Profiling drug-induced cell death pathways in the zebrafish lateral line

Abstract

Programmed cell death (PCD) is an important process in development and disease, as it allows the body to rid itself of unwanted or damaged cells. However, PCD pathways can also be activated in otherwise healthy cells. One such case occurs in sensory hair cells of the inner ear following exposure to ototoxic drugs, resulting in hearing loss and/or balance disorders. The intracellular pathways that determine if hair cells die or survive following this or other ototoxic challenges are incompletely understood. We use the larval zebrafish lateral line, an external hair cell-bearing sensory system, as a platform for profiling cell death pathways activated in response to ototoxic stimuli. In this report the importance of each pathway was assessed by screening a custom cell death inhibitor library for instances when pathway inhibition protected hair cells from the aminoglycosides neomycin or gentamicin, or the chemotherapy agent cisplatin. This screen revealed that each ototoxin likely activated a distinct subset of possible cell death pathways. For example, the proteasome inhibitor Z-LLF-CHO protected hair cells from either aminoglycoside or from cisplatin, while D-methionine, an antioxidant, protected hair cells from gentamicin or cisplatin but not from neomycin toxicity. The calpain inhibitor leupeptin primarily protected hair cells from neomycin, as did a Bax channel blocker. Neither caspase inhibition nor protein synthesis inhibition altered the progression of hair cell death. Taken together, these results suggest that ototoxin-treated hair cells die via multiple processes that form an interactive network of cell death signaling cascades.

Fig. 1

Screening a cell death inhibitor library for compounds that modulate ototoxin-induced hair cell death in the zebrafish lateral line. (A) Screen results for hair cells treated with inhibitor and neomycin. Hair cell survival is represented as fold-change relative to neomycin only, such that 0-fold (the red line) denotes the degree of damage caused by neomycin treatment without an inhibitor present. Inhibitors that protected hair cells from neomycin toxicity are visible as bars extending above the red line. Inhibitor identities are given in Table 1. (B) Heat map of all screen data. Ototoxins are represented in columns, inhibitors in rows. Each box denotes a single ototoxin/inhibitor combination. Black boxes indicate no protection (no change relative to ototoxin only), red boxes are inhibitors that protected hair cells from an ototoxin. Gray boxes denote inhibitor/ototoxin combinations that were toxic to the fish. (C) Venn diagram describing the number of inhibitor “hits” that protected hair cells from damage due to each ototoxin. Some inhibitors protected hair cells from damage due to multiple ototoxins, as indicated in the overlapping regions. The numbers represent confirmed hits that were verified in triplicate. N=7–12 animals per treatment, data in (A) are presented as mean + 1 S.D.

Fig. 2

100 µM GTTR is readily taken up by (A) control hair cells, while (B) 10 µM FUT-175 attenuates GTTR uptake. (C) 5 mM 3-MA does not block GTTR uptake. Scale bar in A is 5 µm and applies to all panels

Fig. 3

The proteasome inhibitor Z-LLF-CHO protects hair cells from ototoxin exposure. (A) Z-LLF-CHO provides dose-dependent protection from 200 µM acute neomycin (1-way ANOVA, F_4,48=37.59, p<0.001). 25 µM Z-LLF-CHO offering optimal protection without any ototoxicity, and there was not a significant difference in protection between 25 and 50 µM Z-LLF-CHO. (B) Direct counts of parvalbumin-labeled hair cells confirm that Z-LLF-CHO treatment protects hair cells from gentamicin toxicity (t-test, p<0.001). Fish were treated with 100 µM continuous gentamicin with or without 25 µM Z-LLF-CHO. Images in B show examples of labeled hair cells, scale bar = 5 µm and applies to both panels. (C–F) 25 µM Z-LLF-CHO robustly protects hair cells from (C) acute neomycin, (D) acute gentamicin, (E) continuous gentamicin, and (F) continuous cisplatin. Statistics for the dose-response analyses shown in C–F are given in Table 2. Significance values for individual comparisons in Bonferroni-corrected posthoc tests are indicated on the figures, where ***p<0.001. Data are presented as mean ± 1 S.D.

Fig. 4

(A) D-methionine significantly protects hair cells from continuous gentamicin damage (1-way ANOVA, F_4,55=7.74, p<0.001), with 5 mM D-met providing optimal protection without overt toxicity. (B) 5 mM D-met does not protect hair cells from neomycin toxicity, while significant protection is offered across much of the dose-response function for acute (C) and continuous (D) gentamicin. Slight but significant protection is also seen from continuous cisplatin exposure (E). 2-way ANOVA statistics are given in Table 2, significance values from Bonferroni-corrected posthoc analysis are indicated on the figure; *p<0.05, **p<0.01, ***p<0.001. Data are presented as mean ± 1 S.D.

Fig. 5

The general caspase inhibitor Z-VAD does not significantly protect hair cells from 200 µM acute neomycin (1-way ANOVA, F_5,52=0.30, p=0.91) or 100 µM continuous gentamicin (1-way ANOVA, F_5,56=0.20, p=0.96). Data are presented as mean ± 1 S.D.

Fig. 6

(A) The protein synthesis inhibitor cycloheximide does not protect hair cells from ototoxic damage. Significant hair cell toxicity is evident following cycloheximide treatment alone (1-way ANOVA, F_3,42=104.7, p<0.001), and cycloheximide increased hair cell loss due to cisplatin toxicity (1-way ANOVA, F_3,31=14.0, p<0.001). Cycloheximide treatment did not influence acute neomycin-induced hair cell death (1-way ANOVA, F_3,40=0.40, p=0.75) nor hair cell death due to continuous gentamicin exposure (1-way ANOVA, F_3,43=1.11, p=0.35). Asterisks indicate significant pairwise differences using Bonferroni-corrected posthoc testing (*p<0.05, ***p<0.001). Data are presented as mean ± SD. (B) 10 µM cycloheximide treatment attenuates GFP activation in the eye of hsp70::GFP transgenic larvae exposed to 1 hr heat shock. The scale bar in the top panel is 0.5 mm and applies to all three panels. Data are presented as mean ± 1 S.D.