Identification of FDA-approved drugs and bioactives that protect hair cells in the zebrafish (Danio rerio) lateral line and mouse (Mus musculus) utricle

Abstract

The hair cells of the larval zebrafish lateral line provide a useful preparation in which to study hair cell death and to screen for genes and small molecules that modulate hair cell toxicity. We recently reported preliminary results from screening a small-molecule library for compounds that inhibit aminoglycoside-induced hair cell death. To potentially reduce the time required for development of drugs and drug combinations that can be clinically useful, we screened a library of 1,040 FDA-approved drugs and bioactive compounds (NINDS Custom Collection II). Seven compounds that protect against neomycin-induced hair cell death were identified. Four of the seven drugs inhibited aminoglycoside uptake, based on Texas-Red-conjugated gentamicin uptake. The activities of two of the remaining three drugs were evaluated using an in vitro adult mouse utricle preparation. One drug, 9-amino-1,2,3,4-tetrahydroacridine (tacrine) demonstrated conserved protective effects in the mouse utricle. These results demonstrate that the zebrafish lateral line can be used to screen successfully for drugs within a library of FDA-approved drugs and bioactives that inhibit hair cell death in the mammalian inner ear and identify tacrine as a promising protective drug for future studies.

FIG. 1

Examples of normal and damaged fluorescently labeled hair cells of the zebrafish lateral line. YO-PRO1 selectively labels hair cell nuclei in normal (A) and neomycin-damaged (B) neuromasts. Hair cell protection can thus be easily assessed during screening. For quantitative hair cell counts, FM1-43FX is used to count normal (C) and neomycin-damaged (B) hair cells. In the undamaged neuromast (C) there are approximately 12 visible hair cells. In the damaged neuromast (D), there are two surviving hair cells. Scale bar in D = 10 μM and applies to all panels.

FIG. 2

Cepharanthine demonstrates significant protection against neomycin-induced hair cell loss. A Five days post-fertilization zebrafish pre-exposed to cepharanthine for 1 h and then treated with 200 μM neomycin demonstrate significant dose-dependent protection by cepharanthine (**p < 0.01, one-way ANOVA). B Five days post-fertilization zebrafish pre-exposed to 100 μM cepharanthine and then treated with 0, 100, 200, or 400 μM neomycin. Solid line Zebrafish pretreated with cepharanthine prior to neomycin. Dotted line Zebrafish with no pretreatment prior to neomycin. Cepharanthine pretreatment led to significant protection against all doses of neomycin (p < 0.0001, two-way ANOVA). For both graphs, data points represent mean hair cell survival of 10–15 fish. Error bars = ±1 SD from the mean. Data for remaining six protective drugs are presented in Tables 2 and 3.

FIG. 3

Twenty-four hour hair cell survival after neomycin. Fluorescently labeled 5 dpf zebrafish were pretreated with each protective drug for 1 h, followed by treatment with 200 μM neomycin for 1 h. Zebrafish then recovered for 24 h, and then were fixed for hair cell counts. All seven drugs demonstrated significant protection 24 h after the neomycin exposure relative to control + neomycin (**p < 0.01, student t-test). Bars represent mean hair cell survival of 10–15 fish. Error bars = ±1 SD from the mean.

FIG. 4

Protection after pre-neomycin washout. Fluorescently labeled 5 dpf zebrafish were pretreated with each protective drug for 1 h. The protective drug was then washed out with multiple rinses in embryo media. Fish were then treated with 200 μM neomycin for 1 h and then fixed for hair cell counts. Only phenoxybenzamine, drofenine, and carvedilol demonstrated significant protection relative to control + neomycin (*p < 0.05; **p < 0.01, student t-test). Bars represent mean hair cell survival of 10–15 fish. Error bars = ±SD from the mean.

FIG. 5

Examples of normal and blocked uptake of Texas-Red conjugated gentamicin (TR-Gent) in the zebrafish lateral line. Hair cells are labeled with YO-PRO1 (green) to label hair cell nuclei, and TR-Gent (red). A Normal uptake of TR-Gent demonstrates multiple double-labeled hair cells. B Uptake of TR-Gent is blocked by pretreatment with the candidate protective drug, phenoxybenzamine. No double-labeled hair cells are seen. Similar findings were seen with carvedilol, hexamethyleneamiloride, and amsacrine. Scale bar in B = 10 μM and applies to both panels.

FIG. 6

Tacrine (THA) protects against neomycin-induced hair cell death in mouse utricle explants. Utricles were pretreated with 10 μM tacrine for 4 h, followed by 2 mM neomycin and THA for 24 h. Utricles were then fixed and labeled with antibodies against calmodulin (green) and calbindin (red). A Control utricle not treated with neomycin or THA. B Utricle pretreated with THA without neomycin exposure. Ten micromolar THA did not cause any hair cell loss. C Utricle without THA pretreatment, but treated with 2 mM neomycin demonstrates marked striolar and extrastriolar hair cell loss with a decrease in calmodulin and calbindin labeling. There is also an increase in cellular debris from dying hair cells. D Utricle pretreated with THA then treated with 2 mM neomycin demonstrates protection against hair cell loss. Scale bar in D = 20 μM and applies to all panels. E Hair cell survival is significantly increased (p < 0.01, one-way ANOVA) in both extrastriolar and striolar hair cells when utricles were pretreated with THA prior to neomycin (Neo+/THA+) compared to control utricles without THA pretreatment (Neo+/THA−). Bars represent the mean hair cell survival (% control) + 1 SEM (n = 11–15 utricles per group).