Yingzi He

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

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.

Wnt activation protects against neomycin-induced hair cell damage in the mouse cochlea

Recent studies have reported the role of Wnt/β-catenin signaling in hair cell (HC) development, regeneration, and differentiation in the mouse cochlea; however, the role of Wnt/β-catenin signaling in HC protection remains unknown. In this study, we took advantage of transgenic mice to specifically knockout or overactivate the canonical Wnt signaling mediator β-catenin in HCs, which allowed us to investigate the role of Wnt/β-catenin signaling in protecting HCs against neomycin-induced damage. We first showed that loss of β-catenin in HCs made them more vulnerable to neomycin-induced injury, while constitutive activation of β-catenin in HCs reduced HC loss both in vivo and in vitro. We then showed that loss of β-catenin in HCs increased caspase-mediated apoptosis induced by neomycin injury, while β-catenin overexpression inhibited caspase-mediated apoptosis. Finally, we demonstrated that loss of β-catenin in HCs led to increased expression of forkhead box O3 transcription factor (Foxo3) and Bim along with decreased expression of antioxidant enzymes; thus, there were increased levels of reactive oxygen species (ROS) after neomycin treatment that might be responsible for the increased aminoglycoside sensitivity of HCs. In contrast, β-catenin overexpression reduced Foxo3 and Bim expression and ROS levels, suggesting that β-catenin is protective against neomycin-induced HC loss. Our findings demonstrate that Wnt/β-catenin signaling has an important role in protecting HCs against neomycin-induced HC loss and thus might be a new therapeutic target for the prevention of HC death.

Trans-2-phenylcyclopropylamine regulates zebrafish lateral line neuromast development mediated by depression of LSD1 activity

The zebrafish mechanosensory lateral line (LL) is a model system for the study of hair cell development, survival and regeneration. Recently, histone modifications have attracted a considerable amount of interest because of their indispensable roles in various kinds of cellular processes including differentiation, proliferation, apoptosis and function. Lysine specific demethylase 1 (LSD1) is an important enzyme that regulates histone methylation. As a transcriptional regulator, this enzyme has broad functional activities and is involved in many biological processes. However, the effects of LSD1 on the early development of zebrafish sensory system have not been fully elucidated. Here, we have found that pharmacological inhibition of LSD1 with the monoamine oxidase (MAO) inhibitor trans-2-phenylcyclopropylamine (referred to as 2-PCPA) reduced the numbers of both sensory hair cells and supporting cells of neuromasts during zebrafish development. Our results showed that the treatment of zebrafish larvae with 2-PCPA caused accumulation of histone methylation and suppressed proliferation of neuromast cells. Finally, acridine orange staining assay demonstrated that 2-PCPA treatment at high concentrations induced an enhancement of cellular apoptosis within neuromasts. Taken together, these results indicate that LSD1 demethylase activity is required for neuromast development in zebrafish larvae.

Cotransfection of Pax2 and Math1 promote in situ cochlear hair cell regeneration after neomycin insult.

The ideal strategy for hair cell regeneration is promoting residual supporting cell proliferation followed by induction of hair cell differentiation. In this study, cultured neonatal mouse organs of Corti were treated with neomycin to eliminate hair cells followed by incubation with recombined adenovirus expressing Pax2 and/or Math1. Results showed that overexpression of Pax2 significantly promoted proliferation of supporting cells. The number of BrdU+/myosin VIIA+ cells increased significantly in hair cell pre-existing region two weeks after adenovirus infection in Ad-Pax2-IRES-Math1 group compared to Ad-Pax2 and Ad-Math1 groups. This indicated that cotransfection of Pax2 and Math1 induced supporting cells to proliferate and differentiate into hair cells in situ. Most new hair cells were labeled by FM1-43 suggesting they acquired certain function. The results also suggest that inducing proliferating cells rather than quiescent cells to differentiate into hair cells by forced expression of Math1 is feasible for mammalian hair cell regeneration.

Effect of histone deacetylase inhibitors trichostatin A and valproic acid on hair cell regeneration in zebrafish lateral line neuromasts.

In humans, auditory hair cells are not replaced when injured. Thus, cochlear hair cell loss causes progressive and permanent hearing loss. Conversely, non-mammalian vertebrates are capable of regenerating lost sensory hair cells. The zebrafish lateral line has numerous qualities that make it well-suited for studying hair cell development and regeneration. Histone deacetylase (HDAC) activity has been shown to have an important role in regenerative processes in vertebrates, but its function in hair cell regeneration in vivo is not fully understood. Here, we have examined the role of HDAC activity in hair cell regeneration in the zebrafish lateral line. We eliminated lateral line hair cells of 5-day post-fertilization larvae using neomycin and then treated the larvae with HDAC inhibitors. To assess hair cell regeneration, we used 5-bromo-2-deoxyuridine (BrdU) incorporation in zebrafish larvae to label mitotic cells after hair cell loss. We found that pharmacological inhibition of HDACs using trichostatin A (TSA) or valproic acid (VPA) increased histone acetylation in the regenerated neuromasts following neomycin-induced damage. We also showed that treatment with TSA or VPA decreased the number of supporting cells and regenerated hair cells in response to hair cell damage. Additionally, BrdU immunostaining and western blot analysis showed that TSA or VPA treatment caused a significant decrease in the percentage of S-phase cells and induced p21Cip1 and p27Kip1 expression, both of which are likely to explain the decrease in the amount of newly regenerated hair cells in treated embryos. Finally, we showed that HDAC inhibitors induced no observable cell death in neuromasts as measured by cleaved caspase-3 immunohistochemistry and western blot analysis. Taken together, our results demonstrate that HDAC activity has an important role in the regeneration of hair cells in the lateral line.

In vivo overexpression of X-linked inhibitor of apoptosis protein protects against neomycin-induced hair cell loss in the apical turn of the cochlea during the ototoxic-sensitive period

Aminoglycoside-induced cochlear ototoxicity causes hair cell (HC) loss and results in hearing impairment in patients. Previous studies have developed the concept of an ototoxicity-sensitive period during which the cochleae of young mice are more vulnerable to auditory trauma than adults. Here, we compared neomycin-induced ototoxicity at the following four developmental ages in mice: postnatal day (P)1–P7, P8–P14, P15–P21, and P60–P66. We found that when neomycin was administered between P8 and P14, the auditory brainstem response threshold increase was significantly higher at low frequencies and HC loss was significantly greater in the apical turn of the cochlea compared to neomycin administration during the other age ranges. Quantitative real-time PCR (qPCR) data revealed that the expression of apoptotic markers, including Casp3 and Casp9, was significantly higher when neomycin was injected from P8 to P14, while the expression of the X-linked inhibitor of apoptosis protein (XIAP) gene was significantly higher when neomycin was injected from P60 to P66. Because XIAP expression was low during the neomycin-sensitive period, we overexpressed XIAP in mice and found that it could protect against neomycin-induced hearing loss at low frequencies and HC loss in the apical turn of the cochlea. Altogether, our findings demonstrate a protective role for XIAP against neomycin-induced hearing loss and HC loss in the apical turn of the cochlea during the ototoxic-sensitive period, and suggest that apoptotic factors mediate the effect of neomycin during the ototoxic-sensitive period.

Role of histone deacetylase activity in the developing lateral line neuromast of zebrafish larvae.

Histone deacetylases are involved in many biological processes and have roles in regulating cell behaviors such as cell cycle entry, cell proliferation and apoptosis. However, the effect of histone deacetylases on the development of hair cells (HCs) has not been fully elucidated. In this study, we examined the influence of histone deacetylases on the early development of neuromasts in the lateral line of zebrafish. Hair cell development was evaluated by fluorescent immunostaining in the absence or presence of histone deacetylase inhibitors. Our results suggested that pharmacological inhibition of histone deacetylases with inhibitors, including trichostatin A, valproic acid and MS-275, reduced the numbers of both HCs and supporting cells in neuromasts. We also found that the treatment of zebrafish larvae with inhibitors caused accumulation of histone acetylation and suppressed proliferation of neuromast cells. Real-time PCR results showed that the expression of both p21 and p27 mRNA was increased following trichostatin A treatment and the increase in p53 mRNA was modest under the same conditions. However, the expression of p53 mRNA was significantly increased by treatment with a high concentration of trichostatin A. A high concentration of trichostatin A also led to increased cell death in neuromasts as detected in a TUNEL assay. Moreover, the nuclei of most of these pyknotic cells were immunohistochemically positive for cleaved caspase-3. These results suggest that histone deacetylase activity is involved in lateral line development in the zebrafish and might have a role in neuromast formation by altering cell proliferation through the expression of cell cycle regulatory proteins.

LSD1 is Required for Hair Cell Regeneration in Zebrafish

Lysine-specific demethylase 1 (LSD1/KDM1A) plays an important role in complex cellular processes such as differentiation, proliferation, apoptosis, and cell cycle progression. It has recently been demonstrated that during development, downregulation of LSD1 inhibits cell proliferation, modulates the expression of cell cycle regulators, and reduces hair cell formation in the zebrafish lateral line, which suggests that LSD1-mediated epigenetic regulation plays a key role in the development of hair cells. However, the role of LSD1 in hair cell regeneration after hair cell loss remains poorly understood. Here, we demonstrate the effect of LSD1 on hair cell regeneration following neomycin-induced hair cell loss. We show that the LSD1 inhibitor trans-2-phenylcyclopropylamine (2-PCPA) significantly decreases the regeneration of hair cells in zebrafish after neomycin damage. In addition, immunofluorescent staining demonstrates that 2-PCPA administration suppresses supporting cell proliferation and alters cell cycle progression. Finally, in situ hybridization shows that 2-PCPA significantly downregulates the expression of genes related to Wnt/β-catenin and Fgf activation. Altogether, our data suggest that downregulation of LSD1 significantly decreases hair cell regeneration after neomycin-induced hair cell loss through inactivation of the Wnt/β-catenin and Fgf signaling pathways. Thus, LSD1 plays a critical role in hair cell regeneration and might represent a novel biomarker and potential therapeutic approach for the treatment of hearing loss.

Inhibition of H3K4me2 Demethylation Protects Auditory Hair Cells from Neomycin-Induced Apoptosis

Aminoglycoside-induced hair cell loss is a major cause of hearing impairment in children and deserves more attention in medical research. Epigenetic mechanisms have been shown to protect hair cells from ototoxic drugs. In this study, we focused on the role of dimethylated histone H3K4 (H3K4me2) in hair cell survival. To investigate the effects of lysine-specific demethylase 1 (LSD1)—the histone demethylase primarily responsible for demethylating H3K4me2—on neomycin-induced hair cell loss, isolated cochleae were pretreated with LSD1 inhibitors followed by neomycin exposure. There was a severe loss of hair cells in the organ of Corti after neomycin exposure, and inhibition of LSD1 significantly protected against neomycin-induced hair cell loss. H3K4me2 expression in the nuclei of hair cells decreased after exposure to neomycin, and blocking the decreased expression of H3K4me2 with LSD1 inhibitors prevented hair cell loss. Local delivery of these inhibitors in vivo also protected hair cells from neomycin-induced ototoxicity and maintained the hearing threshold in mice as determined by auditory brain stem response. This inhibition of neomycin-induced apoptosis occurs via reduced caspase-3 activation. Together, our findings demonstrate the protective role for H3K4me2 against neomycin-induced hair cell loss and hearing loss.

MiR-182-5p protects inner ear hair cells from cisplatin-induced apoptosis by inhibiting FOXO3a

Cisplatin is widely used for chemotherapy of a variety of malignancies. However, the clinical application of cisplatin is hampered by the resultant irreversible hearing loss due to hair cell apoptosis. To date, no practical regimen to resolve this has been developed. Meanwhile, the role of microRNA in protecting hair cells from cisplatin-induced apoptosis in the inner ear has not been extensively investigated. In this study, we monitored miR-183, -96, and -182 turnover in the cochlea during cisplatin treatment in vitro. We found that overexpression of miR-182, but not miR-183 and -96, improved hair cell survival after 3 μM cisplatin treatment in vitro. We demonstrated that overexpression of miR-182 repressed the intrinsic apoptotic pathway by inhibiting the translation of FOXO3a. Our study offers a new therapeutic target for alleviating cisplatin-induced hair cell apoptosis in a rapid and tissue-specific manner.