Integrated scRNAseq analyses of mouse cochlear supporting cells reveal the involvement of Ezh2 in hair cell regeneration.

BACKGROUND: In lower vertebrates like fish, the inner ear and lateral line hair cells (HCs) can regenerate after being damaged by proliferation/differentiation of supporting cells (SCs). However, the HCs of mouse cochlear could only regenerate within one to two weeks after birth but not for adults. METHODS AND RESULTS: To better understand the molecular foundations, we collected several public single-cell RNA sequencing (scRNAseq) data of mouse cochleae from E14 to P33 and extracted the prosensory and supporting cells specifically. Gene Set Enrichment Analysis (GSEA) results revealed a down-regulation of genes in Notch signaling pathway during postnatal stages (P7 and P33). We also identified 107 time-course co-expression genes correlated with developmental stage and predicated that EZH2 and KLF15 may be the key transcriptional regulators for these genes. Expressions of candidate target genes of EZH2 and KLF15 were also found in supporting cells of the auditory epithelia in chick and the neuromasts in zebrafish. Furthermore, inhibiting EZH2 suppressed regeneration of hair cells in zebrafish neuromasts and altered expressions of some developmental stage correlated genes. CONCLUSIONS: Our results extended the understanding for molecular basis of hair cell regeneration ability and revealed the potential role of Ezh2 in it.

Notch1 is required to maintain supporting cell identity and vestibular function during maturation of the mammalian balance organs.

The inner ear houses two sensory modalities: the hearing organ, located in the cochlea, and the balance organs, located throughout the vestibular regions of the ear. Both hearing and vestibular sensory regions are composed of similar cell types, including hair cells and associated supporting cells. Recently, we showed that Notch1 is required for maintaining supporting cell survival postnatally during cochlear maturation. However, it is not known whether Notch1 plays a similar role in the balance organs of the inner ear. To characterize the role of Notch during vestibular maturation, we conditionally deleted Notch1 from Sox2-expressing cells of the vestibular organs in the mouse at P0/P1. Histological analyses showed a dramatic loss of supporting cells accompanied by an increase in type II hair cells without cell death, indicating the supporting cells are converting to hair cells in the maturing vestibular regions. Analysis of 6-week old animals indicate that the converted hair cells survive, despite the reduction of supporting cells. Interestingly, measurements of vestibular sensory evoked potentials (VsEPs), known to be generated in the striolar regions of the vestibular afferents in the maculae, failed to show a response, indicating that NOTCH1 expression is critical for striolar function postnatally. Consistent with this, we find that the specialized type I hair cells in the striola fail to develop the complex calyces typical of these cells. These defects are likely due to the reduction in supporting cells, which have previously been shown to express factors critical for the striolar region. Similar to other mutants that lack proper striolar development, Notch1 mutants do not exhibit typical vestibular behaviors such as circling and head shaking, but do show difficulties in some vestibular tests, including the balance beam and forced swim test. These results indicate that, unlike the hearing organ in which the supporting cells undergo cell death, supporting cells in the balance regions retain the ability to convert to hair cells during maturation, which survive into adulthood despite the reduction in supporting cells.

Comparative biology of the amniote vestibular utricle.

The sensory epithelia of the auditory and vestibular systems of vertebrates have shared developmental and evolutionary histories. However, while the auditory epithelia show great variation across vertebrates, the vestibular sensory epithelia appear seemingly more conserved. An exploration of the current knowledge of the comparative biology of the amniote utricle, a vestibular sensory epithelium that senses linear acceleration, shows interesting instances of variability between birds and mammals. The distribution of sensory hair cell types, the position of the line of hair bundle polarity reversal and the properties of supporting cells show marked differences, likely impacting vestibular function and hair cell regeneration potential.

The Suppression of Ubiquitin C-Terminal Hydrolase L1 Promotes the Transdifferentiation of Auditory Supporting Cells into Hair Cells by Regulating the mTOR Pathway.

In mammals, hearing loss is irreversible due to the lack of the regenerative capacity of the auditory epithelium. However, stem/progenitor cells in mammalian cochleae may be a therapeutic target for hearing regeneration. The ubiquitin proteasome system plays an important role in cochlear development and maintenance. In this study, we investigated the role of ubiquitin C-terminal hydrolase L1 (UCHL1) in the process of the transdifferentiation of auditory supporting cells (SCs) into hair cells (HCs). The expression of UCHL1 gradually decreased as HCs developed and was restricted to inner pillar cells and third-row Deiters' cells between P2 and P7, suggesting that UCHL1-expressing cells are similar to the cells with Lgr5-positive progenitors. UCHL1 expression was decreased even under conditions in which supernumerary HCs were generated with a γ-secretase inhibitor and Wnt agonist. Moreover, the inhibition of UCHL1 by LDN-57444 led to an increase in HC numbers. Mechanistically, LDN-57444 increased mTOR complex 1 activity and allowed SCs to transdifferentiate into HCs. The suppression of UCHL1 induces the transdifferentiation of auditory SCs and progenitors into HCs by regulating the mTOR pathway.

Priming central sound processing circuits through induction of spontaneous activity in the cochlea before hearing onset.

Sensory systems experience a period of intrinsically generated neural activity before maturation is complete and sensory transduction occurs. Here we review evidence describing the mechanisms and functions of this 'spontaneous' activity in the auditory system. Both ex vivo and in vivo studies indicate that this correlated activity is initiated by non-sensory supporting cells within the developing cochlea, which induce depolarization and burst firing of groups of nearby hair cells in the sensory epithelium, activity that is conveyed to auditory neurons that will later process similar sound features. This stereotyped neural burst firing promotes cellular maturation, synaptic refinement, acoustic sensitivity, and establishment of sound-responsive domains in the brain. While sensitive to perturbation, the developing auditory system exhibits remarkable homeostatic mechanisms to preserve periodic burst firing in deaf mice. Preservation of this early spontaneous activity in the context of deafness may enhance the efficacy of later interventions to restore hearing.

Transient Receptor Potential (TRP) Channels in Cochlear Function: Looking Beyond Mechanotransduction.

Transient receptor potential (TRP) channels play key roles in sensory biology as transducers of various stimuli. Although these ion channels are expressed in the cochlea, their functions remain poorly understood. Recent studies by Vélez-Ortega and colleagues indicate that their expression by non-sensory supporting cells helps limit damage from acoustic trauma.

Translocation of telomerase reverse transcriptase coincided with ATP release in postnatal cochlear supporting cells.

The spontaneous bursts of electrical activity in the developing auditory system are derived from the periodic release of adenosine triphosphate (ATP) by supporting cells in the Kölliker's organ. However, the mechanisms responsible for initiating spontaneous ATP release have not been determined. Our previous study revealed that telomerase reverse transcriptase (TERT) is expressed in the basilar membrane during the first postnatal week. Its role in cochlear development remains unclear. In this study, we investigated the expression and role of TERT in postnatal cochlea supporting cells. Our results revealed that in postnatal cochlear Kölliker's organ supporting cells, TERT shifts from the nucleus into the cytoplasm over time. We found that the TERT translocation tendency in postnatal cochlear supporting cells in vitro coincided with that observed in vivo. Further analysis showed that TERT in the cytoplasm was mainly located in mitochondria in the absence of oxidative stress or apoptosis, suggesting that TERT in mitochondria plays roles other than antioxidant or anti-apoptotic functions. We observed increased ATP synthesis, release and activation of purine signaling systems in supporting cells during the first 10 postnatal days. The phenomenon that TERT translocation coincided with changes in ATP synthesis, release and activation of the purine signaling system in postnatal cochlear supporting cells suggested that TERT may be involved in regulating ATP release and activation of the purine signaling system. Our study provides a new research direction for exploring the spontaneous electrical activity of the cochlea during the early postnatal period.

Rac1 and Nectin3 are essential for PCP-directed axon guidance in the peripheral auditory system.

Our sense of hearing is critically dependent on the spiral ganglion neurons (SGNs) that connect the sound receptors in the organ of Corti (OC) to the cochlear nuclei of the hindbrain. Type I SGNs innervate inner hair cells (IHCs) to transmit sound signals, while type II SGNs (SGNIIs) innervate outer hair cells (OHCs) to detect moderate-to-intense sound. During development, SGNII afferents make a characteristic 90-degree turn toward the base of the cochlea and innervate multiple OHCs. It has been shown that the Planar Cell Polarity (PCP) pathway acts non-autonomously to mediate environmental cues in the cochlear epithelium for SGNII afferent turning towards the base. However, the underlying mechanisms are unknown. Here, we present evidence that PCP signaling regulates multiple downstream effectors to influence cell adhesion and the cytoskeleton in cochlear supporting cells (SCs), which serve as intermediate targets of SGNII afferents. We show that the core PCP gene Vangl2 regulates the localization of the small GTPase Rac1 and the cell adhesion molecule Nectin3 at SC-SC junctions through which SGNII afferents travel. Through in vivo genetic analysis, we also show that loss of Rac1 or Nectin3 partially phenocopied SGNII peripheral afferent turning defects in Vangl2 mutants, and that Rac1 plays a non-autonomous role in this process in part by regulating PCP protein localization at the SC-SC junctions. Additionally, epistasis analysis indicates that Nectin3 and Rac1 likely act in the same genetic pathway to control SGNII afferent turning. Together, these experiments identify Nectin3 and Rac1 as novel regulators of PCP-directed SGNII axon guidance in the cochlea.

Effects of lycium barbarum polysaccharides on cis-dichlorodiamineplatinum (II)-induced apoptosis in mouse testis sertoli cells TM4 / 中国临床药理学与治疗学

AIM: To explore the effect of Lycium barbarum polysaccharides (LBP) on cis-dichlorodiamineplatinum (II) (CDDP)-induced apoptosis in mouse testis sertoli cells TM4 and its possible mechanism. METHODS: TM4 cells were cultured in vitro, the effect of LBP on the survival rate of TM4 cells induced by CDDP was detected by MTT assay, the effect of LBP on the expression of apoptosis related genes Bcl-2, Bax and Caspase-3 in TM4 cells induced by CDDP was detected by Western blot, and the change of cell apoptosis rate was detected by flow cytometry. RESULTS: Compared with control group, TM4 cell apoptosis was significantly increased in CDDP group, the expression of anti-apoptotic gene Bcl-2 and pro-caspase-3 in proenzyme state were significantly decreased, the expression of pro-apoptotic gene Bax and caspase-3 were significantly increased. Compared with CDDP group, the apoptosis of TM4 cells in CDDP+LBP group was significantly decreased, the expression levels of anti-apoptotic genes Bcl-2 and Pro-Caspase3 were significantly increased, the expression levels of pro-apoptotic gene Bax and Caspase-3 were significantly decreased. CONCLUSION: LBP, by acting on CDDP induced TM4 cells, can inhibit CDDP induced TM4 cell apoptosis by enhancing the expression of Bcl-2 and inhibiting the expression of Bax and Caspase-3, thus alleviating the damage caused by CDDP to TM4 cells.

Histology and immunocytochemical localization of glial fibrillary acidic protein in the inner ear of Scincella tsinlingensis / Histología y localización inmunocitoquímica de la proteína ácida fibrilar glial en el oído interno de Scincella tsinlingensis

SUMMARY: The microstructure of inner ear in Scincella tsinlingensis was observed by light microscopy and the expression of glial fibrillary acidic protein (GFAP) in membranous labyrinth among the juvenile age group, subadult age group and adult age group were also detected by methods of immunohistochemistry. The inner ear in S. tsinlingensis resembled those in other Scincid lizards in their anatomy and histology. Large and elongate cochlear duct was slightly bowed or arched laterally. There was no hint of limbic modifications and the limbic lip was absent in cochlear recess. The basilar papilla elongated anteroventrally possessed specialized tectorial sallets. GFAP staining was significantly distributed in supporting cells of the sensory epithelia of cochlear duct, while the utricular macula and canal ampullae showed immunopositive for the GFAP antibody, with weaker staining in the saccular macula. The membranous inner ear of three different age groups revealed the similar pattern of GFAP expression, which suggested that the distribution of supporting cells were independent of age in S. tsinlingensis.

RESUMEN: La microestructura del oído interno en Scincella tsinlingensis fue analizada mediante microscopía óptica y por otra parte, fue cuantificada la expresión de la proteína ácida fibrilar glial (GFAP) en el laberinto membranoso, entre los grupos de edad juvenil, subadulto y adulto, utilizándose métodos inmunohistoquímicos. El oído interno de S. tsinlingensis se asemejaba al de otros lagartos Scincid tanto en su anatomía como en su histología. El conducto coclear mayor estaba ligeramente arqueado o arqueado lateralmente. No había indicios de modificaciones límbicas y no se evidenció el labio en el receso coclear. La papila basilar alargada anteroventralmente poseía sallets tectoriales especializados. La tinción de GFAP se distribuyó significativamente en las células del epitelio sensorial del conducto coclear, mientras que la mácula utricular y la ampolla del canal mostraron inmunopositividad para el anticuerpo GFAP, con una tinción más débil en la mácula sacular. El oído interno membranoso de los tres grupos de edad diferentes reveló un patrón similar de expresión de GFAP, lo que sugiere que la distribución de las células de soporte son independiente de la edad en S. tsinlingensis.

Outward Rectifying Current in Isolated Deiters' Cells from Guinea Pig Cochlea / 대한이비인후과학회지

BACKGROUND AND OBJECTIVES: The Deiters' cell is one of the supporting cells in the organ of Corti and is known to possibly regulate the signal transduction pathway in the organ of Corti. The signal transduction process can be modulated by ATP and acetylcholine, the so-called neurotransmitters, in Deiters' cells. Intracellular Ca2+ concentration can be also increased by these neurotransmitters and the control mechanism on the organ of Corti is highly suggested in Deiters' cells. Potassium ion (K+) is known to be important both in hair cells and supporting cells. Through K+ channel, the membrane potential may be controlled and the signal transduction pathway can be regulated. Furthermore, the motility of outer hair cell and the signal transduction from the apical stereocilia are considered to be regulated by this channel. The aim of this study is to record the K+ current in the isolated Deiters' cells from guinea pig cochlea. MATERIALS AND METHODS: Deiters' cells were isolated from the organ of Corti of guinea pig by using collagenase and a pipet. A whole cell patch clamp was performed under the inverted microscope and the current was measured with List-7 amplifier and pClamp 8.0.2 software. RESULTS: The resting membrane potential was -15.02+/-2.66 mV (n=6). When the cell membrane was hyperpolarized into -110 mV from the -40 mV holding potential, the peak current was -227+/-39.9 pA (n=15). After having depolarized to the maximum, (50 mV), the peak current was 7123+/-737 pA, and the reversal potentials of different external K+ concentration changed in the K+-dependent manner. About 80% of this current was inhibited by TEA. When K+ was substituted by Cs+, the peak current was 1788+/-231 pA at 50 mV step pulse. Activation curve of this outward current showed two different Vh (half activation voltage) and K (slope factor). CONCLUSION: Outward rectifying K+ channels exist in Deiters' cells and they can be inhibited by TEA and permeable to Cs+. More than two types of K+ current can exist and they may play a role in the recovery of membrane potential after depolarization,