Sensory Hair Cell Death Research Articles

Sensory hair cell death and regeneration in fishes.

Sensory hair cells are specialized mechanotransductive receptors required for hearing and vestibular function. Loss of hair cells in humans and other mammals is permanent and causes reduced hearing and balance. In the early 1980’s, it was shown that hair cells continue to be added to the inner ear sensory epithelia in cartilaginous and bony fishes. Soon thereafter, hair cell regeneration was documented in the chick cochlea following acoustic trauma. Since then, research using chick and other avian models has led to great insights into hair cell death and regeneration. However, with the rise of the zebrafish as a model organism for studying disease and developmental processes, there has been an increased interest in studying sensory hair cell death and regeneration in its lateral line and inner ears. Advances derived from studies in zebrafish and other fish species include understanding the effect of ototoxins on hair cells and finding otoprotectants to mitigate ototoxin damage, the role of cellular proliferation vs. direct transdifferentiation during hair cell regeneration, and elucidating cellular pathways involved in the regeneration process. This review will summarize research on hair cell death and regeneration using fish models, indicate the potential strengths and weaknesses of these models, and discuss several emerging areas of future studies.

Receptor-interacting protein kinases modulate noise-induced sensory hair cell death.

Receptor-interacting protein (RIP) kinases promote the induction of necrotic cell death pathways. Here we investigated signaling pathways in outer hair cells (OHCs) of adult male CBA/J mice exposed to noise that causes permanent threshold shifts, with a particular focus on RIP kinase-regulated necroptosis. One hour after noise exposure, nuclei of OHCs in the basal region of the cochlea displayed both apoptotic and necrotic features. RIP1 and RIP3 protein levels increased and caspase-8 was activated. Treatment with pan-caspase inhibitor ZVAD blocked the activation of caspase-8 and reduced the number of apoptotic nuclei, while increasing levels of RIP1, RIP3, and necrotic OHCs. Conversely, treatment with necrosis inhibitor necrostatin-1 (Nec-1) or RIP3 siRNA (siRIP3) diminished noise-induced increases in RIP1 and RIP3, and decreased necrotic OHC nuclei. This treatment also increased the number of apoptotic nuclei without increasing activation of caspase-8. Consistent with the elevation of levels of RIP1 and RIP3, noise-induced active AMPKα levels increased with ZVAD treatment, but decreased with Nec-1 and siRIP3 treatment. Furthermore, treatment with siRIP3 did not alter the activation of caspase-8, but instead increased activation of caspase-9 and promoted endonuclease G translocation into OHC nuclei. Finally, auditory brainstem response functional measurements and morphological assessment of OHCs showed that ZVAD treatment reduces noise-induced deficits. This protective function is potentiated when combined with siRIP3 treatment. In conclusion, noise-induced OHC apoptosis and necrosis are modulated by caspases and RIP kinases, respectively. Inhibition of either pathway shifts the prevalence of OHC death to the alternative pathway.

Gene-expression analysis of hair cell regeneration in the zebrafish lateral line

Deafness caused by the terminal loss of inner ear hair cells is one of the most common sensory diseases. However, nonmammalian animals (e.g., birds, amphibians, and fish) regenerate damaged hair cells. To understand better the reasons underpinning such disparities in regeneration among vertebrates, we set out to define at high resolution the changes in gene expression associated with the regeneration of hair cells in the zebrafish lateral line. We performed RNA-Seq analyses on regenerating support cells purified by FACS. The resulting expression data were subjected to pathway enrichment analyses, and the differentially expressed genes were validated in vivo via whole-mount in situ hybridizations. We discovered that cell cycle regulators are expressed hours before the activation of Wnt/β-catenin signaling following hair cell death. We propose that Wnt/β-catenin signaling is not involved in regulating the onset of proliferation but governs proliferation at later stages of regeneration. In addition, and in marked contrast to mammals, our data clearly indicate that the Notch pathway is significantly down-regulated shortly after injury, thus uncovering a key difference between the zebrafish and mammalian responses to hair cell injury. Taken together, our findings lay the foundation for identifying differences in signaling pathway regulation that could be exploited as potential therapeutic targets to promote either sensory epithelium or hair cell regeneration in mammals.

Aminoglycoside-Induced Hair Cell Death of Inner Ear Organs Causes Functional Deficits in Adult Zebrafish (Danio rerio)

Aminoglycoside antibiotics, like gentamicin, kill inner ear sensory hair cells in a variety of species including chickens, mice, and humans. The zebrafish (Danio rerio) has been used to study hair cell cytotoxicity in the lateral line organs of larval and adult animals. Little is known about whether aminoglycosides kill the hair cells within the inner ear of adult zebrafish. We report here the ototoxic effects of gentamicin on hair cells in the saccule, the putative hearing organ, and utricle of zebrafish. First, adult zebrafish received a single 30 mg/kg intraperitoneal injection of fluorescently-tagged gentamicin (GTTR) to determine the distribution of gentamicin within inner ear sensory epithelia. After 4 hours, GTTR was observed in hair cells throughout the saccular and utriclar sensory epithelia. To assess the ototoxic effects of gentamicin, adult zebrafish received a single 250 mg/kg intraperitoneal injection of gentamicin and, 24 hours later, auditory evoked potential recordings (AEPs) revealed significant shifts in auditory thresholds compared to untreated controls. Zebrafish were then euthanized, the inner ear fixed, and labeled for apoptotic cells (TUNEL reaction), and the stereociliary bundles of hair cells labeled with fluorescently-tagged phalloidin. Whole mounts of the saccule and utricle were imaged and cells counted. There were significantly more TUNEL-labeled cells found in both organs 4 hours after gentamicin injection compared to vehicle-injected controls. As expected, significantly fewer hair cell bundles were found along the rostral-caudal axis of the saccule and in the extrastriolar and striolar regions of the utricle in gentamicin-treated animals compared to untreated controls. Therefore, as in other species, gentamicin causes significant inner ear sensory hair cell death and auditory dysfunction in zebrafish.

The miR-183/Taok1 Target Pair Is Implicated in Cochlear Responses to Acoustic Trauma

Acoustic trauma, one of the leading causes of sensorineural hearing loss, induces sensory hair cell damage in the cochlea. Identifying the molecular mechanisms involved in regulating sensory hair cell death is critical towards developing effective treatments for preventing hair cell damage. Recently, microRNAs (miRNAs) have been shown to participate in the regulatory mechanisms of inner ear development and homeostasis. However, their involvement in cochlear sensory cell degeneration following acoustic trauma is unknown. Here, we profiled the expression pattern of miRNAs in the cochlear sensory epithelium, defined miRNA responses to acoustic overstimulation, and explored potential mRNA targets of miRNAs that may be responsible for the stress responses of the cochlea. Expression analysis of miRNAs in the cochlear sensory epithelium revealed constitutive expression of 176 miRNAs, many of which have not been previously reported in cochlear tissue. Exposure to intense noise caused significant threshold shift and apoptotic activity in the cochleae. Gene expression analysis of noise-traumatized cochleae revealed time-dependent transcriptional changes in the expression of miRNAs. Target prediction analysis revealed potential target genes of the significantly downregulated miRNAs, many of which had cell death- and apoptosis-related functions. Verification of the predicted targets revealed a significant upregulation of Taok1, a target of miRNA-183. Moreover, inhibition of miR-183 with morpholino antisense oligos in cochlear organotypic cultures revealed a negative correlation between the expression levels of miR-183 and Taok1, suggesting the presence of a miR-183/Taok1 target pair. Together, miRNA profiling as well as the target analysis and validation suggest the involvement of miRNAs in the regulation of the degenerative process of the cochlea following acoustic overstimulation. The miR-183/Taok1 target pair is likely to play a role in this regulatory process.

Dimethyl Sulfoxide (DMSO) Exacerbates Cisplatin-induced Sensory Hair Cell Death in Zebrafish (Danio rerio)

Inner ear sensory hair cells die following exposure to aminoglycoside antibiotics or chemotherapeutics like cisplatin, leading to permanent auditory and/or balance deficits in humans. Zebrafish (Danio rerio) are used to study drug-induced sensory hair cell death since their hair cells are similar in structure and function to those found in humans. We developed a cisplatin dose-response curve using a transgenic line of zebrafish that expresses membrane-targeted green fluorescent protein under the control of the Brn3c promoter/enhancer. Recently, several small molecule screens have been conducted using zebrafish to identify potential pharmacological agents that could be used to protect sensory hair cells in the presence of ototoxic drugs. Dimethyl sulfoxide (DMSO) is typically used as a solvent for many pharmacological agents in sensory hair cell cytotoxicity assays. Serendipitously, we found that DMSO potentiated the effects of cisplatin and killed more sensory hair cells than treatment with cisplatin alone. Yet, DMSO alone did not kill hair cells. We did not observe the synergistic effects of DMSO with the ototoxic aminoglycoside antibiotic neomycin. Cisplatin treatment with other commonly used organic solvents (i.e. ethanol, methanol, and polyethylene glycol 400) also did not result in increased cell death compared to cisplatin treatment alone. Thus, caution should be exercised when interpreting data generated from small molecule screens since many compounds are dissolved in DMSO.

A new oral otoprotective agent. Part 1: Electrophysiology data from protection against noise-induced hearing loss

SummaryBackgroundData from animal studies show that antioxidants can compensate against noise-induced stress and sensory hair cell death. The aim of this study was to evaluate the otoprotection efficacy of various versions of orally administered Acuval 400® against noise damage in a rat animal model.Material/MethodsFifty-five Sprague Dawley rats were divided into 4 groups: A) noise-exposed animals; B) animals exposed to noise and treated with the Acuval; C) animals exposed to noise and treated with a combination of Coenzyme Q10 and Acuval; D) animals treated only with Acuval and Coenzyme Q10 and with no exposure to noise. All solutions were administered orally 5 times: 24 and 2 hrs prior to noise exposure, and then daily for 3 days. The auditory function was assessed by measuring auditory brainstem responses (ABR) in the range from 2 to 32 kHz at times =1, 7, 14 and 21 days after noise exposure.ResultsAt low frequencies (click and 4 kHz) animals from both A and B groups showed significant threshold shifts in the majority of the tested frequencies and tested times. For the same frequencies, animals from group C presented threshold levels similar to those from group D. At frequencies ≥8 kHz the protective performance of the 2 Acuval groups is more clearly distinguished from the noise group A. At 32 kHz the 2 Acuval groups perform equally well in terms of otoprotection. Animals in Group D did not show any significant differences in the hearing threshold during the experiment.ConclusionsThe data of this study suggest that a solution containing Coenzyme Q10 and Acuval 400®, administered orally, protects from noise-induced hearing loss.

Functional Hair Cell Mechanotransducer Channels Are Required for Aminoglycoside Ototoxicity

Aminoglycosides (AG) are commonly prescribed antibiotics with potent bactericidal activities. One main side effect is permanent sensorineural hearing loss, induced by selective inner ear sensory hair cell death. Much work has focused on AG's initiating cell death processes, however, fewer studies exist defining mechanisms of AG uptake by hair cells. The current study investigated two proposed mechanisms of AG transport in mammalian hair cells: mechanotransducer (MET) channels and endocytosis. To study these two mechanisms, rat cochlear explants were cultured as whole organs in gentamicin-containing media. Two-photon imaging of Texas Red conjugated gentamicin (GTTR) uptake into live hair cells was rapid and selective. Hypocalcemia, which increases the open probability of MET channels, increased AG entry into hair cells. Three blockers of MET channels (curare, quinine, and amiloride) significantly reduced GTTR uptake, whereas the endocytosis inhibitor concanavalin A did not. Dynosore quenched the fluorescence of GTTR and could not be tested. Pharmacologic blockade of MET channels with curare or quinine, but not concanavalin A or dynosore, prevented hair cell loss when challenged with gentamicin for up to 96 hours. Taken together, data indicate that the patency of MET channels mediated AG entry into hair cells and its toxicity. Results suggest that limiting permeation of AGs through MET channel or preventing their entry into endolymph are potential therapeutic targets for preventing hair cell death and hearing loss.

Mechanically‐induced hair cell death using the zebrafish lateral line as a model system.

Noise‐induced hearing loss is a major type of hearing impairment that affects the quality of life for millions of Americans. The cause of noise‐induced hearing loss is the death of sensory hair cells in the ear. The primary goal of the present study is to have a better understanding of mechanisms underlying noise‐induced hearing and balance deficits. Mechanically‐induced hair cell degeneration was investigated using the zebrafish (Danio rerio), which has become a model system for biomedical research because of a combination of powerful genetics, excellent embryology, and exceptional in vivo visualization in one organism. Zebrafish have the lateral line system containing superficial neuromasts in the head and trunk, and each neuromast has a cluster of hair cells that structurally and functionally resemble the hair cells in the ear. In this study, hair cells in single neuromasts of zebrafish larvae of 5 to 10 days post fertilization were stimulated by a piezoelectric actuator and the time course of degeneration of hair cells exposed to overstimulation was obtained using light microscopy. Microphonic potentials of neuromast hair cells were also recorded to show effects of intense mechanical stimulation on the function of hair cells. [Work supported by NIH/NIDCD R21DC009879 and Gabelli Fellowship.]

Life and Death of Sensory Hair Cells Expressing Constitutively Active TRPML3

The varitint-waddler mutation A419P renders TRPML3 constitutively active, resulting in cationic overload, particularly in sustained influx of Ca(2+). TRPML3 is expressed by inner ear sensory hair cells, and we were intrigued by the fact that hair cells are able to cope with expressing the TRPML3(A419P) isoform for weeks before they ultimately die. We hypothesized that the survival of varitint-waddler hair cells is linked to their ability to deal with Ca(2+) loads due to the abundance of plasma membrane calcium ATPases (PMCAs). Here, we show that PMCA2 significantly reduced [Ca(2+)](i) increase and apoptosis in HEK293 cells expressing TRPML3(A419P). The deaf-waddler isoform of PMCA2, operating at 30% efficacy, showed a significantly decreased ability to rescue the Ca(2+) loading of cells expressing TRPML3(A419P). When we combined mice heterozygous for the varitint-waddler mutant allele with mice heterozygous for the deaf-waddler mutant allele, we found severe hair bundle defects as well as increased hair cell loss compared with mice heterozygous for each mutant allele alone. Furthermore, 3-week-old double mutant mice lacked auditory brainstem responses, which were present in their respective littermates containing single mutant alleles. Likewise, heterozygous double mutant mice exhibited severe circling behavior, which was not observed in mice heterozygous for TRPML3(A419P) or PMCA2(G283S) alone. Our results provide a molecular rationale for the delayed hair cell loss in varitint-waddler mice. They also show that hair cells are able to survive for weeks with sustained Ca(2+) loading, which implies that Ca(2+) loading is an unlikely primary cause of hair cell death in ototoxic stress situations.

Extracellular divalent cations modulate aminoglycoside-induced hair cell death in the zebrafish lateral line

Aminoglycoside antibiotics cause death of sensory hair cells. Research over the past decade has identified several key players in the intracellular cascade. However, the role of the extracellular environment in aminoglycoside ototoxicity has received comparatively little attention. The present study uses the zebrafish lateral line to demonstrate that extracellular calcium and magnesium ions modulate hair cell death from neomycin and gentamicin in vivo, with high levels of either divalent cation providing significant protection. Imaging experiments with fluorescently-tagged gentamicin show that drug uptake is reduced under high calcium conditions. Treating fish with the hair cell transduction blocker amiloride also reduces aminoglycoside uptake, preventing the toxicity, and experiments with variable calcium and amiloride concentrations suggest complementary effects between the two protectants. Elevated magnesium, in contrast, does not appear to significantly attenuate drug uptake, suggesting that the two divalent cations may protect hair cells from aminoglycoside damage through different mechanisms. These results provide additional evidence for calcium- and transduction-dependent aminoglycoside uptake. Divalent cations provided differential protection from neomycin and gentamicin, with high cation concentrations almost completely protecting hair cells from neomycin and acute gentamicin toxicity, but offering reduced protection from continuous (6 h) gentamicin exposure. These experiments lend further support to the hypothesis that aminoglycoside toxicity occurs via multiple pathways in a both a drug and time course-specific manner.

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.

JNK signaling in neomycin-induced vestibular hair cell death

Mechanosensory hair cells are susceptible to apoptotic death in response to exposure to ototoxic drugs, including aminoglycoside antibiotics. The c-Jun n-terminal kinase (JNK) is a stress-activated protein kinase that can promote apoptotic cell death in a variety of systems. Inhibition of the JNK signaling pathway can prevent aminoglycoside-induced death of cochlear and vestibular sensory hair cells. We used an in vitro preparation of utricles from adult mice to examine the role of JNK activation in aminoglycoside-induced hair cell death. CEP-11004 was used as an indirect inhibitor of JNK signaling. Immunohistochemistry showed that both JNK and its downstream target c-Jun are phosphorylated in hair cells of utricles exposed to neomycin. CEP-11004 inhibited neomycin-induced phosphorylation of both JNK and c-Jun. CEP-11004 inhibited hair cell death in utricles exposed to moderate doses of neomycin. However, the results were not uniform across the dose–response function; CEP-11004 did not inhibit hair cell death in utricles exposed to high-dose neomycin. The CEP-11004-induced protective effect was not due to inhibition of PKC or p38, since neither Chelerythrine nor SB203580 could mimic the protective effect of CEP-11004. In addition, inhibition of JNK inhibited the activation of caspase-9 in hair cells. These results indicate that JNK plays an important role in neomycin-induced vestibular hair cell death and caspase-9 activation.

Heat shock inhibits both aminoglycoside- and cisplatin-induced sensory hair cell death.

Human hearing and balance impairments are often attributable to the death of sensory hair cells in the inner ear. These cells are hypersensitive to death induced by noise exposure, aging, and some therapeutic drugs. Two major classes of ototoxic drugs are the aminoglycoside antibiotics and the antineoplastic agent cisplatin. Exposure to these drugs leads to hair cell death that is mediated by the activation of specific apoptotic proteins, including caspases. The induction of heat shock proteins (HSPs) in response to cellular stress is a ubiquitous and highly conserved response that can significantly inhibit apoptosis in some systems by inhibiting apoptotic proteins. Induction of HSPs occurs in hair cells in response to a variety of stimuli. Given that HSPs can directly inhibit apoptosis, we hypothesized that heat shock may inhibit apoptosis in hair cells exposed to ototoxic drugs. To test this hypothesis, we developed a method for inducing HSP expression in the adult mouse utricle in vitro. In vitro heat shock reliably produces a robust up-regulation of HSP-70 mRNA and protein, as well as more modest up-regulation of HSP-90 and HSP-27. The heat shock does not result in death of hair cells. Heat shock has a significant protective effect against both aminoglycoside- and cisplatin-induced hair cell death in the utricle preparation in vitro. These data indicate that heat shock can inhibit ototoxic drug-induced hair cell death, and that the utricle preparation can be used to examine the molecular mechanism(s) underlying this protective effect.

The adult mouse utricle as an in vitro preparation for studies of ototoxic-drug-induced sensory hair cell death

Sensory hair cells of the inner ear are susceptible to death from a variety of stresses including aging, noise trauma, genetic disorders, and exposure to certain therapeutic drugs. Ototoxic drugs include the aminoglycoside antibiotics and the antineoplastic agent cisplatin. This is a short technical report describing the dissection and culture of the adult mouse utricle. This in vitro preparation allows for detailed studies of ototoxic-drug-induced hair cell death in an adult mammalian system. In addition, this preparation allows for examination of the effects of specific gene products through the use of transgenic and knockout mouse models.

末梢前庭器から前庭神経核へ―最近の知見―３．有毛細胞死におけるシグナル伝達と保護機構

It is well-known that the inner ear hair cells are easily damaged by ototoxic stresses (e.g. aminoglycosides, ischemia). Hair cell death often results in permanent hearing loss and vestibular dysfunction. To treat the inner ear diseases with loss of hair cells, we should understand the signaling pathway of sensory hair cell death and protection. In this article, I would like to discuss some recent findings about inner ear hair cell death and protection. There are many reports that hair cell death is induced in the form of apoptosis. The key molecules are caspases and c -Jun N2 terminal kinase (JNK) in the signaling pathway of hair cell death. The inhibition of these molecules can protect the hair cells against ototoxic chemicals.The heat shock response is a fundamental mechanism of cells to cope with adverse environments, and is regulated mainly at the level of transcription by heat shock transcription factor (HSF1) in mammals. The activations of caspases and JNK are inhibited by heat shock proteins, and result in the survival of cells. Therefore, we performed experiments to analyze the role of the heat shock response in the inner ear. Our results showed that the heat shock response protected the inner era against intense noise, and that the heat shock response was regulated by HSF1. The heat shock response is the one of the potential candidates for the protection of the inner ear. But more in vivo experiments will be required for the clinical treatment of inner ear diseases.

New pharmacological strategies to restore hearing and treat tinnitus

Recent advances in molecular pharmacology of the cochlea have lead to a much better understanding of the physiology, and especially the pathophysiology, of the sensorineural structures of the organ of Corti. Knowledge of the intimate molecular mechanisms of cellular dysfunction is of considerable use in the development of new therapeutic strategies. Herein, we summarize the mechanisms of sensory hair cell death after various injuries. Based on these molecular mechanisms, we propose novel therapeutic strategies to restore hearing. In addition to permanent hearing loss, exposure to noise or ototoxic drugs also induces tinnitus. We thus review recent findings obtained from a behavioral model of tinnitus in rats. In addition to providing evidence for the site and mechanism of generation of tinnitus induced by salicylate, these results support the idea that targeting cochlear N-methyl-D-aspartate receptors may represent a promising therapeutic strategy for treating tinnitus.

Hair cell death in the avian basilar papilla: characterization of the in vitro model and caspase activation.

Caspases are a family of proteases that have been implicated as key mediators of cell death. Although nonspecific inhibition of caspase activation has been reported to prevent mammalian sensory hair cell death, the exact roles of individual caspases during hair cell death are unclear. In other systems, the activation of initiator caspases, such as caspase-8 and caspase-9, can lead to the activation of the effector caspase-3. We have begun to systematically characterize hair cell death in an in vitro system by examining the activation of these specific caspases in degenerating hair cells after acutely damaging the whole avian basilar papilla with gentamicin. Basilar papillae (BP) displayed a dose-dependent hair cell loss after a 24-h treatment with gentamicin at concentrations of 0.1, 0.5, and 2.0 mM. When treated with 0.5 mM gentamicin for 6, 12, or 24 h, hair cells first began to degenerate in the basal third of the BP and damage progressed apically. Supplementation of z-VAD-fmk, a general caspase inhibitor, provided short-term protection against gentamicin-induced hair cell death. Treatment with gentamicin for 6 or 12 h promoted the expression of active caspase-3 and active caspase-9 in many hair cells along the BP as shown by immunohistochemistry. At these time-points, specific fluorescent-labeled peptide substrates detected more active caspase-3, caspase-8, and caspase-9 in gentamicin-treated hair cells relative to controls. Our data indicate that auditory hair cells degenerate as a result of gentamicin exposure in a caspase-dependent manner. Specifically, the upstream caspases, caspase-8 and caspase-9, and the downstream caspase-3 are activated in aminoglycoside-damaged hair cells.