Identification of Genetic and Chemical Modulators of Zebrafish Mechanosensory Hair Cell Death

Department of Biological Structure, University of Washington, Seattle, Washington, United States of America.
PLoS Genetics (Impact Factor: 7.53). 03/2008; 4(2):e1000020. DOI: 10.1371/journal.pgen.1000020
Source: PubMed


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.

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    • "To identify candidate genes and pathways regulating hair cell susceptibility to ototoxic drugs, we have used the zebrafish lateral line system to screen for mutants involved in aminoglycoside toxicity (Owens et al., 2008). To date, our studies have identified three novel genes involved in ototoxicity: cc2d2a (Owens et al., 2008), slc4a1b (Hailey et al., 2012) and gcm2 (this work). Two of these genes, slc4a1b and gcm2, are involved in pH regulation and both genes cause a decrease in whole body proton extrusion when knocked down in zebrafish (Chang et al., 2009; Lee et al., 2011). "
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    ABSTRACT: Control of the extracellular environment of inner ear hair cells by ionic transporters is crucial for hair cell function. In addition to inner ear hair cells, aquatic vertebrates have hair cells on the surface of their body in the lateral line system. The ionic environment of these cells also appears to be regulated, although the mechanisms of this regulation are less understood than those of the mammalian inner ear. We identified the merovingian mutant through genetic screening in zebrafish for genes involved in drug-induced hair cell death. Mutants show complete resistance to neomycin-induced hair cell death and partial resistance to cisplatin-induced hair cell death. This resistance is probably due to impaired drug uptake as a result of reduced mechanotransduction ability, suggesting that the mutants have defects in hair cell function independent of drug treatment. Through genetic mapping we found that merovingian mutants contain a mutation in the transcription factor gcm2. This gene is important for the production of ionocytes, which are cells crucial for whole body pH regulation in fish. We found that merovingian mutants showed an acidified extracellular environment in the vicinity of both inner ear and lateral line hair cells. We believe that this acidified extracellular environment is responsible for the defects seen in hair cells of merovingian mutants, and that these mutants would serve as a valuable model for further study of the role of pH in hair cell function.
    Disease Models and Mechanisms 07/2014; 7(7):847-56. DOI:10.1242/dmm.016576 · 4.97 Impact Factor
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    • "In a recent paper, a phenotype-based screen identified clemizole (an FDA-approved compound) as a potential lead compound for Dravet syndrome because it effectively inhibited spontaneous convulsive behaviours and electrographic seizures in zebrafish scn1Lab mutants (Baraban et al., 2013). In another successful in vivo screen study, using the zebrafish lateral line sensory system as a model system for mammalian auditory hair cells, the collaborative work of the Rubel and Raibel labs identified two small molecules (named PROTO-1 and PROTO-2) (Owens et al., 2008) and an FDA-approved compound (named tacrine) (Ou et al., 2009) that protect hair cells from aminoglycoside-induced death. These compounds also inhibited hair cell death in the mammalian inner ear and subsequently preserved hearing. "
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    ABSTRACT: Motor neuron disorders (MNDs) are a clinically heterogeneous group of neurological diseases characterized by progressive degeneration of motor neurons, and share some common pathological pathways. Despite remarkable advances in our understanding of these diseases, no curative treatment for MNDs exists. To better understand the pathogenesis of MNDs and to help develop new treatments, the establishment of animal models that can be studied efficiently and thoroughly is paramount. The zebrafish (Danio rerio) is increasingly becoming a valuable model for studying human diseases and in screening for potential therapeutics. In this Review, we highlight recent progress in using zebrafish to study the pathology of the most common MNDs: spinal muscular atrophy (SMA), amyotrophic lateral sclerosis (ALS) and hereditary spastic paraplegia (HSP). These studies indicate the power of zebrafish as a model to study the consequences of disease-related genes, because zebrafish homologues of human genes have conserved functions with respect to the aetiology of MNDs. Zebrafish also complement other animal models for the study of pathological mechanisms of MNDs and are particularly advantageous for the screening of compounds with therapeutic potential. We present an overview of their potential usefulness in MND drug discovery, which is just beginning and holds much promise for future therapeutic development.
    Disease Models and Mechanisms 07/2014; 7(7):799-809. DOI:10.1242/dmm.015719 · 4.97 Impact Factor
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    • "Another advantage of using the zebrafish lateral line system is the ability of the neuromast to produce newly differentiated HCs primarily from the continuous proliferation of supporting cells after the original HCs have been destroyed.4 Therefore, neuromasts provide a powerful tool to investigate mechanosensory HC development and to screen for potential ototoxic and otoprotective drugs.5 "
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    ABSTRACT: Histone deacetylases are involved in many biological processes and have roles in regulating cell behaviors such as cell cycle entry, cell proliferation and apoptosis. However, the effect of histone deacetylases on the development of hair cells (HCs) has not been fully elucidated. In this study, we examined the influence of histone deacetylases on the early development of neuromasts in the lateral line of zebrafish. Hair cell development was evaluated by fluorescent immunostaining in the absence or presence of histone deacetylase inhibitors. Our results suggested that pharmacological inhibition of histone deacetylases with inhibitors, including trichostatin A, valproic acid and MS-275, reduced the numbers of both HCs and supporting cells in neuromasts. We also found that the treatment of zebrafish larvae with inhibitors caused accumulation of histone acetylation and suppressed proliferation of neuromast cells. Real-time PCR results showed that the expression of both p21 and p27 mRNA was increased following trichostatin A treatment and the increase in p53 mRNA was modest under the same conditions. However, the expression of p53 mRNA was significantly increased by treatment with a high concentration of trichostatin A. A high concentration of trichostatin A also led to increased cell death in neuromasts as detected in a TUNEL assay. Moreover, the nuclei of most of these pyknotic cells were immunohistochemically positive for cleaved caspase-3. These results suggest that histone deacetylase activity is involved in lateral line development in the zebrafish and might have a role in neuromast formation by altering cell proliferation through the expression of cell cycle regulatory proteins.
    05/2014; 46(5):e94. DOI:10.1038/emm.2014.18
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