Inhibition of H3K9 methyltransferases G9a/GLP prevents ototoxicity and ongoing hair cell death

Abstract

Sensorineural hearing loss (SNHL) is one of the most common sensory defects in humans. Hair cells are vulnerable to various ototoxic insults. Effective prevention of hair cell loss remains an unmet medical need. Apoptotic hair cell death, which involves active regulation of transcription, accounts for the majority of aminoglycoside-induced hair cells loss. As one of the important epigenetic covalent modifications, histone methylation is involved in the regulation of gene expression, development and reaction to injury. In particular, H3K9 dimethylation (H3K9me2) is critical for euchromatin gene silencing. In the present study, we examined the roles of two highly homologous histone methyltransfereases responsible for this modification, G9a/G9a-like protein (GLP), in the reaction to aminoglycoside-induced hair cell damage. We observed a rapid increase of H3K9me2 upon hair cell damage in organotypic cochlear cultures. Treatment with the G9a/GLP-specific inhibitors, BIX01294 or UNC0638, reduced the level of H3K9me2 and prevented hair cells from death. Local delivery of BIX01294 also prevented neomycin-induced in vivo auditory hair cell loss in the organ of Corti in a mouse damage model. It is unlikely that BIX01294 functions through blocking aminoglycoside absorption as it does not interfere with aminoglycoside uptaking by hair cells in the organotypic cochlear cultures. Our data revealed a novel role of histone methylation in otoprotection, which is of potential therapeutic value for SNHL management.

Figure 1

The global H3K9me2 levels increased rapidly upon hair cell damage. (a) H3K9me2 immunofluorescence was detected as punctuate staining in normal hair cells cultured as organ culture in serum-free medium. (b and c) The relative fluorescent intensity of H3K9me2 increased significantly after the addition of 1 mM neomycin for 15 min or 3 h when compared with the untreated group. (d) The H3K9me2 level increased after all of the following treatments: 100 μM cisplatin treatment for 3 h, 50 μM copper (CU) treatment for 3 h, ultraviolet (UV) irradiation for 15 min, or 1 mM neomycin treatment for 3 h, as confirmed by western blot analysis (n=12 per group in each trial) and quantified by the grey-degree numerical value in the form of mean±S.E. values obtained from three replicates. Shown in the images is the middle turn segment. The red spots represent the H3K9me2 signal, the green fluorescence shows staining of the hair cells and the nuclei are labelled with 4,6-diamidino-2-phenylindole (DAPI). Bar=10 μm. **P<0.01. DDP; cisplatin; CON, control group

Figure 2

The H3K9me2 level decreased with BIX01294 treatment in hair cells. (a) H3K9me2 level is relatively uniform across the hair cell epithelium within the organs of Corti cultured in serum-free medium. Bar=20 μm. (b) H3K9me2 significantly decreased with the addition of 2 μM BIX01294 for 24 h. Bar=20 μm. (c) Hair cell loss was observed in the presence of 10 μM BIX01294 for 24 h in the basal segment. Bar=20 μm. (d) The dose-dependent inhibition of H3K9me2 in cultured organ of Corti was confirmed by western blot analysis (n=12 per group) and quantified by the grey-degree numerical value from three replicates. Analysis of variance and SNK-q test, **P<0.01. DAPI, 4,6-diamidino-2-phenylindole. CON, control group

Figure 3

Assessment of hair cell survival and apoptosis upon neomycin treatment with or without BIX01294. (a–l) Organs of Corti were cultured in serum-free medium. They were either treated with neomycin alone (Neo), neomycin with BIX01294 pre-treatment (BIX-Neo), neomycin and BIX01294 co-treatment (co-treatment), or neomycin with BIX01294 post treatment (Neo-BIX). Shown here are the confocal images of the apical, middle, and basal turns from these four groups, respectively. The hair cells were labelled with Math1-GFP (Math1-green fluorescent protein; green), and the nuclei were DAPI (4,6-diamidino-2-phenylindole) stained (blue). Arrows in f, h, i, k, and l indicate the apoptotic bodies with condensed or fragmented nuclei. Bar=5 μm. (m) Quantitative analysis of hair cell protection by BIX01294. The samples were divided into six segments of equal length, which was called segment 1, 2, 3, 4, 5, and 6 from apex to base. We counted the number of hair cells in each visual field with the same length of each segment for each treatment group, data are expressed as mean±S.E. (the length is 360 μm, n=8 per group, *P<0.05, **P<0.01.). (n) Quantification of the apoptotic bodies (the length is 360 μm, n=8 per group, *P<0.05, **P<0.01)

Figure 4

Quantification of hair cells and apoptotic bodies induced by neomycin with or without UNC0638 pre-conditioning. (a–f) Confocal images of the apical, middle, and basal turns in the control group and UNC0638 pre-conditioned group. The hair cells were labelled with Math1-GFP (Math1-green fluorescent protein), and the nuclei were DAPI (4,6-diamidino-2-phenylindole) stained (blue). Bar=5 μm. (g and h) Quantification of the hair cells and apoptotic bodies was performed the same as above (the length is 360 μm, n=10 per group, *P<0.05, **P<0.01). CON, control group

Figure 5

Quantification of hair cells and apoptotic bodies induced by cisplatin with or without BIX01294 pre-conditioning. (a and b) Confocal images of the middle turns in the control group and BIX01294 pre-conditioned group. The hair cells were labelled with Math1-GFP (Math1-green fluorescent protein) and the nuclei were DAPI (4,6-diamidino-2-phenylindole) stained (blue). Arrows in a and b indicate the apoptotic bodies with condensed or fragmented nuclei. Bar=5 μm. (c and d) Quantification of the hair cells and apoptotic bodies was performed the same as described in Figure 4 (the length is 360 μm, n=10 per group, *P<0.05, **P<0.01)

Figure 6

BIX01294 does not interfere with the uptake of the gentamicin and FM1-43FX. (a and b) Explants were incubated with GTTR for 30 min in the absence or presence of BIX01294. The red fluorescence represents GTTR, the green fluorescence represents the hair cells, and blue represents DAPI (4,6-diamidino-2-phenylindole) staining of the nuclei. Shown here are the confocal images of the middle segments of each group. Bar=20 μm. (c and d) The hair cell function was not affected when measuring the uptake of FM1-43FX in the presence or absence of 2 μM BIX01294. GFP, green fluorescent protein

Figure 7

BIX01294 treatment affects mitochondrial apoptosis pathway. (a and b) Mitochondrial membrane permeability (MMP) staining of the organs of Corti in neomycin-induced hair cell damage model with or without BIX01294. The red fluorescence shows the live-cell imaging of the MMP tagged with TMRM, the green fluorescence represents the hair cells labelled with Math1-GFP (Math1-green fluorescent protein), and blue represents DAPI (4,6-diamidino-2-phenylindole) staining of the nuclei. Shown here are the confocal images of the middle segments of each group. The TMRM staining in the hair cells decreased significantly with the neomycin-only treatment, whereas it only slightly decreased in the presence of BIX01294. Bar=20 μm. (c) The relative fluorescence intensity of TMRM in the control group and BIX01294 pre-conditioned group (n=3 per group). (d and e) The level of cleaved caspase-3 decreased in the presence of 2 μM BIX01294 when compared with the neomycin-only-treated organ of Corti. Shown here are the confocal images of the middle segments of each group. Bar=20 μm. (f) The average level of cleaved caspase-3 was measured using western blot (n=12 per group in each trial) and plotted in the form of mean±S.E. values obtained from three replicates. β-actin was used as the loading control

Figure 8

Inhibition of G9a/GLP protects auditory hair cell death in the organ of Corti in vivo. (a–f) SEM examination of the stereocilia in the organ of Corti was performed from the apical turn to the basal turn in the neomycin-exposed animal with or without the BIX01294 pre-treatment. There is less severe hair cells loss in the BIX01294 pre-treatment group as compared with the untreated group. Bar=5 μm. (g–l) Similar results were obtained using confocal microscopy of Myosin7a (red) and nuclear (blue) staining. Bar=10 μm. (m) Quantification of the surviving hair cells at the apical, middle, and basal turns of the cochlea of each group. The length used for the statistical analysis was 100 μm, n=9 in each group. Data are expressed as mean±S.E. (Student's t-test, *P<0.05, **P<0.01). (n) A sketch was drawn to show the ‘transitional' zone for the different conditions: BIX-Neo (#) versus Neo (*). (o) The mean ABR threshold was compared between left and right ears, that is, the ears with or without BIX01294 pre-treatment, n=10 in each group. Data are expressed as mean±S.E. (Student's t-test, *P<0.05, **P<0.01)