; however, little is known about how Notch functions later during cochlear maturation. Uniquely amongst Notch ligands, Jagged1 (JAG1) is localized to supporting cells during cell fate acquisition and continues to be expressed into adulthood. Here, we demonstrate that JAG1 in maturing cochlear supporting cells is essential for normal cochlear function. Specifically, we show that deletion of JAG1 during cochlear maturation disrupts the inner hair cell pathway and leads to a type of deafness clinically similar to auditory neuropathy. Common pathologies associated with disruptions in inner hair cell function, including loss of hair cells, synapses, or auditory neurons, were not observed in JAG1 mutant cochleae. Instead, RNA-seq analysis of JAG1-deficient cochleae identified dysregulation of the Rho GTPase pathway, known to be involved in stereocilia development and maintenance. Interestingly, the overexpression of one of the altered genes, Diaph3, is responsible for autosomal dominant auditory neuropathy-1 (AUNA1) in humans and mice, and is associated with defects in the inner hair cell stereocilia. Strikingly, ultrastructural analyses of JAG1-deleted cochleae revealed stereocilia defects in inner hair cells, including fused and elongated bundles, that were similar to those stereocilia defects reported in AUNA1 mice. Taken together, these data indicate a novel role for Notch signaling in normal hearing development through maintaining stereocilia integrity of the inner hair cells during cochlear maturation., (© 2022. The Author(s).)

Background: Uncoupling protein 2 (UCP2), activated by excessive reactive oxygen species (ROS) in vivo, has the dual effect of reducing ROS to protect against oxidative stress and reducing ATP production to regulate cellular metabolism. Both the UCP2 and ROS are increased in cochleae in age-related hearing loss (ARHL). However, the role of UCP2 in sensory hair cells in ARHL remains unclear., Methods: Male C57BL/6 J mice were randomly assigned to an 8-week-old group (Group 1), a 16-week-old group (Group 2), a 16-week-old + adeno-associated virus-inner ear (AAV-ie) group (Group 3), and a 16-week-old + AAV-ie-UCP2 group (Group 4). Mice aged 8 weeks were administrated with AAV-ie-GFP or AAV-ie-UCP2 via posterior semicircular canal injection. Eight weeks after this viral intervention, hearing thresholds and wave-I amplitudes were tested by auditory brainstem response (ABR). Subsequently, the cochlear basilar membrane was dissected for investigation. The number of hair cells and inner hair cell (IHC) synapses, the level of ROS, and the expression of AMP-activated protein kinase α (AMPKα), were assessed by immunofluorescence staining. In addition, mitochondrial function was determined, and the expression of AMPKα and UCP2 proteins was further evaluated using western blotting., Results: Mice with early-onset ARHL exhibited enhanced oxidative stress and loss of outer hair cells and IHC synapses, while UCP2 overexpression aggravated hearing loss and cochlear pathophysiological changes in mice. UCP2 overexpression resulted in a notable decrease in the number of IHCs and IHC synapses, caused ATP depletion and excessive ROS generation, increased AMPKα protein levels, and promoted IHC apoptosis, especially in the apical and middle turns of the cochlea., Conclusion: Collectively, our data suggest that UCP2 overexpression may cause mitochondrial dysfunction via energy metabolism, which activates mitochondrion-dependent cellular apoptosis and leads to IHC loss, ultimately exacerbating ARHL., (© 2022. The Author(s).)

Peroxisome biogenesis disorders (due to PEX gene mutations) are associated with symptoms that range in severity and can lead to early childhood death, but a common feature is hearing impairment. In this study, mice carrying Pex3 mutations were found to show normal auditory development followed by an early-onset progressive increase in auditory response thresholds. The only structural defect detected in the cochlea at four weeks old was the disruption of synapses below inner hair cells. A conditional approach was used to establish that Pex3 expression is required locally within the cochlea for normal hearing, rather than hearing loss being due to systemic effects. A lipidomics analysis of the inner ear revealed a local reduction in plasmalogens in the Pex3 mouse mutants, comparable to the systemic plasmalogen reduction reported in human peroxisome biogenesis disorders. Thus, mice with Pex3 mutations may be a useful tool to understand the physiological basis of peroxisome biogenesis disorders.

Tubby mice exhibit hearing impairment due to the loss of stereocilin from the tip regions that connect the tallest stereocilia of the outer hair cells (OHCs) to the tectorial membrane. Stereocilin is an essential stereociliary protein in the OHCs, the mutation of which in humans causes autosomal recessive non-syndromic deafness. Map1a is a modifier of tubby hearing (moth1), and its wild-type allele, rather than the moth1 allele from the C57BL/6 J strain, restores stereocilin localization to the stereocilia and rescues the hearing impairment of tubby mice. The mechanism by which MAP1A accomplishes this is unclear, partly due to ambiguity regarding whether the tubby mutation is a true null. We therefore generated Tub-null (Tub -/- ) mice by deleting exon 3 and found that they exhibit hearing impairment like that of tubby mice, suggesting the tubby mutation is a loss-of-function mutation with regard to hearing. When we crossed Tub -/- mice with AKR mice that have wild-type Map1a alleles, we found that wild-type MAP1A restores stereocilin localization to the tips of stereocilia and rescues hearing impairment. These data suggest MAP1A does not require interaction with tubby protein in maintaining stereocilin at the tips of stereocilia and that OHCs use two independent molecules-MAP1A and tubby-to doubly ensure proper stereocilin localization., (© 2022. The Author(s).)

Sensorineural hearing loss often results from damaged or deficient inner ear hair cells. Mitochondrial 12S rRNA 1555A>G mutation has been associated with hearing loss in many families. The m.1555A>G mutation is a primary factor underlying the development of hearing loss and TRMU allele (c.28G>T, p.Ala10Sser) encoding tRNA thiouridylase interact with m.1555A>G mutation to cause hearing loss. However, the tissue specificity of mitochondrial dysfunction remains elusive and there is no highly effective therapy for mitochondrial deafness. We report here the generation of induced pluripotent stem cells (iPSCs) from lymphoblastoid cell lines derived from members of an Arab-Israeli family (asymptomatic individual carrying only m.1555A>G mutation, symptomatic individual bearing both m.1555A>G and c.28G>T mutations, and control subject). The c.28G>T mutation in iPSC lines from a hearing-impaired subject was corrected by CRISPR/Cas9. These iPSCs were differentiated into otic epithelial progenitor (OEP) cells and subsequent inner ear hair cell (HC)-like cells. The iPSCs bearing m.1555A>G mutation exhibited mildly deficient differentiation into OEP and resultant HC-like cells displayed mild defects in morphology and electrophysiological properties. Strikingly, those HC-like cells harboring m.1555A>G and TRMU c.28G>T mutations displayed greater defects in the development, morphology and functions than those in cells bearing only m.1555A>G mutation. Transcriptome analysis of patients-derived HC-like cells revealed altered expressions of genes vital for mechanotransduction of hair cells. Genetic correction of TRMU c.28G>T mutation yielded morphologic and functional recovery of patient derived HC-like cells. These findings provide new insights into pathophysiology of maternally inherited hearing loss and a step toward therapeutic interventions for this disease., (© The Author(s) 2022. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)