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The mechanosensory hair cell of the vertebrate inner ear responds to the mechanical deflections that result from hearing or change in the acceleration due to gravity, to allow us to perceive and interpret sounds, maintain balance and spatial orientation. In mammals, ototoxic compounds, disease, and acoustic trauma can result in damage and extrusion of hair cells, without replacement, resulting in hearing loss. In contrast, non-mammalian vertebrates can regenerate sensory hair cells. Upon damage, hair cells are extruded and an associated cell type, the supporting cell is transformed into a hair cell. The mechanisms that can trigger regeneration are not known. Using mosaic deletion of the hair cell master gene, Atoh1, in the embryonic avian inner ear, we find that despite hair cells depletion at E9, by E12, hair cell number is restored in sensory epithelium. Our study suggests a homeostatic mechanism can restores hair cell number in the basilar papilla, that is activated when juxtracrine signalling is disrupted. Restoration of hair cell numbers during development may mirror regenerative processes, and our work provides insights into the mechanisms that trigger regeneration., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Notch signaling patterns the cochlear organ of Corti, and individuals with the JAG1/NOTCH2-related genetic disorder Alagille syndrome can thus experience hearing loss. We investigated the function of Jag1 in cochlear patterning and signaling using Jag1Ndr/Ndr mice, which are a model of Alagille syndrome. Jag1Ndr/Ndr mice exhibited expected vestibular and auditory deficits, a dose-dependent increase in ectopic inner hair cells, and a reduction in outer hair cells. Single cell RNA sequencing of the organ of Corti demonstrated a global dysregulation of genes associated with inner ear development and deafness. Analysis of individual cell types further revealed that Jag1 represses Notch activation in lateral supporting cells and demonstrated a function for Jag1 in gene regulation and development of outer hair cells. Surprisingly, ectopic 'outer hair cell-like' cells were present in the medial compartment and pillar cell region of Jag1Ndr/Ndr cochleae, yet they exhibited location-dependent expression of the inner hair cell fate-determinant Tbx2, suggesting Jag1 is required for Tbx2 to drive inner hair cell commitment. This study thus identifies new roles for Jag1 in supporting cells, and in outer hair cell specification and positioning., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Background: Gentamicin is a commonly used aminoglycoside antibiotic, with ototoxicity as a significant side effect. Ferroptosis, an iron-dependent form of cell death, has been implicated in a variety of disorders. Whether ferroptosis impacts gentamicin ototoxicity is not yet known. The current work used an in-vitro model to examine the influence of gentamicin-induced ferroptosis on cochlear hair cell damage and probable molecular biological pathways., Methods: House Ear Institute-Organ of Corti 1 (HEI-OC1) cells were treated with different concentrations of gentamicin for 24 hours, with or without ferrostatin-1 pretreatment, to observe gentamicin-induced ferroptosis. The role of p53/solute carrier family 7 member 11 (SLC7A11)/glutathione peroxidase 4 (GPX4) signaling in gentamicin-induced ferroptosis was explored by pretreating cells with the p53 inhibitor pifithrin-α (PFT-α). We investigated the effect of gentamicin on cells by assessing cell viability. Cellular proteins were isolated and Western blots were performed to detect changes in the expression of p53, SLC7A11, and GPX4. Fluorescence staining was used to assess levels of reactive oxygen species. An enzymatic detection kit was used to detect glutathione, Fe, and malondialdehyde markers., Results: Gentamicin reduced cell viability, glutathione content, and SLC7A11 and GPX4 protein levels, and increased levels of p53 protein, reactive oxygen species, malondialdehyde, and Fe. These effects were largely blocked by pretreatment with ferrostatin-1. Pretreatment with the p53 inhibitor PFT-α prevented the gentamicin-induced reduction in SLC7A11 and GPX4, which alleviated several features of ferroptosis including glutathione depletion, iron overload, and lipid peroxidation build-up., Conclusion: Gentamicin induces ferroptosis in the HEI-OC1 cell line, and the mechanism may be related to the p53/SLC7A11/GPX4 signaling pathway., Competing Interests: Conflict of interest statement: 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 © 2024 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of Otology & Neurotology, Inc.)

Following up on our previous observation that early B cell factor (EBF) sites are enriched in open chromatin of the developing sensory epithelium of the mouse cochlea, we investigated the effect of deletion of Ebf1 on inner ear development. We used a Cre driver to delete Ebf1 at the otocyst stage before development of the cochlea. We examined the cochlea at postnatal day (P) 1 and found that the sensory epithelium had doubled in size but the length of the cochlear duct was unaffected. We also found that deletion of Ebf1 led to ectopic sensory patches in the Kölliker's organ. Innervation of the developing organ of Corti was disrupted with no obvious spiral bundles. The ectopic patches were also innervated. All the extra hair cells (HCs) within the sensory epithelium and Kölliker's organ contained mechanoelectrical transduction channels, as indicated by rapid uptake of FM1-43. The excessive numbers of HCs were still present in the adult Ebf1 conditional knockout (cKO) animal. The animals had significantly elevated auditory brainstem response thresholds, suggesting that this gene is essential for hearing development., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Gasdermin E (GSDME), a member of the gasdermin protein family, is associated with post-lingual hearing loss. All GSDME pathogenic mutations lead to skipping exon 8; however, the molecular mechanisms underlying hearing loss caused by GSDME mutants remain unclear. GSDME was recently identified as one of the mediators of programmed cell death, including apoptosis and pyroptosis. Therefore, in this study, we injected mice with GSDME mutant (MT) and examined the expression levels to assess its effect on hearing impairment. We observed loss of hair cells in the organ of Corti and spiral ganglion neurons. Further, the N-terminal release from the GSDME mutant in HEI-OC1 cells caused pyroptosis, characterized by cell swelling and rupture of the plasma membrane, releasing lactate dehydrogenase and cytokines such as interleukin-1β. We also observed that the N-terminal release from GSDME mutants could permeabilize the mitochondrial membrane, releasing cytochromes and activating the mitochondrial apoptotic pathway, thereby generating possible positive feedback on the cleavage of GSDME. Furthermore, we found that treatment with disulfiram or dimethyl fumarate might inhibit pyroptosis and apoptosis by inhibiting the release of GSDME-N from GSDME mutants. In conclusion, this study elucidated the molecular mechanism associated with hearing loss caused by GSDME gene mutations, offering novel insights for potential treatment strategies., (© 2024. The Author(s).)

The sensory epithelium of the cochlea, the organ of Corti, has complex cytoarchitecture consisting of mechanosensory hair cells intercalated by epithelial support cells. The support cells provide important trophic and structural support to the hair cells. Thus, the support cells must be stiff yet compliant enough to withstand and modulate vibrations to the hair cells. Once the sensory cells are properly patterned, the support cells undergo significant remodeling from a simple epithelium into a structurally rigid epithelium with fluid-filled spaces in the murine cochlea. Cell adhesion molecules such as cadherins are necessary for sorting and connecting cells in an intact epithelium. To create the fluid-filled spaces, cell adhesion properties of adjoining cell membranes between cells must change to allow the formation of spaces within an epithelium. However, the dynamic localization of cadherins has not been properly analyzed as these spaces are formed. There are three cadherins that are reported to be expressed during the first postnatal week of development when the tunnel of Corti forms in the cochlea. In this study, we characterize the dynamic localization of cadherins that are associated with cytoskeletal remodeling at the contacting membranes of the inner and outer pillar cells flanking the tunnel of Corti., (© 2024 American Association for Anatomy.)

Ribbon synapses between inner hair cells (IHCs) and type I spiral ganglion neurons (SGNs) in the inner ear are damaged by noise trauma and with aging, causing "synaptopathy" and hearing loss. Cocultures of neonatal denervated organs of Corti and newly introduced SGNs have been developed to find strategies for improving IHC synapse regeneration, but evidence of the physiological normality of regenerated synapses is missing. This study utilizes IHC optogenetic stimulation and SGN recordings, showing that, when P3-5 denervated organs of Corti are cocultured with SGNs, newly formed IHC/SGN synapses are indeed functional, exhibiting glutamatergic excitatory postsynaptic currents. When using older organs of Corti at P10-11, synaptic activity probed by deconvolution showed more mature release properties, closer to the specialized mode of IHC synaptic transmission crucial for coding the sound signal. This functional assessment of newly formed IHC synapses developed here, provides a powerful tool for testing approaches to improve synapse regeneration., Competing Interests: Competing interests statement:The authors declare no competing interest.

In the mammalian auditory system, frequency discrimination depends on numerous morphological and physiological properties of the organ of Corti, which gradually change along the apex-to-base (tonotopic) axis of the organ. For example, the basilar membrane stiffness changes tonotopically, thus affecting the tuning properties of individual hair cells. At the molecular level, those frequency-specific characteristics are mirrored by gene expression gradients; however, the molecular mechanisms controlling tonotopic gene expression in the mouse cochlea remain elusive. Through analyzing single-cell RNA sequencing (scRNA-seq) data from E12.5 and E14.5 time points, we predicted that morphogens, rather than a cell division-associated mechanism, confer spatial identity in the extending cochlea. Subsequently, we reconstructed the developing cochlea in 3D space from scRNA-seq data to investigate the molecular pathways mediating positional information. The retinoic acid (RA) and hedgehog pathways were found to form opposing apex-to-base gradients, and functional interrogation using mouse cochlear explants suggested that both pathways jointly specify the longitudinal axis., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Age-related hearing loss (ARHL) is associated with hair cell apoptosis, but the underlying mechanism of hair cell apoptosis remains unclear. Here, we investigated the expression profiles of long noncoding RNAs (lncRNAs) and mRNAs in an ARHL model created with C57BL/6 J mice using RNA sequencing and found that the expression of several lncRNAs was significantly correlated with apoptosis-associated mRNAs in the cochlear tissues of old mice compared to young mice. We found that lncRNA Mirg was upregulated in the cochlear tissues of old mice compared to young mice and its overexpression promoted apoptosis in House Ear Institute-Organ of Corti 1 (HEI-OC1). H 2 O 2 -induced oxidative stress increased HEI-OC1 cell apoptosis by upregulating lncRNA Mirg. Furthermore, the expression of lncRNA Mirg and Foxp1 showed the highest correlation coefficient in the cochlear tissues of old mice, and lncRNA Mirg promoted HEI-OC1 cell apoptosis by increasing Foxp1 expression. In conclusion, our findings suggest that lncRNA Mirg expression correlates with cell apoptosis-associated mRNAs in the ARHL model created using C57BL/6 J mice and that oxidative stress-induced lncRNA Mirg promotes HEI-OC1 cell apoptosis by increasing Foxp1 expression. These data suggest the potential therapeutic significance of targeting lncRNA Mirg/Foxp1 signaling in ARHL., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2023 Elsevier B.V. All rights reserved.)

ATP, as a paracrine signalling molecule, induces intracellular Ca 2+ elevation via the activation of purinergic receptors on the surface of glia-like cochlear supporting cells. These cells, including the Deiters' cells (DCs), are also coupled by gap junctions that allow the propagation of intercellular Ca 2+ waves via diffusion of Ca 2+ mobilising second messenger IP 3 between neighbouring cells. We have compared the ATP-evoked Ca 2+ transients and the effect of two different gap junction (GJ) blockers (octanol and carbenoxolone, CBX) on the Ca 2+ transients in DCs located in the apical and middle turns of the hemicochlea preparation of BALB / c mice (P14-19). Octanol had no effect on Ca 2+ signalling, while CBX inhibited the ATP response, more prominently in the middle turn. Based on astrocyte models and using our experimental results, we successfully simulated the Ca 2+ dynamics in DCs in different cochlear regions. The mathematical model reliably described the Ca 2+ transients in the DCs and suggested that the tonotopical differences could originate from differences in purinoceptor and Ca 2+ pump expressions and in IP 3 -Ca 2+ release mechanisms. The cochlear turn-dependent effect of CBX might be the result of the differing connexin isoform composition of GJs along the tonotopic axis. The contribution of IP 3 -mediated Ca 2+ signalling inhibition by CBX cannot be excluded.

The auditory function of the mammalian cochlea relies on two types of mechanosensory hair cells and various non-sensory supporting cells. Recent studies identified the transcription factors INSM1 and IKZF2 as regulators of outer hair cell (OHC) fate. However, the transcriptional regulation of the differentiation of inner hair cells (IHCs) and their associated inner supporting cells (ISCs) has remained enigmatic. Here, we show that the expression of the transcription factor TBX2 is restricted to IHCs and ISCs from the onset of differentiation until adulthood and examine its function using conditional deletion and misexpression approaches in the mouse. We demonstrate that TBX2 acts in prosensory progenitors as a patterning factor by specifying the inner compartment of the sensory epithelium that subsequently gives rise to IHCs and ISCs. Hair cell-specific inactivation or misexpression causes transdifferentiation of hair cells indicating a cell-autonomous function of TBX2 in inducing and maintaining IHC fate., (© 2022. The Author(s).)

Different serum thyroxine levels may influence the morphology of the inner ear during development. A well-developed organ of Corti (OC) is considered to be critical to the function of hearing. In our study, we treated mice with triiodothyronine (T3) and found that the opening of the OC occurred sooner than in control mice. We also observed an increased formation of acetylated microtubules and a decrease in the adhesion junction molecule P-cadherin the during opening of the OC. Our investigation indicates that thyroxin affects P-cadherin expression and microtubule acetylation to influence the opening of the OC.

Background: Cisplatin (CP) is an effective anticancer drug broadly used for various types of cancers, but it has shown ototoxicity that results from oxidative stress. Berberine has been reported for its anti-oxidative stress suggesting its therapeutic potential for many diseases such as colitis, diabetes, and vascular dementia., Objective: Organ of Corti of postnatal day 3 mouse cochlear explants were used to compare hair cells after the treatment with cisplatin alone or with berberine chloride (BC) followed by CP., Methods: We investigated the potential of the anti-oxidative effect of BC against the cisplatin-induced ototoxicity. We observed a reduced aberrant bundle of stereocilia in hair cells in CP with BC pre-treated group. Caspase-3 immunofluorescence and TUNEL assay supported the hypothesis that BC attenuates the apoptotic signals induced by CP. Reactive oxygen species level in the mitochondria were investigated by MitoSOX Red staining and the mitochondrial membrane potentials were compared by JC-1 assay., Results: BC decreased ROS generation with preserved mitochondrial membrane potentials in mitochondria as well as reduced DNA fragmentation in hair cells. In summary, our data indicate that BC might act as antioxidant against CP by reducing the stress in mitochondria resulting in cell survival., Conclusion: Our result suggests the therapeutic potential of BC for prevention of the detrimental effect of CP-induced ototoxicity., (© 2021. The Author(s).)

In the vertebrate cochlea, the reticular lamina seals the organ of Corti against the endolymph filled scala media. After noise exposure, fast alterations in the endothelial nitric oxide synthase (eNOS) expression level were identified in this cochlear structure. Minor amounts of nitric oxide (NO) produced by eNOS or applied by NO donors such as S-nitroso- N -acetyl-penicillamine (SNAP) might protect this vulnerable part of the organ of Corti, on the line of gap junctions of supporting cells and cochlear microcirculation. In n =5 anesthetized guinea pigs, SNAP was intravenously applied in two concentrations. Six untreated animals served as controls. The cochleae were removed and prepared for immunoelectron microscopy using specific gold-labeled anti-eNOS antibodies. The density of the gold particles was quantified for seven cellular regions in the reticular lamina at the ultrastructural level. Following SNAP application, a significant increase in eNOS expression (+176%) was detected compared with controls ( p =0.012). The increase occurred mainly in actin-rich cuticular structures and the prominent microtubules bundles. Correlation analysis revealed three clear and five moderate cellular associations for controls, whereas only one clear and one moderate after SNAP application. Thus, application of the NO donor SNAP resulted in an increase in eNOS expression in distinct regions of the reticular lamina.

Single-cell RNA-sequencing (scRNA-seq) technologies have revolutionized modern biomedical sciences. A fundamental challenge is to incorporate spatial information to study tissue organization and spatial gene expression patterns. Here, we describe a detailed protocol for using novoSpaRc, a computational framework that probabilistically assigns cells to tissue locations. At the core of this framework lies a structural correspondence hypothesis, that cells in physical proximity share similar gene expression profiles. Given scRNA-seq data, novoSpaRc spatially reconstructs tissues based on this hypothesis, and optionally, by including a reference atlas of marker genes to improve reconstruction. We describe the novoSpaRc algorithm, and its implementation in an open-source Python package ( https://pypi.org/project/novosparc ). NovoSpaRc maps a scRNA-seq dataset of 10,000 cells onto 1,000 locations in <5 min. We describe results obtained using novoSpaRc to reconstruct the mouse organ of Corti de novo based on the structural correspondence assumption and human osteosarcoma cultured cells based on marker gene information, and provide a step-by-step guide to Drosophila embryo reconstruction in the Procedure to demonstrate how these two strategies can be combined., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Objective: Age-related hearing loss (AHL), characterized by degeneration of cochlea structures, is the most common sensory disorder among the elderly worldwide. The calcium channel is considered to contribute to normal hearing. However, the role of the T-type voltage-activated calcium channel, Cav3.1, remains unclear in AHL. Here, we investigate the age-related change of Cav3.1 expression in the cochlea and D-gal-induced senescent HEI-OC1 cells., Methods: Cochleae from C57BL/6 mice at 2 months and 12 months of age were assessed. Senescence in House Ear Institute-Organ of Corti 1 (HEI-OC1) cells was induced by D-gal treatment. The immunofluorescence technique was employed to investigate the distribution of Cav3.1 in vivo and in vitro. Quantitative assessment was achieved by Western blotting and real-time PCR., Results: In comparison with 2-month-old animals, 12-month old C57BL/6 mice exhibited great loss of hair cells and elevated auditory brainstem threshold. The Cav3.1 was located in hair cells, spiral ganglion cells, lateral walls, and the expression of Cav3.1 protein and mRNA decreased in the aged cochleae. D-gal-induced senescence assay confirmed the down-regulation of Cav3.1 expression in senescent HEI-OC1 cells., Conclusion: Our results show that age-related down-regulated expression of Cav3.1 in the cochleae is associated with AHL and may contribute to the pathogenesis of AHL., (© 2021. Huazhong University of Science and Technology.)

Background: Fibroblast Growth Factor 20 (FGF20)-FGF receptor 1 (FGFR1) signaling is essential for cochlear hair cell (HC) and supporting cell (SC) differentiation. In other organ systems, FGFR1 signals through several intracellular pathways including MAPK (ERK), PI3K, phospholipase C ɣ (PLCɣ), and p38. Previous studies implicated MAPK and PI3K pathways in HC and SC development. We hypothesized that one or both would be important downstream mediators of FGF20-FGFR1 signaling for HC differentiation., Results: By inhibiting pathways downstream of FGFR1 in cochlea explant cultures, we established that both MAPK and PI3K pathways are required for HC differentiation while PLCɣ and p38 pathways are not. Examining the canonical PI3K pathway, we found that while AKT is necessary for HC differentiation, it is not sufficient to rescue the Fgf20 -/- phenotype. To determine whether PI3K functions downstream of FGF20, we inhibited Phosphatase and Tensin Homolog (PTEN) in Fgf20 -/- explants. Overactivation of PI3K resulted in a partial rescue of the Fgf20 -/- phenotype, demonstrating a requirement for PI3K downstream of FGF20. Consistent with a requirement for the MAPK pathway for FGF20-regulated HC differentiation, we show that treating Fgf20 -/- explants with FGF9 increased levels of dpERK., Conclusions: Together, these data provide evidence that both MAPK and PI3K are important downstream mediators of FGF20-FGFR1 signaling during HC and SC differentiation., (© 2020 Wiley Periodicals LLC.)

Background: Understanding the mechanisms that regulate hair cell (HC) differentiation in the organ of Corti (OC) is essential to designing genetic therapies for hearing loss due to HC loss or damage. We have previously identified Fibroblast Growth Factor 20 (FGF20) as having a key role in HC and supporting cell differentiation in the mouse OC. To investigate the genetic landscape regulated by FGF20 signaling in OC progenitors, we employ Translating Ribosome Affinity Purification combined with Next Generation RNA Sequencing (TRAPseq) in the Fgf20 lineage., Results: We show that TRAPseq targeting OC progenitors effectively enriched for RNA from this rare cell population. TRAPseq identified differentially expressed genes (DEGs) downstream of FGF20, including Etv4, Etv5, Etv1, Dusp6, Hey1, Hey2, Heyl, Tectb, Fat3, Cpxm2, Sall1, Sall3, and cell cycle regulators such as Cdc20. Analysis of Cdc20 conditional-null mice identified decreased cochlea length, while analysis of Sall1-null and Sall1-ΔZn2-10 mice, which harbor a mutation that causes Townes-Brocks syndrome, identified a decrease in outer hair cell number., Conclusions: We present two datasets: genes with enriched expression in OC progenitors, and DEGs downstream of FGF20 in the embryonic day 14.5 cochlea. We validate select DEGs via in situ hybridization and in vivo functional studies in mice., (© 2020 Wiley Periodicals LLC.)

Hair cell (HC) degeneration causes hearing loss in millions of people worldwide. Aminoglycoside exposure is one major cause of sensory HC damage. Aminoglycosides generate free radicals within the inner ear, permanently damaging sensory cells, and thus causing hearing loss. Hearing protection requires strategies to overcome the apparently irreversible loss of HCs in mammals. The nuclear factor of activated T cells (NFAT) inhibitor 11R-VIVIT reportedly protects HCs from gentamicin toxicity. Here we investigated whether the combination of 11R-VIVIT with the antioxidant L-carnitine or N-acetylcysteine could protect mouse cochlear HCs from gentamicin damage. Compared to single-component treatment, combined treatment with 11R-VIVIT plus L-carnitine yielded significant protection from gentamicin, and 11R-VIVIT plus N-acetylcysteine provided almost complete protection of HCs from gentamicin. Caspase activity in organ of Corti was significantly reduced by combined treatment with 11R-VIVIT + N-acetylcysteine + gentamicin, compared to 11R-VIVIT + gentamicin or gentamicin alone. Analysis of relative gene expression by qPCR revealed down-regulation of the pro-apoptotic genes Fasl and Casp9, and up-regulation of the antioxidant genes Hmox1 and Nrf2 after treatment with 11R-VIVIT + N-acetylcysteine + gentamicin, compared to single-compound treatment or gentamicin alone in cultures. Selective NFAT inhibition by 11R-VIVIT may be a good strategy for preventing gentamicin-induced HC damage. L-carnitine and N-acetylcysteine, with their ROS-reducing properties, contribute to the synergistic effectiveness with 11R-VIVIT by decreasing ROS-induced NFAT translocation. Our data suggest that a combined approach of NFAT inhibition together with an antioxidant, like N-acetylcysteine, could be useful for hearing loss treatment and/or prevention. Cover Image for this issue: https://doi.org/10.1111/jnc.14759., (© 2019 International Society for Neurochemistry.)

An increased number of patients with residual hearing are undergoing cochlear implantation. A subset of these experience delayed hearing loss post-implantation, and the aetiology of this loss is not well understood. Our previous studies suggest that electrical stimulation can induce damage to hair cells in organ of Corti (OC) organotypic cultures. Dexamethasone has the potential to protect residual hearing due to its multiple effects on cells and tissue (e.g., anti-inflammatory, free radical scavenger). We therefore hypothesized that dexamethasone treatment could prevent electrical stimulation induced changes in the OC. Organ of Corti explants from neonatal rats (P2-4) were cultured for 24 h with two different concentrations of dexamethasone. Thereafter, OC were subjected to a charge-balanced biphasic pulsed electrical stimulation (0.44-2 mA) for a further 24 h. Unstimulated dexamethasone-treated OC served as controls. Outcome analysis included immunohistochemical labelling of ribbon synapses, histochemical analysis of free reactive oxygen species and morphological analysis of stereocilia bundles. Overall, the protective effects of dexamethasone on electrically induced damage in cochlear explants were moderate. High-dose dexamethasone protected bundle integrity at higher current levels. Low-dose dexamethasone tended to increase ribbon density in the apical region.

The cochlea, a coiled structure located in the ventral region of the inner ear, acts as the primary structure for the perception of sound. Along the length of the cochlear spiral is the organ of Corti, a highly derived and rigorously patterned sensory epithelium that acts to convert auditory stimuli into neural impulses. The development of the organ of Corti requires a series of inductive events that specify unique cellular characteristics and axial identities along its three major axes. Here, we review recent studies of the cellular and molecular processes regulating several aspects of cochlear development, such as axial patterning, cochlear outgrowth and cellular differentiation. We highlight how the precise coordination of multiple signaling pathways is required for the successful formation of a complete organ of Corti., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

Precise control of organ growth and patterning is executed through a balanced regulation of progenitor self-renewal and differentiation. In the auditory sensory epithelium-the organ of Corti-progenitor cells exit the cell cycle in a coordinated wave between E12.5 and E14.5 before the initiation of sensory receptor cell differentiation, making it a unique system for studying the molecular mechanisms controlling the switch between proliferation and differentiation. Here we identify the Yap/Tead complex as a key regulator of the self-renewal gene network in organ of Corti progenitor cells. We show that Tead transcription factors bind directly to the putative regulatory elements of many stemness- and cell cycle-related genes. We also show that the Tead coactivator protein, Yap, is degraded specifically in the Sox2-positive domain of the cochlear duct, resulting in down-regulation of Tead gene targets. Further, conditional loss of the Yap gene in the inner ear results in the formation of significantly smaller auditory and vestibular sensory epithelia, while conditional overexpression of a constitutively active version of Yap , Yap5SA , is sufficient to prevent cell cycle exit and to prolong sensory tissue growth. We also show that viral gene delivery of Yap5SA in the postnatal inner ear sensory epithelia in vivo drives cell cycle reentry after hair cell loss. Taken together, these data highlight the key role of the Yap/Tead transcription factor complex in maintaining inner ear progenitors during development, and suggest new strategies to induce sensory cell regeneration., Competing Interests: J.F.M. is a founder and owns shares in Yap therapeutics.

Previous studies have shown histone modifications being present in cochlear hair cells in animal models of hearing loss. Our past studies have shown that ATP depletion, histone deacetylase (HDAC) upregulation, and histone deacetylation occur in cochlea after noise exposure, and these are linked to hair cell death. Whether ATP depletion correlates with the expression level of HDACs and acetylation of histones is still unknown. In this study, we investigated the changes in the expression of HDACs and the level of histone acetylation in cochlear hair cells using an ATP-depleted explant culture of mouse organ of Corti. We found that the expression of HDAC3 and HDAC6 increased and hair cells were lost after oligomycin A (OA) treatment. Meanwhile, the acetylation level of histone H2B reduced. However, when oligomycin was combined with an HDAC inhibitor, trichostatin A (TSA), the acetylation level of histone H3 was restored. Moreover, combined treatment of oligomycin and TSA or sodium butyrate (NaB) attenuated oligomycin-induced cochlear hair cell loss. In conclusion, our results indicated that ATP depletion led to histone deacetylation and eventually resulted in hair cell death., Competing Interests: Declaration of Competing Interest The authors declare no conflicts of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Acid-base homeostasis is critical for normal growth, development, and hearing function. The sodium-hydrogen exchanger 6 (NHE6), a protein mainly expressed in early and recycling endosomes, plays an important role in regulating organellar pH. Mutations in NHE6 cause complex, slowly progressive neurodegeneration. Little is known about NHE6 function in the mouse cochlea. Here, we found that all NHE isoforms were expressed in wild-type (WT) mouse cochlea. Nhe6 knockout (KO) mice showed significant hearing loss compared to WT littermates. Immunohistochemistry in WT mouse cochlea showed that Nhe6 was localized in the organ of Corti (OC), spiral ganglion (SG), stria vascularis (SV), and afferent nerve fibres. The middle and the inner ears of WT and Nhe6 KO mice were not different morphologically. Given the putative role of NHE6 in early endosomal function, we examined Rab GTPase expression in early and late endosomes. We found no change in Rab5, significantly lower Rab7, and higher Rab11 levels in the Nhe6 KO OC, compared to WT littermates. Because Rabs mediate TrkB endosomal signalling, we evaluated TrkB phosphorylation in the OCs of both strains. Nhe6 KO mice showed significant reductions in TrkB and Akt phosphorylation in the OC. In addition, we examined genes used as markers of SG type I (Slc17a7, Calb1, Pou4f1, Cal2) and type II neurons (Prph, Plk5, Cacna1g). We found that all marker gene expression levels were significantly elevated in the SG of Nhe6 KO mice, compared to WT littermates. Anti-neurofilament factor staining showed axon loss in the cochlear nerves of Nhe6 KO mice compared to WT mice. These findings indicated that BDNF/TrkB signalling was disrupted in the OC of Nhe6 KO mice, probably due to TrkB reduction, caused by over acidification in the absence of NHE6. Thus, our findings demonstrated that NHEs play important roles in normal hearing in the mammalian cochlea.

Several factors trigger apoptosis in cochlear hair cells. Previous studies have shown that mitochondria play key roles in apoptosis, but the role of mitochondrial deoxyribonucleic acid (mtDNA) copy number in the pathogenesis of hair cell apoptosis remains largely unknown. We used mouse cochlear hair cells and House Ear Institute‑Organ of Corti 1 (HEI‑OC1) cells to explore the relationship between mtDNA copy number and cell apoptosis. We found that the mtDNA copy number of hair cells was reduced relative to mitochondrial mass and hypothesized that increasing it might have a protective effect. We then increased the mtDNA copy number of the hair and HEI‑OC1 cells by transfecting them with an adeno‑associated virus (AAV) vector containing mitochondrial transcription factor A (TFAM). We found that the apoptosis rates decreased upon inducing apoptosis with neomycin or cisplatin (DDP). To elucidate the mechanisms, we analyzed the mitochondrial‑membrane permeability and mitochondrial function of HEI‑OC1 cells. Our results suggested that the increase in mtDNA copy number could protect hair cells and HEI‑OC1 cells against drug‑induced apoptosis by stabilizing the permeability of the mitochondrial membrane and mitochondrial function.

Sensorineural hearing loss due to aging, noise exposure, trauma or drug ototoxicity is irreversible because cochlear hair cells and neurons cannot regenerate. Recently, therapeutic strategies involving nanoparticles have been developed as innovative drug delivery systems. Thermodynamically stable liquid crystalline nanoparticles based on the polar lipid glycerol monooleate (GMO NP, cubosomes), nontoxic and able to encapsulate both hydrophilic and hydrophobic compounds, were produced and tested for biocompatibility in an immortalized Organ of Corti derived cell line (OC-k3), through cell viability and cytomorphological assays, and Western blot expression profiles of apoptotic markers. Overall, the GMO NP were biocompatible in OC-k3 at the doses and time tested, supporting previous data obtained in a neuronal cell line (PC12). The results encourage further tests on GMO NP-mediated drug release with improved target specificity and could be useful to develop innovative therapies against sensorineural hearing loss., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Fragile X syndrome (FXS) is the most common form of inherited intellectual disability and the leading monogenetic cause of autism. One symptom of FXS and autism is sensory hypersensitivity (also called sensory over-responsivity). Perhaps related to this, the audiogenic seizure (AGS) is arguably the most robust behavioral phenotype in the FXS mouse model-the Fmr1 knock-out (KO) mouse. Therefore, the AGS may be considered a mouse model of sensory hypersensitivity. Hyperactive circuits are hypothesized to underlie dysfunction in a number of brain regions in patients with FXS and Fmr1 KO mice, and the AGS may be a result of this. But the specific cell types and brain regions underlying AGSs in the Fmr1 KO are unknown. We used conditional deletion or expression of Fmr1 in different cell populations to determine whether Fmr1 deletion in those cells was sufficient or necessary, respectively, for the AGS phenotype in males. Our data indicate that Fmr1 deletion in glutamatergic neurons that express vesicular glutamate transporter 2 (VGlut2) and are located in subcortical brain regions is sufficient and necessary to cause AGSs. Furthermore, the deletion of Fmr1 in glutamatergic neurons of the inferior colliculus is necessary for AGSs. When we demonstrate necessity, we show that Fmr1 expression in either the larger population of VGlut2-expressing glutamatergic neurons or the smaller population of inferior collicular glutamatergic neurons-in an otherwise Fmr1 KO mouse-eliminates AGSs. Therefore, targeting these neuronal populations in FXS and autism may be part of a therapeutic strategy to alleviate sensory hypersensitivity. SIGNIFICANCE STATEMENT Sensory hypersensitivity in fragile X syndrome (FXS) and autism patients significantly interferes with quality of life. Audiogenic seizures (AGSs) are arguably the most robust behavioral phenotype in the FXS mouse model-the Fmr1 knockout-and may be considered a model of sensory hypersensitivity in FXS. We provide the clearest and most precise genetic evidence to date for the cell types and brain regions involved in causing AGSs in the Fmr1 knockout and, more broadly, for any mouse mutant. The expression of Fmr1 in these same cell types in an otherwise Fmr1 knockout eliminates AGSs indicating possible cellular targets for alleviating sensory hypersensitivity in FXS and other forms of autism., (Copyright © 2019 the authors.)

Several studies have documented the early development of OHC electromechanical behavior. The mechanical response (electromotility, eM) and its electrical correlate (nonlinear capacitance, NLC), resulting from prestin's voltage-sensor charge movement, increase over the course of several postnatal days in altricial animals. They increase until about p18, near the time of peripheral auditory maturity. The correspondence of auditory capabilities and prestin function indicates that mature activity of prestin occurs at this time. One of the major requirements of eM is its responsiveness across auditory frequencies. Here we evaluate the frequency response of prestin charge movement in mice over the course of development up to 8 months. We find that in apical turn OHCs prestin's frequency response increases during postnatal development and stabilizes when mature hearing is established. The low frequency component of NLC, within in situ explants, agrees with previously reported results on isolated cells. If prestin activity is independent of cochlear place, as might be expected, then these observations suggest that prestin activity somehow influences cochlear amplification at high frequencies in spite of its low pass behavior.

Development of multicellular organs requires the coordination of cell differentiation and patterning. Critical for sound detection, the mammalian organ of Corti contains functional units arranged tonotopically along the cochlear turns. Each unit consists of sensory hair cells intercalated by nonsensory supporting cells, both specified and radially patterned with exquisite precision during embryonic development. However, how cell identity and radial patterning are jointly controlled is poorly understood. Here we show that β-catenin is required for specification of hair cell and supporting cell subtypes and radial patterning of the cochlea in vivo. In 2 mouse models of conditional β-catenin deletion, early specification of Myosin7-expressing hair cells and Prox1-positive supporting cells was preserved. While β-catenin-deficient cochleae expressed FGF8 and FGFR3, both of which are essential for pillar cell specification, the radial patterning of organ of Corti was disrupted, revealed by aberrant expression of cadherins and the pillar cell markers P75 and Lgr6. Moreover, β-catenin ablation caused duplication of FGF8-positive inner hair cells and reduction of outer hair cells without affecting the overall hair cell density. In contrast, in another transgenic model with suppressed transcriptional activity of β-catenin but preserved cell adhesion function, both specification and radial patterning of the organ of Corti were intact. Our study reveals specific functions of β-catenin in governing cell identity and patterning mediated through cell adhesion in the developing cochlea., Competing Interests: The authors declare no competing interest.

Purinergic signaling is deeply involved in the development, functions and protective mechanisms of the cochlea. Release of ATP and activation of purinergic receptors on sensory and supporting/epithelial cells play a substantial role in cochlear (patho)physiology. Both the ionotropic P2X and the metabotropic P2Y receptors are widely distributed on the inner and outer hair cells as well as on the different supporting cells in the organ of Corti and on other epithelial cells in the scala media. Among others, they are implicated in the sensitivity adjustment of the receptor cells by a K + shunt and can attenuate the cochlear amplification by modifying cochlear micromechanics acting on outer hair cells and supporting cells. Cochlear blood flow is also regulated by purines. Sensorineural hearing losses currently lack any specific or efficient pharmacotherapy. Decreasing hearing sensitivity and increasing cochlear blood supply by pharmacological targeting of purinergic signaling in the cochlea are potential new therapeutic approaches in these hearing disabilities, especially in the noise-induced ones., (Copyright © 2019 Elsevier Inc. All rights reserved.)

JAK/STAT pathway is one among the several oxidative stress-responsive signaling pathways that play a critical role in facilitating cisplatin-induced ototoxicity. Cisplatin treatment decreases the levels of cochlear LMO4, which acts as a scaffold for IL6-GP130 protein complex. Cisplatin-induced nitration and degradation of LMO4 could destabilize this protein complex, which in turn could compromise the downstream STAT3-mediated cellular defense mechanism. Here, we investigated the link between cisplatin-induced nitrative stress and STAT3-mediated apoptosis by using organ of Corti cell cultures. SRI110, a peroxynitrite decomposition catalyst that prevented cisplatin-induced decrease in LMO4 levels and ototoxicity, was used to inhibit nitrative stress. Immunoblotting and immunostaining indicated that cisplatin treatment decreased the expression levels, phosphorylation, and nuclear localization of STAT3 in UB/OC1 cells. Inhibition of nitration by SRI110 co-treatment prevented cisplatin-induced inactivation of STAT3 and promoted its nuclear localization. SRI110 co-treatment reversed the cisplatin-induced changes in the expression levels of Bcl2l1, Ccnd1, Jak2, Jak3, and Src and significantly attenuated the changes in the expression levels of Cdkn1a, Egfr, Fas, Il6st, Jak1, Stat3, and Tyk2. Collectively, these results suggest that the inhibition of cisplatin-induced nitration prevents the inactivation of STAT3, which in turn enables the transcription of anti-apoptotic genes and thereby helps to mitigate cisplatin-induced toxicity., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The mouse organ of Corti, housed inside the cochlea, contains hair cells and supporting cells that transduce sound into electrical signals. These cells develop in two main steps: progenitor specification followed by differentiation. Fibroblast Growth Factor (FGF) signaling is important in this developmental pathway, as deletion of FGF receptor 1 (Fgfr1) or its ligand, Fgf20, leads to the loss of hair cells and supporting cells from the organ of Corti. However, whether FGF20-FGFR1 signaling is required during specification or differentiation, and how it interacts with the transcription factor Sox2, also important for hair cell and supporting cell development, has been a topic of debate. Here, we show that while FGF20-FGFR1 signaling functions during progenitor differentiation, FGFR1 has an FGF20-independent, Sox2-dependent role in specification. We also show that a combination of reduction in Sox2 expression and Fgf20 deletion recapitulates the Fgfr1-deletion phenotype. Furthermore, we uncovered a strong genetic interaction between Sox2 and Fgf20, especially in regulating the development of hair cells and supporting cells towards the basal end and the outer compartment of the cochlea. To explain this genetic interaction and its effects on the basal end of the cochlea, we provide evidence that decreased Sox2 expression delays specification, which begins at the apex of the cochlea and progresses towards the base, while Fgf20-deletion results in premature onset of differentiation, which begins near the base of the cochlea and progresses towards the apex. Thereby, Sox2 and Fgf20 interact to ensure that specification occurs before differentiation towards the cochlear base. These findings reveal an intricate developmental program regulating organ of Corti development along the basal-apical axis of the cochlea., Competing Interests: The authors have declared that no competing interests exist.

Background: The study aimed to investigate the effect of oxidative stress on Prestin expression, and explore the transcription factors (TFs) that are involved in regulating the expression of Prestin in House Ear Institute-Organ of Corti 1 (HEI-OC1) cells upon oxidative stress., Methods: Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot were used to detect the expression level of Prestin. Reverse chromatin immunoprecipitation (reverse ChIP) assay was performed to identify proteins that could bind to the Prestin gene. Small interfering RNA (siRNA) and chromatin immunoprecipitation (ChIP) experiments were used to further verify the results. HEI-OC1 cells were incubated with four different concentrations of tert-butyl hydroperoxide (t-BHP) for 24 h or 48 h to construct the oxidative stress model., Results: Oxidative stress induced Prestin increase at the mRNA level but with a concomitant decrease at the protein level. TF activating enhancer binding protein-2δ (AP-2δ) screened by reverse ChIP assay was demonstrated to bind to transcriptional start site 1441 of the Prestin promoter region and negatively regulate the expression of Prestin by siRNA and ChIP experiments. Furthermore, AP-2δ was down-regulated under oxidative stress., Conclusions: In conclusion, oxidative stress inhibits the expression of Prestin protein, and the transcription mechanism is triggered to compensate for the loss of Prestin protein. AP-2δ is one of the important TFs that suppresses transcription of the Prestin gene, and AP-2δ suppression further boosted Prestin mRNA activation under oxidative stress., Competing Interests: Competing interestsThe authors declare that they have no competing interests.

Class III Semaphorin (Sema) secreted ligands are known to repel neurites expressing Neuropilin (Nrp) and/or Plexin (Plxn) receptors. There is, however, a growing body of literature supporting that Sema signaling also has alternative roles in development such as synaptogenesis, boundary formation, and vasculogenesis. To evaluate these options during inner ear development, we used in situ hybridization or immunohistochemistry to map the expression of Sema3D, Sema3F, Nrp1, Nrp2, and PlxnA1 in the chicken (Gallus gallus) inner ear from embryonic day (E)5-E10. The resulting expression patterns in either the otic epithelium or its surrounding mesenchyme suggest that Sema signaling could be involved in each of the varied functions reported for other tissues. Sema3D expression flanking the sensory tissue in vestibular organs suggests that it may repel Nrp2- and PlxnA1-expressing neurites of the vestibular ganglion away from nonsensory epithelia, thus channeling them into the sensory domains at E5-E8. Expression of Sema signaling genes in the sensory hair cells of both the auditory and vestibular organs on E8-E10 may implicate Sema signaling in synaptogenesis. In the nonsensory regions of the cochlea, Sema3D in the future tegmentum vasculosum opposes Nrp1 and PlxnA1 in the future cuboidal cells; the abutment of ligand and receptors in adjacent domains may enforce or maintain the boundary between them. In the mesenchyme, Nrp1 colocalized with capillary-rich tissue. Sema3D immediately flanks this Nrp1-expressing tissue, suggesting a role in endothelial cell migration towards the inner ear. In summary, Sema signaling may play multiple roles in the developing inner ear., (© 2018 Wiley Periodicals, Inc.)

Single-cell RNA sequencing is a powerful tool by which to characterize the transcriptional profile of low-abundance cell types, but its application to the inner ear has been hampered by the bony labyrinth, tissue sparsity, and difficulty dissociating the ultra-rare cells of the membranous cochlea. Herein, we present a method to isolate individual inner hair cells (IHCs), outer hair cells (OHCs), and Deiters' cells (DCs) from the murine cochlea at any post-natal time point. We harvested more than 200 murine IHCs, OHCs, and DCs from post-natal days 15 (p15) to 228 (p228) and leveraged both short- and long-read single-cell RNA sequencing to profile transcript abundance and structure. Our results provide insights into the expression profiles of these cells and document an unappreciated complexity in isoform variety in deafness-associated genes. This refined view of transcription in the organ of Corti improves our understanding of the biology of hearing and deafness., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

In the inner ear sensory epithelia, stereociliary hair bundles atop sensory hair cells are mechanosensory apparatus with planar polarized structure and orientation. This is established during development by the concerted action of tissue-level, intercellular planar cell polarity (PCP) signaling and a hair cell-intrinsic, microtubule-mediated machinery. However, how various polarity signals are integrated during hair bundle morphogenesis is poorly understood. Here, we show that the conserved cell polarity protein Par3 is essential for planar polarization of hair cells. Par3 deletion in the inner ear disrupted cochlear outgrowth, hair bundle orientation, kinocilium positioning, and basal body planar polarity, accompanied by defects in the organization and cortical attachment of hair cell microtubules. Genetic mosaic analysis revealed that Par3 functions both cell-autonomously and cell-nonautonomously to regulate kinocilium positioning and hair bundle orientation. At the tissue level, intercellular PCP signaling regulates the asymmetric localization of Par3, which in turn maintains the asymmetric localization of the core PCP protein Vangl2. Mechanistically, Par3 interacts with and regulates the localization of Tiam1 and Trio, which are guanine nucleotide exchange factors (GEFs) for Rac, thereby stimulating Rac-Pak signaling. Finally, constitutively active Rac1 rescued the PCP defects in Par3 -deficient cochleae. Thus, a Par3-GEF-Rac axis mediates both tissue-level and hair cell-intrinsic PCP signaling., Competing Interests: The authors declare no conflict of interest.

Purpose: The aim of the present study was to evaluate the effect of acetyl-l-carnitine (ALC) and N-acetyl cysteine (NAC) on ionizing radiation (IR)-induced cytotoxicity and change in DNA damage-related genes in House Ear Institute-Organ of Corti 1 (HEI-OC1) cells., Methods: HEI-OC1 cells were irradiated with 5 Gy radiation and treated by eight combinations of NAC and/or ALC: control, NAC, ALC, IR, NAC + IR, ALC + NAC, ALC + IR, and ALC + NAC + IR. Cell viability, apoptotic cell death, and DNA damage were measured at the 72nd hour. Eighty-four IR-induced DNA-damage-related genes were determined by RT-PCR gene array and >10-fold changes were considered significant., Results: IR decreased cell viability by about 50% at 72 hours of incubation. In particular, the ALC and/or NAC combination before IR protected the HEI-OC1 cells (p < .05). Single and combination treatment prior to IR led to lower apoptotic cell death (p < .05). There was a significant lower DNA damage in ALC + NAC + IR group compared to IR group (p < .05). Expressions of Brca2, Xpc, Mlh3, Rad51, Xrcc2, Hus1, Rad9a, Cdkn1a, Gadd45a which are the DNA-repair genes were found to be significantly higher in NAC + ALC + IR group than those in individual treatment of ALC or NAC., Conclusions: ALC and/or NAC treatment prior to IR led to higher cell viability and lower apoptotic cell damage compared to the IR group. The results of the study show that the ALC + NAC combination treatment inhibits DNA damage and induces DNA-repair genes to repair radiation damage, and this combination treatment is more effective against radiation-induced DNA damage than NAC or ALC therapy individually.

Damage or loss of auditory hair cells leads to irreversible sensorineural hearing loss in human, thus regeneration of these cells to reconstruct auditory sensory epithelium holds the promise for the treatment of deafness. Regulatory factors involved in the development of auditory sensory epithelium play crucial roles in hair cell regeneration and hearing restoration. Here, we first focus on the transcription factor Atoh1 which is critical for hair cell development and regeneration, and comprehensively summarize the current understanding of the protein structure, target binding motif, developmental expression pattern, functional role, and upstream and downstream regulatory mechanism of Atoh1 in the context of controlling the cell fate commitment to hair cells or transdifferentiation from supporting cells. We also discuss cellular context dependency of Atoh1 in hair cell induction which should be taken into consideration when using Atoh1 gene therapy for hair cell regeneration. Next, we review the roles of Gfi1, Pou4f3, and Barhl1 in hair cell maturation and maintenance, and suggest that manipulation of these genes and their downstream targets will be helpful for the generation of functional hair cells with long-term viability. Finally, we provide an overview of the interplay between Notch, Wnt, Shh, and FGF signaling pathways during auditory sensory epithelium development. By analyzing crosstalk between these pathways, we suggest that combination of Wnt signaling activation with Hey1 and Hey2 inhibition will be crucial for hair cell regeneration and hearing restoration. Furthermore, this review highlights the importance of deeper understanding of the cellular context for hair cell development and the interconnection between these key regulators in developing new strategies to treat sensorineural hearing loss., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Ca 2+ is an important intracellular messenger and regulator in both physiological and pathophysiological mechanisms in the hearing organ. Investigation of cellular Ca 2+ homeostasis in the mature cochlea is hampered by the special anatomy and high vulnerability of the organ. A quick, straightforward and reliable Ca 2+ imaging method with high spatial and temporal resolution in the mature organ of Corti is missing. Cell cultures or isolated cells do not preserve the special microenvironment and intercellular communication, while cochlear explants are excised from only a restricted portion of the organ of Corti and usually from neonatal pre-hearing murines. The hemicochlea, prepared from hearing mice allows tonotopic experimental approach on the radial perspective in the basal, middle and apical turns of the organ. We used the preparation recently for functional imaging in supporting cells of the organ of Corti after bulk loading of the Ca 2+ indicator. However, bulk loading takes long time, is variable and non-selective, and causes the accumulation of the indicator in the extracellular space. In this study we show the improved labeling of supporting cells of the organ of Corti by targeted single-cell electroporation in mature mouse hemicochlea. Single-cell electroporation proved to be a reliable way of reducing the duration and variability of loading and allowed subcellular Ca 2+ imaging by increasing the signal-to-noise ratio, while cell viability was retained during the experiments. We demonstrated the applicability of the method by measuring the effect of purinergic, TRPA1, TRPV1 and ACh receptor stimulation on intracellular Ca 2+ concentration at the cellular and subcellular level. In agreement with previous results, ATP evoked reversible and repeatable Ca 2+ transients in Deiters', Hensen's and Claudius' cells. TRPA1 and TRPV1 stimulation by AITC and capsaicin, respectively, failed to induce any Ca 2+ response in the supporting cells, except in a single Hensen's cell in which AITC evoked transients with smaller amplitude. AITC also caused the displacement of the tissue. Carbachol, agonist of ACh receptors induced Ca 2+ transients in about a third of Deiters' and fifth of Hensen's cells. Here we have presented a fast and cell-specific indicator loading method allowing subcellular functional Ca 2+ imaging in supporting cells of the organ of Corti in the mature hemicochlea preparation, thus providing a straightforward tool for deciphering the poorly understood regulation of Ca 2+ homeostasis in these cells., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Serine/threonine kinase 33 (STK33), a member of the calcium/calmodulin-dependent kinase (CAMK), plays vital roles in a wide spectrum of cell processes. The present study was designed to investigate whether STK33 expressed in the mammalian cochlea and, if so, what effect STK33 exerted on aminoglycoside-induced ototoxicity in House Ear Institute-Organ of Corti 1 (HEI-OC1) cells. Immunofluorescence staining and western blotting were performed to investigate STK33 expression in cochlear hair cells (HCs) and HEI-OC1 cells with or without gentamicin treatment. CCK8, flow cytometry, immunofluorescence staining and western blotting were employed to detect the effects of STK33 knockdown, and/or U0126, and/or N-acetyl-L-cysteine (NAC) on the sensitivity to gentamicin-induced ototoxicity in HEI-OC1 cells. We found that STK33 was expressed in both mice cochlear HCs and HEI-OC1 cells, and the expression of STK33 was significantly decreased in cochlear HCs and HEI-OC1 cells after gentamicin exposure. STK33 knockdown resulted in an increase in the cleaved caspase-3 and Bax expressions as well as cell apoptosis after gentamicin damage in HEI-OC1 cells. Mechanistic studies revealed that knockdown of STK33 led to activated mitochondrial apoptosis pathway as well as augmented reactive oxygen species (ROS) accumulation after gentamicin damage. Moreover, STK33 was involved in extracellular signal-regulated kinase 1/2 pathway in primary culture of HCs and HEI-OC1 cells in response to gentamicin insult. The findings from this work indicate that STK33 decreases the sensitivity to the apoptosis dependent on mitochondrial apoptotic pathway by regulating ROS generation after gentamicin treatment, which provides a new potential target for protection from the aminoglycoside-induced ototoxicity., (© 2018 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Sensory hair cells located in the organ of Corti are essential for cochlear mechanosensation. Their loss is irreversible in humans resulting in permanent hearing loss. The development of therapeutic interventions for hearing loss requires fundamental knowledge about similarities and potential differences between animal models and human development as well as the establishment of human cell based-assays. Here we analyze gene and protein expression of the developing human inner ear in a temporal window spanning from week 8 to 12 post conception, when cochlear hair cells become specified. Utilizing surface markers for the cochlear prosensory domain, namely EPCAM and CD271, we purify postmitotic hair cell progenitors that, when placed in culture in three-dimensional organoids, regain proliferative potential and eventually differentiate to hair cell-like cells in vitro. These results provide a foundation for comparative studies with otic cells generated from human pluripotent stem cells and for establishing novel platforms for drug validation.

Advances in microelectromechanical systems (MEMS) technologies are enhancing the development of intracochlear delivery devices for the treatment of hearing loss with emerging pharmacological therapies. Direct intracochlear delivery addresses the limitations of systemic and intratympanic delivery. However, optimization of delivery parameters for these devices requires pharmacokinetic assessment of the spatiotemporal drug distribution inside the cochlea. Robust methods of measuring drug concentration in the perilymph have been developed, but lack spatial resolution along the tonotopic axis or require complex physiological measurements. Here we describe an approach for quantifying distribution of fluorescent drug-surrogate probe along the cochlea's sensory epithelium with high spatial resolution enabled by confocal fluorescence imaging. Fluorescence from FM 1-43 FX, a fixable endocytosis marker, was quantified using confocal fluorescence imaging of whole mount sections of the organ of Corti from cochleae resected and fixed at several time points after intracochlear delivery. Intracochlear delivery of FM 1-43 FX near the base of the cochlea produces a base-apex gradient of fluorescence in the row of inner hair cells after 1 h post-delivery that is consistent with diffusion-limited transport along the scala tympani. By 3 h post-delivery there is approximately an order of magnitude decrease in peak average fluorescence intensity, suggesting FM 1-43 FX clearance from both the perilymph and inner hair cells. The increase in fluorescence intensity at 72 h post-delivery compared to 3 h post-delivery may implicate a potential radial transport pathway into the scala media., (Copyright © 2018 Elsevier B.V. All rights reserved.)

GJB2 mutations are the leading cause of non-syndromic inherited hearing loss. GJB2 encodes connexin-26 (CX26), which is a connexin (CX) family protein expressed in cochlea, skin, liver, and brain, displaying short cytoplasmic N-termini and C-termini. We searched for CX26 C-terminus binding partners by affinity capture and identified 12 unique proteins associated with cell junctions or cytoskeleton (CGN, DAAM1, FLNB, GAPDH, HOMER2, MAP7, MAPRE2 (EB2), JUP, PTK2B, RAI14, TJP1, and VCL) by using mass spectrometry. We show that, similar to other CX family members, CX26 co-fractionates with TJP1, VCL, and EB2 (EB1 paralogue) as well as the membrane-associated protein ASS1. The adaptor protein CGN (cingulin) co-immuno-precipitates with CX26, ASS1, and TJP1. In addition, CGN co-immunoprecipitation with CX30, CX31, and CX43 indicates that CX association is independent on the CX C-terminus length or sequence. CX26, CGN, FLNB, and DAMM1 were shown to distribute to the organ of Corti and hepatocyte plasma membrane. In the mouse liver, CX26 and TJP1 co-localized at the plasma membrane. In conclusion, CX26 associates with components of other membrane junctions that integrate with the cytoskeleton.

The mammalian hearing organ is a stereotyped cellular assembly with orderly innervation: two types of spiral ganglion neurons (SGNs) innervate two types of differentially distributed hair cells (HCs). HCs and SGNs evolved from single neurosensory cells through gene multiplication and diversification. Independent regulation of HCs and neuronal differentiation through expression of basic helix-loop-helix transcription factors (bHLH TFs: Atoh1, Neurog1, Neurod1) led to the evolution of vestibular HC assembly and their unique type of innervation. In ancestral mammals, a vestibular organ was transformed into the organ of Corti (OC) containing a single row of inner HC (IHC), three rows of outer HCs (OHCs), several unique supporting cell types, and a peculiar innervation distribution. Restoring the OC following long-term hearing loss is complicated by the fact that the entire organ is replaced by a flat epithelium and requires reconstructing the organ from uniform undifferentiated cell types, recapitulating both evolution and development. Finding the right sequence of gene activation during development that is useful for regeneration could benefit from an understanding of the OC evolution. Toward this end, we report on Foxg1 and Lmx1a mutants that radically alter the OC cell assembly and its innervation when mutated and may have driven the evolutionary reorganization of the basilar papilla into an OC in ancestral Therapsids. Furthermore, genetically manipulating the level of bHLH TFs changes HC type and distribution and allows inference how transformation of HCs might have happened evolutionarily. We report on how bHLH TFs regulate OHC/IHC and how misexpression (Atoh1-Cre; Atoh1f/kiNeurog1) alters HC fate and supporting cell development. Using mice with altered HC types and distribution, we demonstrate innervation changes driven by HC patterning. Using these insights, we speculate on necessary steps needed to convert a random mixture of post-mitotic precursors into the orderly OC through spatially and temporally regulated critical bHLH genes in the context of other TFs to restore normal innervation patterns.

The exocyst, an octameric protein complex consisting of Exoc1 through Exoc8, was first determined to regulate exocytosis by targeting vesicles to the plasma membrane in yeast to mice. In addition to this fundamental role, the exocyst complex has been implicated in other cellular processes. In this study, we investigated the role of the exocyst in cochlear development and hearing by targeting EXOC5, a central exocyst component. Deleting Exoc5 in the otic epithelium with widely used Cre lines resulted in early lethality. Thus, we generated two different inner ear-specific Exoc5 knockout models by crossing Gfi1 Cre mice with Exoc5 f/f mice for hair cell-specific deletion (Gfi1 Cre/+ ;Exoc5 f/f ) and by in utero delivery of rAAV-iCre into the otocyst of embryonic day 12.5 for deletion throughout the otic epithelium (rAAV2/1-iCre;Exoc5 f/f ). Gfi1 Cre/+ ;Exoc5 f/f mice showed relatively normal hair cell morphology until postnatal day 20, after which hair cells underwent apoptosis accompanied by disorganization of stereociliary bundles, resulting in progressive hearing loss. rAAV2/1-iCre;Exoc5 f/f mice exhibited abnormal neurite morphology, followed by apoptotic degeneration of spiral ganglion neurons (SGNs) and hair cells, which led to profound and early-onset hearing loss. These results demonstrate that Exoc5 is essential for the normal development and survival of cochlear hair cells and SGNs, as well as the functional maintenance of hearing.

Autophagy is a highly evolutionary conserved quality control defense mechanism within cells, which has also been implicated in cell death processes. In the mammalian inner ear, autophagy has been shown to play a role during early morphogenesis as well as in adult cochlear hair cells exposed to ototoxic insults. Mitophagy, a selective autophagic cell process targeting mitochondria, hasn't been studied in the inner ear so far. On this work, we searched for molecular indicators of mitophagy within House Ear Institute-Organ of Corti-1 (HEI-OC1) cells as well as in the organ of Corti (OC). We first tested for the expression of Pink1/Park2 mRNA in 5-day-old C57BL/6 mice's cochleae using RT-PCR. We focused on the induction of mitophagy in HEI-OC1 cells as well as in the OC and investigated a possible mitophagic potential of the aminoglycoside agent gentamicin. The induction of mitophagy in HEI-OC1 cells was detected by objectivizing the translocation of fluorescence-tagged LC3 to mitochondria using confocal microscopy after a 6-h incubation with a well-described mitochondrial uncoupler and mitophagy-inducing agent: carbonyl cyanide m-chlorophenyl hydrazone (CCCP). Incubation with gentamicin generated no mitochondrial translocation of LC3. Protein levels of COXIV, Atg5/12 and LC3 were evaluated by an immunoblot analysis after a 24-h CCCP treatment as well as gentamicin. We demonstrated mitophagy after CCCP exposure in HEI-OC1 cells by showing a downregulation of COXIV. A downregulation of COXIV could also be visualized in the OC after CCCP. A significant oxygen consumption rate (OCR) changed in cells treated with CCCP as well as significant morphological changes of mitochondria by electron microscopy (EM) strengthen this assumption. Gentamicin exposure generated no impact on OCR or mitochondrial morphological changes by EM. Finally, we demonstrated changes in the expression of Atg12 and LC3 proteins in both the OC and HEI-OC1 cells after CCCP exposure but not after gentamicin. Our data indicate that gentamicin had no impact in the activation of mitophagy-neither in the HEI-OC1 cell line nor in the OC. Therefore, we speculate that mitophagic-independent mechanisms may underly aminoglycoside ototoxicity., (Copyright © 2018 Elsevier B.V. All rights reserved.)

To date, the mechanism (s) underlying the cisplatin-elicited ototoxicity has not been elucidated fully. Nucleotide-binding domain and leucine-rich-repeat-containing family member ×1 (NLRX1), a cytoplasmic pattern recognition receptor, is tightly related to mitochondrial function, reactive oxygen species (ROS) production, and autophagy. In this work, autophagy alteration, NLRX1 expression, ROS generation and cell injury were investigated correspondingly by immunofluorescence staining, western-blot, TEM, flow cytometry and MTT in HEI-OC1 cells of both NLRX1 overexpression and silencing in response to cisplatin stimulus. We found that NLRX1 expression was increased concurrent with the increase of autophagy activation in HEI-OC1 cells under the cisplatin insult. NLRX1 overexpression led to the amount of accumulation of autophagsomes in HEI-OC1 cells in normal condition and a higher activation of autophagy concurrent with cell injury in HEI-OC1 cells treated with cisplatin, whereas, NLRX1 silencing decreased the activation level of autophagy concurrent with increased cell viability in HEI-OC1 cells treated with cisplatin. Mechanistic studies showed that NLRX1 potentiated mitochondrial-derived ROS generation in response to cisplatin exposure. Inhibition of ROS generation significantly prevented autophagy activation and apoptosis both in HEI-OC1cells and cochlear explants treated with cisplatin. The findings from this work reveal that NLRX1 sensitizes auditory cells in vitro to cisplatin-induced ototoxity via autophagic cell death pathway, providing another strategy against cisplatin-induced ototoxity., (Copyright © 2018. Published by Elsevier Inc.)

Transmembrane proteins that mediate the cellular uptake or efflux of thyroid hormone potentially provide a key level of control over neurodevelopment. In humans, defects in one such protein, solute carrier SLC16A2 (MCT8) are associated with psychomotor retardation. Other proteins that transport the active form of thyroid hormone triiodothyronine (T3) or its precursor thyroxine (T4) have been identified in vitro but the wider significance of such transporters in vivo is unclear. The development of the auditory system requires thyroid hormone and the cochlea is a primary target tissue. We have proposed that the compartmental anatomy of the cochlea would necessitate transport mechanisms to convey blood-borne hormone to target tissues. We report hearing loss in mice with mutations in Slc16a2 and a related gene Slc16a10 (Mct10, Tat1). Deficiency of both transporters results in retarded development of the sensory epithelium similar to impairment caused by hypothyroidism, compounded with a progressive degeneration of cochlear hair cells and loss of endocochlear potential. Administration of T3 largely restores the development of the sensory epithelium and limited auditory function, indicating the T3-sensitivity of defects in the sensory epithelium. The results indicate a necessity for thyroid hormone transporters in cochlear development and function.

Survival of cochlear sensory epithelial cells may be regulated by inhibitor of differentiation-1 (Id1) and the N-methyl-D-aspartic acid (NMDA) receptor. However, it is unclear whether Id1 and the NMDA receptor are involved in the radiation-mediated survival of rat cochlear sensory epithelial cells. Here, we show that the percentage of apoptotic cells increased, the percentage of cells in the S phase decreased, Id1 mRNA and protein expression decreased and the NMDA receptor subtype 2B (NR2B) mRNA and protein level increased in OC1 cells after radiation. Cells infected with the Id1 gene exhibited higher Id1 mRNA and protein levels and lower NR2B mRNA and protein levels than the control cells. In contrast, after transfection of the Id1 siRNA into OC1 cells, Id1 mRNA and protein expression decreased and NR2B mRNA and protein expression increased relative to that of the control group. Additionally, treatment with ifenprodil for 24 h before radiation reduced apoptosis and increased the percentage of cells in the S phase. Our results suggest that Id1 and NR2B might regulate the survival of OC1 cells following radiation., (© 2018 S. Karger AG, Basel.)

A K + -Cl - cotransport system was documented for the first time during the mid-seventies in sheep and goat red blood cells. It was then described as a Na + -independent and ouabain-insensitive ion carrier that could be stimulated by cell swelling and N-ethylmaleimide (NEM), a thiol-reacting agent. Twenty years later, this system was found to be dispensed by four different isoforms in animal cells. The first one was identified in the expressed sequence tag (EST) database by Gillen et al. based on the assumption that it would be homologous to the Na + -dependent K + -Cl - cotransport system for which the molecular identity had already been uncovered. Not long after, the three other isoforms were once again identified in the EST databank. Among those, KCC4 has generated much interest a few years ago when it was shown to sustain distal renal acidification and hearing development in mouse. As will be seen in this review, many additional roles were ascribed to this isoform, in keeping with its wide distribution in animal species. However, some of them have still not been confirmed through animal models of gene inactivation or overexpression. Along the same line, considerable knowledge has been acquired on the mechanisms by which KCC4 is regulated and the environmental cues to which it is sensitive. Yet, it is inferred to some extent from historical views and extrapolations., (Copyright © 2017 Elsevier B.V. All rights reserved.)