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, 23 (24), 8596-607

A Peptide Inhibitor of c-Jun N-terminal Kinase Protects Against Both Aminoglycoside and Acoustic Trauma-Induced Auditory Hair Cell Death and Hearing Loss

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A Peptide Inhibitor of c-Jun N-terminal Kinase Protects Against Both Aminoglycoside and Acoustic Trauma-Induced Auditory Hair Cell Death and Hearing Loss

J Wang et al. J Neurosci.

Abstract

Hearing loss can be caused by a variety of insults, including acoustic trauma and exposure to ototoxins, that principally effect the viability of sensory hair cells via the MAP kinase (MAPK) cell death signaling pathway that incorporates c-Jun N-terminal kinase (JNK). We evaluated the otoprotective efficacy of D-JNKI-1, a cell permeable peptide that blocks the MAPK-JNK signal pathway. The experimental studies included organ cultures of neonatal mouse cochlea exposed to an ototoxic drug and cochleae of adult guinea pigs that were exposed to either an ototoxic drug or acoustic trauma. Results obtained from the organ of Corti explants demonstrated that the MAPK-JNK signal pathway is associated with injury and that blocking of this signal pathway prevented apoptosis in areas of aminoglycoside damage. Treatment of the neomycin-exposed organ of Corti explants with D-JNKI-1 completely prevented hair cell death initiated by this ototoxin. Results from in vivo studies showed that direct application of D-JNKI-1 into the scala tympani of the guinea pig cochlea prevented nearly all hair cell death and permanent hearing loss induced by neomycin ototoxicity. Local delivery of D-JNKI-1 also prevented acoustic trauma-induced permanent hearing loss in a dose-dependent manner. These results indicate that the MAPK-JNK signal pathway is involved in both ototoxicity and acoustic trauma-induced hair cell loss and permanent hearing loss. Blocking this signal pathway with D-JNKI-1 is of potential therapeutic value for long-term protection of both the morphological integrity and physiological function of the organ of Corti during times of oxidative stress.

Figures

Figure 1.
Figure 1.
A, Schematic illustration of JIP1 and its association with components of the MAPK–JNK signaling module. The JIP1 group of scaffold proteins selectively facilitates the sequential phosphorylation cascade by a mixed-lineage kinase (MLK) to MAP kinase kinase 7 (MKK7) to c-Jun NH2-terminal kinase (JNK). The location of the JNK binding domain (JBD) of JIP1 is indicated by hatched lines. This scaffold assembly is thought to organize the MAPK–JNK signal module to respond to an oxidative stress insult to a cell. B, A schematic representation of the structural organization of the D-JNKI-1 peptide. D-JNKI-1 (30 aa) is a chimeric peptide starting with a 10 aa TAT transporter sequence, followed by a 20 aa minimal JBD sequence from the JIP1 molecule. The D-JNKI-1 peptide functions by strongly interacting with JNK, thereby preventing its interaction with and subsequent phosphorylation of c-Jun. This prevents c-Jun from interacting with itself (homodimerization) or other immediate early proteins such as c-Fos (heterodimerization) to form activator protein-1 complexes.
Figure 2.
Figure 2.
D-JNKI-1 is rapidly taken up by the hair cells in the organ of Corti explants. A–C, Three-day-old (P3) mouse organ of Corti explants incubated with FITC-conjugated D-JNKI-1 (1 μmol/l; FITC, green label) and then labeled with phalloidin-TRIC (red label) to identify the hair cells. Images were taken at 1 hr (A), 4 hr (B), and 24 hr (C) after the addition of FITC-labeled D-JNKI-1 to the medium. Green fluorescent-labeled D-JNKI-1 was initially visible at the level of a few supporting cell nuclei at 1 hr (A, arrows), was present in some hair cells (B, arrow) and cells in the greater epithelial ridge area (B, arrowheads) by 4 hr, and was present in almost all IHCs and OHCs by 24 hr (C). Scale bars, 20 μm.
Figure 3.
Figure 3.
D-JNKI-1 treatment prevents apoptosis and loss of neomycin-exposed hair cells. A–C, Confocal images of anti-myosin VIIa (red) and TUNEL (green) double-labeled P3 organ of Corti explants. A, Untreated, control explant. B, Explant exposed to 1 mm neomycin for 48 hr. C, Explant exposed to1 mm neomycin in the presence of 2 μm D-JNKI-1 for 48 hr. Neomycin exposure resulted in a severe loss of hair cells and the presence of many TUNEL-labeled nuclei within the region of the damaged auditory sensory epithelium (B). Most hair cells were already missing, but a few remaining damaged hair cells that were in the process of apoptosis are indicated by TUNEL labeling of their nuclei (arrows). D-JNKI-1 completely prevented neomycin-induced apoptotic cell death of both IHCs and OHCs; no TUNEL-positive cells were present (C). Arrowhead, IHC; rows 1–3, OHCs. Scale bars, 20 μm. D, Results of quantitative analysis of hair cell counts to determine the protective effect of D-JNKI-1 on IHCs, OHCs, and total hair cells (Total HCs) against neomycin induced loss in organ of Corti explants obtained from the middle turns of P3 mouse cochleae. Hair cell counts are presented as means ± SD (bars) from five separate experiments (n = 3–5 explants/condition). D-JNKI-1 treatment of explants provided protection against neomycin-induced hair cell loss that was highly significant (p < 0.001).
Figure 4.
Figure 4.
Neomycin exposure caused both hair cell loss and c-Jun phosphorylation in sensory cell nuclei in organ of Corti explants. Transverse plane tissue sections of P3 mouse organ of Corti explants. A, D, Untreated, control explants. B, E, Explants exposed to 1 mm neomycin for 24 hr. C, F, Explants exposed to 1 mm neomycin in the presence of D-JNKI-1 (2 μm) for 24 hr. A–C, Sections stained with hematoxylin and eosin. Nuclear condensations were present in some of the remaining hair cells in the neomycin-exposed explant (B) but were not present in either control explant hair cells (A) or in the hair cells of neomycin-exposed cultures that were protected by D-JNKI-1 (C). D–F, Sections immunostained with an anti-phospho-c-Jun antibody. Phospho-c-Jun antibody-immunolabeled hair cells were present in the neomycin-exposed explant (E) but were not detected in either control explants (D) or in the explants that had been coincubated with neomycin and D-JNKI-1 peptide for 24 hr. (F). Large arrowheads indicate the area of the IHCs, and the small arrowheads indicate the area of the OHCs. Scale bars, 5 μm. G, Results from real-time RT-PCR analysis of c-fos expression in cochlear explants. Bar graphs show relative levels of c-fos expression normalized to tubulin mRNA (n = 3). RNAs were extracted from untreated control, neomycin-exposed (24 hr), and neomycin-exposed, D-JNKI-1-treated (24hr) P3 cochlear explants, as described in Materials and Methods. Note that c-fos expression was significantly upregulated in cochlear explants after neomycin exposure and was at a near untreated, control explant level of expression in the neomycin-exposed, D-JNKI-1-treated explants.
Figure 5.
Figure 5.
Local delivery of D-JNKI-1 into the cochlea strongly protected against neomycin-induced hearing and hair cell losses. A, B, A comparison of hearing threshold shifts of contralateral unperfused left cochleae (A) to those of the D-JNKI-1-perfused right cochleae (B) from the same neomycin-treated animals (n=7). Hearing loss was calculated as the difference in decibels between auditory thresholds before neomycin treatment and after 1 d (black circles), 3 d (white circles), and 6 d (white triangle). Changes in hearing thresholds are expressed as mean values ± SEM. Note that the neomycin treatment (300 mg/kg/d during 5 d) induced dose-dependent cumulative hearing losses in the contralateral unperfused left cochleae (A) but there were no significant hearing losses in the neomycin-exposed, D-JNKI-1-perfused inner ears (B). C, D, Protective effect of 10 μm solution of D-JNKI-1 against neomycin ototoxicity on amplitude–intensity function of the CAP evoked by stimulation with 8 kHz tone bursts. Shown are the results obtained before neomycin treatment (white circles) and at 6 d after neomycin (black circles). In the contralateral, unperfused cochleae, neomycin treatment caused a reduction in the CAP amplitudes predominantly in the low portion of amplitude–intensity function (C). In contrast, there was no reduction in CAP amplitude seen in the cochleae perfused with a 10μm solution of D-JNKI-1 (D). AD, All points represent mean ± SEM values calculated from seven animals. E, F, Scanning electron micrographs from the basal turns of cochleae from the same neomycin-exposed animal. Note the extensive loss of OHCs from the organ of Corti of the basal turn of the contralateral unperfused, neomycin-exposed left cochlea. Only four OHCs remain in the area viewed in E as apposed to the 29 OHCs present in the image presented in F, in which perfusion of a 10 μm solution of D-JNKI-1 prevented loss of OHCs, O, Area of all three rows of OHCs. I, Single row of IHCs. Scale bar, 15μm. G, H, Cytocochleograms obtained from contralateral unperfused left cochleae (G; n = 3) and D-JNKI-1-perfused right cochleae (H; n = 3) of the same neomycin-treated animals 10 d after the start of neomycin injections. Cytocochleograms show the percentage of surviving IHCs (white circles) and OHCs from the first (black circles), second (dark gray circles), and third (light gray circles) rows as a function of the distance from the cochlear apex (millimeter). Note the extensive loss of OHCs in the basal turns and loss of a few IHCs in both the basal and apical turns of the contralateral unperfused left cochleae. An average of only 58.2% of the OHCs remained intact in the damaged area of the basal turns of contralateral unperfused cochleae (G). In contrast, the D-JNKI-1-perfused cochleae show only minimal losses of OHCs (i.e., 4%), which occur predominantly in the basal turns (H).
Figure 6.
Figure 6.
Perfusion of D-JNKI-1 into the scala tympani protected against acoustic trauma-induced permanent hearing loss. A, B, Hearing thresholds from contralateral noise-exposed, unperfused left cochleae (A; n = 6) and the noise-exposed right cochleae perfused with a 10 μm solution of D-JNKI-1 (B; n = 6) from the same animals. Hearing loss was calculated as the difference in decibels between auditory thresholds before acoustic trauma, 20 min (black circles) and 30 d (white circles) after noise exposure. Acoustic trauma (6 kHz, 120 dB SPL, 30 min) induced a maximum hearing loss of 60 dB when measured 20 min after exposure. Note the spontaneous, but incomplete, recovery of thresholds in the contralateral unperfused cochleae (A). Protection against a permanent hearing loss was clearly observed for the 10μm D-JNKI-1-treated cochleae, with an initial hearing loss (TTS) that was similar to the contralateral unperfused cochleae at 20 min but with a near complete recovery of hearing function by 30 d after exposure (B). C, D, Protective effect of 10 μm D-JNKI-1 against acoustic trauma on amplitude–intensity function of the CAP evoked by stimulation with 8 kHz tone bursts. Shown are the results obtained before (white circles) and 6 d after exposure to the acoustic trauma paradigm (black circles). Acoustic trauma induced a drastic decrease in the CAP amplitude for all intensity levels of sound stimulation (8 kHz) in the contralateral noise-exposed, unperfused left cochleae by day 6 (C). Note a near complete recovery of the amplitude–intensity function by 6 d after exposure in cochleae perfused with 10 μm D-JNKI-1 (D). A–D, All points represent mean ± SEM values calculated from six animals.
Figure 7.
Figure 7.
Intracochlear perfusion with D-JNKI-1, but not with D-TAT-empty or inactive JNKI-1-mut peptides, prevented the hearing loss induced by exposure to the acoustic trauma paradigm. A, Shown are the functional recoveries over time of CAP threshold shifts, for a 8 kHz pure tone from contralateral control cochleae (black circles) and cochleae perfused with artificial perilymph alone (i.e., 0 μm; white circles) or with artificial perilymph containing either 10 μm (white squares) or 100 μm (white triangles) D-JNKI-1 peptide. Note the initial rapid phase of recovery within the first 2 d, followed by a slower recovery phase in all animals. D-JNKI-1 both accelerated and improved the final functional recovery of the CAP. Time-response data have been fitted to exponential curves. B, The dose-dependent effects of D-JNKI-1 at 6 d after acoustic trauma with hearing loss at 8 kHz expressed as the percentage of recovery is shown. Dose–response data were then fitted to a curve using a nonlinear least-square logistic equation. The Boltzman equation was used for fitting sigmoid curves. The EC50 was calculated as 2.31 μm. C, A comparison a 10 μm D-TAT-empty-perfused right cochleae (white circles) with contralateral unperfused left cochleae (black circles) in the same animals exposed to acoustic trauma (n = 3) showed no significant differences (i.e., both ears showed both the same level and pattern of hearing loss). D, A comparison of a 10 μm JNKI-1-mut-perfused right cochleae (black circles) with contralateral unperfused left cochleae (white circles) in the same animals exposed to acoustic trauma (n = 3) showed similar patterns of hearing losses.
Figure 8.
Figure 8.
Local delivery of D-JNKI-1 into the scala tympani protected against acoustic trauma-induced hair cell loss. A, B, Scanning electron micrographs of areas of acoustic trauma damage in cochleae from the same noise exposed animal. In the damaged area of the contralateral unperfused cochleae, the most severe damage was observed in the row of IHCs (I) and the first row of OHCs (O), with a gradation of damage in the second and the third rows of OHCs (A). Note that direct delivery of 10 μm D-JNKI-1 into the scala tympani of the cochlea effectively prevented acoustic trauma-induced hair cell loss (B). Scale bar, 15μm. C, D, Quantitative analysis of hair cell damage consisted of counting all hair cells along the entire length of the cochlear ducts. Cochleograms represent the mean survival of hair cells as the function of the distance from the apex (in millimeters) in contralateral unperfused cochleae (C; n = 3) and in the 10μm D-JNKI-1-perfused cochleae (D; n = 3) of the same animals. Noise exposure caused a narrow band of hair cell trauma in the cochlea located 14–16 mm from the apex of the cochlea. Ninety-one percent of the IHCs (white circles) and 43% of the OHCs were lost from this area by 30 d after the initial acoustic trauma in the unprotected cochleae (C). Note the typical gradient of loss from the first row (black circles) to the second (dark gray circles) and third (light gray circles) rows of OHCs. In contrast, only 6% OHCs and 11.9% IHCs were lost as a consequence of acoustic trauma if cochleae that were treated with local application of a 10 μm solution of D-JNKI-1 (D).

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