Fish in a dish: drug discovery for hearing habilitation

doi:10.1016/j.ddmod.2012.02.001

Drug Discovery Today: Disease Models

Volume 10, Issue 1, Spring 2013, Pages e23-e29

https://doi.org/10.1016/j.ddmod.2012.02.001 Get rights and content

The majority of hearing loss is caused by the permanent loss of inner ear hair cells. The identification of drugs that modulate the susceptibility to hair cell loss or spur their regeneration is often hampered by the difficulties of assaying for such complex phenomena in mammalian models. The zebrafish has emerged as a powerful animal model for chemical screening in many contexts. Several characteristics of the zebrafish, such as its small size and external location of sensory hair cells, uniquely position it as an ideal model organism for the study of hair cell toxicity, protection, and regeneration. We have used this model to screen for drugs that affect each of these aspects of hair cell biology and have identified compounds that affect each of these processes. The identification of such drugs and drug-like compounds holds promise in the future ability to stem hearing loss in the human population.

Section snippets

Chemical screens in the zebrafish lateral line

The high fecundity, small size and optical transparency of larval zebrafish facilitate medium- or high-throughput screening. Larvae are distributed in 96-well plates, administered a wide range of compounds dissolved in water, and assayed for the desired phenotype. Screens of this type have been conducted for a wide range of purposes, from basic biology to drug safety testing [21, 25, 26, 27, 28, 29, 30, 31, 32, 33]. Since 2000, our group has used the lateral line in larval zebrafish for drug

Identification of compounds with ototoxic potential

The attribution of ototoxic properties to a therapeutic compound typically occurs only after the frequency of anecdotal reports of hearing or vestibular impairment warrants further study in animal models. This is unfortunate from both financial and human suffering points of view. While ototoxicity is most widely recognized for aminoglycosides and platinum-based anticancer drugs, as well as some nonsteroidal anti-inflammatory agents and loop diurectics, the inherent nature of some effective

Identification of compounds that protect against hair cell loss

To screen for compounds that protect hair cells against ototoxic drugs, only minor modifications are necessary to our toxicity screens. Our initial screens for hair cell protectants were designed to identify compounds that conferred robust protection to neomycin-treated hair cells [26]. We initially focused on small molecules within the Chembridge Diverset E library. To efficiently screen a sample of 10,960 compounds from the library, compounds were multiplexed five per well and then reassessed

Repurposing existing FDA-approved drugs

There are numerous economic challenges to de novo drug development. FDA approval of new drugs requires approximately 10–12 years and often hundreds of millions of dollars to identify effective compounds with optimal pharmacokinetic profiles [42, 43]. The odds of a newly minted drug being brought to market are approximately 1:5000 [44], making drug synthesis a far less efficient endeavor than identification of new uses for therapeutics that have already been shown to be safe and effective for

Blocking ototoxin entry into hair cells: the ultimate protectant?

Perhaps the most appealing way to block hair cell loss associated with ototoxin exposure is by preventing entry into the hair cell before it can induce cellular damage. In the case of aminoglycoside antibiotics, several genetic and chemical studies indicate that the major route of entry occurs through mechanotransduction channels [34, 45, 46, 47, 48, 49, 50, 51, 52, 53], which are channels of unknown molecular identify that allow hair cells to respond to vibrational (sound) stimuli. In our

Screening for compounds that alter hair cell regeneration

While hair cell loss, and the resulting hearing loss, is permanent in humans and other mammals, birds, zebrafish, and other non-mammalian vertebrates possess the remarkable ability to regenerate hair cells and restore sensory function [23, 24]. Hair cells in the larval zebrafish lateral line completely regenerate by 72 hours after toxic neomycin damage [35], making this an ideal system in which to quickly evaluate compounds that alter regenerative potential. This regeneration process is

Intersection between chemical and genetic screens

Zebrafish are particularly amenable for traditional forward genetic screens, with the first large-scale screens conducted to identify genes important for early vertebrate development [56, 57, 58] and more recent screens aimed at uncovering genes involved in the development of specific tissues or modification of behavioral phenotypes [59, 60, 61, 62]. Our group developed a mutagenesis screen to identify genes that modify hair cell responses to neomycin exposure as a complement to the chemical

Conclusion

The zebrafish lateral line provides a powerful tool to discover drugs, potential drugs, and genes that affect hearing in the human population. Beyond their translational aspects, molecules that induce or promote hair cell survival possess the power to provide information about the pathways involved in these processes. A better understanding of the pathways involved in ototoxin-induced hair cell death and regeneration will maximize efficiency at which we can predictively design drugs based on

Acknowledgements

Work in the laboratories of H.O., E.W.R., J.A.S., and D.W.R. are supported by NIDCD grants DC05987, DC011269, DC009807, DC009631, as well as others from the Action on Hearing Loss Foundation, the Deafness Research Fund, and V.M. Bloedel Hearing Research Center. A.B.C. is supported by a NIDCD grant DC11344. R.E. is supported by a Ruth Kirschstein NRSA fellowship DC012244.

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Evaluation of the hair cell regeneration and claudin b and phoenix gene expression during exposure to low concentrations of cadmium and zinc in early developing zebrafish larvae
2021, Comparative Biochemistry and Physiology Part - C: Toxicology and Pharmacology
Citation Excerpt :
While in mammals the loss of cochlear hair cells leads to irreversible hearing disorders, as no new hair cells are produced after birth (Cheng et al., 2005; Schacht et al., 2012; Yamasoba et al., 2013; Kurabi et al., 2017; Czajkowski et al., 2019), other non-mammals vertebrates, including fish, possess the remarkable ability to continuously regenerate sensory hair cells even during post embryonic life (Corwin and Oberholtzer, 1997; Groves, 2010). Zebrafish (Danio rerio) hair cells share cellular, physiological and molecular properties with those of mammalian inner ear, providing an excellent tool for studying the molecular and cellular basis of hearing disorders (Duncan and Fritzsch, 2012; Whitfield, 2002; Brignull et al., 2009; Esterberg et al., 2013; Thomas et al., 2015). In zebrafish, hair cells are found in the lateral line (LL) system located in the apical part of the mechanosensory organs known as neuromasts.
Zebrafish possess hair cells on the body surface similar to that of mammals inner hear, in particular in the neuromasts, and due to its ability in regenerating damaged hair cells, is regularly used as a powerful animal model to study in vivo cytotoxicity.
Among the factors leading to hair cell disruption, metal ions are of particular concern since they are important environmental pollutants. To date, several studies on zebrafish hair cell regeneration after metal exposure exist, while no data on regeneration during continuous metal exposure are available. In the present study, neuromast hair cell disruption and regeneration were assessed in zebrafish larvae for the first time during zinc (Zn) and cadmium (Cd) continuous exposure and a visual and molecular approach was adopted.
Fluorescent vital dye DASPEI was used to assess hair cell regeneration and the gene expression of claudin b (cldnb) and phoenix (pho), was analyzed. Metallotionein-2 (mt2) gene expression was used as standard molecular marker of metal toxicity and confirmed the higher toxicity of Cd compared to Zn. In addition, Cd caused a delay in hair cell regeneration compared to Zn. Molecular analysis showed cldnb gene expression increased in relation to the metal concentrations used, confirming the involvement of this gene in hair cell regeneration. On the contrary, a dramatic decrease of pho gene expression was observed in Cd exposed groups, suggesting a negative impact of Cd on pho expression, thus negatively interfering with hair cell regeneration in zebrafish larvae exposed to this metal.
Glucococorticoid receptor activation exacerbates aminoglycoside-induced damage to the zebrafish lateral line
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Citation Excerpt :
In all experiments described below, zebrafish larvae were treated with cortisol or a specific GC signaling modulator for up to 24 h prior to treatment (except initial experiments with neomycin and variable cortisol, which used incubation times up to 30 h), with either the aminoglycoside neomycin (30 min with 1-hr recovery) or gentamicin (6-hr continuous exposure) according to published protocols (Coffin et al., 2009; Owens et al., 2009). Neomycin and gentamicin act with different time courses and activate somewhat distinct cell death mechanisms in the lateral line, hence the use of multiple aminoglycosides and exposure paradigms (Owens et al., 2009; Pickles, 2012; Coffin et al., 2013a; Esterberg et al., 2013). Pharmacologic inhibitors were diluted in defined E2 embryo medium (EM; Westerfield, 2007) and dissolved in MeOH or DMSO, as described below.
Aminoglycoside antibiotics have potent antibacterial properties but cause hearing loss in up to 25% of patients. These drugs are commonly administered in patients with high glucocorticoid stress hormone levels and can be combined with exogenous glucocorticoid treatment. However, the interaction of stress and aminoglycoside-induced hearing loss has not been fully explored. In this study, we investigated the effect of the glucocorticoid stress hormone cortisol on hair cells in the zebrafish lateral line as an important step toward understanding how physiological stressors modulate hair cell survival. We found that 24-hr cortisol incubation sensitized hair cells to neomycin damage. Pharmacological and genetic manipulation demonstrates that sensitization depended on the action of the glucocorticoid receptor but not the mineralocorticoid receptor. Blocking endogenous cortisol production reduced hair cell susceptibility to neomycin, further evidence that glucocorticoids modulate aminoglycoside ototoxicity. Glucocorticoid transcriptional activity was apparent in lateral line hair cells, suggesting a direct action of cortisol in these aminoglycoside-sensitive cells. Our work shows that the stress hormone cortisol can increase hair cell sensitivity to aminoglycoside damage, which highlights the importance of recognizing stress and the impacts of glucocorticoid signaling in both ototoxicity research and clinical practice.
Anatomical basis of drug delivery to the inner ear
2018, Hearing Research
Citation Excerpt :
Gradients in susceptibility for ototoxic substances and degeneration pattern following acoustic trauma are common across different mammalian species (Lee et al., 2013; Ohlemiller and Gagnon, 2004; Sha et al., 2001). HC receptor cells seem to be evolutionarily quite conserved in several aspects, so that even toxicity studies from fish lateral line neuromast HCs may predict vulnerability in inner ear HCs (Esterberg et al., 2013; Ou et al., 2012). Marked differences imply their regeneration capacity that distinguish the primal features of fish lateral line HCs.
The isolated anatomical position and blood-labyrinth barrier hampers systemic drug delivery to the mammalian inner ear. Intratympanic placement of drugs and permeation via the round- and oval window are established methods for local pharmaceutical treatment. Mechanisms of drug uptake and pathways for distribution within the inner ear are hard to predict. The complex microanatomy with fluid-filled spaces separated by tight- and leaky barriers compose various compartments that connect via active and passive transport mechanisms. Here we provide a review on the inner ear architecture at light- and electron microscopy level, relevant for drug delivery. Focus is laid on the human inner ear architecture. Some new data add information on the human inner ear fluid spaces generated with high resolution microcomputed tomography at 15 μm resolution. Perilymphatic spaces are connected with the central modiolus by active transport mechanisms of mesothelial cells that provide access to spiral ganglion neurons. Reports on leaky barriers between scala tympani and the so-called cortilymph compartment likely open the best path for hair cell targeting. The complex barrier system of tight junction proteins such as occludins, claudins and tricellulin isolates the endolymphatic space for most drugs. Comparison of relevant differences of barriers, target cells and cell types involved in drug spread between main animal models and humans shall provide some translational aspects for inner ear drug applications.
Neuromast hair cells retain the capacity of regeneration during heavy metal exposure
2018, Annals of Anatomy
Citation Excerpt :
The support cells play an important role in the formation of new hair cells during development and regeneration because they are multipotent progenitor cells (Ghysen and Dambly-Chaudiere, 2007; Lush and Piotrowski, 2014; Wada and Kawakami, 2015). In zebrafish, neuromast hair cells are very similar to those present in the inner ear of higher vertebrates and can be easily studied for their accessibility and visibility, facilitating the study of cellular and molecular mechanisms of development and differentiation (Brignull et al., 2009; Esterberg et al., 2013; Thomas et al., 2015). In recent years the zebrafish LL has become an excellent experimental model for toxicological studies and especially to investigate the regenerative processes considering the ability of zebrafish hair cells to regenerate after the damage.
The neuromast is the morphological unit of the lateral line of fishes and is composed of a cluster of central sensory cells (hair cells) surrounded by support and mantle cells. Heavy metals exposure leads to disruption of hair cells within the neuromast. It is well known that the zebrafish has the ability to regenerate the hair cells after damage caused by toxicants. The process of regeneration depends on proliferation, differentiation and cellular migration of sensory and non-sensory progenitor cells. Therefore, our study was made in order to identify which cellular types are involved in the complex process of regeneration during heavy metals exposure. For this purpose, adult zebrafish were exposed to various heavy metals (Arsenic, cadmium and zinc) for 72 h. After acute (24 h) exposure, immunohistochemical localization of S100 (a specific marker for hair cells) in the neuromasts highlighted the hair cells loss. The immunoreaction for Sox2 (a specific marker for stem cells), at the same time, was observed in the support and mantle cells, after exposure to arsenic and cadmium, while only in the support cells after exposure to zinc. After chronic (72 h) exposure the hair cells were regenerated, showing an immunoreaction for S100 protein. At the same exposure time to the three metals, a Sox2 immunoreaction was expressed in support and mantle cells. Our results showed for the first time the regenerative capacity of hair cells, not only after, but also during exposure to heavy metals, demonstrated by the presence of different stem cells that can diversify in hair cells.
Advancements in zebrafish applications for 21st century toxicology
2016, Pharmacology and Therapeutics
The zebrafish model is the only available high-throughput vertebrate assessment system, and it is uniquely suited for studies of in vivo cell biology. A sequenced and annotated genome has revealed a large degree of evolutionary conservation in comparison to the human genome. Due to our shared evolutionary history, the anatomical and physiological features of fish are highly homologous to humans, which facilitates studies relevant to human health. In addition, zebrafish provide a very unique vertebrate data stream that allows researchers to anchor hypotheses at the biochemical, genetic, and cellular levels to observations at the structural, functional, and behavioral level in a high-throughput format. In this review, we will draw heavily from toxicological studies to highlight advances in zebrafish high-throughput systems. Breakthroughs in transgenic/reporter lines and methods for genetic manipulation, such as the CRISPR-Cas9 system, will be comprised of reports across diverse disciplines.
Methods to study the development, anatomy, and function of the zebrafish inner ear across the life course
2016, Methods in Cell Biology
The inner ear is a remarkably intricate structure able to detect sound, motion, and gravity. During development of the zebrafish embryo, the ear undergoes dynamic morphogenesis from a simple epithelial vesicle into a complex labyrinth, consisting of three semicircular canals and three otolithic sensory organs, each with an array of differentiated cell types. This microcosm of biology has led to advances in understanding molecular and cellular changes in epithelial patterning and morphogenesis, through to mechanisms of mechanosensory transduction and the origins of reflexive behavior. In this chapter, we describe different methods to study the zebrafish ear, including high-speed imaging of otic cilia, confocal microscopy, and light-sheet fluorescent microscopy. Many dyes, antibodies, and transgenic lines for labeling the ear are available, and we provide a comprehensive review of these resources. The developing ear is amenable to genetic, chemical, and physical manipulations, including injection and transplantation. Chemical modulation of developmental signaling pathways has paved the way for zebrafish to be widely used in drug discovery. We describe two chemical screens with relevance to the ear: a fluorescent-based screen for compounds that protect against ototoxicity, and an in situ–based screen for modulators of a signaling pathway involved in semicircular canal development. We also describe methods for dissection and imaging of the adult otic epithelia. We review both manual and automated methods to test the function of the inner ear and lateral line, defects in which can lead to altered locomotor behavior. Finally, we review a collection of zebrafish models that are generating new insights into human deafness and vestibular disorders.

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⁶: These authors contributed equally to this work.

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Zebrafish as a platform for in vivo drug discoveryFish in a dish: drug discovery for hearing habilitation

Section snippets

Chemical screens in the zebrafish lateral line

Identification of compounds with ototoxic potential

Identification of compounds that protect against hair cell loss

Repurposing existing FDA-approved drugs

Blocking ototoxin entry into hair cells: the ultimate protectant?

Screening for compounds that alter hair cell regeneration

Intersection between chemical and genetic screens

Conclusion

Acknowledgements

Hear Res

Hear Res

Kidney Int

Hear Res

Drug Discov Today

J Neurobiol

Hear Res

Hear Res

Hear Res

Brain Res

Hear Res

J Pharmacol Toxicol Methods

Hear Res

Hear Res

Hear Res

Hear Res

Curr Biol

Curr Biol

Curr Biol

Mechanisms of hair cell death and protection

Curr Opin Otolaryngol Head Neck Surg

Aminoglycoside antibiotics

Audiol Neurootol

Synergistic ototoxicity due to noise exposure and aminoglycoside antibiotics

Noise Health

Mechanisms of cisplatin ototoxicity and progress in otoprotection

Curr Opin Otolaryngol Head Neck Surg

Streptomycin in treatment of clinical tuberculosis: a preliminary report

Mayo Clin Proc

Cellular mechanisms of aminoglycoside ototoxicity

Curr Opin Otolaryngol Head Neck Surg

Chemical screening for hair cell loss and protection in the zebrafish lateral line

Zebrafish

Organization of the lateral line system in embryonic zebrafish

J Comp Neurol

The functioning and significance of the lateral-line organs

Biol Rev Camb Philos Soc

Sensory tuning of lateral line receptors in antarctic fish to the movements of planktonic prey

Science

The Mechanosensory Lateral Line: Neurobiology and Evolution.

Neomycin-induced hair cell death and rapid regeneration in the lateral line of zebrafish (Danio rerio)

J Assoc Res Otolaryngol

PROTO1 provides robust protection against kanamycin-induced hearing loss in rats

Zebrafish as a platform for in vivo drug discovery
Fish in a dish: drug discovery for hearing habilitation