Wenli Ni

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.

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.

Bmi1 regulates auditory hair cell survival by maintaining redox balance

Reactive oxygen species (ROS) accumulation are involved in noise- and ototoxic drug-induced hair cell loss, which is the major cause of hearing loss. Bmi1 is a member of the Polycomb protein family and has been reported to regulate mitochondrial function and ROS level in thymocytes and neurons. In this study, we reported the expression of Bmi1 in mouse cochlea and investigated the role of Bmi1 in hair cell survival. Bmi1 expressed in hair cells and supporting cells in mouse cochlea. Bmi1−/− mice displayed severe hearing loss and patched outer hair cell loss from postnatal day 22. Ototoxic drug-induced hair cells loss dramatically increased in Bmi1−/− mice compared with that in wild-type controls both in vivo and in vitro, indicating Bmi1−/− hair cells were significantly more sensitive to ototoxic drug-induced damage. Cleaved caspase-3 and TUNEL staining demonstrated that apoptosis was involved in the increased hair cell loss of Bmi1−/− mice. Aminophenyl fluorescein and MitoSOX Red staining showed the level of free radicals and mitochondrial ROS increased in Bmi1−/− hair cells due to the aggravated disequilibrium of antioxidant–prooxidant balance. Furthermore, the antioxidant N-acetylcysteine rescued Bmi1−/− hair cells from neomycin injury both in vitro and in vivo, suggesting that ROS accumulation was mainly responsible for the increased aminoglycosides sensitivity in Bmi1−/− hair cells. Our findings demonstrate that Bmi1 has an important role in hair cell survival by controlling redox balance and ROS level, thus suggesting that Bmi1 may work as a new therapeutic target for the prevention of hair cell death.

Wnt activation followed by Notch inhibition promotes mitotic hair cell regeneration in the postnatal mouse cochlea.

Hair cell (HC) loss is the main cause of permanent hearing loss in mammals. Previous studies have reported that in neonatal mice cochleae, Wnt activation promotes supporting cell (SC) proliferation and Notch inhibition promotes the trans-differentiation of SCs into HCs. However, Wnt activation alone fails to regenerate significant amounts of new HCs, Notch inhibition alone regenerates the HCs at the cost of exhausting the SC population, which leads to the death of the newly regenerated HCs. Mitotic HC regeneration might preserve the SC number while regenerating the HCs, which could be a better approach for long-term HC regeneration. We present a two-step gene manipulation, Wnt activation followed by Notch inhibition, to accomplish mitotic regeneration of HCs while partially preserving the SC number. We show that Wnt activation followed by Notch inhibition strongly promotes the mitotic regeneration of new HCs in both normal and neomycin-damaged cochleae while partially preserving the SC number. Lineage tracing shows that the majority of the mitotically regenerated HCs are derived specifically from the Lgr5+ progenitors with or without HC damage. Our findings suggest that the co-regulation of Wnt and Notch signaling might provide a better approach to mitotically regenerate HCs from Lgr5+ progenitor cells.

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.

Extensive Supporting Cell Proliferation and Mitotic Hair Cell Generation by In Vivo Genetic Reprogramming in the Neonatal Mouse Cochlea.

The generation of hair cells (HCs) from the differentiation of proliferating supporting cells (SCs) appears to be an ideal approach for replacing lost HCs in the cochlea and is promising for restoring hearing after damage to the organ of Corti. We show here that extensive proliferation of SCs followed by mitotic HC generation is achieved through a genetic reprogramming process involving the activation of β-catenin to upregulate Wnt signaling, the deletion of Notch1 to downregulate Notch signaling, and the overexpression of Atoh1 in Sox2+ SCs in neonatal mouse cochleae. We used RNA sequencing to compare the transcripts of the cochleae from control mice and from mice with β-catenin activation, Notch1 deletion, and β-catenin activation combined with Notch1 deletion in Sox2+ SCs. We identified the genes involved in the proliferation and transdifferentiation process that are either controlled by individual signaling pathways or by the combination of Wnt and Notch signaling. Moreover, the proliferation of SCs induced by Notch1 deletion disappears after deleting β-catenin in Notch1 knock-out Sox2+ cells, which further demonstrates that Notch signaling is an upstream and negative regulator of Wnt signaling. SIGNIFICANCE STATEMENT We show here that the extensive proliferation of supporting cells (SCs) and the subsequent mitotic hair cell (HC) generation is achieved through a genetic reprogramming process involving activation of β-catenin to upregulate Wnt signaling, deletion of Notch1 to downregulate Notch signaling, and overexpression of Atoh1 in Sox2+ SCs in neonatal mice cochleae. By comparing the transcripts of the cochleae among controls, β-catenin activation, Notch1 deletion, and β-catenin activation combined with Notch1 deletion group, we identified multiple genes involved in the proliferation and transdifferentiation process that are either controlled by individual signaling pathways or by the combination of Wnt and Notch signaling. This provides a better understanding of the mechanisms behind mitotic HC generation and might provide new approaches to stimulating mitotic HC regeneration.

Dynamic expression of Lgr6 in the developing and mature mouse cochlea

The Wnt/β-catenin signaling pathway plays important roles in mammalian inner ear development. Lgr5, one of the downstream target genes of the Wnt/β-catenin signaling pathway, has been reported to be a marker for inner ear hair cell progenitors. Lgr6 shares approximately 50% sequence homology with Lgr5 and has been identified as a stem cell marker in several organs. However, the detailed expression profiles of Lgr6 have not yet been investigated in the mouse inner ear. Here, we first used Lgr6-EGFP-Ires-CreERT2 mice to examine the spatiotemporal expression of Lgr6 protein in the cochlear duct during embryonic and postnatal development. Lgr6-EGFP was first observed in one row of prosensory cells in the middle and basal turn at embryonic day 15.5 (E15.5). From E18.5 to postnatal day 3 (P3), the expression of Lgr6-EGFP was restricted to the inner pillar cells (IPCs). From P7 to P15, the Lgr6-EGFP expression level gradually decreased in the IPCs and gradually increased in the inner border cells (IBCs). At P20, Lgr6-EGFP was only expressed in the IBCs, and by P30 Lgr6-EGFP expression had completely disappeared. Next, we demonstrated that Wnt/β-catenin signaling is required to maintain the Lgr6-EGFP expression in vitro. Finally, we demonstrated that the Lgr6-EGFP-positive cells isolated by flow cytometry could differentiate into myosin 7a-positive hair cells after 10 days in-culture, and this suggests that the Lgr6-positive cells might serve as the hair cell progenitor cells in the cochlea.

Mir-27a promotes apoptosis of cochlear sensory epithelium in Cx26 knockout mice.

To investigate the underlying molecular mechanism for connexin 26 (Cx26) knockout-induced apoptosis, we performed TUNEL assays to detect apoptosis in the cochlear sensory epithelium in Cx26 knockout mice. We also compared the miRNA expression profiles of Cx26 knockout and wild-type mice using microarray technology and bioinformatic analyses. Real-time PCR, luciferase reporter gene assays, and scala media microinjections were performed to identify the effect of a specific miRNA and its targets. The results showed that apoptosis increased in the cochlear sensory epithelium of Cx26 knockout mice. The abnormal expression of mir-27a and sgk1 in Cx26 knockout mice was verified with real-time PCR. Luciferase reporter gene assays showed that overexpression of mir-27a significantly decreased sgk1 reporter gene activity; an inhibitor of mir-27a blocked the effect. Mir-27a lentivirus also inhibited sgk1 expression in cultured cochlear tissue. Mir-27a shRNA treatment inhibited Cx26 knockout-induced apoptosis in the cochlear sensory epithelium of mice and increased the expression of sgk1 mRNA. Thus, mir-27a was identified as an apoptotic molecule that participates in Cx26 knockout-induced apoptosis in the cochlear sensory epithelium of mice by downregulating sgk1 expression.

Hedgehog Signaling Promotes the Proliferation and Subsequent Hair Cell Formation of Progenitor Cells in the Neonatal Mouse Cochlea

Hair cell (HC) loss is the major cause of permanent sensorineural hearing loss in mammals. Unlike lower vertebrates, mammalian cochlear HCs cannot regenerate spontaneously after damage, although the vestibular system does maintain limited HC regeneration capacity. Thus HC regeneration from the damaged sensory epithelium has been one of the main areas of research in the field of hearing restoration. Hedgehog signaling plays important roles during the embryonic development of the inner ear, and it is involved in progenitor cell proliferation and differentiation as well as the cell fate decision. In this study, we show that recombinant Sonic Hedgehog (Shh) protein effectively promotes sphere formation, proliferation, and differentiation of Lgr5+ progenitor cells isolated from the neonatal mouse cochlea. To further explore this, we determined the effect of Hedgehog signaling on cell proliferation and HC regeneration in cultured cochlear explant from transgenic R26-SmoM2 mice that constitutively activate Hedgehog signaling in the supporting cells of the cochlea. Without neomycin treatment, up-regulation of Hedgehog signaling did not significantly promote cell proliferation or new HC formation. However, after injury to the sensory epithelium by neomycin treatment, the over-activation of Hedgehog signaling led to significant supporting cell proliferation and HC regeneration in the cochlear epithelium explants. RNA sequencing and real-time PCR were used to compare the transcripts of the cochleae from control mice and R26-SmoM2 mice, and multiple genes involved in the proliferation and differentiation processes were identified. This study has important implications for the treatment of sensorineural hearing loss by manipulating the Hedgehog signaling pathway.

Notch Signaling Regulates Lgr5+ Olfactory Epithelium Progenitor/Stem Cell Turnover and Mediates Recovery of Lesioned Olfactory Epithelium in Mouse Model

The Notch signaling pathway regulates stem cell proliferation and differentiation in multiple tissues and organs, and is required for tissue maintenance. However, the role of Notch in regulation of olfactory epithelium (OE) progenitor/stem cells to maintain tissue function is still not clear. A recent study reported that leucine‐rich repeat‐containing G‐protein‐coupled receptor 5 (Lgr5) is expressed in globose basal cells (GBCs) localized in OE. Through lineage tracing in vivo, we found that Lgr5+ cells act as progenitor/stem cells in OE. The generation of daughter cells from Lgr5+ progenitor/stem cells is delicately regulated by the Notch signaling pathway, which not only controls the proliferation of Lgr5+ cells and their immediate progenies but also affects their subsequent terminal differentiation. In conditionally cultured OE organoids in vitro, inhibition of Notch signaling promotes neuronal differentiation. Besides, OE lesion through methimazole administration in mice induces generation of more Notch1+ cells in the horizontal basal cell (HBC) layer, and organoids derived from lesioned OE possesses more proliferative Notch1+ HBCs. In summary, we concluded that Notch signaling regulates Lgr5+ GBCs by controlling cellular proliferation and differentiation as well as maintaining epithelial cell homeostasis in normal OE. Meanwhile, Notch1 also marks HBCs in lesioned OE and Notch1+ HBCs are transiently present in OE after injury. This implies that Notch1+ cells in OE may have dual roles, functioning as GBCs in early development of OE and HBCs in restoring the lesioned OE. Stem Cells 2018;36:1259–1272