Simultaneous gentamicin-mediated damage and Atoh1 overexpression promotes hair cell regeneration in the neonatal mouse utricle.

Loss of hair cells from vestibular epithelium results in balance dysfunction. The current therapeutic regimen for vestibular diseases is limited. Upon injury or Atoh1 overexpression, hair cell replacement occurs rapidly in the mammalian utricle, suggesting a promising approach to induce vestibular hair cell regeneration. In this study, we applied simultaneous gentamicin-mediated hair cell ablation and Atoh1 overexpression to induce neonatal utricular hair cell formation in vitro. We confirmed that type I hair cells were the primary targets of gentamicin. Furthermore, injury and Atoh1 overexpression promoted hair cell regeneration in a timely and efficient manner through robust viral transfection. Hair cells regenerated with type II characteristics in the striola and type I/II characteristics in non-sensory regions. Rare EdU+/myosin7a+ cells in sensory regions and robust EdU+/myosin7a+ signals in ectopic regions indicate that transdifferentiation of supporting cells in situ, and mitosis and differentiation of non-sensory epithelial cells in ectopic regions, are sources of regenerative hair cells. Distinct regeneration patterns in in situ and ectopic regions suggested robust plasticity of vestibular non-sensory epithelium, generating more developed hair cell subtypes and thus providing a promising stem cell-like source of hair cells. These findings suggest that simultaneously causing injury and overexpressing Atoh1 promotes hair cell regeneration efficacy and maturity, thus expanding the understanding of ectopic plasticity in neonatal vestibular organs.

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.

EGF and a GSK3 Inhibitor Deplete Junctional E-cadherin and Stimulate Proliferation in the Mature Mammalian Ear.

Sensory hair cell losses underlie the vast majority of permanent hearing and balance deficits in humans, but many nonmammalian vertebrates can fully recover from hearing impairments and balance dysfunctions because supporting cells (SCs) in their ears retain lifelong regenerative capacities that depend on proliferation and differentiation as replacement hair cells. Most SCs in vertebrate ears stop dividing during embryogenesis; and soon after birth, vestibular SCs in mammals transition to lasting quiescence as they develop massively thickened circumferential F-actin bands at their E-cadherin-rich adherens junctions. Here, we report that treatment with EGF and a GSK3 inhibitor thinned the circumferential F-actin bands throughout the sensory epithelium of cultured utricles that were isolated from adult mice of either sex. That treatment also caused decreases in E-cadherin, ß-catenin, and YAP in the striola, and stimulated robust proliferation of mature, normally quiescent striolar SCs. The findings suggest that E-cadherin-rich junctions, which are not present in the SCs of the fish, amphibians, and birds which readily regenerate hair cells, are responsible in part for the mammalian ear's vulnerability to permanent balance and hearing deficits.SIGNIFICANCE STATEMENT Millions of people are affected by hearing and balance deficits that arise when loud sounds, ototoxic drugs, infections, and aging cause hair cell losses. Such deficits are permanent for humans and other mammals, but nonmammals can recover hearing and balance after supporting cells regenerate replacement hair cells. Mammalian supporting cells lose the capacity to proliferate around the time they develop unique, exceptionally reinforced, E-cadherin-rich intercellular junctions. Here, we report the discovery of a pharmacological treatment that thins F-actin bands, depletes E-cadherin, and stimulates proliferation in long-quiescent supporting cells within a balance epithelium from adult mice. The findings suggest that high E-cadherin in those supporting cell junctions may be responsible, in part, for the permanence of hair cell loss in mammals.

Toxic Effects of 3,3'-Iminodipropionitrile on Vestibular System in Adult C57BL/6J Mice In Vivo.

The utricle is one of the five sensory organs in the mammalian vestibular system, and while the utricle has a limited ability to repair itself, this is not sufficient for the recovery of vestibular function after hair cell (HC) loss induced by ototoxic drugs. In order to further explore the possible self-recovery mechanism of the adult mouse vestibular system, we established a reliable utricle epithelium injury model for studying the regeneration of HCs and examined the toxic effects of 3,3'-iminodiproprionitrile (IDPN) on the utricle in vivo in C57BL/6J mice, which is one of the most commonly used strains in inner ear research. This work focused on the epithelial cell loss, vestibular dysfunction, and spontaneous cell regeneration after IDPN administration. HC loss and supporting cell (SC) loss after IDPN treatment was dose-dependent and resulted in dysfunction of the vestibular system, as indicated by the swim test and the rotating vestibular ocular reflex (VOR) test. EdU-positive SCs were observed only in severely injured utricles wherein above 47% SCs were dead. No EdU-positive HCs were observed in either control or injured utricles. RT-qPCR showed transient upregulation of Hes5 and Hey1 and fluctuating upregulation of Axin2 and ß-catenin after IDPN administration. We conclude that a single intraperitoneal injection of IDPN is a practical way to establish an injured utricle model in adult C57BL/6J mice in vivo. We observed activation of Notch and Wnt signaling during the limited spontaneous HC regeneration after vestibular sensory epithelium damage, and such signaling might act as the promoting factors for tissue self-repair in the inner ear.

Characterization of the Transcriptome of Hair Cell Regeneration in the Neonatal Mouse Utricle.

BACKGROUND/AIMS: Hearing and balance deficits are mainly caused by loss of sensory inner ear hair cells. The key signals that control hair cell regeneration are of great interest. However, the molecular events by which the cellular signals mediate hair cell regeneration in the mouse utricle are largely unknown. METHODS: In the present study, we investigated gene expression changes and related molecular pathways using RNA-seq and qRT-PCR in the newborn mouse utricle in response to neomycin-induced damage. RESULTS: There were 302 and 624 genes that were found to be up-regulated and down-regulated in neomycin-treated samples. GO and KEGG pathway analyses of these genes revealed many deregulated cellular components, molecular functions, biological processes and signaling pathways that may be related to hair cell development. More importantly, the differentially expressed genes included 9 transcription factors from the zf-C2H2 family, and eight of them were consistently down-regulated during hair cell damage and subsequent regeneration. CONCLUSION: Our results provide a valuable source for future studies and highlighted some promising genes, pathways or processes that may be useful for therapeutic applications.

Cisplatin exposure damages resident stem cells of the mammalian inner ear.

BACKGROUND: Cisplatin is a widely used chemotherapeutic agent that can also cause ototoxic injury. One potential treatment for cisplatin-induced hearing loss involves the activation of endogenous inner ear stem cells, which may then produce replacement hair cells. In this series of experiments, we examined the effects of cisplatin exposure on both hair cells and resident stem cells of the mouse inner ear. RESULTS: Treatment for 24 hr with 10 µM cisplatin caused significant loss of hair cells in the mouse utricle, but such damage was not evident until 4 days after the cisplatin exposure. In addition to killing hair cells, cisplatin treatment also disrupted the actin cytoskeleton in remaining supporting cells, and led to increased histone H2AX phosphorylation within the sensory epithelia. Finally, treatment with 10 µM cisplatin appeared to have direct toxic effects on resident stem cells in the mouse utricle. Exposure to cisplatin blocked the proliferation of isolated stem cells and prevented sphere formation when those cells were maintained in suspension culture. CONCLUSION: The results suggest that inner ear stem cells may be injured during cisplatin ototoxicity, thus limiting their ability to mediate sensory repair.

Pharmaceutical countermeasures have opposite effects on the utricles and semicircular canals in man.

INTRODUCTION: Sensory conflicts in the vestibular system lead to motion sickness of which space motion sickness (SMS) is a special case. SMS affects up to 70% of the astronauts during the first 3 days in space. The search for effective countermeasures has led to several nonpharmacological and pharmacological approaches. The current study focuses on the effects of lorazepam (1 mg), meclizine (25 mg), promethazine (25 mg), and scopolamine (0.4 mg) on the vestibular system, with special focus on the canal and otolith functions separately. METHODS: The study had a placebo-controlled, single blind, repeated measures design. Sixteen healthy volunteers were subjected to a total of 7 test sessions, the first and last being without intake of medication. Semicircular canal function was evaluated by means of electronystagmography and otolith function with unilateral centrifugation. The horizontal semicircular canal function was characterized by the vestibulo-ocular reflex (VOR) gain measured during earth vertical axis rotation as well as the total caloric response. The function of the utricles was represented by the utricular sensitivity, reflecting the ocular counter roll relative to the virtual induced head tilt. RESULTS: Promethazine significantly decreased the semicircular canal and utricular parameters. Both scopolamine and lorazepam caused only a decrease in the utricular sensitivity, whereas meclizine only decreased the semicircular canal-induced VOR gain. DISCUSSION: The results show that the drugs affected different areas of the vestibular system and that the effects can thus be attributed to the specific pharmacological properties of each drug. Meclizine, as an antihistaminergic and weak anticholinergic drug, only affected the VOR gain, suggesting a central action on the medial vestibular nucleus. The same site of action is suggested for the anticholinergic scopolamine since acetylcholine receptors are present and utricular fibers terminate here. The global vestibular suppression caused by promethazine is probably a consequence of its anticholinergic, antihistaminergic, and antidopaminergic properties. Based on the fact that lorazepam increased the affinity of gamma-aminobutyric acid (GABA) for the GABA(A)-receptor and its effects on the utriculi, the site of action seems to be the lateral vestibular nucleus. CONCLUSION: Meclizine, scopolamine, and lorazepam selectively suppress specific parts of the vestibular system. Selective suppression of different parts of the vestibular system may be more beneficial for alleviating (space) motion sickness than general suppressive agents. Additionally, this knowledge may help the clinician in his therapeutic management of patients with either semicircular canal or otolith dysfunction.

Identification of genetic and chemical modulators of zebrafish mechanosensory hair cell death.

Inner ear sensory hair cell death is observed in the majority of hearing and balance disorders, affecting the health of more than 600 million people worldwide. While normal aging is the single greatest contributor, exposure to environmental toxins and therapeutic drugs such as aminoglycoside antibiotics and antineoplastic agents are significant contributors. Genetic variation contributes markedly to differences in normal disease progression during aging and in susceptibility to ototoxic agents. Using the lateral line system of larval zebrafish, we developed an in vivo drug toxicity interaction screen to uncover genetic modulators of antibiotic-induced hair cell death and to identify compounds that confer protection. We have identified 5 mutations that modulate aminoglycoside susceptibility. Further characterization and identification of one protective mutant, sentinel (snl), revealed a novel conserved vertebrate gene. A similar screen identified a new class of drug-like small molecules, benzothiophene carboxamides, that prevent aminoglycoside-induced hair cell death in zebrafish and in mammals. Testing for interaction with the sentinel mutation suggests that the gene and compounds may operate in different pathways. The combination of chemical screening with traditional genetic approaches is a new strategy for identifying drugs and drug targets to attenuate hearing and balance disorders.

Effects of L-Serine Against Cisplatin-Mediated Reactive Oxygen Species Generation in Zebrafish Vestibular Tissue Culture and HEI-OC1 Auditory Hybridoma Cells.

Cisplatin is a platinum-based chemotherapy compound effective against a variety of cancers. However, it can cause increased reactive oxygen species (ROS) production in auditory and vestibular tissue leading to permanent hearing and balance loss. The amino acid, L-serine, has been shown to reduce ROS in some tissue types. In this project, we first investigated whether L-serine could reduce cisplatin-mediated ROS generation in zebrafish utricular tissue culture using spectrophotometry and the fluorescent ROS detector dye, H2DCFDA. Then, we examined whether L-serine could prevent the effect of cisplatin against cellular viability in the mouse auditory hybridoma cell line, HEI-OC1, using the spectrophotometric (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT) assay. As a final step, we used H2DCFDA dye and flow cytometry analysis to determine if L-serine could counteract the effect of cisplatin on ROS production in this cell line. We found that cisplatin and L-serine treatment may influence ROS production in utricular tissue. Further, although L-serine did not counteract the effect of cisplatin against HEI-OC1 cellular viability, the amino acid did prevent the platinum compound's effect to increase ROS in these cells. These results suggest that L-serine may act in auditory and vestibular tissues as an effective protectant against cisplatin-mediated toxicity.

An in vitro model to assess the peripheral vestibulotoxicity of aromatic solvents.

Epidemiological and experimental studies indicate that a number of aromatic solvents widely used in the industry can affect hearing and balance following chronic exposure. Animal studies demonstrated that long-term exposure to aromatic solvents directly damages the auditory receptor within the inner ear: the cochlea. However, no information is available on their effect on the vestibular receptor, which shares many structural features with the cochlea and is also localized in inner ear. The aim of this study was to use an in vitro approach to assess and compare the vestibular toxicity of different aromatic solvents (toluene, ethylbenzene, styrene and ortho-, meta-, para-xylene), all of which have well known cochleotoxic properties. We used a three-dimensional culture model of rat utricles ("cysts") with preserved functional sensory and secretory epithelia, and containing a potassium-rich (K+) endolymph-like fluid for this study. Variations in K+ concentrations in this model were considered as biomarkers of toxicity of the substances tested. After 72 h exposure, o-xylene, ethylbenzene and styrene decreased the K+ concentration by 78 %, 37 % and 28 %, respectively. O- xylene and styrene both caused histopathological alterations in secretory and sensory epithelial areas after 72 h exposure, whereas no anomalies were observed in ethylbenzene-exposed samples. These in vitro results suggest that some widely used aromatic solvents might have vestibulotoxic properties (o-xylene, styrene and ethylbenzene), whereas others may not (p-xylene, m-xylene, toluene). Our results also indicate that variations in endolymphatic K+ concentration may be a more sensitive marker of vestibular toxicity than histopathological events. Finally, this study suggests that cochleotoxic solvents might not be necessarily vestibulotoxic, and vice versa.

Cisplatin-induced hair cell death requires STAT1 and is attenuated by epigallocatechin gallate.

Cisplatin is a chemotherapy drug that frequently causes auditory impairment due to the death of mechanosensory hair cells. Cisplatin ototoxicity may result from oxidative stress, DNA damage, and inflammatory cytokines. The transcription factor STAT1, an important mediator of cell death, can regulate all of these processes in other cell types. We used cultured utricles from mature Swiss Webster mice to investigate the role of STAT1 in cisplatin-induced hair cell death. We show that STAT1 phosphorylation is an early event in both hair cells and support cells after exposure of utricles to cisplatin. STAT1 phosphorylation peaked after 4 h of cisplatin exposure and returned to control levels by 8 h of exposure. The STAT1 inhibitor epigallocatechin gallate (EGCG) attenuated STAT1 phosphorylation in cisplatin-treated utricles and resulted in concentration-dependent increases in hair cell survival at 24 h postexposure. Furthermore, we show that utricular hair cells from STAT1-deficient mice are resistant to cisplatin toxicity. EGCG failed to provide additional protection from cisplatin in STAT1-deficient mice, further supporting the hypothesis that the protective effects of EGCG are due to its inhibition of STAT1. Treatment with IFN-gamma, which also causes STAT1 activation, also induced hair cell death in wild-type but not STAT1-deficient mice. These results show that STAT1 is required for maximal cisplatin-induced hair cell death in the mouse utricle and suggest that treatment with EGCG may be a useful strategy for prevention of cisplatin ototoxicity.

Spikes and membrane potential oscillations in hair cells generate periodic afferent activity in the frog sacculus.

To look for membrane potential oscillations that may contribute to sensory coding or amplification in the ear, we made whole-cell and perforated-patch recordings from hair cells and postsynaptic afferent neurites in the explanted frog sacculus, with mechanoelectrical transduction (MET) blocked. Small depolarizing holding currents, which may serve to replace the in vivo resting MET current, evoked all-or-none calcium spikes (39-75 mV amplitude) in 37% of hair cells tested, and continuous membrane potential oscillations (14-28 mV; 15-130 Hz) in an additional 14% of cells. Spiking hair cells were on average taller and thinner than nonspiking hair cells, and had smaller outward currents through delayed rectifier channels (I(KV)) and noninactivating calcium-activated potassium channels (I(BK,steady)), and larger inward rectifier currents (I(K1)). Some spiking hair cells fired only a brief train at the onset of a current step, but others could sustain repetitive firing (3-70 Hz). Partial blockade of I(BK) changed the amplitude and frequency of oscillations and spikes, and converted some nonspiking cells into spiking cells. Oscillatory hair cells preferentially amplified sinusoidal stimuli at frequencies near their natural oscillation frequency. Postsynaptic recordings revealed regularly timed bursts of EPSPs in some afferent neurites. EPSP bursts were able to trigger afferent spikes, which may be initiated at the sodium channel cluster located adjacent to the afferent axon's most peripheral myelin segment. These results show that some frog saccular hair cells can generate spontaneous rhythmic activity that may drive periodic background activity in afferent axons.

Release and elementary mechanisms of nitric oxide in hair cells.

The enzyme nitric oxide (NO) synthase, that produces the signaling molecule NO, has been identified in several cell types in the inner ear. However, it is unclear whether a measurable quantity of NO is released in the inner ear to confer specific functions. Indeed, the functional significance of NO and the elementary cellular mechanism thereof are most uncertain. Here, we demonstrate that the sensory epithelia of the frog saccule release NO and explore its release mechanisms by using self-referencing NO-selective electrodes. Additionally, we investigated the functional effects of NO on electrical properties of hair cells and determined their underlying cellular mechanism. We show detectable amounts of NO are released by hair cells (>50 nM). Furthermore, a hair-cell efferent modulator acetylcholine produces at least a threefold increase in NO release. NO not only attenuated the baseline membrane oscillations but it also increased the magnitude of current required to generate the characteristic membrane potential oscillations. This resulted in a rightward shift in the frequency-current relationship and altered the excitability of hair cells. Our data suggest that these effects ensue because NO reduces whole cell Ca(2+) current and drastically decreases the open probability of single-channel events of the L-type and non L-type Ca(2+) channels in hair cells, an effect that is mediated through direct nitrosylation of the channel and activation of protein kinase G. Finally, NO increases the magnitude of Ca(2+)-activated K(+) currents via direct NO nitrosylation. We conclude that NO-mediated inhibition serves as a component of efferent nerve modulation of hair cells.

Thyroid hormone mediates otolith growth and development during flatfish metamorphosis.

Flatfish begin life as bilaterally symmetrical larvae that swim up-right, then abruptly metamorphose into asymmetrically shaped juveniles with lateralized swimming postures. Flatfish metamorphosis is mediated entirely by thyroid hormone (TH). Changes in flatfish swim posture are thought to be regulated via vestibular remodeling, although the influence of TH on teleost inner ear development remains unclear. This study addresses the role of TH on the development of the three otolith end-organs (sacculus, utricle, and lagena) during southern flounder (Paralichthys lethostigma) metamorphosis. Compared with pre-metamorphosis, growth rates of the sacculus and utricle otoliths increase dramatically during metamorphosis in a manner that is uncoupled from general somatic growth. Treatment of P. lethostigma larvae with methimazol (a pharmacological inhibitor of endogenous TH production) inhibits growth of the sacculus and utricle, whereas treatment with TH dramatically accelerates their growth. In contrast with the sacculus and utricle otoliths that begin to form and mineralize during embryogenesis, a non-mineralized lagena otolith is first visible 10-12 days after hatching. The lagena grows during pre- and pro-metamorphosis, then abruptly mineralizes during metamorphic climax. Mineralization of the lagena, but not growth, can be induced with TH treatment, whereas treatment with methimazol completely inhibits lagena mineralization without inhibiting its growth. These findings suggest that during southern flounder metamorphosis TH exerts differential effects on growth and development among the three types of otolith.

Differential ablation of sensory receptors underlies ototoxin-induced shifts in auditory thresholds of the goldfish (Carassius auratus).

In recent years, fish models have become popular for investigations of ototoxic agents. However, the vast majority of such studies have focused on anatomical changes in lateral line hair cells after drug administration. Using the goldfish (Carassius auratus), we confirm that the acquisition of auditory evoked potentials offers a rapid and non-invasive method for quantifying ototoxin-induced changes in hearing sensitivity. Gentamicin (100 mg ml(-1)) was the drug of choice as it is a well-studied human ototoxin. Auditory threshold elevation was observed between 300 and 600 Hz and was accompanied by significant reductions in hair cell ciliary bundle densities in specific regions of the utricle and saccule. The correlations between structure and function suggest that differential susceptibility of sensory hair cells to acute gentamicin treatment underlies the frequency-specific elevation of auditory thresholds. We propose that fish auditory systems should be used alongside the lateral line, for the assessment of ototoxicity in new-developed drugs.

Styrene alters potassium endolymphatic concentration in a model of cultured utricle explants.

Despite well-documented neurotoxic and ototoxic properties, styrene remains commonly used in industry. Its effects on the cochlea have been extensively studied in animals, and epidemiological and animal evidence indicates an impact on balance. However, its influence on the peripheral vestibular receptor has yet to be investigated. Here, we assessed the vestibulotoxicity of styrene using an in vitro model, consisting of three-dimensional cultured newborn rat utricles filled with a high­potassium (K+) endolymph-like fluid, called "cysts". K+ entry in the cyst ("influx") and its exit ("efflux") are controlled by secretory cells and hair cells, respectively. The vestibular epithelium's functionality is thus linked to K+ concentration, measured using a microelectrode. Known inhibitors of K+ efflux and influx validated the model. Cysts were subsequently exposed to styrene (0.25; 0.5; 0.75 and 1 mM) for 2 h or 72 h. The decrease in K+ concentration measured after both exposure durations was dose-dependent, and significant from 0.75 mM styrene. Vacuoles were visible in the cytoplasm of epithelial cells from 0.5 mM after 2 h and from 0.25 mM after 72 h. The results presented here are the first evidence that styrene may deregulate K+ homeostasis in the endolymphatic space, thereby altering the functionality of the vestibular receptor.

Ultrastructural Characterization of Stem Cell-Derived Replacement Vestibular Hair Cells Within Ototoxin-Damaged Rat Utricle Explants.

The auditory apparatus of the inner ear does not show turnover of sensory hair cells (HCs) in adult mammals; in contrast, there are many observations supporting low-level turnover of vestibular HCs within the balance organs of mammalian inner ears. This low-level renewal of vestibular HCs exists during normal conditions and it is further enhanced after trauma-induced loss of these HCs. The main process for renewal of HCs within mammalian vestibular epithelia is a conversion/transdifferentiation of existing supporting cells (SCs) into replacement HCs.In earlier studies using long-term organ cultures of postnatal rat macula utriculi, HC loss induced by gentamicin resulted in an initial substantial decline in HC density followed by a significant increase in the proportion of HCs to SCs indicating the production of replacement HCs. In the present study, using the same model of ototoxic damage to study renewal of vestibular HCs, we focus on the ultrastructural characteristics of SCs undergoing transdifferentiation into new HCs. Our objective was to search for morphological signs of SC plasticity during this process. In the utricular epithelia, we observed immature HCs, which appear to be SCs transdifferentiating into HCs. These bridge SCs have unique morphological features characterized by formation of foot processes, basal accumulation of mitochondria, and an increased amount of connections with nearby SCs. No gap junctions were observed on these transitional cells. The tight junction seals were morphologically intact in both control and gentamicin-exposed explants. Anat Rec, 303:506-515, 2020. © 2019 The Authors. The Anatomical Record published by Wiley Periodicals, Inc. on behalf of American Association of Anatomists.

Knockdown of Foxg1 in Sox9+ supporting cells increases the trans-differentiation of supporting cells into hair cells in the neonatal mouse utricle.

Foxg1 plays important roles in regeneration of hair cell (HC) in the cochlea of neonatal mouse. Here, we used Sox9-CreER to knock down Foxg1 in supporting cells (SCs) in the utricle in order to investigate the role of Foxg1 in HC regeneration in the utricle. We found Sox9 an ideal marker of utricle SCs and bred Sox9CreER/+Foxg1loxp/loxp mice to conditionally knock down Foxg1 in utricular SCs. Conditional knockdown (cKD) of Foxg1 in SCs at postnatal day one (P01) led to increased number of HCs at P08. These regenerated HCs had normal characteristics, and could survive to at least P30. Lineage tracing showed that a significant portion of newly regenerated HCs originated from SCs in Foxg1 cKD mice compared to the mice subjected to the same treatment, which suggested SCs trans-differentiate into HCs in the Foxg1 cKD mouse utricle. After neomycin treatment in vitro, more HCs were observed in Foxg1 cKD mice utricle compared to the control group. Together, these results suggest that Foxg1 cKD in utricular SCs may promote HC regeneration by inducing trans-differentiation of SCs. This research therefore provides theoretical basis for the effects of Foxg1 in trans-differentiation of SCs and regeneration of HCs in the mouse utricle.

P2RX2 and P2RX4 receptors mediate cation absorption in transitional cells and supporting cells of the utricular macula.

Purinergic receptors protect the cochlea during high-intensity stimulation by providing a parallel shunt pathway through non-sensory neighboring epithelial cells for cation absorption. So far, there is no direct functional evidence for the presence and type/subunit of purinergic receptors in the utricle of the vestibular labyrinth. The goal of the present study was to investigate which purinergic receptors are expressed and carry cation-absorption currents in the utricular transitional cells and macula. Purinergic agonists induced cation-absorption currents with a potency order of ATP > bzATP = αßmeATP â‰« ADP = UTP = UDP. ATP and bzATP are full agonists, whereas αßmeATP is a partial agonist. ATP-induced currents were partially inhibited by 100 µM suramin, 10 µM pyridoxal-phosphate-6-azo-(benzene-2,4-disulfonic acid (PPADS), or 5 µM 5-(3-bromophenyl)-1,3-dihydro-2H-benzofuro[3,2-e]-1, 4-diazepin-2-one (5-BDBD), and almost completely blocked by 100 µM Gd3+ or by a combination of 10 µM PPADS and 5 µM 5-BDBD. Expression of the P2RX2 and P2RX4 receptor was detected by immunocytochemistry in transitional cells and macular supporting cells. This is the first study to demonstrate that ATP induces cation currents carried by a combination of P2RX2 and P2RX4 in utricular transitional and macular epithelial cells, and supporting the hypothesis that purinergic receptors protect utricular hair cells during elevated stimulus intensity levels.

Using the zebrafish lateral line to screen for ototoxicity.

The zebrafish is a valuable model for studying hair cell development, structure, genetics, and behavior. Zebrafish and other aquatic vertebrates have hair cells on their body surface organized into a sensory system called the lateral line. These hair cells are highly accessible and easily visualized using fluorescent dyes. Morphological and functional similarities to mammalian hair cells of the inner ear make the zebrafish a powerful preparation for studying hair cell toxicity. The ototoxic potential of drugs has historically been uncovered by anecdotal reports that have led to more formal investigation. Currently, no standard screen for ototoxicity exists in drug development. Thus, for the vast majority of Food and Drug Association (FDA)-approved drugs, the ototoxic potential remains unknown. In this study, we used 5-day-old zebrafish larvae to screen a library of 1,040 FDA-approved drugs and bioactives (NINDS Custom Collection II) for ototoxic effects in hair cells of the lateral line. Hair cell nuclei were selectively labeled using a fluorescent vital dye. For the initial screen, fish were exposed to drugs from the library at a 100-muM concentration for 1 h in 96-well tissue culture plates. Hair cell viability was assessed in vivo using fluorescence microscopy. One thousand forty drugs were rapidly screened for ototoxic effects. Seven known ototoxic drugs included in the library, including neomycin and cisplatin, were positively identified using these methods, as proof of concept. Fourteen compounds without previously known ototoxicity were discovered to be selectively toxic to hair cells. Dose-response curves for all 21 ototoxic compounds were determined by quantifying hair cell survival as a function of drug concentration. Dose-response relationships in the mammalian inner ear for two of the compounds without known ototoxicity, pentamidine isethionate and propantheline bromide, were then examined using in vitro preparations of the adult mouse utricle. Significant dose-dependent hair cell loss in the mouse utricle was demonstrated for both compounds. This study represents an important step in validating the use of the zebrafish lateral line as a screening tool for the identification of potentially ototoxic drugs.