JNK signaling in neomycin-induced vestibular hair cell death

Abstract

Mechanosensory hair cells are susceptible to apoptotic death in response to exposure to ototoxic drugs, including aminoglycoside antibiotics. The c-Jun n-terminal kinase (JNK) is a stress-activated protein kinase that can promote apoptotic cell death in a variety of systems. Inhibition of the JNK signaling pathway can prevent aminoglycoside-induced death of cochlear and vestibular sensory hair cells. We used an in vitro preparation of utricles from adult mice to examine the role of JNK activation in aminoglycoside-induced hair cell death. CEP-11004 was used as an indirect inhibitor of JNK signaling. Immunohistochemistry showed that both JNK and its downstream target c-Jun are phosphorylated in hair cells of utricles exposed to neomycin. CEP-11004 inhibited neomycin-induced phosphorylation of both JNK and c-Jun. CEP-11004 inhibited hair cell death in utricles exposed to moderate doses of neomycin. However, the results were not uniform across the dose-response function; CEP-11004 did not inhibit hair cell death in utricles exposed to high-dose neomycin. The CEP-11004-induced protective effect was not due to inhibition of PKC or p38, since neither Chelerythrine nor SB203580 could mimic the protective effect of CEP-11004. In addition, inhibition of JNK inhibited the activation of caspase-9 in hair cells. These results indicate that JNK plays an important role in neomycin-induced vestibular hair cell death and caspase-9 activation.

Fig. 1. CEP-11004 inhibits phosphorylation of both JNK and c-Jun

Utricles were cultured for 12 hours without neomycin (A, D), with 1mM neomycin (B, E), or with both neomycin and CEP-11004 (C, F). Hair cells were labeled with anti-calmodulin (green). Phosphorylated JNK (A, B, C) and c-Jun (D, E, F) were detected using phospho-specific antibodies (red). Neomycin exposure resulted in phosphorylation of both JNK (B) and c-Jun (E) in hair cells. CEP-11004 inhibited neomycin-induced phosphorylation of both JNK (C) and c-Jun (F). Scale bar in F = 10 µm.

Fig. 2. CEP-11004 inhibits neomycin-induced hair cell death

Utricles were cultured for 24 hours without neomycin (A, E), with 1mM neomycin (B, F), or with both neomycin and CEP-11004 (C, G, D, H). Utricles were double-labeled for calmodulin (green) and calbindin (red) immunoreactivity. Control utricles appeared normal and had normal numbers of hair cells in both the striolar (A) and extrastriolar (E) regions. Neomycin (B, F) resulted in significant hair cell death, particularly in the striolar region. At a concentration of 1µM, CEP-11004 inhibited neomycin-induced death of both striolar (C) and extrastriolar (G) hair cells. CEP-11004 had no protective effect at 0.1µM (D, H). Results are quantified in panel I: Hair cells were counted in calmodulin/calbindin double-labeled utricles. CEP-11004 significantly inhibited hair cell death (Two-way ANOVA, p<0.0001). CEP-11004 was most protective at 1µM (Tukey’s post hoc test, p<0.01 for both the striolar and extrastriolar regions). At 0.5µM, CEP 11004 was significantly protective only in the extrastriolar region (Tukey’s post hoc test, p<0.05 for the extrastriolar region, p> 0.1 for the striolar region). Both higher (2µM) and lower (0.2µM, 0.1µM) concentrations of CEP-11004 failed to significantly inhibit neomycin-induced hair cell death. Shown are mean (±SEM) hair cell densities for both the striolar and extrastriolar regions for n = 5–8 utricles per experimental condition. Asterisks indicate significance (Tukey’s post hoc test p<0.05).

Fig. 3. CEP-11004 is protective against moderate doses of neomycin

Utricles were cultured for 24 hours in the presence (or absence) of neomycin alone or neomycin plus 1.0µM CEP-11004. Shown are hair cell densities for the extrastriolar region. CEP-11004 significantly inhibited hair cell death when the neomycin dose was near the middle of the dose-response curve (1mM and 2mM neomycin, two-wayANOVA, p<0.001). CEP-11004 was not effective at inhibiting hair cell death in response to high-dose neomycin (4 mM). In the striolar region, the protective effect of CEP-11004 was significant only at 1mM neomycin (data not shown). Shown are normalized hair cell densities (mean ± SEM) for n= 5–8 utricles per condition. Asterisks indicate significance (Tukey’s post-hoc tests, p<0.001).

Fig. 4. Neomycin-induced hair cell death is not inhibited by inhibition of PKC or p38

Utricles were cultured for 24 hours in control conditions, neomycin alone (1mM), or neomycin plus either CEP-11004, 13µM Chelerythrine (an inhibitor of PKC), or 10µM SB203580 (an inhibitor of p38). Shown are hair cell densities for both the extrastriolar and striolar regions. Neither Chelerythrine nor SB203580 showed a protective effect against neomycin-induced hair cell death. Shown are mean ± SEM hair cell densities for n=5–8 utricles per condition.

Fig. 5. CEP-11004 inhibits neomycin-induced caspase-9 activation

Utricles were cultured for 12 hours without neomycin (A, D, G), with 1mM neomycin (B, E, H), or with both neomycin and CEP-11004 (C, F, I). Caspase-9 activation was examined using the fluorescent caspase-9 substrate FAM-LEHD-fmk (green). Hair cell stereocilia were labeled using rhodamine phalloidin (red). Very little caspase-9 activation is detected in the control utricles (A, D, G). Robust caspase-9 activation is present in utricles cultured in neomycin alone (B, E, H). CEP-11004 inhibits neomycin-induced activation of caspase-9 (C, F, I). Scale bar in I = 10 µm.

Fig. 6. Quantification of CEP-11004 inhibition of neomycin-induced caspase-9 activation

Caspase-9-positive hair cells were counted in utricles that were prepared as described in Figure 5. Utricles cultured in control conditions showed few hair cells with activated caspase-9. Neomycin exposure resulted in robust (but variable) activation of caspase-9. CEP-11004 inhibited neomycin-induced caspase-9 activation in hair cells. Shown are mean ± SEM caspase-9 positive hair cells for n=5–6 utricles per condition. Asterisk indicates significance (one-way ANOVA with Newman-Keuls post-hoc test, p < 0.05).