Ear Hair Cells Research Articles

Emerging Roles of RNA-Binding Proteins in Inner Ear Hair Cell Development and Regeneration.

RNA-binding proteins (RBPs) regulate gene expression at the post-transcriptional level. They play major roles in the tissue- and stage-specific expression of protein isoforms as well as in the maintenance of protein homeostasis. The inner ear is a bi-functional organ, with the cochlea and the vestibular system required for hearing and for maintaining balance, respectively. It is relatively well documented that transcription factors and signaling pathways are critically involved in the formation of inner ear structures and in the development of hair cells. Accumulating evidence highlights emerging functions of RBPs in the post-transcriptional regulation of inner ear development and hair cell function. Importantly, mutations of splicing factors of the RBP family and defective alternative splicing, which result in inappropriate expression of protein isoforms, lead to deafness in both animal models and humans. Because RBPs are critical regulators of cell proliferation and differentiation, they present the potential to promote hair cell regeneration following noise- or ototoxin-induced damage through mitotic and non-mitotic mechanisms. Therefore, deciphering RBP-regulated events during inner ear development and hair cell regeneration can help define therapeutic strategies for treatment of hearing loss. In this review, we outline our evolving understanding of the implications of RBPs in hair cell formation and hearing disease with the aim of promoting future research in this field.

The heterogeneity of mammalian utricular cells over the course of development.

BackgroundThe inner ear organ is a delicate tissue consisting of hair cells (HCs) and supporting cells (SCs).The mammalian inner ear HCs are terminally differentiated cells that cannot spontaneously regenerate in adults. Epithelial non‐hair cells (ENHCs) in the utricle include HC progenitors and SCs, and the progenitors share similar characteristics with SCs in the neonatal inner ear.MethodsWe applied single‐cell sequencing to whole mouse utricles from the neonatal period to adulthood, including samples from postnatal day (P)2, P7 and P30 mice. Furthermore, using transgenic mice and immunostaining, we traced the source of new HC generation.ResultsWe identified several sensory epithelial cell clusters and further found that new HCs arose mainly through differentiation from Sox9+ progenitor cells and that only a few cells were produced by mitotic proliferation in both neonatal and adult mouse utricles. In addition, we identified the proliferative cells using the marker UbcH10 and demonstrated that in adulthood the mitotically generated HCs were primarily found in the extrastriola. Moreover, we observed that not only Type II, but also Type I HCs could be regenerated by either mitotic cell proliferation or progenitor cell differentiation.ConclusionsOverall, our findings expand our understanding of ENHC cell fate and the characteristics of the vestibular organs in mammals over the course of development.

Pitavastatin protects against neomycin-induced ototoxicity through inhibition of endoplasmic reticulum stress.

Irreversible injury to inner ear hair cells induced by aminoglycoside antibiotics contributes to the formation of sensorineural hearing loss. Pitavastatin (PTV), a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, has been reported to exert neuroprotective effects. However, its role in aminoglycoside-induced hearing loss remains unknown. The objectives of this study were to investigate the beneficial effects, as well as the mechanism of action of PTV against neomycin-induced ototoxicity. We found that PTV remarkably reduced hair cell loss in mouse cochlear explants and promoted auditory HEI-OC1 cells survival after neomycin stimulation. We also observed that the auditory brainstem response threshold that was increased by neomycin was significantly reduced by pretreatment with PTV in mice. Furthermore, neomycin-induced endoplasmic reticulum stress in hair cells was attenuated by PTV treatment through inhibition of PERK/eIF2α/ATF4 signaling. Additionally, we found that PTV suppressed the RhoA/ROCK/JNK signal pathway, which was activated by neomycin stimulation in HEI-OC1 cells. Collectively, our results showed that PTV might serve as a promising therapeutic agent against aminoglycoside-induced ototoxicity.

Is there an unmet medical need for improved hearing restoration?

Hearing impairment, the most prevalent sensory deficit, affects more than 466 million people worldwide (WHO). We presently lack causative treatment for the most common form, sensorineural hearing impairment; hearing aids and cochlear implants (CI) remain the only means of hearing restoration. We engaged with CI users to learn about their expectations and their willingness to collaborate with health care professionals on establishing novel therapies. We summarize upcoming CI innovations, gene therapies, and regenerative approaches and evaluate the chances for clinical translation of these novel strategies. We conclude that there remains an unmet medical need for improving hearing restoration and that we are likely to witness the clinical translation of gene therapy and major CI innovations within this decade.

Deciphering the Molecular Interaction Between the Adhesion G Protein-Coupled Receptor ADGRV1 and its PDZ-Containing Regulator PDZD7.

Hearing relies on the transduction of sound-evoked vibrations into electrical signals, occurring in the stereocilia bundle of inner ear hair cells. The G protein-coupled receptor (GPCR) ADGRV1 and the multi-PDZ protein PDZD7 play a critical role in the formation and function of stereocilia through their scaffolding and signaling properties. During hair cell development, the GPCR activity of ADGRV1 is specifically inhibited by PDZD7 through an unknown mechanism. Here, we describe the key interactions mediated by the two N-terminal PDZ domains of PDZD7 and the cytoplasmic domain of ADGRV1. Both PDZ domains can bind to the C-terminal PDZ binding motif (PBM) of ADGRV1 with the critical contribution of atypical C-terminal β extensions. The two PDZ domains form a supramodule in solution, stabilized upon PBM binding. Interestingly, we showed that the stability and binding properties of the PDZ tandem are affected by two deafness-causing mutations located in the binding grooves of PDZD7 PDZ domains.

Single-cell RNA-sequencing of zebrafish hair cells reveals novel genes potentially involved in hearing loss.

Hair cells play key roles in hearing and balance, and hair cell loss would result in hearing loss or vestibular dysfunction. Cellular and molecular research in hair cell biology provides us a better understanding of hearing and deafness. Zebrafish, owing to their hair cell-enriched organs, have been widely applied in hair cell-related research worldwide. Similar to mammals, zebrafish have inner ear hair cells. In addition, they also have lateral line neuromast hair cells. These different types of hair cells vary in morphology and function. However, systematic analysis of their molecular characteristics remains lacking. In this study, we analyzed the GFP+ cells isolated from Tg(Brn3c:mGFP) larvae with GFP expression in all hair cells using single-cell RNA-sequencing (scRNA-seq). Three subtypes of hair cells, namely macula hair cell (MHC), crista hair cell (CHC), and neuromast hair cell (NHC), were characterized and validated by whole-mount in situ hybridization analysis of marker genes. The hair cell scRNA-seq data revealed hair cell-specific genes, including hearing loss genes that have been identified in humans and novel genes potentially involved in hair cell formation and function. Two novel genes were discovered to specifically function in NHCs and MHCs, corresponding to their specific expression in NHCs and MHCs. This study allows us to understand the specific genes in hair cell subpopulations of zebrafish, which will shed light on the genetics of both human vestibular and cochlear hair cell function.

Graphene Substrates Promote the Differentiation of Inner Ear Lgr5+ Progenitor Cells Into Hair Cells.

The ideal treatment for sensory hearing loss is to regenerate inner ear hair cells (HCs) through stem cell therapy, thereby restoring the function and structure of the cochlea. Previous studies have found that Lgr5+ supporting cells (SCs) in the inner ear can regenerate HCs, thus being considered inner ear progenitor cells. In addition to traditional biochemical factors, physical factors such as electrical conductivity also play a crucial role in the regulation of stem cell proliferation and differentiation. In this study, the graphene substrates were used to culture Lgr5+ progenitor cells and investigated their regulatory effects on cells. It was demonstrated that the graphene substrates displayed great cytocompatibility for Lgr5+ progenitors and promoted their sphere-forming ability. Moreover, more Myosin7a+ cells were found on the graphene substrates compared with tissue culture polystyrene (TCPS). These results suggest that graphene is an efficient interface that can promote the differentiation of Lgr5+ progenitors into HCs, which is great significance for its future application in combination with Lgr5+ cells to regenerate HCs in the inner ear.

Abstract 2360: The role of the neurogenic transcription factor ATOH1 in small cell lung cancer

Abstract SCLC is an aggressive neuroendocrine (NE) cancer with a 7% 5-year survival rate. SCLC is treated as a clinically homogenous disease, but evidence of its intra- and inter-tumor heterogeneity is increasing. This is partly attributed to expression of neurogenic transcription factors (TFs) ASCL1 and NEUROD1, which correlate with SCLC pathogenesis and NE status, and are now included in a consensus for SCLC classification1. Our Circulating tumor cell patient Derived eXplant (CDX) models recapitulate disease heterogeneity in terms of neurogenic TF expression; furthermore, we recently identified a previously unrecognized subset within our CDX biobank expressing another neurogenic TF, ATOH12. ATOH1 function has been inferred from mouse embryonic and neonatal developmental mouse models, where Atoh1 governs the formation of cerebellar granule cells, auditory hair cells and Merkel cells. In human settings, ATOH1 reprograms pluripotent stem cells to a neuronal lineage and has context-dependent activity in cancer, being considered an oncogene in medulloblastoma and a tumor suppressor in the intestine3. I am investigating the role of ATOH1 in SCLC, to refine disease classification and explore new therapeutic approaches stratified to SCLC phenotypes. To interrogate the role of ATOH1 in SCLC, we developed an in-house antibody and established stable SCLC CDX and cell lines in which ATOH1 can be inducibly depleted in vitro and in vivo. To understand the transcriptional profile mediated by ATOH1, we performed ChIP-Seq and RNA-Seq in ATOH1-competent and depleted cells and integrated the datasets to infer ATOH1 direct transcriptional targets. In concordance with its role in development, ATOH1 direct transcriptional targets are enriched for processes regulating neurogenesis and inner ear hair cell differentiation. ATOH1 depletion in vitro in CDX17P resulted in elevated expression of Non-NE marker genes and NE to Non-NE phenotype transition, confirmed by gene set enrichment analysis, which implicated a possible role for ATOH1 in regulating neuroendocrine plasticity in SCLC. ATOH1 depletion resulted in a significant increase in apoptosis-independent cell death (p < 0.003), and whilst ATOH1 depletion did not affect cell proliferation in CDX17P, there was decreased S-phase progression in CDX30 and HCC33 cells. Depletion of ATOH1 in CDX17P In vivo did result in slower tumor growth and further studies will investigate the impact of ATOH1 on cell death, proliferation and NE to Non-NE transition in vivo. In summary, we have characterized a rare subset of SCLC expressing ATOH1 and found that ATOH1 is necessary for cell survival in vitro and ablation of ATOH1 slows tumor growth in vivo. Ongoing studies focus on investigating possible therapeutic approaches to modulate ATOH1 expression. Citation Format: Alessia Catozzi, Mitchell Revill, Jordan Roebuck, Sam Humphrey, Melanie Galvin, Fiona Blackhall, Kathryn Simpson, Alastair Kerr, Kristopher Frese, Caroline Dive. The role of the neurogenic transcription factor ATOH1 in small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2360.

Critical roles of FGF, RA, and WNT signalling in the development of the human otic placode and subsequent lineages in a dish

IntroductionEfficient induction of the otic placode, the developmental origin of the inner ear from human pluripotent stem cells (hPSCs), provides a robust platform for otic development and sensorineural hearing loss modelling. Nevertheless, there remains a limited capacity of otic lineage specification from hPSCs by stepwise differentiation methods, since the critical factors for successful otic cell differentiation have not been thoroughly investigated. In this study, we developed a novel differentiation system involving the use of a three-dimensional (3D) floating culture with signalling factors for generating otic cell lineages via stepwise differentiation of hPSCs.MethodsWe differentiated hPSCs into preplacodal cells under a two-dimensional (2D) monolayer culture. Then, we transferred the induced preplacodal cells into a 3D floating culture under the control of the fibroblast growth factor (FGF), bone morphogenetic protein (BMP), retinoic acid (RA) and WNT signalling pathways. We evaluated the characteristics of the induced cells using immunocytochemistry, quantitative PCR (qPCR), population averaging, and single-cell RNA-seq (RNA-seq) analysis. We further investigated the methods for differentiating otic progenitors towards hair cells by overexpression of defined transcription factors.ResultsWe demonstrated that hPSC-derived preplacodal cells acquired the potential to differentiate into posterior placodal cells in 3D floating culture with FGF2 and RA. Subsequent activation of WNT signalling induced otic placodal cell formation. By single-cell RNA-seq (scRNA-seq) analysis, we identified multiple clusters of otic placode- and otocyst marker-positive cells in the induced spheres. Moreover, the induced otic cells showed the potential to generate hair cell-like cells by overexpression of the transcription factors ATOH1, POU4F3 and GFI1.ConclusionsWe demonstrated the critical role of FGF2, RA and WNT signalling in a 3D environment for the in vitro differentiation of otic lineage cells from hPSCs. The induced otic cells had the capacity to differentiate into inner ear hair cells with stereociliary bundles and tip link-like structures. The protocol will be useful for in vitro disease modelling of sensorineural hearing loss and human inner ear development and thus contribute to drug screening and stem cell-based regenerative medicine.

Stiffening of Circumferential F-Actin Bands Correlates With Regenerative Failure and May Act as a Biomechanical Brake in the Mammalian Inner Ear.

The loss of inner ear hair cells causes permanent hearing and balance deficits in humans and other mammals, but non-mammals recover after supporting cells (SCs) divide and replace hair cells. The proliferative capacity of mammalian SCs declines as exceptionally thick circumferential F-actin bands develop at their adherens junctions. We hypothesized that the reinforced junctions were limiting regenerative responses of mammalian SCs by impeding changes in cell shape and epithelial tension. Using micropipette aspiration and atomic force microscopy, we measured mechanical properties of utricles from mice and chickens. Our data show that the epithelial surface of the mouse utricle stiffens significantly during postnatal maturation. This stiffening correlates with and is dependent on the postnatal accumulation of F-actin and the cross-linker Alpha-Actinin-4 at SC-SC junctions. In chicken utricles, where SCs lack junctional reinforcement, the epithelial surface remains compliant. There, SCs undergo oriented cell divisions and their apical surfaces progressively elongate throughout development, consistent with anisotropic intraepithelial tension. In chicken utricles, inhibition of actomyosin contractility led to drastic SC shape change and epithelial buckling, but neither occurred in mouse utricles. These findings suggest that species differences in the capacity for hair cell regeneration may be attributable in part to the differences in the stiffness and contractility of the actin cytoskeletal elements that reinforce adherens junctions and participate in regulation of the cell cycle.

Genetic correction of TRMU allele restored the mitochondrial dysfunction-induced deficiencies in iPSCs-derived hair cells of hearing-impaired patients.

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.

Early onset adult deafness in the Rhodesian Ridgeback dog is associated with an in-frame deletion in the EPS8L2 gene.

Domestic dogs exhibit diverse types of both congenital and non-congenital hearing losses. Rhodesian Ridgebacks can suffer from a progressive hearing loss in the early stage of their life, a condition known as early onset adult deafness (EOAD), where they lose their hearing ability within 1-2 years after birth. In order to investigate the genetic basis of this hereditary hearing disorder, we performed a genome-wide association study (GWAS) by using a sample of 23 affected and 162 control Rhodesian Ridgebacks. We identified a genomic region on canine chromosome 18 (CFA18) that is strongly associated with EOAD, and our subsequent targeted Sanger sequencing analysis identified a 12-bp inframe deletion in EPS8L2 (CFA18:25,868,739-25,868,751 in the UMICH_Zoey_3.1/canFam5 reference genome build). Additional genotyping confirmed a strong association between the 12-bp deletion and EOAD, where all affected dogs were homozygous for the deletion, while none of the control dogs was a deletion homozygote. A segregation pattern of this deletion in a 2-generation nuclear family indicated an autosomal recessive mode of inheritance. Since EPS8L2 plays a critical role in the maintenance and integrity of the inner ear hair cells in humans and other mammals, the inframe deletion found in this study represents a strong candidate causal mutation for EOAD in Rhodesian Ridgebacks. Genetic and clinical similarities between childhood deafness in humans and EOAD in Rhodesian Ridgebacks emphasizes the potential value of this dog breed in translational research in hereditary hearing disorders.

Loud noise in early development wrecks zebrafish hearing

It used to be rare to see people in noisy industrial situations wearing ear defenders. Now, bulky ear protection is everywhere, as we appreciate the damage that can be done to hearing even after short periods of loud sound. But are other species also falling prey to human noise pollution encroaching on their environments? ‘Few studies have evaluated long-term noise effects on animal health and even less have focused on early critical periods for the development and establishment of adult characteristics’, says Raquel Vasconcelos from the University of Saint Joseph, Macao, China, who, with Rafael Lara (University of Saint Joseph) began wondering what impact loud sound might have on the developing ears of zebrafish larvae. ‘Zebrafish have become an important model to investigate the mechanisms of inner ear development and hair cell development’, says Vasconcelos, adding that this made the embryos an ideal candidate for the investigation.Lara played loud (150 dB) white noise to the developing embryos for the first 3 and 5 days post-fertilisation (dpf) of the eggs, then collected the zebrafish larvae around the time of hatching (3 dpf) and 2 days later (5 dpf), the age at which they begin to flee when startled. ‘150 dB is a sound amplitude [volume] representative of freshwater habitats characterized by anthropogenic noise activity such as shipping’, says Vasconcelos. Then, Lara tested the youngsters’ hearing by recording the electrical signals produced by their ears as they listened to 100, 200, 300 and 400 Hz beeps. Although the hearing of the 3 dpf embryos didn't suffer too badly, that of the 5 dpf embryos was quite badly affected, especially when listening to the two deeper beeps; their hearing sensitivity plummeted by ∼7 dB. But what impact would this hearing loss have on an embryo's ability to escape a threat?This time, Lara, with colleagues from Macao and from South University of Science and Technology in mainland China, played the 5 dpf larvae a series of soft beeps, at either 100 or 200 Hz, before startling them with a loud (150 dB) beep at the same pitch while filming their reactions. The team realised that the loud noise experienced by the larvae as they developed had made them more sensitive to startling sounds, nipping off 41% faster (∼6 mm s−1 at 100 Hz and ∼8 mm s−1 at 200 Hz), compared with larvae that had developed without the persistent loud noise (∼4 mm s−1 at 100 Hz and ∼6 mm s−1 at 200 Hz).Finally, the team was curious to find out how loud noise affected the structure of the larvae's inner ears. When they viewed the sound-sensitive hair cells in the tiny fish inner ears, in addition to losing ∼21% of their hair cells at 3 dpf (down from 49 hair cells to 38 in fish that developed in a noisy tank) the fish also lost 19% of their hair cells at 5 dpf. The total area of the inner ear that can sense sound was also smaller in the fish from a noisy tank.The young fish that developed in a noisy environment had definitely suffered hearing loss as a result of their loud upbringing. ‘The results reveal noise-induced effects on inner ear structure–function in a larval fish’, says Vasconcelos, adding that it is essential to find out how noise impacts developing youngsters, as they may suffer the consequences for the rest of their lives.

Noise-induced hearing loss correlates with inner ear hair cell decrease in larval zebrafish.

Anthropogenic noise can be hazardous for the auditory system and wellbeing of animals, including humans. However, very limited information is known on how this global environmental pollutant affects auditory function and inner ear sensory receptors in early ontogeny. The zebrafish (Danio rerio) is a valuable model in hearing research, including investigations of developmental processes of the vertebrate inner ear. We tested the effects of chronic exposure to white noise in larval zebrafish on inner ear saccular sensitivity and morphology at 3 and 5 days post-fertilization (dpf), as well as on auditory-evoked swimming responses using the prepulse inhibition (PPI) paradigm at 5 dpf. Noise-exposed larvae showed a significant increase in microphonic potential thresholds at low frequencies, 100 and 200 Hz, while the PPI revealed a hypersensitization effect and a similar threshold shift at 200 Hz. Auditory sensitivity changes were accompanied by a decrease in saccular hair cell number and epithelium area. In aggregate, the results reveal noise-induced effects on inner ear structure-function in a larval fish paralleled by a decrease in auditory-evoked sensorimotor responses. More broadly, this study highlights the importance of investigating the impact of environmental noise on early development of sensory and behavioural responsiveness to acoustic stimuli.

Merkel Cell Carcinoma Sensitivity to EZH2 Inhibition Is Mediated by SIX1 Derepression

Polycomb repressive complex 2 has a critical role in the maintenance of bivalent promoters and is often perturbed in cancer, including neuroendocrine tumors. In this study, we investigated the susceptibility of Merkel cell carcinoma (MCC), a neuroendocrine carcinoma of the skin, to inhibitors of the Polycomb repressive complex 2 catalytic subunit EZH2. We show that a subset of MCC cell lines is sensitive to EZH2 inhibitor-induced cell viability loss. We find that inhibitor treatment of susceptible cells derepresses the Polycomb repressive complex 2 target SIX1, a transcription factor in the PAX-SIX-EYA-DACH network normally involved in inner ear hair cell development, and that PAX-SIX-EYA-DACH network transcription factors are critical contributors to EZH2 inhibitor-induced MCC cell viability loss. Furthermore, we show the EZH2 inhibitor tazemetostat slows the growth of MCC xenografts and derepresses SIX1 and its downstream inner ear transcriptional target MYO6 invivo. We propose that EZH2 inhibition in MCC leads to SIX1 derepression with dysregulation of hearing-related transcriptional programs and growth inhibition. This study provides evidence that MCC tumors may be specifically susceptible to EZH2 inhibitors, while giving mechanistic insight into the transcriptional programs these inhibitors perturb in MCC, and potentially in other neuroendocrine cancers.

Small activating RNA activation of ATOH1 promotes regeneration of human inner ear hair cells

ABSTRACT The loss of inner ear hair cells leads to irreversible acoustic injury in mammals, and regeneration of inner ear hair cells to restore hearing loss is challenging. ATOH1 is a key gene in the development and regeneration of hair cells. Small activating RNAs (saRNAs) can target a gene to specifically upregulate its expression. This study aimed to explore whether small activating RNAs could induce the differentiation of human adipose-derived mesenchymal stem cells into hair cell-like cells with a combination of growth factors in vitro and thus provide a new strategy for hair cell regeneration and the treatment of sensorineural hearing loss. Fifteen small activating RNAs targeting the human ATOH1 gene were designed and screened in 293 T and human adipose-derived mesenchymal stem cells, and 3 of these candidates were found to be capable of effectively and stably activating ATOH1 gene expression. The selected small activating RNAs were then transfected into hair cell progenitor cells, and hair cell markers were examined 10 days after transfection. After transfection of the selected small activating RNAs, the expression of the characteristic markers of inner ear hair cells, POU class 4 homeobox 3 (POU4F3) and myosin VIIA (MYO7A), was detected. Human adipose-derived mesenchymal stem cells have the potential to differentiate into human hair cell progenitor cells. In vitro, small activating RNAs were able to induce the differentiation of hair cell progenitor cells into hair cell-like cells. Therefore, RNA activation technology has the potential to provide a new strategy for the regeneration of hair cells.

Inhibition of PRMT6 reduces neomycin-induced inner ear hair cell injury through the restraint of FoxG1arginine methylation.

Hair cells in the inner ear have been demonstrated to be sensitive to the ototoxicity from some beneficial pharmaceutical drugs. This study aimed to explore the role of protein arginine methyltransferase 6 (PRMT6) in the process of neomycin-induced hearing loss and the underlying mechanism. The neomycin-induced hearing loss mouse model and hair cell injury in vitro model were established. We took advantage of the HEI-OC1 cell line to evaluate PRMT6 expression in neomycin-induced hair cells, and the effect of PRMT6 on mitochondrial function and FoxG1 arginine methylation. Apoptotic cells were assessed and apoptotic marker cleaved caspase-3 level was detected. Reactive oxygen species (ROS) level and mitochondrial membrane potential (MMP) were subsequently measured. The result showed that PRMT6 was significantly upregulated in neomycin-induced HEI-OC-1 cells, and PRMT6 silencing prevented MMP loss, reduced ROS production, as well as decreased cell apoptosis under neomycin treatment. Further results showed that FoxG1 was downregulated in neomycin-induced HEI-OC-1 cells, and PRMT6 promoted the FoxG1-mediated luciferase activity, while PRMT6 silencing reversed this effect. Mechanistic experiments revealed that PRMT6 silencing reduced the arginine methylation level of FoxG1 protein. In vivo, neomycin-induced upregulation of hearing thresholds and increased cell apoptosis, whereas PRMT6 inhibitor partly reversed these effects. Our findings suggested that inhibition of PRMT6 reduced neomycin-induced inner ear hair cell injury through the restraint of FoxG1arginine methylation.

Calcium dependence of MYO3B motor function

Class III myosins are actin based motors that localize to the tips of stereocilia in inner ear hair cells. In vertebrates, class III myosins exist in two isoforms, MYO3A and MYO3B. Mutations in MYO3A are associated with delayed-onset deafness, and MYO3B is proposed to partially compensate for the lack of functional MYO3A. Both MYO3A and MYO3B have been shown to bind espin, a stereocilia actin bundling protein, which may help facilitate elongation at the actin protrusion tips. In this study we discovered that MYO3B is regulated by micromolar concentrations of calcium, while MYO3A is not.

Promotion of In Vitro Hair Cell-like Cell Differentiation from Human Embryonic Stem Cells through the Regulation of Notch Signaling.

The Notch signaling pathway plays an important role in otic neurogenesis by regulating the differentiation of inner ear hair cells and supporting cells. Notch-regulated differentiation is required for the regeneration of hair cells in the inner ear. The temporal expression pattern of Notch ligands and receptors during in vitro hair cell-like cell differentiation from human embryonic stem cells (hESCs) was detected by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Subsequently, pAJ-U6-shRNA-CMV-Puro/GFP recombinant lentiviral vectors encoding short hairpin RNAs were used to silence JAG-1, JAG-2, and DLL-1, according to the temporal expression pattern of Notch ligands. Then, the effect of each ligand on the in vitro differentiation of hair cells was examined by RT-PCR, immunofluorescence, and scanning electron microscopy (SEM). The results showed that the individual deletion of JAG-2 or DLL-1 had no significant effect on the differentiation of hair cell-like cells. However, the simultaneous inhibition of both DLL-1 and JAG-2 increased the number of hair cell-like cells and decreased the number of supporting cells. JAG-2 and DLL-1 may have a synergistic role in in vitro hair cell differentiation.

Current Response in Ca V 1.3-/- Mouse Vestibular and Cochlear Hair Cells.

Signal transmission by sensory auditory and vestibular hair cells relies upon Ca2+-dependent exocytosis of glutamate. The Ca2+ current in mammalian inner ear hair cells is predominantly carried through CaV1.3 voltage-gated Ca2+ channels. Despite this, CaV1.3 deficient mice (CaV1.3–/–) are deaf but do not show any obvious vestibular phenotype. Here, we compared the Ca2+ current (ICa) in auditory and vestibular hair cells from wild-type and CaV1.3–/– mice, to assess whether differences in the size of the residual ICa could explain, at least in part, the two phenotypes. Using 5 mM extracellular Ca2+ and near-body temperature conditions, we investigated the cochlear primary sensory receptors inner hair cells (IHCs) and both type I and type II hair cells of the semicircular canals. We found that the residual ICa in both auditory and vestibular hair cells from CaV1.3–/– mice was less than 20% (12–19%, depending on the hair cell type and age investigated) compared to controls, indicating a comparable expression of CaV1.3 Ca2+ channels in both sensory organs. We also showed that, different from IHCs, type I and type II hair cells from CaV1.3–/– mice were able to acquire the adult-like K+ current profile in their basolateral membrane. Intercellular K+ accumulation was still present in CaV1.3–/– mice during IK,L activation, suggesting that the K+-based, non-exocytotic, afferent transmission is still functional in these mice. This non-vesicular mechanism might contribute to the apparent normal vestibular functions in CaV1.3–/– mice.