Spherical harmonics analysis reveals cell shape-fate relationships in zebrafish lateral line neuromasts.

Cell shape is a powerful readout of cell state, fate and function. We describe a custom workflow to perform semi-automated, 3D cell and nucleus segmentation, and spherical harmonics and principal components analysis to distill cell and nuclear shape variation into discrete biologically meaningful parameters. We apply these methods to analyze shape in the neuromast cells of the zebrafish lateral line system, finding that shapes vary with cell location and identity. The distinction between hair cells and support cells accounted for much of the variation, which allowed us to train classifiers to predict cell identity from shape features. Using transgenic markers for support cell subpopulations, we found that subtypes had different shapes from each other. To investigate how loss of a neuromast cell type altered cell shape distributions, we examined atoh1a mutants that lack hair cells. We found that mutant neuromasts lacked the cell shape phenotype associated with hair cells, but did not exhibit a mutant-specific cell shape. Our results demonstrate the utility of using 3D cell shape features to characterize, compare and classify cells in a living developing organism.

Differential regulation of hair cell actin cytoskeleton mediated by SRF and MRTFB.

The MRTF-SRF pathway has been extensively studied for its crucial role in driving the expression of a large number of genes involved in actin cytoskeleton of various cell types. However, the specific contribution of MRTF-SRF in hair cells remains unknown. In this study, we showed that hair cell-specific deletion of Srf or Mrtfb, but not Mrtfa, leads to similar defects in the development of stereocilia dimensions and the maintenance of cuticular plate integrity. We used fluorescence-activated cell sorting-based hair cell RNA-Seq analysis to investigate the mechanistic underpinnings of the changes observed in Srf and Mrtfb mutants, respectively. Interestingly, the transcriptome analysis revealed distinct profiles of genes regulated by Srf and Mrtfb, suggesting different transcriptional regulation mechanisms of actin cytoskeleton activities mediated by Srf and Mrtfb. Exogenous delivery of calponin 2 using Adeno-associated virus transduction in Srf mutants partially rescued the impairments of stereocilia dimensions and the F-actin intensity of cuticular plate, suggesting the involvement of Cnn2, as an Srf downstream target, in regulating the hair bundle morphology and cuticular plate actin cytoskeleton organization. Our study uncovers, for the first time, the unexpected differential transcriptional regulation of actin cytoskeleton mediated by Srf and Mrtfb in hair cells, and also demonstrates the critical role of SRF-CNN2 in modulating actin dynamics of the stereocilia and cuticular plate, providing new insights into the molecular mechanism underlying hair cell development and maintenance.

Mechanical overstimulation causes acute injury and synapse loss followed by fast recovery in lateral-line neuromasts of larval zebrafish.

Excess noise damages sensory hair cells, resulting in loss of synaptic connections with auditory nerves and, in some cases, hair-cell death. The cellular mechanisms underlying mechanically induced hair-cell damage and subsequent repair are not completely understood. Hair cells in neuromasts of larval zebrafish are structurally and functionally comparable to mammalian hair cells but undergo robust regeneration following ototoxic damage. We therefore developed a model for mechanically induced hair-cell damage in this highly tractable system. Free swimming larvae exposed to strong water wave stimulus for 2 hr displayed mechanical injury to neuromasts, including afferent neurite retraction, damaged hair bundles, and reduced mechanotransduction. Synapse loss was observed in apparently intact exposed neuromasts, and this loss was exacerbated by inhibiting glutamate uptake. Mechanical damage also elicited an inflammatory response and macrophage recruitment. Remarkably, neuromast hair-cell morphology and mechanotransduction recovered within hours following exposure, suggesting severely damaged neuromasts undergo repair. Our results indicate functional changes and synapse loss in mechanically damaged lateral-line neuromasts that share key features of damage observed in noise-exposed mammalian ear. Yet, unlike the mammalian ear, mechanical damage to neuromasts is rapidly reversible.

Differential regulation of mammalian and avian ATOH1 by E2F1 and its implication for hair cell regeneration in the inner ear.

The mammalian inner ear has a limited capacity to regenerate its mechanosensory hair cells. This lack of regenerative capacity underlies the high incidence of age-related hearing loss in humans. In contrast, non-mammalian vertebrates can form new hair cells when damage occurs, a mechanism that depends on re-activation of expression of the pro-hair cell transcription factor Atoh1. Here, we show that members of the E2F transcription factor family, known to play a key role in cell cycle progression, regulate the expression of Atoh1. E2F1 activates chicken Atoh1 by directly interacting with a cis-regulatory region distal to the avian Atoh1 gene. E2F does not activate mouse Atoh1 gene expression, since this regulatory element is absent in mammals. We also show that E2F1 expression changes dynamically in the chicken auditory epithelium during ototoxic damage and hair cell regeneration. Therefore, we propose a model in which the mitotic regeneration of non-mammalian hair cells is due to E2F1-mediated activation of Atoh1 expression, a mechanism which has been lost in mammals.

Key Signaling Pathways Regulate the Development and Survival of Auditory Hair Cells.

The loss of auditory sensory hair cells (HCs) is the most common cause of sensorineural hearing loss (SNHL). As the main sound transmission structure in the cochlea, it is necessary to maintain the normal shape and survival of HCs. In this review, we described and summarized the signaling pathways that regulate the development and survival of auditory HCs in SNHL. The role of the mitogen-activated protein kinase (MAPK), phosphoinositide-3 kinase/protein kinase B (PI3K/Akt), Notch/Wnt/Atoh1, calcium channels, and oxidative stress/reactive oxygen species (ROS) signaling pathways are the most relevant. The molecular interactions of these signaling pathways play an important role in the survival of HCs, which may provide a theoretical basis and possible therapeutic interventions for the treatment of hearing loss.

Deletion of Clusterin Protects Cochlear Hair Cells against Hair Cell Aging and Ototoxicity.

Hearing loss is a debilitating disease that affects 10% of adults worldwide. Most sensorineural hearing loss is caused by the loss of mechanosensitive hair cells in the cochlea, often due to aging, noise, and ototoxic drugs. The identification of genes that can be targeted to slow aging and reduce the vulnerability of hair cells to insults is critical for the prevention of sensorineural hearing loss. Our previous cell-specific transcriptome analysis of adult cochlear hair cells and supporting cells showed that Clu, encoding a secreted chaperone that is involved in several basic biological events, such as cell death, tumor progression, and neurodegenerative disorders, is expressed in hair cells and supporting cells. We generated Clu-null mice (C57BL/6) to investigate its role in the organ of Corti, the sensory epithelium responsible for hearing in the mammalian cochlea. We showed that the deletion of Clu did not affect the development of hair cells and supporting cells; hair cells and supporting cells appeared normal at 1 month of age. Auditory function tests showed that Clu-null mice had hearing thresholds comparable to those of wild-type littermates before 3 months of age. Interestingly, Clu-null mice displayed less hair cell and hearing loss compared to their wildtype littermates after 3 months. Furthermore, the deletion of Clu is protected against aminoglycoside-induced hair cell loss in both in vivo and in vitro models. Our findings suggested that the inhibition of Clu expression could represent a potential therapeutic strategy for the alleviation of age-related and ototoxic drug-induced hearing loss.

Residual Hair Cell Responses in Electric-Acoustic Stimulation Cochlear Implant Users with Complete Loss of Acoustic Hearing After Implantation.

Changes in cochlear implant (CI) design and surgical techniques have enabled the preservation of residual acoustic hearing in the implanted ear. While most Nucleus Hybrid L24 CI users retain significant acoustic hearing years after surgery, 6-17 % experience a complete loss of acoustic hearing (Roland et al. Laryngoscope. 126(1):175-81. (2016), Laryngoscope. 128(8):1939-1945 (2018); Scheperle et al. Hear Res. 350:45-57 (2017)). Electrocochleography (ECoG) enables non-invasive monitoring of peripheral auditory function and may provide insight into the pathophysiology of hearing loss. The ECoG response is evoked using an acoustic stimulus and includes contributions from the hair cells (cochlear microphonic-CM) as well as the auditory nerve (auditory nerve neurophonic-ANN). Seven Hybrid L24 CI users with complete loss of residual hearing months after surgery underwent ECoG measures before and after loss of hearing. While significant reductions in CMs were evident after hearing loss, all participants had measurable CMs despite having no measurable acoustic hearing. None retained measurable ANNs. Given histological data suggesting stable hair cell and neural counts after hearing loss (e.g., Quesnel et al. Hear Res. 333:225-234. (2016)), the loss of ECoG and audiometric hearing may reflect reduced synaptic input. This is consistent with the theory that residual CM responses coupled with little to no ANN responses reflect a "disconnect" between hair cells and auditory nerve fibers (Fontenot et al. Ear Hear. 40(3):577-591. 2019). This "disconnection" may prevent proper encoding of auditory stimulation at higher auditory pathways, leading to a lack of audiometric responses, even in the presence of viable cochlear hair cells.

AAV8-mediated Atoh1 overexpression induces dose-dependent regeneration of vestibular hair cells in adult mice.

Vestibular hair cells (HCs) are mechanoreceptors for the detection of head movement. Vestibular HCs of adult mammals never completely regenerate after damage, resulting in vestibular dysfunction. Overexpression of Atoh1 is effective for inducing HC regeneration. However, method of clinical feasibility and improvement of regenerative extent are both in need. Here we used an adeno-associated virus (AAV) serotype 8 vector of two different titers to overexpress Atoh1 in the injured utricles of adult mice. One month after virus inoculation, abundant myosin VIIa-positive cells and immature stereocilia were observed. Quantitative analyses revealed that Atoh1 overexpression replenished vestibular HCs in a dose-dependent manner. Vectors of a higher titer increased the number of myosin VIIa-positive cells compared to those of lower titer. Moreover, only Atoh1 overexpression in the higher titer group enhanced stereocilium regeneration, which is an important step in the maturation of regenerated HCs. Although the current treatment failed to initiate functional recovery of the animals, our results prompt further improvements in the recovery of vestibular dysfunction by AAV.

Purinergic Signaling Controls Spontaneous Activity in the Auditory System throughout Early Development.

Spontaneous bursts of electrical activity in the developing auditory system arise within the cochlea before hearing onset and propagate through future sound-processing circuits of the brain to promote maturation of auditory neurons. Studies in isolated cochleae revealed that this intrinsically generated activity is initiated by ATP release from inner supporting cells (ISCs), resulting in activation of purinergic autoreceptors, K+ efflux, and subsequent depolarization of inner hair cells. However, it is unknown when this activity emerges or whether different mechanisms induce activity during distinct stages of development. Here we show that spontaneous electrical activity in mouse cochlea from both sexes emerges within ISCs during the late embryonic period, preceding the onset of spontaneous correlated activity in inner hair cells and spiral ganglion neurons, which begins at birth and follows a base to apex developmental gradient. At all developmental ages, pharmacological inhibition of P2Y1 purinergic receptors dramatically reduced spontaneous activity in these three cell types. Moreover, in vivo imaging within the inferior colliculus revealed that auditory neurons within future isofrequency zones exhibit coordinated neural activity at birth. The frequency of these discrete bursts increased progressively during the postnatal prehearing period yet remained dependent on P2RY1. Analysis of mice with disrupted cholinergic signaling in the cochlea indicate that this efferent input modulates, rather than initiates, spontaneous activity before hearing onset. Thus, the auditory system uses a consistent mechanism involving ATP release from ISCs and activation of P2RY1 autoreceptors to elicit coordinated excitation of neurons that will process similar frequencies of sound.SIGNIFICANCE STATEMENT In developing sensory systems, groups of neurons that will process information from similar sensory space exhibit highly correlated electrical activity that is critical for proper maturation and circuit refinement. Defining the period when this activity is present, the mechanisms responsible and the features of this activity are crucial for understanding how spontaneous activity influences circuit development. We show that, from birth to hearing onset, the auditory system relies on a consistent mechanism to elicit correlate firing of neurons that will process similar frequencies of sound. Targeted disruption of this activity will increase our understanding of how these early circuits mature and may provide insight into processes responsible for developmental disorders of the auditory system.

Generation of Cochlear Hair Cells from Sox2 Positive Supporting Cells via DNA Demethylation.

Regeneration of auditory hair cells in adult mammals is challenging. It is also difficult to track the sources of regenerated hair cells, especially in vivo. Previous paper found newly generated hair cells in deafened mouse by injecting a DNA methyltransferase inhibitor 5-azacytidine into the inner ear. This paper aims to investigate the cell sources of new hair cells. Transgenic mice with enhanced green fluorescent protein (EGFP) expression controlled by the Sox2 gene were used in the study. A combination of kanamycin and furosemide was applied to deafen adult mice, which received 4 mM 5-azacytidine injection into the inner ear three days later. Mice were followed for 3, 5, 7 and 14 days after surgery to track hair cell regeneration. Immunostaining of Myosin VIIa and EGFP signals were used to track the fate of Sox2-expressing supporting cells. The results show that (i) expression of EGFP in the transgenic mice colocalized the supporting cells in the organ of Corti, and (ii) the cell source of regenerated hair cells following 5-azacytidine treatment may be supporting cells during 5-7 days post 5-azacytidine injection. In conclusion, 5-azacytidine may promote the conversion of supporting cells to hair cells in chemically deafened adult mice.

Developmental and Functional Hair Cell-Like Cells Induced by Atoh1 Overexpression in the Adult Mammalian Cochlea In Vitro.

Hair cells (HCs) in the mammalian cochleae cannot spontaneously regenerate once damaged, resulting in permanent hearing loss. It has been shown that Atoh1 overexpression induces hair cell-like cells (HCLCs) in the cochlea of newborn rodents, but this is hard to achieve in adult mammals. In this study, we used a three-dimensional cochlear culture system and an adenoviral-mediated delivery vector to overexpress Atoh1 in adult mouse cochleae. HCLCs were successfully induced from 3 days after virus infection (3 DVI) in vitro, and the number increased with time. HCLCs were myosin7a positive and distinguishable from remnant HCs in a culture environment. Meanwhile, patch-clamp results showed that noninactive outward potassium currents (sustained outward potassium currents) could be recorded in HCLCs and that their magnitude increased with time, similar to normal HCs. Furthermore, transient HCN currents were recorded in some HCLCs, indicating that the HCLCs experienced a developmental stage similar to normal HCs. We also compared the electrophysiological features of HCLCs from adult mice with native HCs and found the HCLCs gradually matured, similar to the normal HCs. Meanwhile, HCLCs from adult mice possessed the same bundles as developmental HCs. However, these HCLCs did not express prestin, which is a special marker for outer hair cells (OHCs), even at 13 DVI. These results demonstrate that Atoh1 overexpression induces HCLC formation in the adult mammalian cochlea and that these HCLCs were functional and experienced a developmental process similar to that of normal HCs.

Biological insights from multi-omic analysis of 31 genomic risk loci for adult hearing difficulty.

Age-related hearing impairment (ARHI), one of the most common medical conditions, is strongly heritable, yet its genetic causes remain largely unknown. We conducted a meta-analysis of GWAS summary statistics from multiple hearing-related traits in the UK Biobank (n = up to 330,759) and identified 31 genome-wide significant risk loci for self-reported hearing difficulty (p < 5x10-8), of which eight have not been reported previously in the peer-reviewed literature. We investigated the regulatory and cell specific expression for these loci by generating mRNA-seq, ATAC-seq, and single-cell RNA-seq from cells in the mouse cochlea. Risk-associated genes were most strongly enriched for expression in cochlear epithelial cells, as well as for genes related to sensory perception and known Mendelian deafness genes, supporting their relevance to auditory function. Regions of the human genome homologous to open chromatin in epithelial cells from the mouse were strongly enriched for heritable risk for hearing difficulty, even after adjusting for baseline effects of evolutionary conservation and cell-type non-specific regulatory regions. Epigenomic and statistical fine-mapping most strongly supported 50 putative risk genes. Of these, 39 were expressed robustly in mouse cochlea and 16 were enriched specifically in sensory hair cells. These results reveal new risk loci and risk genes for hearing difficulty and suggest an important role for altered gene regulation in the cochlear sensory epithelium.

Calcium-induced calcium release in proximity to hair cell BK channels revealed by PKA activation.

Large-conductance calcium-activated potassium (BK) channels play a critical role in electrical resonance, a mechanism of frequency selectivity in chicken hair cells. We determine that BK currents are dependent on inward flow of Ca2+ , and intracellular buffering of Ca2+ . Entry of Ca2+ is further amplified locally by calcium-induced Ca2+ release (CICR) in close proximity to plasma membrane BK channels. Ca2+ imaging reveals peripheral clusters of high concentrations of Ca2+ that are suprathreshold to that needed to activate BK channels. Protein kinase A (PKA) activation increases the size of BK currents likely by recruiting more BK channels due to spatial spread of high Ca2+ concentrations in turn from increasing CICR. STORM imaging confirms the presence of nanodomains with ryanodine and IP3 receptors in close proximity to the Slo subunit of BK channels. Together, these data require a rethinking of how electrical resonance is brought about and suggest effects of CICR in synaptic release. Both genders were included in this study.

Stem Cell-Based Therapeutic Approaches to Restore Sensorineural Hearing Loss in Mammals.

The hair cells that reside in the cochlear sensory epithelium are the fundamental sensory structures responsible for understanding the mechanical sound waves evoked in the environment. The intense damage to these sensory structures may result in permanent hearing loss. The present strategies to rehabilitate the hearing function include either hearing aids or cochlear implants that may recover the hearing capability of deaf patients to a limited extent. Therefore, much attention has been paid on developing regenerative therapies to regenerate/replace the lost hair cells to treat the damaged cochlear sensory epithelium. The stem cell therapy is a promising approach to develop the functional hair cells and neuronal cells from endogenous and exogenous stem cell pool to recover hearing loss. In this review, we specifically discuss the potential of different kinds of stem cells that hold the potential to restore sensorineural hearing loss in mammals and comprehensively explain the current therapeutic applications of stem cells in both the human and mouse inner ear to regenerate/replace the lost hair cells and spiral ganglion neurons.

YAP Mediates Hair Cell Regeneration in Balance Organs of Chickens, But LATS Kinases Suppress Its Activity in Mice.

Loss of sensory hair cells causes permanent hearing and balance deficits in humans and other mammals, but for nonmammals such deficits are temporary. Nonmammals recover hearing and balance sensitivity after supporting cells proliferate and differentiate into replacement hair cells. Evidence of mechanical differences between those sensory epithelia and their supporting cells prompted us to investigate whether the capacity to activate YAP, an effector in the mechanosensitive Hippo pathway, correlates with regenerative capacity in acceleration-sensing utricles of chickens and mice of both sexes. After hair cell ablation, YAP accumulated in supporting cell nuclei in chicken utricles and promoted regenerative proliferation, but YAP remained cytoplasmic and little proliferation occurred in mouse utricles. YAP localization in supporting cells was also more sensitive to shape change and inhibition of MST1/2 in chicken utricles than in mouse utricles. Genetic manipulations showed that in vivo expression of the YAP-S127A variant caused robust proliferation of neonatal mouse supporting cells, which produced progeny that expressed hair cell markers, but proliferative responses declined postnatally. Expression of YAP-5SA, which more effectively evades inhibitory phosphorylation, resulted in TEAD-dependent proliferation of striolar supporting cells, even in adult utricles. Conditional deletion of LATS1/2 kinases abolished the inhibitory phosphorylation of endogenous YAP and led to striolar proliferation in adult mouse utricles. The findings suggest that damage overcomes inhibitory Hippo signaling and facilitates regenerative proliferation in nonmammalian utricles, whereas constitutive LATS1/2 kinase activity suppresses YAP-TEAD signaling in mammalian utricles and contributes to maintaining the proliferative quiescence that appears to underlie the permanence of sensory deficits.SIGNIFICANCE STATEMENT Loud sounds, ototoxic drugs, infections, and aging kill sensory hair cells in the ear, causing irreversible hearing loss and balance deficits for millions. In nonmammals, damage evokes shape changes in supporting cells, which can divide and regenerate hair cells. Such shape changes are limited in mammalian ears, where supporting cells develop E-cadherin-rich apical junctions reinforced by robust F-actin bands, and the cells fail to divide. Here, we find that damage readily activates YAP in supporting cells within balance epithelia of chickens, but not mice. Deleting LATS kinases or expressing YAP variants that evade LATS-mediated inhibitory phosphorylation induces proliferation in supporting cells of adult mice. YAP signaling eventually may be harnessed to overcome proliferative quiescence that limits regeneration in mammalian ears.

The Atoh1 expression levels are correlated with the arrangement, ciliary morphology, and electrophysiological characteristics of ectopic hair cell-like cells.

Previous reports have suggested that the level and duration of Atoh1 expression are correlated with the survival, arrangement and stereociliary bundle-related morphology of hair cells during development, but whether Atoh1 expression levels are correlated with the arrangement, bundle formation and electrophysiological characteristics of newly formed hair cells is unknown. To address this question, cultured cochlear explants obtained from neonatal rats were treated with different titers of a human adenovirus serotype 5 (Ad5) vector encoding Atoh1 and/or EGFP (EGFP-Atoh1+/-). The results showed that higher EGFP-Atoh1 concentrations led to higher initial Atoh1 mRNA expression levels and induced greater numbers of ectopic hair cell-like cells (EHCLCs) in the lesser epithelial ridge (LER). Furthermore, gradual increases in the number of EHCLCs were associated with the progressive conversion of the LER region similarly to that of hair cells during development. Some of the cilia on EHCLCs with higher Atoh1 expression were regularly arranged in a manner similar to that of normal hair bundles. As demonstrated through patch clamp recordings, high Atoh1 expression was associated with significantly decreased proportions of cells with Ih currents, significantly reduced proportions of transient potassium channel currents, and potassium channel currents with a greatly increased mean amplitude, which indicated that EHCLCs with high Atoh1 expression were more mature than those with low Atoh1 expression. Overall, the evidence suggests that the Atoh1 expression levels affect not only the arrangement and ciliary morphology of hair cells but also the electrophysiological characteristics of Atoh1-induced EHCLCs, and these findings provide important guidance for future therapies aimed at treating deafness.

Ectopic expression of ROR1 prevents cochlear hair cell loss in guinea pigs with noise-induced hearing loss.

Noise-induced hearing loss (NIHL) is one of the most frequent disabilities in industrialized countries. Evidence shows that hair cell loss in the auditory end organ is responsible for the majority of various ear pathological conditions. The functional roles of the receptor tyrosine kinase ROR1 have been underscored in various tumours. In this study, we evaluated the ability of ROR1 to influence cochlear hair cell loss of guinea pigs with NIHL. The NIHL model was developed in guinea pigs, with subsequent measurement of the auditory brainstem response (ABR). Gain-of-function experiments were employed to explore the role of ROR1 in NIHL. The interaction between ROR1 and Wnt5a and their functions in the cochlear hair cell loss were further analysed in response to alteration of ROR1 and Wnt5a. Guinea pigs with NIHL demonstrated elevated ABR threshold and down-regulated ROR1, Wnt5a and NF-κB p65. The up-regulation of ROR1 was shown to decrease the cochlear hair cell loss and the expression of pro-apoptotic gene (Bax, p53) in guinea pig cochlea, but promoted the expression of anti-apoptotic gene (Bcl-2) and the fluorescence intensity of cleaved-caspase-3. ROR1 interacted with Wnt5a to activate the NF-κB signalling pathway through inducing phosphorylation and translocation of p65. Furthermore, Wnt5a overexpression decreased the cochlear hair cell loss. Collectively, this study suggested the protection of overexpression of ROR1 against cochlear hair cell loss in guinea pigs with NIHL via the Wnt5a-dependent NF-κB signalling pathway.

A model for reticular dysgenesis shows impaired sensory organ development and hair cell regeneration linked to cellular stress.

Mutations in the gene AK2 are responsible for reticular dysgenesis (RD), a rare and severe form of primary immunodeficiency in children. RD patients have a severely shortened life expectancy and without treatment die, generally from sepsis soon after birth. The only available therapeutic option for RD is hematopoietic stem cell transplantation (HSCT). To gain insight into the pathophysiology of RD, we previously created zebrafish models for Ak2 deficiencies. One of the clinical features of RD is hearing loss, but its pathophysiology and causes have not been determined. In adult mammals, sensory hair cells of the inner ear do not regenerate; however, their regeneration has been observed in several non-mammalian vertebrates, including zebrafish. Therefore, we used our RD zebrafish models to determine whether Ak2 deficiency affects sensory organ development and/or hair cell regeneration. Our studies indicated that Ak2 is required for the correct development, survival and regeneration of sensory hair cells. Interestingly, Ak2 deficiency induces the expression of several oxidative stress markers and it triggers an increased level of cell death in the hair cells. Finally, we show that glutathione treatment can partially rescue hair cell development in the sensory organs in our RD models, pointing to the potential use of antioxidants as a therapeutic treatment supplementing HSCT to prevent or ameliorate sensorineural hearing deficits in RD patients.

TMC1 is an essential component of a leak channel that modulates tonotopy and excitability of auditory hair cells in mice.

Hearing sensation relies on the mechano-electrical transducer (MET) channel of cochlear hair cells, in which transmembrane channel-like 1 (TMC1) and transmembrane channel-like 2 (TMC2) have been proposed to be the pore-forming subunits in mammals. TMCs were also found to regulate biological processes other than MET in invertebrates, ranging from sensations to motor function. However, whether TMCs have a non-MET role remains elusive in mammals. Here, we report that in mouse hair cells, TMC1, but not TMC2, provides a background leak conductance, with properties distinct from those of the MET channels. By cysteine substitutions in TMC1, we characterized four amino acids that are required for the leak conductance. The leak conductance is graded in a frequency-dependent manner along the length of the cochlea and is indispensable for action potential firing. Taken together, our results show that TMC1 confers a background leak conductance in cochlear hair cells, which may be critical for the acquisition of sound-frequency and -intensity.

[The study of AAV9 expression in cochleae of mice at different ages].

Objective: To determine the extent of transfection and expression of adeno-associated virus (AAV) serotype 9 (AAV9) in the cochleae of mice at different ages. Methods: AAV9-green fluorescent protein (GFP) was inoculated into the cochlea of mice via the round window membrane (RWM) or through cochleostomy at different ages. Four groups were divided according to ages and injection sites: P1SM group, AAV9-GFP was delivered to the scala media by cochleostomy at postnatal day 1; P1RW group, AAV9-GFP was delivered to the scala tympani via RWM at postnatal day 1; P9RW group: AAV9-GFP was injected through RWM at postnatal day 9; and P30RW group, adult mice (P30) were injected through RWM. GFP expression in cochlear whole mount was analyzed and auditory brainstem response (ABR) tests were conducted one month after virus injection (for each animal, only left cochlea was injected and the right side was used as a control). GraphPad Prism 5 statistical software was used for data analysis. Results: All of inner hair cells (IHCs) and most of outer hair cells (OHCs) were transfected via two approaches at P1 injection. There was no significant difference in ABR threshold between injected ears and untreated ears (P>0.05). All of the IHCs and parts of OHCs (69% in apical turn) were transfected via RWM at P9. The strongest GFP expression was observed near the apical turn. Cochlear inoculation via RWM at P30 led to transgene expression in only IHCs. The ABR threshold of injected ears in P9RW group and P30RW group was significantly higher than that of contralateral ears (P<0.01). Conclusions: AAV9 can be highly expressed in the inner and outer hair cells of the cochlea and hearing sensitivity can be preserved if virus injections are performed in neonatal mice. After AAV9 is transfected into the inner ear of adult mice, it is only expressed in the inner hair cells, which leads to the increase of the ABR response threshold of mice. Transfection efficiency is significant higher in neonate mice than in P9 and adult mice.