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. 2013 Apr 24;33(17):7513-25.
doi: 10.1523/JNEUROSCI.4559-12.2013.

Disruption of intracellular calcium regulation is integral to aminoglycoside-induced hair cell death

Robert Esterberg  1 , Dale W Hailey, Allison B Coffin, David W Raible, Edwin W Rubel
Affiliations

Disruption of intracellular calcium regulation is integral to aminoglycoside-induced hair cell death

Robert Esterberg et al. J Neurosci. .

Abstract

Intracellular Ca(2+) is a key regulator of life or death decisions in cultured neurons and sensory cells. The role of Ca(2+) in these processes is less clear in vivo, as the location of these cells often impedes visualization of intracellular Ca(2+) dynamics. We generated transgenic zebrafish lines that express the genetically encoded Ca(2+) indicator GCaMP in mechanosensory hair cells of the lateral line. These lines allow us to monitor intracellular Ca(2+) dynamics in real time during aminoglycoside-induced hair cell death. After exposure of live larvae to aminoglycosides, dying hair cells undergo a transient increase in intracellular Ca(2+) that occurs shortly after mitochondrial membrane potential collapse. Inhibition of intracellular Ca(2+) elevation through either caged chelators or pharmacological inhibitors of Ca(2+) effectors mitigates toxic effects of aminoglycoside exposure. Conversely, artificial elevation of intracellular Ca(2+) by caged Ca(2+) release agents sensitizes hair cells to the toxic effects of aminoglycosides. These data suggest that alterations in intracellular Ca(2+) homeostasis play an essential role in aminoglycoside-induced hair cell death, and indicate several potential therapeutic targets to stem ototoxicity.

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Figures

Figure 1.
Figure 1.
Calibration of cytoGCaMP within lateral line hair cells. A, Change in fluorescence (ΔF/baseline) of living Tg(myo6b:GCaMP3) lateral line hair cells during exposure to 5 μm ionomycin and 70 nm, 270 nm, or 610 nm extracellular Ca2+. Baseline is taken in 1 nm Ca2+/5 mm EGTA and 5 μm ionomycin. Gray lines indicate data from individual cells, and red lines indicate the mean response; n = 10 cells from one neuromast. B, Mean responses of living Tg(myo6b:GCaMP3) lateral line hair cells during exposure to 5 μm ionomycin and varying levels of extracellular Ca2+. Baseline is taken in 1 nm Ca2+/5 mm EGTA and 5 μm ionomycin, and values are expressed as SDs around baseline (signal-to-noise ratio [SNR]). C, Frequency of SDs around baseline (expressed as maximum SNR) of the mean cytoGCaMP response after addition of indicated extracellular [Ca2+] and EGTA in the presence of ionomycin. D, Plot of maximal fluorescence [(ΔF/baseline)max] and extracellular Ca2+. Error bars indicate SEM; n = 7–40 cells from ≥3 neuromasts. E, Hair cell survival after 30 min exposure to extracellular Ca2+. Error bars indicate SEM; n >7 neuromasts. Dying cells were not used in data acquired in A to D.
Figure 2.
Figure 2.
Cytoplasmic Ca2+ transients precede cell death in lateral line hair cells exposed to aminoglycoside antibiotics. Heat-mapped, time-lapse image of a Tg(myo6b:GCaMP3) anterior lateral line neuromast exposed to 50 μm neomycin. Time (min:sec) after neomycin administration is indicated. Cells that die and are extruded from the neuromast are highlighted with dashed lines. Note that cytoGCaMP fluorescence in living cells remains largely static, whereas that of dying cells peaks shortly before clearance from the neuromast.
Figure 3.
Figure 3.
Cytoplasmic Ca2+ dynamics in lateral line hair cells after aminoglycoside exposure. A, B, E, F, Transformed (ΔF/baseline) fluorescence intensity data of individual dying (A, E) or living (B, F) Tg(myo6b:GCaMP3) hair cells exposed to 50 μm neomycin (A, B) or 50 μm gentamicin (E, F). Three cells each from three neuromasts are depicted; traces are color-coded to illustrate neuromast of origin. Dying cells were chosen to highlight variability in the timing of cell death. C, G, Mean intensity data of living (green) or dying (red) Tg(myo6b:GCaMP3) hair cells exposed to 50 μm neomycin (C) or 50 μm gentamicin (G) aligned to the time point at which they are cleared from the neuromast. Because living cells are not cleared, they are aligned to the end of imaging (i.e., the last 60 min). D, H, Plot of the time at which half-maximal [(ΔF/baseline)half-max] Tg(myo6b:GCaMP3) intensity ratios or cell clearance occur in response to increasing neomycin (D) or gentamicin (H) concentrations. p value indicates significance of the correlation coefficient. In all grouped data, error bars indicate SEM; n = 15 from >5 neuromasts and experimental runs.
Figure 4.
Figure 4.
Early cytoplasmic Ca2+ response is indistinguishable between cells that are resistant or susceptible to aminoglycosides. A, B, Mean cytoGCaMP response of cells that are resistant (gray) or susceptible (black) for the initial 10 min after 50 μm neomycin (A) or 50 μm gentamicin (B) exposure. C, Cumulative cytoGCaMP fluorescence (expressed as cumulative ΔF/baseline) of aminoglycoside resistant (gray) or susceptible (black) cells. Error bars indicate SEM; n = 18 and 20 neomycin-treated resistant and susceptible cells, respectively, from >6 neuromasts, and 15 each of gentamicin-treated resistant or susceptible cells from 5 neuromasts.
Figure 5.
Figure 5.
Initial cytoplasmic Ca2+ levels are not indicative of cell position within a neuromast or aminoglycoside susceptibility. A, Percentage of dying hair cells within a neuromast exposed to 50 μm neomycin. Central hair cells were defined as containing nuclei within a 5 μm radius from the center of the stereocilia bundle cluster, and peripheral hair cells containing nuclei 5–10 μm away. Error bars indicate SEM; n = 7 neuromasts. B, Mean (inner bar) and range (outer bars) of mean baseline intensity of all centrally and peripherally located hair cells of 5 neuromasts. C, Mean (inner bar) and range (outer bars) of mean baseline intensity of all dying and living hair cells of 5 neuromasts exposed to 50 μm neomycin.
Figure 6.
Figure 6.
Dose-independent response of cytoplasmic Ca2+ within dying lateral line hair cells to aminoglycoside exposure. A, Maximal intensity ratios of Tg(myo6b:GCaMP3) compared between living and dying cells exposed to increasing concentrations of neomycin and gentamicin. Corresponding signal-to-noise ratios (SNR) are indicated on the right axis. Data are mean ± SEM indicated for each group. Cells expressing cpGFP die after exposure to 50 μm neomycin. Note that there is little variability in [Ca2+]i response in hair cells that live (or in cpGFP-expressing cells that die) regardless of condition, whereas the variability of the GCaMP3.0 response is much greater, although the level of the response does not depend on neomycin concentration. ***p < 0.0001 (one-way ANOVA, Dunnett post-test). B, Linear regression analysis of maximal intensity ratios in response to increasing neomycin concentrations. Error bars indicate SEM; n = 15 from >5 experimental runs.
Figure 7.
Figure 7.
Timing of cytoplasmic Ca2+ peaks relative to loss of mitochondrial membrane potential. A, Heat-mapped, time-lapse image of Tg(myo6b:GCaMP3) anterior lateral line neuromasts colabeled with the potentiometric vital dye TMRE and exposed to 400 μm neomycin. Time indicates seconds relative to TMREhalf-min of the outlined cell, corresponding with TMRE redistribution from mitochondria into cytoplasm. Note that TMRE redistribution occurs before elevated cytoGCaMP fluorescence. B, Transformed (ΔF/baseline) fluorescence intensity data of individual dying Tg(myo6b:GCaMP3) hair cells (solid lines) labeled with TMRE (dashed lines) and exposed to 400 μm neomycin. Cells were chosen to highlight variability in both TMRE response and cell death. Note the reduction in TMRE fluorescence, corresponding with cytoplasmic TMRE redistribution, before cytoGCaMP peaks. C, Mean fluorescent intensity data of Tg(myo6b:GCaMP3) in dying anterior lateral line hair cells colabeled with TMRE and exposed to 400 μm neomycin. Data are aligned to TMREhalf-min, corresponding with TMRE redistribution from mitochondria into cytoplasm. D, Comparison of the timing at which cytoGCaMP reaches half-maximal intensity relative to TMREhalf-min as they occur within the same cells. Error bars indicate SEM; n = 10 from >3 experimental runs.
Figure 8.
Figure 8.
Effectiveness of caged Ca2+ modulators at altering intracellular Ca2+ within lateral line hair cells. A, ΔF/baseline of Tg(myo6b:GCaMP3) 1 min after exposure to UV light. Where indicated, larvae were injected with caged diazo2 or caged EGTA at the one cell stage, and then uncaged at 5 dpf. ***p < 0.0001 between uncaged cells and controls (one-way ANOVA, Bonferroni post-tests). UV/diazo-2 cells are also as expected significantly different from UV/EGTA cells (p < 0.0001). Error bars indicate SEM. B, Heat-mapped, time-lapse images of the lateral line neuromast of a Tg(myo6b:GCaMP3) larvae injected with caged EGTA at one cell stage and exposed at 5 dpf to UV light. cytoGCaMP fluorescence increases after UV exposure (uncaging). Time indicates seconds elapsed in relation to UV exposure.
Figure 9.
Figure 9.
Ca2+ modulation alters toxicity of aminoglycoside antibiotics. A, B, Lateral line hair cell survival after injection and activation of caged EGTA preloaded with Ca2+ (A) or the caged Ca2+ chelator diazo2 (B) after exposure to increasing concentrations of neomycin. Hair cell counts are of parvalbumin-positive hair cells. Error bars indicate SD; n = 6 neuromasts from each of five treated larvae. *p < 0.05 (two-way ANOVA, Tukey post-test). ***p < 0.0001 (two-way ANOVA, Tukey post-test). C, Hair cells of 5 dpf zebrafish larvae stained with antisera against parvalbumin after UV activation of the caged Ca2+ chelator diazo-2 and exposure to 200 μm neomycin. Lateral views of the zebrafish lateral line system under low magnification (10× magnification). Red clusters (such as the one labeled in the box) label anterior lateral line neuromasts. The anterior of the zebrafish larvae faces left. High-magnification (63× magnification) views are of boxed neuromasts. Lateral and apical views are indicated. Experimental treatments are as indicated.
Figure 10.
Figure 10.
Pharmacological inhibitors of calmodulin protect lateral line hair cells from aminoglycoside-induced hair cell death. A, Optimal doses of A7 and W7 were determined by pretreatment of inhibitor followed by coadministration with 200 μm neomycin. B, Lateral line hair cell survival after pretreatment of the calmodulin inhibitors A7 (30 μm) and W7 (20 μm) and subsequent coadministration with increasing doses of neomycin. Hair cell counts are of parvalbumin-positive hair cells. Error bars indicate SD; n = 6 neuromasts from each of 5 treated larvae. ***p < 0.0001 (two-way ANOVA, Tukey post-test).
Figure 11.
Figure 11.
Pharmacological inhibitors of Ca2+-activated serine proteases protect lateral line hair cells from aminoglycoside-induced hair cell death. A, Optimal doses of leupeptin determined by pretreatment of inhibitor followed by coadministration with 200 μm neomycin. B, Lateral line hair cell survival after pretreatment of leupeptin (500 μm) and subsequent coadministration with increasing doses of neomycin. Hair cell survival was assessed with DASPEI scoring (Harris et al., 2003). Error bars indicate SD; n = 10 neuromasts from each of 12 treated larvae. ***p < 0.0001 (two-way ANOVA, Tukey post-test).
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References

    1. Akerboom J, Chen TW, Wardill TJ, Tian L, Marvin JS, Mutlu S, Calderon NC, Esposti F, Borghuis BG, Sun XR, Gordus A, Orger MB, Portugues R, Engert F, Macklin JJ, Filosa A, Aggarwal A, Kerr RA, Takagi R, Kracun S, et al. Optimization of a GCaMP calcium indicator for neural activity imaging. J Neurosci. 2012;32:13819–13840. doi: 10.1523/JNEUROSCI.2601-12.2012. - DOI - PMC - PubMed
    1. Alharazneh A, Luk L, Huth M, Monfared A, Steyger PS, Cheng AG, Ricci AJ. Functional hair cell mechanotransducer channels are required for aminoglycoside ototoxicity. PLoS One. 2011;6:e22347. doi: 10.1371/journal.pone.0022347. - DOI - PMC - PubMed
    1. Ali BH, Al Za'abi M, Blunden G, Nemmar A. Experimental gentamicin nephrotoxicity and agents that modify it: a mini-review of recent research. Basic Clin Pharmacol Toxicol. 2011;109:225–232. doi: 10.1111/j.1742-7843.2011.00728.x. - DOI - PubMed
    1. Amr S, Heisey C, Zhang M, Xia XJ, Shows KH, Ajlouni K, Pandya A, Satin LS, El-Shanti H, Shiang R. A homozygous mutation in a novel zinc-finger protein, ERIS, is responsible for Wolfram syndrome 2. Am J Hum Genet. 2007;81:673–683. doi: 10.1086/520961. - DOI - PMC - PubMed
    1. Audo I, Warchol ME. Retinal and cochlear toxicity of drugs: new insights into mechanisms and detection. Curr Opin Neurol. 2012;25:76–85. doi: 10.1097/WCO.0b013e32834ed882. - DOI - PubMed

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