Hair cells of the mammalian cochlea are specialized for the dynamic coding of sound stimuli. The transduction of sound waves into electrical signals depends upon mechanosensitive hair bundles that project from the cell's apical surface. Each stereocilium within a hair bundle is composed of uniformly polarized and tightly packed actin filaments. Several stereociliary proteins have been shown to be associated with hair bundle development and function and are known to cause deafness in mice and humans when mutated. The growth of the stereociliar actin core is dynamically regulated at the actin filament barbed ends in the stereociliary tip. We show that Eps8, a protein with actin binding, bundling, and barbed-end capping activities in other systems, is a novel component of the hair bundle. Eps8 is localized predominantly at the tip of the stereocilia and is essential for their normal elongation and function. Moreover, we have found that Eps8 knockout mice are profoundly deaf and that IHCs, but not OHCs, fail to mature into fully functional sensory receptors. We propose that Eps8 directly regulates stereocilia growth in hair cells and also plays a crucial role in the physiological maturation of mammalian cochlear IHCs. Together, our results indicate that Eps8 is critical in coordinating the development and functionality of mammalian auditory hair cells.

Hearing loss affects millions of people worldwide. Yet, there are still no curative therapies for sensorineural hearing loss. Frequent causes of sensorineural hearing loss are due to damage or loss of the sensory hair cells, the spiral ganglion neurons, or the synapses between them. Culturing the organ of Corti allows the study of all these structures in an experimental model, which is easy to manipulate. Therefore, the in vitro culture of the neonatal mammalian organ of Corti remains a frequently used experimental system, in which hair cell survival is routinely assessed. However, the analysis of the surviving hair cells is commonly performed via manual counting, which is a time-consuming process and the inter-rater reliability can be an issue. Here, we describe a deep learning approach to quantify hair cell survival in the murine organ of Corti explants. We used StarDist, a publicly available platform and plugin for Fiji (Fiji is just ImageJ), to train and apply our own custom deep learning model. We successfully validated our model in untreated, cisplatin, and gentamicin treated organ of Corti explants. Therefore, deep learning is a valuable approach for quantifying hair cell survival in organ of Corti explants. Moreover, we also demonstrate how the publicly available Fiji plugin StarDist can be efficiently used for this purpose.

Aminoglycosides are toxic to sensory hair cells (HCs). Macroautophagy/autophagy is an essential and highly conserved self-digestion pathway that plays important roles in the maintenance of cellular function and viability under stress. However, the role of autophagy in aminoglycoside-induced HC injury is unknown. Here, we first found that autophagy activity was significantly increased, including enhanced autophagosome-lysosome fusion, in both cochlear HCs and HEI-OC-1 cells after neomycin or gentamicin injury, suggesting that autophagy might be correlated with aminoglycoside-induced cell death. We then used rapamycin, an autophagy activator, to increase the autophagy activity and found that the ROS levels, apoptosis, and cell death were significantly decreased after neomycin or gentamicin injury. In contrast, treatment with the autophagy inhibitor 3-methyladenine (3-MA) or knockdown of autophagy-related (ATG) proteins resulted in reduced autophagy activity and significantly increased ROS levels, apoptosis, and cell death after neomycin or gentamicin injury. Finally, after neomycin injury, the antioxidant N-acetylcysteine could successfully prevent the increased apoptosis and HC loss induced by 3-MA treatment or ATG knockdown, suggesting that autophagy protects against neomycin-induced HC damage by inhibiting oxidative stress. We also found that the dysfunctional mitochondria were not eliminated by selective autophagy (mitophagy) in HEI-OC-1 cells after neomycin treatment, suggesting that autophagy might not directly target the damaged mitochondria for degradation. This study demonstrates that moderate ROS levels can promote autophagy to recycle damaged cellular constituents and maintain cellular homeostasis, while the induction of autophagy can inhibit apoptosis and protect the HCs by suppressing ROS accumulation after aminoglycoside injury.

Age-related hearing loss is an increasing sensorineural hearing loss. But the pathogenesis of ARHL has not been clarified. Herein, we studied the role and significance of PIN1 in regulating autophagy activity in senescence HEI-OC1cells and HCs.

Aminoglycoside-induced hair cell (HC) loss is one of the most important causes of hearing loss. After entering the inner ear, aminoglycosides induce the production of high levels of reactive oxygen species (ROS) that subsequently activate apoptosis in HCs. Citicoline, a nucleoside derivative, plays a therapeutic role in central nervous system injury and in neurodegenerative disease models, including addictive disorders, stroke, head trauma, and cognitive impairment in the elderly, and has been widely used in the clinic as an FDA approved drug. However, its effect on auditory HCs remains unknown. Here, we used HC-like HEI-OC-1 cells and whole organ explant cultured mouse cochleae to explore the effect of citicoline on aminoglycoside-induced HC damage. Consistent with previous reports, both ROS levels and apoptosis were significantly increased in neomycin-induced cochlear HCs and HEI-OC-1 cells compared to undamaged controls. Interestingly, we found that co-treatment with citicoline significantly protected against neomycin-induced HC loss in both HEI-OC-1 cells and whole organ explant cultured cochleae. Furthermore, we demonstrated that citicoline could significantly reduce neomycin-induced mitochondrial dysfunction and inhibit neomycin-induced ROS accumulation and subsequent apoptosis. Thus, we conclude that citicoline can protect against neomycin-induced HC loss by inhibiting ROS aggregation and thus preventing apoptosis in HCs, and this suggests that citicoline might serve as a potential therapeutic drug in the clinic to protect HCs.

SignificanceGeckos are lizards capable of vocalization and can detect frequencies up to 5 kHz, but the mechanism of frequency discrimination is incompletely understood. The gecko's auditory papilla has a unique arrangement over the high-frequency zone, with rows of mechanically sensitive hair bundles covered with gelatinous sallets. Lower-frequency hair cells are tuned by an electrical resonance employing Ca2+-activated K+ channels, but hair cells tuned above 1 kHz probably rely on a mechanical resonance of the sallets. The resonance may be boosted by an electromotile force from hair bundles found to be evoked by changes in hair cell membrane potential. This unusual mechanism operates independently of mechanotransduction and differs from mammals which amplify the mechanical input using the motor protein prestin.

Transmembrane channel-like protein isoform 1 (TMC1) is a major component of the mechano-electrical transducer (MET) channel in cochlear hair cells and is subject to numerous mutations causing deafness. We report a new dominant human deafness mutation, TMC1 p.T422K, and have characterized the homologous mouse mutant, Tmc1 p.T416K, which caused deafness and outer hair cell (OHC) loss by the fourth postnatal week. MET channels showed decreased Ca2+ permeability and resting open probability, but no change in single-channel conductance or expression. Three adjacent deafness mutations are TMC1 p.L416R, p.G417R, and p.M418K, the last homologous to the mouse Beethoven that exhibits similar channel effects. All substitute a positive for a neutral residue, which could produce charge screening in the channel pore or influence binding of an accessory subunit. Channel properties were compared in mice of both sexes between dominant (Tmc1 p.T416K, Tmc1 p.D569N) and recessive (Tmc1 p.W554L, Tmc1 p.D528N) mutations of residues near the putative pore of the channel. Tmc1 p.W554L and p.D569N exhibit reduced maximum current with no effect on single-channel conductance, implying a smaller number of channels transported to the stereociliary tips; this may stem from impaired TMC1 binding to LHFPL5. Tmc1 p.D528N, located in the pore's narrowest region, uniquely caused large reductions in MET channel conductance and block by dihydrostreptomycin (DHS). For Tmc1 p.T416K and Tmc1 p.D528N, transduction loss occurred between P15 and P20. We propose two mechanisms linking channel mutations and deafness: decreased Ca2+ permeability, common to all mutants, and decreased resting open probability in low Ca2+, confined to dominant mutations.SIGNIFICANCE STATEMENT Transmembrane channel-like protein isoform 1 (TMC1) is thought to be a major component of the mechanotransducer channel in auditory hair cells, but the protein organization and channel structure are still uncertain. We made four mouse lines harboring Tmc1 point mutations that alter channel properties, causing hair cell degeneration and deafness. These include a mouse homolog of a new human deafness mutation pT416K that decreased channel Ca2+ permeability by introducing a positively-charged amino acid in the putative pore. All mutations are consistent with the channel structure predicted from modeling, but only one, p.D528N near the external face of the pore, substantially reduced channel conductance and Ca2+ permeability and virtually abolished block by dihydrostreptomycin (DHS), strongly endorsing its siting within the pore.

Millions of Americans experience hearing or balance disorders due to loss of hair cells in the inner ear. The hair cells are mechanosensory receptors used in the auditory and vestibular organs of all vertebrates as well as the lateral line systems of aquatic vertebrates. In zebrafish and other non-mammalian vertebrates, hair cells turnover during homeostasis and regenerate completely after being destroyed or damaged by acoustic or chemical exposure. However, in mammals, destroying or damaging hair cells results in permanent impairments to hearing or balance. We sought an improved method for studying hair cell damage and regeneration in adult aquatic vertebrates by generating a transgenic zebrafish with the capacity for targeted and inducible hair cell ablation in vivo. This model expresses the human diphtheria toxin receptor (hDTR) gene under the control of the myo6b promoter, resulting in hDTR expressed only in hair cells. Cell ablation is achieved by an intraperitoneal injection of diphtheria toxin (DT) in adult zebrafish or DT dissolved in the water for larvae. In the lateral line of 5 days post fertilization (dpf) zebrafish, ablation of hair cells by DT treatment occurred within 2 days in a dose-dependent manner. Similarly, in adult utricles and saccules, a single intraperitoneal injection of 0.05 ng DT caused complete loss of hair cells in the utricle and saccule by 5 days post-injection. Full hair cell regeneration was observed for the lateral line and the inner ear tissues. This study introduces a new method for efficient conditional hair cell ablation in adult zebrafish inner ear sensory epithelia (utricles and saccules) and demonstrates that zebrafish hair cells will regenerate in vivo after this treatment.

Sound detection by inner ear hair cells requires tip links that interconnect mechanosensory stereocilia and convey force to yet unidentified transduction channels. Current models postulate a static composition of the tip link, with protocadherin 15 (PCDH15) at the lower and cadherin 23 (CDH23) at the upper end of the link. In terminally differentiated mammalian auditory hair cells, tip links are subjected to sound-induced forces throughout an organism's life. Although hair cells can regenerate disrupted tip links and restore hearing, the molecular details of this process are unknown. We developed a novel implementation of backscatter electron scanning microscopy to visualize simultaneously immuno-gold particles and stereocilia links, both of only a few nanometers in diameter. We show that functional, mechanotransduction-mediating tip links have at least two molecular compositions, containing either PCDH15/CDH23 or PCDH15/PCDH15. During regeneration, shorter tip links containing nearly equal amounts of PCDH15 at both ends appear first. Whole-cell patch-clamp recordings demonstrate that these transient PCDH15/PCDH15 links mediate mechanotransduction currents of normal amplitude but abnormal Ca(2+)-dependent decay (adaptation). The mature PCDH15/CDH23 tip link composition is re-established later, concomitant with complete recovery of adaptation. Thus, our findings provide a molecular mechanism for regeneration and maintenance of mechanosensory function in postmitotic auditory hair cells and could help identify elusive components of the mechanotransduction machinery.

In the ear, inner hair cells (IHCs) employ sophisticated glutamatergic ribbon synapses with afferent neurons to transmit auditory information to the brain. The presynaptic machinery responsible for neurotransmitter release in IHC synapses includes proteins such as the multi-C2-domain protein otoferlin and the vesicular glutamate transporter 3 (VGluT3). Yet, much of this likely unique molecular machinery remains to be deciphered. The scarcity of material has so far hampered biochemical studies which require large amounts of purified samples. We developed a subcellular fractionation workflow combined with immunoisolation of VGluT3-containing membrane vesicles, allowing for the enrichment of glutamatergic organelles that are likely dominated by synaptic vesicles (SVs) of IHCs. We have characterized their protein composition in mice before and after hearing onset using mass spectrometry and confocal imaging and provide a fully annotated proteome with hitherto unidentified proteins. Despite the prevalence of IHC marker proteins across IHC maturation, the profiles of trafficking proteins differed markedly before and after hearing onset. Among the proteins enriched after hearing onset were VAMP-7, syntaxin-7, syntaxin-8, syntaxin-12/13, SCAMP1, V-ATPase, SV2, and PKCα. Our study provides an inventory of the machinery associated with synaptic vesicle-mediated trafficking and presynaptic activity at IHC ribbon synapses and serves as a foundation for future functional studies.

In contrast to mammals, the avian cochlea, specifically the basilar papilla, can regenerate sensory hair cells, which involves fate conversion of supporting cells to hair cells. To determine the mechanisms for converting supporting cells to hair cells, we used single-cell RNA sequencing during hair cell regeneration in explant cultures of chick basilar papillae. We identified dynamic changes in the gene expression of supporting cells, and the pseudotime trajectory analysis demonstrated the stepwise fate conversion from supporting cells to hair cells. Initially, supporting cell identity was erased and transition to the precursor state occurred. A subsequent gain in hair cell identity progressed together with downregulation of precursor-state genes. Transforming growth factor β receptor 1-mediated signaling was involved in induction of the initial step, and its inhibition resulted in suppression of hair cell regeneration. Our data provide new insights for understanding fate conversion from supporting cells to hair cells in avian basilar papillae.

Auditory hair cells in the amphibian papilla (APHCs) of the leopard frog, Rana pipiens pipiens, have a significantly higher permeability to water than that observed in mammalian hair cells. The insensitivity of water permeability in frog hair cells to extracellular mercury suggests that an amphibian homologue of the water channel aquaporin-4 (AQP4) may mediate water transport in these cells. Using immunocytochemistry, we show that an AQP4-like protein is found in APHCs. Rabbit anti-AQP4 antibody was used in multiple-immunohistochemical staining experiments along with AP hair cell and hair bundle markers in leopard frog and mouse tissue. AQP4 immunoreactivity was found in the basal and apical poles of the APHCs and shows uniform immunoreactivity. This study provides the first identification and localization of an AQP4-like protein in the amphibian inner ear. We also report a more direct measure of hyperosmotically-induced volume changes in APHCs that confirms previous findings. The presence of water channels in anuran APHCs constitutes a novel physiological difference between amphibian and mammalian hair cell structure and function.

The mechanotransduction (MT) complex in auditory hair cells converts the mechanical stimulation of sound waves into neural signals. Recently, the MT complex has been suggested to contain at least four distinct integral membrane proteins: protocadherin 15 (PCDH15), transmembrane channel-like protein 1 (TMC1), lipoma HMGIC fusion partner-like 5 (LHFPL5), and transmembrane inner ear protein (TMIE). However, the composition, function, and regulation of the MT-complex proteins remain incompletely investigated. Here, we report previously undescribed splicing isoforms of TMC1, LHFPL5, and TMIE. We identified four alternative splicing events for the genes encoding these three proteins by analyzing RNA-seq libraries of auditory hair cells from adult mice [over postnatal day (P)28], and we then verified the alternative splicing events by using RT-PCR and Sanger sequencing. Moreover, we examined the tissue-specific distribution, developmental expression patterns, and tonotopic gradient of the splicing isoforms by performing semiquantitative and quantitative real-time PCR (qRT-PCR), and we found that the alternative splicing of TMC1 and LHFPL5 is cochlear-specific and occurs in both neonatal and adult mouse cochleae. Our findings not only reveal the potential complexity of the MT-complex composition, but also provide critical insights for guiding future research on the function, regulation, and trafficking of TMC1, LHFPL5, and TMIE and on the clinical diagnosis of hearing loss related to aberrant splicing of these three key genes in hearing.

Hearing loss is the most common and costly sensory defect in humans and genetic causes underlie a significant proportion of affected individuals. In mammals, sound is detected by hair cells (HCs) housed in the cochlea of the inner ear, whose function depends on a highly specialized mechanotransduction organelle, the hair bundle. Understanding the factors that regulate the development and functional maturation of the hair bundle is crucial for understanding the pathophysiology of human deafness. Genetic analysis of deafness genes in animal models, together with complementary forward genetic screens and conditional knock-out mutations in essential genes, have provided great insights into the molecular machinery underpinning hair-bundle development and function. In this review, we highlight recent advances in our understanding of hair-bundle morphogenesis, with an emphasis on the molecular pathways governing hair-bundle polarity and orientation. We next discuss the proteins and structural elements important for hair-cell mechanotransduction as well as hair-bundle cohesion and maintenance. In addition, developmental signals thought to regulate tonotopic features of HCs are introduced. Finally, novel approaches that complement classic genetics for studying the molecular etiology of human deafness are presented. WIREs Dev Biol 2016, 5:85-101. doi: 10.1002/wdev.202 For further resources related to this article, please visit the WIREs website.

Sensorineural hearing loss has been a global burden of diseases for decades. However, according to recent progress in experimental studies on hair cell regeneration and protection, clinical trials of pharmacotherapy for sensorineural hearing loss have rapidly progressed. In this review, we focus on recent clinical trials for hair cell protection and regeneration and outline mechanisms based on associated experimental studies. Outcomes of recent clinical trials provided valuable data regarding the safety and tolerability of intra-cochlear and intra-tympanic applications as drug delivery methods. Recent findings in molecular mechanisms of hair cell regeneration suggested the realization of regenerative medicine for sensorineural hearing loss in the near future.

We show that a cage-shaped F-actin network is essential for maintaining a tight spatial organization of Cav1.3 Ca(2+) channels at the synaptic ribbons of auditory inner hair cells. This F-actin network is also found to provide mechanosensitivity to the Cav1.3 channels when varying intracellular hydrostatic pressure. Furthermore, this F-actin mesh network attached to the synaptic ribbons directly influences the efficiency of otoferlin-dependent exocytosis and its sensitivity to intracellular hydrostatic pressure, independently of its action on the Cav1.3 channels. We propose a new mechanistic model for vesicle exocytosis in auditory hair cells where the rate of vesicle recruitment to the ribbons is directly controlled by a synaptic F-actin network and changes in intracellular hydrostatic pressure.

Sound coding at the auditory inner hair cell synapse requires graded changes in neurotransmitter release, triggered by sustained activation of presynaptic Ca(v)1.3 voltage-gated Ca(2+) channels. Central to their role in this regard, Ca(v)1.3 channels in inner hair cells show little Ca(2+)-dependent inactivation, a fast negative feedback regulation by incoming Ca(2+) ions, which depends on calmodulin association with the Ca(2+) channel alpha(1) subunit. Ca(2+)-dependent inactivation characterizes nearly all voltage-gated Ca(2+) channels including Ca(v)1.3 in other excitable cells. The mechanism underlying the limited autoregulation of Ca(v)1.3 in inner hair cells remains a mystery. Previously, we established calmodulin-like Ca(2+)-binding proteins in the brain and retina (CaBPs) as essential modulators of voltage-gated Ca(2+) channels. Here, we demonstrate that CaBPs differentially modify Ca(2+) feedback to Ca(v)1.3 channels in transfected cells and explore their significance for Ca(v)1.3 regulation in inner hair cells. Of multiple CaBPs detected in inner hair cells (CaBP1, CaBP2, CaBP4 and CaBP5), CaBP1 most efficiently blunts Ca(2+)-dependent inactivation of Ca(v)1.3. CaBP1 and CaBP4 both interact with calmodulin-binding sequences in Ca(v)1.3, but CaBP4 more weakly inhibits Ca(2+)-dependent inactivation than CaBP1. Ca(2+)-dependent inactivation is marginally greater in inner hair cells from CaBP4(-/-) than from wild-type mice, yet CaBP4(-/-) mice are not hearing-impaired. In contrast to CaBP4, CaBP1 is strongly localized at the presynaptic ribbon synapse of adult inner hair cells both in wild-type and CaBP4(-/-) mice and therefore is positioned to modulate native Ca(v)1.3 channels. Our results reveal unexpected diversity in the strengths of CaBPs as Ca(2+) channel modulators, and implicate CaBP1 rather than CaBP4 in conferring the anomalous slow inactivation of Ca(v)1.3 Ca(2+) currents required for auditory transmission.

mTOR broadly controls cell growth, but little is known about the role of mTOR complex 2 (mTORC2) in the inner ear. To investigate the role of mTORC2 in sensory hair cells (HCs), we generated HC-specific Rictor knockout (HC-RicKO) mice. HC-RicKO mice exhibited early-onset, progressive, and profound hearing loss. Increased DPOAE thresholds indicated outer HC dysfunction. HCs are lost, but this occurs after hearing loss. Ultrastructural analysis revealed stunted and absent stereocilia in outer HCs. In inner HCs, the number of synapses was significantly decreased and the remaining synapses displayed a disrupted actin cytoskeleton and disorganized Ca2+ channels. Thus, the mTORC2 signaling pathway plays an important role in regulating auditory HC structure and function via regulation of the actin cytoskeleton. These results provide molecular insights on a central regulator of cochlear HCs and thus hearing.

Hair cell calcium channels regulate membrane excitability and control synaptic transmission. The present investigations focused on determining whether calcium channels vary between hair cells of different characteristic frequencies or if multiple channel types exist within a hair cell, each serving a different function. To this end, turtle auditory hair cells from high- (317 +/- 27 Hz) and low-frequency (115 +/- 6 Hz) positions were voltage clamped using the whole-cell recording technique, and calcium currents were characterized based on activation, inactivation and pharmacological properties. Pharmacological sensitivity to dihydropyridines (nimodipine, Bay K 8644), benzothiazepines (diltiazem) and acetonitrile derivatives (verapamil, D600) and the insensitivity to non-L-type calcium channel antagonists support the conclusion that only L-type calcium channels were present. Fast activation rise times (< 0.5 ms), hyperpolarized half-activation potentials and a relative insensitivity to nimodipine suggest the channels were of the alpha1D (CaV1.3) variety. Although no pharmacological differences were found between calcium currents obtained from high- and low-frequency cells, low-frequency cells activated slightly faster and at hyperpolarized potentials, with half-activating voltages of -43 +/- 1 mV compared to -35 +/- 1 mV. Inactivation was observed in both high- and low-frequency cells. The time course of inactivation required three time constants for a fit. Long depolarizations could result in complete inactivation. The voltage of half-inactivation was -40 +/- 2 mV for high-frequency cells and -46 +/- 2 mV for low-frequency cells. Calcium channel inactivation did not significantly alter hair cell electrical resonant properties elicited from protocols where the membrane potential was hyperpolarized or depolarized prior to characterizing the resonance. A bell-shaped voltage dependence and modest sensitivities to intracellular calcium chelators and external barium ions suggest that inactivation was calcium dependent.

Sensorineural hearing loss (SNHL) poses a major threat to both physical and mental health; however, there is still a lack of effective drugs to treat the disease. Recently, novel biological therapies, such as mesenchymal stem cells (MSCs) and their products, namely, exosomes, are showing promising therapeutic potential due to their low immunogenicity, few ethical concerns, and easy accessibility. Nevertheless, the precise mechanisms underlying the therapeutic effects of MSC-derived exosomes remain unclear.