Efferent neurons control hearing sensitivity and protect hearing from noise through the regulation of gap junctions between cochlear supporting cells

It is critical for hearing that the descending cochlear efferent system provide a negative feedback to hair cells to regulate hearing sensitivity and provide the protection of hearing from noise. Here, we report that the medial olivocochlear (MOC) efferent nerves, which project to outer hair cells (OHCs), also could innervate OHC surrounding supporting cells (SCs) to regulate hearing sensitivity. MOC nerve fibers are cholinergic and acetylcholine (ACh) is a primary neurotransmitter. MOC nerve endings, presynaptic vesicular acetylcholine transporters (VAChT), and postsynaptic ACh receptors were visible in SCs and the SC area. Application of ACh in the SC could evoke a typical inward current, which reduced gap junctions (GJs) between SCs and consequently declined OHC electromotility, which is an active cochlear amplification and can increase hearing sensitivity. This indirect, GJ-mediated inhibition enhanced the direct inhibition of ACh on OHC electromotility but had long-lasting influence. In vivo experiments further demonstrated that deficiency of this GJ-mediated efferent pathway declined the regulation of active cochlear amplification and compromised the protection against noise. In particular, distortion production otoacoustic emission (DPOAE) showed a delayed reduction after noise exposure. Our findings reveal a new pathway for the MOC efferent system via innervating SCs to control active cochlear amplification and hearing sensitivity. These data also suggest that this GJ-mediated efferent pathway may play a critical role in the long-term efferent inhibition and is required for protecting hearing from noise trauma.

Virus-infection in cochlear supporting cells induces audiosensory receptor hair cell death by TRAIL-induced necroptosis

Although sensorineural hearing loss (SHL) is relatively common, its cause has not been identified in most cases. Previous studies have suggested that viral infection is a major cause of SHL, especially sudden SHL, but the system that protects against pathogens in the inner ear, which is isolated by the blood-labyrinthine barrier, remains poorly understood. We recently showed that, as audiosensory receptor cells, cochlear hair cells (HCs) are protected by surrounding accessory supporting cells (SCs) and greater epithelial ridge (GER or Kölliker’s organ) cells (GERCs) against viral infections. Here, we found that virus-infected SCs and GERCs induce HC death via production of the tumour necrosis factor-related apoptosis-inducing ligand (TRAIL). Notably, the HCs expressed the TRAIL death receptors (DR) DR4 and DR5, and virus-induced HC death was suppressed by TRAIL-neutralizing antibodies. TRAIL-induced HC death was not caused by apoptosis, and was inhibited by necroptosis inhibitors. Moreover, corticosteroids, the only effective drug for SHL, inhibited the virus-induced transformation of SCs and GERCs into macrophage-like cells and HC death, while macrophage depletion also inhibited virus-induced HC death. These results reveal a novel mechanism underlying virus-induced HC death in the cochlear sensory epithelium and suggest a possible target for preventing virus-induced SHL.

Lineage-tracing and translatomic analysis of damage-inducible mitotic cochlear progenitors identifies candidate genes regulating regeneration

Cochlear supporting cells (SCs) are glia-like cells critical for hearing function. In the neonatal cochlea, the greater epithelial ridge (GER) is a mitotically quiescent and transient organ, which has been shown to nonmitotically regenerate SCs. Here, we ablated Lgr5+ SCs using Lgr5-DTR mice and found mitotic regeneration of SCs by GER cells in vivo. With lineage tracing, we show that the GER houses progenitor cells that robustly divide and migrate into the organ of Corti to replenish ablated SCs. Regenerated SCs display coordinated calcium transients, markers of the SC subtype inner phalangeal cells, and survive in the mature cochlea. Via RiboTag, RNA-sequencing, and gene clustering algorithms, we reveal 11 distinct gene clusters comprising markers of the quiescent and damaged GER, and damage-responsive genes driving cell migration and mitotic regeneration. Together, our study characterizes GER cells as mitotic progenitors with regenerative potential and unveils their quiescent and damaged translatomes.

Cochlear organoids reveal epigenetic and transcriptional programs of postnatal hair cell differentiation from supporting cells

We explored the transcriptional and epigenetic programs underlying the differentiation of hair cells from postnatal progenitor cells in cochlear organoids. Heterogeneity in the cells including cells with the transcriptional signatures of mature hair cells allowed a full picture of possible cell fates. Construction of trajectories identified Lgr5+ cells as progenitors for hair cells and the genomic data revealed gene regulatory networks leading to hair cells. We validated these networks, demonstrating dynamic changes both in expression and predicted binding sites of these transcription factors during organoid differentiation. We identified known regulators of hair cell development, Atoh1, Pou4f3, and Gfi1, and predicted novel regulatory factors, Tcf4, an E-protein and heterodimerization partner of Atoh1, and Ddit3, a CCAAT/enhancer-binding protein (C/EBP) that represses Hes1 and activates transcription of Wnt signaling-related genes. Deciphering the signals for hair cell regeneration from mammalian cochlear supporting cells reveals candidates for HC regeneration which is limited in the adult.

Dual expression of Atoh1 and Ikzf2 promotes transformation of adult cochlear supporting cells into outer hair cells

Mammalian cochlear outer hair cells (OHCs) are essential for hearing. Severe hearing impairment follows OHC degeneration. Previous attempts at regenerating new OHCs from cochlear supporting cells (SCs) have been unsuccessful, notably lacking expression of the key OHC motor protein, Prestin. Thus, regeneration of Prestin+ OHCs represents a barrier to restore auditory function in vivo. Here, we reported the successful in vivo conversion of adult mouse cochlear SCs into Prestin+ OHC-like cells through the concurrent induction of two key transcriptional factors known to be necessary for OHC development: Atoh1 and Ikzf2. Single-cell RNA sequencing revealed the upregulation of 729 OHC genes and downregulation of 331 SC genes in OHC-like cells. The resulting differentiation status of these OHC-like cells was much more advanced than previously achieved. This study thus established an efficient approach to induce the regeneration of Prestin+ OHCs, paving the way for in vivo cochlear repair via SC transdifferentiation.

GATA3 maintains the quiescent state of cochlear supporting cells by regulating p27kip1

Haplo-insufficiency of the GATA3 gene causes hypoparathyroidism, sensorineural hearing loss, and renal disease (HDR) syndrome. Previous studies have shown that Gata3 is required for the development of the prosensory domain and spiral ganglion neurons (SGNs) of the mouse cochlea during embryogenesis. However, its role in supporting cells (SCs) after cell fate specification is largely unknown. In this study, we used tamoxifen-inducible Sox2CreERT2 mice to delete Gata3 in SCs of the neonatal mouse cochlea and showed that loss of Gata3 resulted in the proliferation of SCs, including the inner pillar cells (IPCs), inner border cells (IBCs), and lateral greater epithelium ridge (GER). In addition, loss of Gata3 resulted in the down-regulation of p27kip1, a cell cycle inhibitor, in the SCs of Gata3-CKO neonatal cochleae. Chromatin immunoprecipitation analysis revealed that GATA3 directly binds to p27kip1 promoter and could maintain the quiescent state of cochlear SCs by regulating p27kip1 expression. Furthermore, RNA-seq analysis revealed that loss of Gata3 function resulted in the change in the expression of genes essential for the development and function of cochlear SCs, including Tectb, Cyp26b1, Slitrk6, Ano1, and Aqp4.

Dual expression of Atoh1 and Ikzf2 promotes transformation of adult cochlear supporting cells into outer hair cells

ABSTRACTMammalian cochlear outer hair cells (OHCs) are essential for hearing. OHC degeneration causes severe hearing impairment. Previous attempts of regenerating new OHCs from cochlear supporting cells (SCs) had yielded cells lacking Prestin, a key motor protein for OHC function. Thus, regeneration of Prestin+ OHCs remains a challenge for repairing OHC damage in vivo. Here, we reported that successful in vivo conversion of adult cochlear SCs into Prestin+ OHC-like cells could be achieved by simultaneous expression of Atoh1 and Ikzf2, two key transcriptional factors necessary for OHC development. New OHC-like cells exhibited upregulation of hundreds of OHC genes and downregulation of SC genes. Single cell transcriptomic analysis demonstrated that the differentiation status of these OHC-like cells was much more advanced than previously achieved. Thus, we have established an efficient approach to promote regeneration of Prestin+ OHCs and paved the way for repairing damaged cochlea in vivo via transdifferentiation of SCs.

Efferent control of hearing sensitivity and protection via inner ear supporting cells

It is critical for hearing that the descending cochlear efferent system provide negative feedback to hair cells to regulate hearing sensitivity and provide protection from noise. The medial olivocochlear (MOC) efferent nerves project to outer hair cells (OHCs) and inhibit OHC electromotility, which is an active cochlear amplification and can increase hearing sensitivity. Here, we report that the MOC efferent nerves also have functional innervation with the cochlear supporting cells to regulate hearing sensitivity. The MOC efferent nerve fibers and the corresponding MOC neurotransmitter acetylcholine (ACh) receptors were visible in the cochlear supporting cells. Application of ACh in the cochlear supporting cells could also evoke inward currents in a dose-dependent manner and reduced gap junctional (GJ) coupling between the cochlear supporting cells, which consequently declined electromotility in OHCs. This indirect inhibitory effect through the mediated GJs between the cochlear supporting cells on OHC electromotility was consistent and enhanced the direct inhibition of ACh on OHC electromotility but had long-lasting influence. In vivo experiments further demonstrated that deficiency of this GJ-mediated efferent control pathway declined the regulation of active cochlear amplification and impaired the protection from noise trauma. Our findings reveal a new pathway for the cochlear efferent system to control hearing sensitivity, and also demonstrate that this supporting cell GJ-mediated efferent pathway is critical for control of hearing sensitivity and the protection of hearing from noise trauma.

Combinatorial Atoh1 and Gfi1 induction enhances hair cell regeneration in the adult cochlea

Mature mammalian cochlear hair cells (HCs) do not spontaneously regenerate once lost, leading to life-long hearing deficits. Attempts to induce HC regeneration in adult mammals have used over-expression of the HC-specific transcription factor Atoh1, but to date this approach has yielded low and variable efficiency of HC production. Gfi1 is a transcription factor important for HC development and survival. We evaluated the combinatorial effects of Atoh1 and Gfi1 over-expression on HC regeneration using gene transfer methods in neonatal cochlear explants, and in vivo in adult mice. Adenoviral over-expression of Atoh1 and Gfi1 in cultured neonatal cochlear explants resulted in numerous ectopic HC-like cells (HCLCs), with significantly more cells in Atoh1 + Gfi1 cultures than Atoh1 alone. In vitro, ectopic HCLCs emerged in regions medial to inner HCs as well as in the stria vascularis. In vivo experiments were performed in mature Pou4f3DTR mice in which HCs were completely and specifically ablated by administration of diphtheria toxin. Adenoviral expression of Atoh1 or Atoh1 + Gfi1 in cochlear supporting cells induced appearance of HCLCs, with Atoh1 + Gfi1 expression leading to 6.2-fold increase of new HCLCs after 4 weeks compared to Atoh1 alone. New HCLCs were detected throughout the cochlea, exhibited immature stereocilia and survived for at least 8 weeks. Combinatorial Atoh1 and Gfi1 induction is thus a promising strategy to promote HC regeneration in the mature mammalian cochlea.