Mouse Cochlea Research Articles

Wnt signalling facilitates neuronal differentiation of cochlear Frizzled10-positive cells in mouse cochlea via glypican 6 modulation

Degeneration of cochlear spiral ganglion neurons (SGNs) leads to irreversible sensorineural hearing loss (SNHL), as SGNs lack regenerative capacity. Although cochlear glial cells (GCs) have some neuronal differentiation potential, their specific identities remain unclear. This study identifies a distinct subpopulation, Frizzled10 positive (FZD10+) cells, as an important type of GC responsible for neuronal differentiation in mouse cochlea. FZD10 + cells can differentiate into various SGN subtypes in vivo, adhering to natural proportions. Wnt signaling enhances the ability of FZD10 + cells to function as neural progenitors and increases the neuronal excitability of the FZD10–derived neurons. Single-cell RNA sequencing analysis characterizes FZD10-derived differentiating cell populations, while crosstalk network analysis identifies multiple signaling pathways and target genes influenced by Wnt signaling that contribute to the function of FZD10 + cells as neural progenitors. Pseudotime analysis maps the differentiation trajectory from proliferated GCs to differentiating neurons. Further experiments indicate that glypican 6 (GPC6) may regulate this neuronal lineage, while GPC6 deficiency diminishes the effects of Wnt signaling on FZD10–derived neuronal differentiation and synapse formation. These findings suggest the critical role of Wnt signaling in the neuronal differentiation derived from cochlear FZD10 + cells and provide insights into the mechanisms potentially involved in this process.

MiR-145b/AP2B1 Axis Contributes to Noise-induced Sensorineural Hearing Loss In a Male Mouse Model.

Sensorineural hearing loss (SNHL) is an increasingly prevalent sensory disorder, but the underlying mechanisms remain poorly understood. Adaptor related protein complex 2 subunit beta 1 (AP2B1) has been indicated to be detectable in mature cochleae. Nonetheless, it is unclear whether AP2B1 is implicated in the progression of SNHL. Male CBA/J mice were exposed to 2-20 kHz broadband noise at 96 or 101 dB SPL to induce temporary or permanent threshold shifts (TTS or PTS). Auditory brainstem responses were measured for hearing loss evaluation. Bioinformatics analysis was used to predict the upstream miRNAs of Ap2b1. RT-qPCR and western blotting were utilized to determine miR-145b and AP2B1 expression in mouse cochleae. Luciferase reporter assay was implemented to verify the interaction between Ap2b1 and miR-145b. Bioinformatics analysis identified miR-145b as an upstream miRNA of Ap2b1. AP2B1 expression was decreased and miR-145b expression was increased in mouse cochleae after PTS noise exposure. miR-145b targeted and negatively regulated Ap2b1 in PTS noise-exposed mice. Depletion of miR-145b alleviated auditory threshold shifts and outer hair cell loss in mice with exposure to PTS noise. In conclusion, inhibition of miR-145b ameliorates noise-induced SNHL in mice by upregulating AP2B1 expression.

A rare haplotype of the GJD3 gene segregating in familial Meniere’s disease interferes with connexin assembly

BackgroundFamilial Meniere’s disease (FMD) is a rare polygenic disorder of the inner ear. Mutations in the connexin gene family, which encodes gap junction proteins, can also cause hearing loss, but their role in FMD is largely unknown.MethodsWe retrieved exome sequencing data from 94 individuals in 70 Meniere's disease (MD) families. Through gene burden analysis, we calculated the enrichment of rare variants (allele frequency < 0.05) in connexins genes in FMD individuals compared with the reference population. The connexin monomer and the homomeric connexon structural models were predicted using AlphaFold2 and HDOCK. RT-qPCR and immunofluorescence were done in mice cochleae to identify expression of the mouse ortholog candidate gene Gjd3.ResultsWe found an enrichment of rare missense variants in the GJD3 gene when comparing allelic frequencies in FMD (N = 94) with the Spanish reference population (OR = 3.9[1.92–7.91], FDR = 2.36E-03). In the GJD3 sequence, we identified a rare haplotype (TGAGT) composed of two missense, two synonymous, and one downstream variant. This haplotype was found in five individuals with FMD, segregating in three unrelated families with a total of ten individuals; and in another eight MD individuals. GJD3 encodes the gap junction protein delta 3, also known as human connexin 31.9 (Cx31.9). The protein model predicted that the NP_689343.3:p.(His175Tyr) missense variant could modify the interaction between connexins and the connexon assembly, affecting the homotypic GJD3 gap junction between cells. Our studies in mice revealed that Gjd3—encoding Gjd3 or mouse connexin 30.2 (Cx30.2)—was expressed in the organ of Corti and vestibular organs, particularly in the tectorial membrane, the base of inner and outer hair cells and the nerve fibers.ConclusionsThe present results describe a novel association between GJD3 and FMD, providing evidence that FMD is related to changes in the inner ear channels, and supporting a new role of tectorial membrane proteins in MD.

Complexes of vertebrate TMC1/2 and CIB2/3 proteins form hair-cell mechanotransduction cation channels.

Calcium and integrin-binding protein 2 (CIB2) and CIB3 bind to transmembrane channel-like 1 (TMC1) and TMC2, the pore-forming subunits of the inner-ear mechano-electrical transduction (MET) apparatus. These interactions have been proposed to be functionally relevant across mechanosensory organs and vertebrate species. Here, we show that both CIB2 and CIB3 can form heteromeric complexes with TMC1 and TMC2 and are integral for MET function in mouse cochlea and vestibular end organs as well as in zebrafish inner ear and lateral line. Our AlphaFold 2 models suggest that vertebrate CIB proteins can simultaneously interact with at least two cytoplasmic domains of TMC1 and TMC2 as validated using nuclear magnetic resonance spectroscopy of TMC1 fragments interacting with CIB2 and CIB3. Molecular dynamics simulations of TMC1/2 complexes with CIB2/3 predict that TMCs are structurally stabilized by CIB proteins to form cation channels. Overall, our work demonstrates that intact CIB2/3 and TMC1/2 complexes are integral to hair-cell MET function in vertebrate mechanosensory epithelia.

Cochlear Organ Dissection, Immunostaining, and Confocal Imaging in Mice.

The organ of Corti, located in the inner ear, is the primary organ responsible for animal hearing. Each hair cell has a V-shaped or U-shaped hair bundle composed of actin-filled stereocilia and a kinocilium supported by true transport microtubules. Damage to these structures due to noise exposure, drug toxicity, aging, or environmental factors can lead to hearing loss and other disorders. The challenge when examining auditory organs is their location within the bony labyrinth and their small and fragile nature. This protocol describes the dissection procedure for the cochlear organ, followed by confocal imaging of immunostained endogenous and fluorescent proteins. This approach can be used to understand hair cell physiology and the molecular mechanisms required for normal hearing. Key features • Protocol for the microdissection of the organ of Corti and suitable preparation for later immunostaining. • This technique involves the evaluation of mouse cochlea for planar-cell-polarity protein. • Quantitative and qualitative analysis of hair cell cilia in different dimensions.

Conditional Overexpression of Net1 Enhances the Trans-Differentiation of Lgr5+ Progenitors into Hair Cells in the Neonatal Mouse Cochlea.

Sensorineural hearing loss is mainly caused by damage to hair cells (HC), which cannot be regenerated spontaneously in adult mammals once damaged. Cochlear Lgr5+ progenitors are characterised by HC regeneration capacity in neonatal mice, and we previously screened several new genes that might induce HC regeneration from Lgr5+ progenitors. Net1, a guanine nucleotide exchange factor, is one of the screened new genes and is particularly active in cancer cells and is involved in cell proliferation and differentiation. Here, to explore invivo roles of Net1 in HC regeneration, Net1loxp/loxp mice were constructed and crossed with Lgr5CreER/+ mice to conditionally overexpress (cOE) Net1 in cochlear Lgr5+ progenitors. We observed a large number of ectopic HCs in Lgr5CreER/+Net1loxp/loxp mouse cochlea, which showed a dose-dependent effect. Moreover, the EdU assay was unable to detect any EdU+/Sox2+ supporting cells, while lineage tracing showed significantly more regenerated tdTomato+ HCs in Lgr5CreER/+Net1loxp/loxptdTomato mice, which indicated that Net1 cOE enhanced HC regeneration by inducing the direct trans-differentiation of Lgr5+ progenitors rather than mitotic HC regeneration. Additionally, qPCR results showed that the transcription factors related to HC regeneration, including Atoh1, Gfi1 and Pou4f3, were significantly upregulated and are probably the mechanism behind the HC regeneration induced by Net1. In conclusion, our study provides new evidence for the role of Net1 in enhancing HC regeneration in the neonatal mouse cochlea.

Rescue of cochlear vascular pathology prevents sensory hair cell loss in Norrie disease

Variants in the gene NDP cause Norrie disease, a severe dual-sensory disorder characterized by congenital blindness due to disrupted retinal vascular development and progressive hearing loss accompanied by sensory hair cell death. NDP encodes the secreted signaling molecule norrin. The role of norrin in the cochlea is incompletely understood. We investigated whether the Norrie disease cochlear pathology can be ameliorated in an Ndp-knockout (Ndp-KO) mouse model by conditional activation of stabilized β-catenin in vascular endothelial cells. We hypothesized that in the cochlea microvasculature, β-catenin is the primary downstream intracellular effector of norrin binding to endothelial cell surface receptors and that restoration of this signaling pathway is sufficient to prevent sensory hair cell death and hearing loss. We show that tamoxifen induction of Cdh5CreERT2;Ctnnb1flex3/+;Ndp-KO mice stabilizing β-catenin in vascular endothelial cells alone rescued defects in cochlear vascular barrier function, restored dysregulated expression of endothelial cell disease biomarkers (Cldn5, Abcb1a, Slc7a1, and Slc7a5), and prevented progressive outer hair cell death and hearing loss. Single-cell transcriptome profiling of human cochleas showed NDP expression by fibrocytes and glial cells while receptor gene expression (FZD4, TSPAN12, LRP5, and LRP6) coincided in vascular endothelial cells. Our findings support the conclusion that vascular endothelial cells are a primary target of norrin signaling in the cochlea of mice and humans and restoration of β-catenin regulation of target gene expression within cochlear endothelial cells is sufficient to maintain a cochlear microenvironment critical for hair cell survival.

AAV-regulated Serpine2 overexpression promotes hair cell regeneration

Inner ear hair cell (HC) damage is irreversible in mammals, but is has been shown that supporting cells (SCs) have the potential to differentiate into HCs. Serpine2, a serine protease inhibitor, encodes protease nexin 1, and this has been suggested to be a factor that promotes HC regeneration. In this study, we overexpressed Serpine2 in inner ear SCs cultured in two- and three-dimensional (2D and 3D) systems using the Adeno-associated virus-inner ear (AAV-ie) vector, which promoted organoid expansion and HC differentiation. Overexpression of Serpine2 in the mouse cochlea through the round window membrane (RWM) injection promoted SC proliferation and HC regeneration, and the regenerated HCs were found to be derived from Lgr5+ SCs. Regenerated HCs have electrophysiological properties that are similar to those of native HCs. Notably, Serpine2 overexpression promoted HC survival and restored hearing of neomycin-damaged mice. In conclusion, our findings indicate that Serpine2 overexpression promotes HC regeneration and suggest that the utilization of inner ear progenitor cells in combination with AAVs might be a promising therapeutic target for hearing restoration.

Developmental expression of calretinin in the mouse cochlea.

This study investigated the expression of calretinin (CR) in the mouse cochlea from embryonic day 17 (E17) to adulthood through immunofluorescence. At E17, CR immunoreactivity was only detected in the inner hair cells (IHCs). At E19, the IHCs and spiral ganglion neurons (SGNs) begin to express CR. At birth, CR immunoreactivity was confined primarily to the IHCs and the majority of the SGNs, as identified by TUJ1, both the cytoplasm and the nucleus of SGNs exhibited CR positivity. At postnatal day 2 (P2), auditory nerve fibers reaching the IHCs were stained for CR. CR continued to be expressed in the IHCs, whereas only single row of outer hair cells (OHCs) were positive for CR. By P5, CR expression was evident in IHCs and the three rows of OHCs, with SGNs soma and their neurite projections also displaying CR immunoreactivity. From P8 through adulthood, CR expression persisted in the SGNs and their afferent neurite projections to the IHCs, as well as in IHCs and OHCs. Dual labeling of CR with afferent nerve marker neurofilament 200 (NF200) demonstrated that NF 200-positive SGN somas were encompassed by CR-labeled plasma membrane of SGNs, and NF 200 was co-localized with CR in the afferent nerve fibers innervating the IHCs. We also described the expression of peripherin, a marker for type II SGNs, in the mouse cochlea at various postnatal stages. Peripherin showed a distinct spatio-temporal expression compared to CR in auditory nerve fibers. No co-expression of peripherin and CR was detected in adult. Dynamic expression patterns of CR in the embryonic and postnatal cochlea supported its roles in cochlear development.

Oxidative and endoplasmic reticulum stress in diabetes-related hearing loss: Protective effects of thioredoxin

Diabetes mellitus (DM) induces complex physiological changes in the inner ear environment. This study investigates the roles of oxidative stress (OS) and endoplasmic reticulum stress (ERS) in diabetes-related hearing loss (DRHL) and explores the potential of thioredoxin (Trx) in regulating OS, ERS, and apoptosis-related factors to mitigate the progression of hearing impairment. We conducted auditory and serological assessments in 63 patients with type 2 diabetes and 30 healthy controls. Type 2 diabetes models were induced in wild-type and Trx transgenic (Tg) mice, with auditory brainstem response (ABR) used to evaluate hearing changes. Cochlear tissues were isolated to analyse markers of apoptosis, OS, and ERS. Both patients with diabetes and mouse models exhibited hearing loss, alongside increased serum levels of Trx1, TXNIP, and AOPP, indicating oxidative damage. H&E and succinate dehydrogenase (SDH) staining revealed varying degrees of hair cell loss from the base to the apex of the cochlea in diabetic mice, with decreased expression of the hair cell protein prestin gene. Notably, Tg mice showed significant delay in hearing loss progression. In vitro, advanced glycation end-products (AGEs) induced OS and ERS in cochlear-like HEI-OC1 cells, while Trx overexpression enhanced Nrf2 activity, alleviating AGE-induced cellular stress. In conclusion, Trx exhibits protective effects against DRHL, potentially by enhancing Nrf2/HO-1/SOD2 function to reduce OS and ERS.

Morphological phenotyping of the aging cochlea in inbred C57BL/6N and outbred CD1 mouse strains.

Morphological mouse phenotyping plays a pivotal role in the translational setting and even more in the area of auditory research, where mouse is a central model organism due to the evolutionary genetic relationship and morpho-functional analogies with the human auditory system. However, some results obtained in murine models cannot be translated to humans due to the inadequate description of experimental conditions underlying poor reproducibility. We approach the characterization of the aging process of the mouse cochlea in animals up to 18 months of age belonging to two of the most used outbred (CD1) and inbred (C57BL/6N) strains. Striving to reduce any environmental variable we performed our study compliantly to the ARRIVE guidelines. We integrated instrumental data (auditory brainstem response test), with morphological analyses to correlate functional discrepancies to morphological changes and track the differences in the evolution of sensorineural hearing loss in the two strains. We featured the localization of Gipc3, Myosin VIIa, and TMC1 in hair cells of the Corti organ as well as NF 200 and the density of type I neuron in the spiral ganglion. We outlined age-related hearing loss (ARHL) in both strains, and a clear drop in the selected marker localization. However, in CD1 we detected a different trend allowing the identification of potential strain-specific mechanisms, namely an increase in myosin VIIa in 6 months aging mice in comparison to 2 months old animals. Our findings represent an asset to investigate the strain-dependent physiological trigger of ARHL providing new insights in the translational area.

Endolymphatic hydrops and cochlear synaptopathy after noise exposure are distinct sequelae of hair cell stereociliary bundle trauma

Endolymphatic hydrops, increased endolymphatic fluid within the cochlea, is the key pathologic finding in patients with Meniere’s disease, a disease of episodic vertigo, fluctuating hearing loss, tinnitus, and aural fullness. Endolymphatic hydrops also can occur after noise trauma and its presence correlates with cochlear synaptopathy, a form of hearing loss caused by reduced numbers of synapses between hair cells and auditory nerve fibers. Here we tested whether there is a mechanistic link between these two phenomena by using multimodal imaging techniques to analyze the cochleae of transgenic mice exposed to blast and osmotic challenge. In vivo cochlear imaging after blast exposure revealed dynamic increases in endolymph that involved hair cell mechanoelectrical transduction channel block but not the synaptic release of glutamate at the hair cell–auditory nerve synapse. In contrast, ex vivo and in vivo auditory nerve imaging revealed that synaptopathy requires glutamate release from hair cells but not endolymphatic hydrops. Thus, although endolymphatic hydrops and cochlear synaptopathy are both observed after noise exposure, one does not cause the other. They are simply co-existent sequelae that derive from the traumatic stimulation of hair cell stereociliary bundles. Importantly, these data argue that Meniere’s disease derives from hair cell transduction channel blockade.

The Role of Connexin26 and Connexin30 in the Mouse Cochlea of Noise-Induced Hearing Loss.

We aimed to explore the role of connexin26 (Cx26) and connexin30 (Cx30) in the cochlea in noise-induced permanent threshold shifts (PTS) and temporary threshold shift (TTS). Prospective, controlled. Laboratory. A mouse model of noise-induced PTS and TTS was constructed. Western blots were used to detect the expression of Cx26 and Cx30 in the cochlea. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were used to assess the potential biological pathways. Both the expression of Cx26 and Cx30 showed a trend of first rising and then falling in noise-induced PTS. The expression of Cx26 increased greatly in the 24 hours noise exposure (P < .05) and reached the highest level in the 4 hours after noise exposure (P < .05), then decreased gradually and returned to the control level on the seventh day after the noise exposure, when compared with the control group. The expression of Cx30 showed a similar trend in noise-induced PTS. However, both the expression of Cx26 and Cx30 showed a trend of first falling and then rising in noise induced TTS. The expression of Cx26/Cx30 reached its lowest level in the 4 hours after noise exposure (P < .05), and then increased to the control level on the second day after noise exposure (P > .05), compared with the control group. The first KEGG and GO pathway may be related with oxidative phosphorylation. Cx26 and Cx30 may have an effect in noise induced PTS and TTS. Future studies are needed to confirm the results.

Conductance properties of the α9α10 nicotinic acetylcholine receptor of neonatal mouse inner and outer hair cells

In the developing cochlea, just before the onset of hearing on postnatal day 12, the medial olivocochlear efferent axons in synaptic contact with the inner hair cells (IHCs) start withdrawing and new efferent synaptic connections are formed on the outer hair cells (OHCs), thereby progressing towards the adult pattern of medial olivocochlear efferent innervation. The synapses are inhibitory, calcium influx through the α9α10 nicotinic acetylcholine receptors (nAChRs) driving opening of calcium-dependent potassium channels. The nAChRs appear to function similarly in IHCs and OHCs, although with probable kinetic differences. Our aim was to assess their functional similarity in the neonatal mouse cochlea by making whole-cell recordings from both hair cell types between postnatal day 7 and 10 when nAChRs are expressed. ACh was applied to voltage-clamped hair cells by pressure-ejection from a pipette. The cells were dialysed with a Cs+-based solution designed to eliminate calcium-dependent potassium currents. There were differences in amplitude, voltage-sensitivity and reversal potential of the nAChR currents between IHCs and OHCs. There was also some indication that IHC nAChRs have slower activation and desensitization kinetics, although the relatively slow ACh application limited interpretation of this result. These differences, particularly concerning the reversal potential, might indicate the presence of different auxiliary protein subunits of the α9α10 receptor in neonatal IHCs and OHCs.

Stub1 promotes degradation of the activated Diaph3: A negative feedback regulatory mechanism of the actin nucleator

The formin protein Diaph3 is an actin nucleator that regulates numerous cytoskeleton-dependent cellular processes through the activation of actin polymerization. Expression and activity of Diaph3 is tightly regulated: lack of Diaph3 results in developmental defects and embryonic lethality in mice, while overexpression of Diaph3 causes auditory neuropathy. It is known that Diaph3 homophilic interactions include the intramolecular interaction of its DID-DAD domains and the intermolecular interactions of DD-DD domains or FH2-FH2 domains. However, the physiological significance of these interactions in Diaph3 protein stability and activity is not fully understood. In this study, we show that FH2-FH2 interaction promotes Diaph3 activity, while DID-DAD and DD-DD interactions inhibit Diaph3 activity through distinct mechanisms. DID-DAD interaction is responsible for the autoinhibition of Diaph3 protein, which is disrupted by binding of Rho GTPases. Interestingly, we find that DID-DAD interaction stabilizes the expression of each DID or DAD domain against proteasomal-mediated degradation. Disruption of DID-DAD interaction by RhoA binding or M1041A mutation causes increased Diaph3 activity and accelerated degradation of the activated Diaph3 protein. Further, the activated Diaph3 is ubiquitinated at K1142/1143/1144 lysine residues by the E3 ligase Stub1. Expression of Stub1 is causally related to the stability and activity of Diaph3. Knockdown of Stub1 in mouse cochlea results in hair cell stereocilia defects, neuronal degeneration and hearing loss, resembling the phenotypes of mice overexpressing Diaph3. Thus, our study reports a novel regulatory mechanism of Diaph3 protein expression and activity whereby the active but not inactive Diaph3 is readily degraded to prevent excessive actin polymerization.

The role of GDF15 in attenuating noise-induced hidden hearing loss by alleviating oxidative stress

Noise-induced hidden hearing loss (HHL) is a newly uncovered form of hearing impairment that causes hidden damage to the cochlea. Patients with HHL do not have significant abnormalities in their hearing thresholds, but they experience impaired speech recognition in noisy environments. However, the mechanisms underlying HHL remain unclear. In this study, we developed single-cell transcriptome profiles of the cochlea of mice with HHL, detailing changes in individual cell types. Our study revealed a transient threshold shift, reduced auditory brainstem response wave I amplitude, and decreased number of ribbon synapses in HHL mice. Our findings suggest elevated oxidative stress and GDF15 expression in cochlear hair cells of HHL mice. Notably, the upregulation of GDF15 attenuated oxidative stress and auditory impairment in the cochlea of HHL mice. This suggests that a therapeutic strategy targeting GDF15 may be efficacious against HHL.Graphical HHL mice had a transient threshold shift, reduced ABR wave I amplitude, and decreased number of ribbon synapses.HHL mice's cochlear hair cells exhibited increased oxidative stress and elevated GDF15 expression.Upregulation of GDF15 attenuated oxidative stress and auditory damage in the cochlea of HHL mice, implying that GDF15-targeted treatment techniques may be useful for HHL.

Calcitriol alleviates noise-induced hearing loss by regulating the ATF3/DUSP1 signalling pathway

BackgroundCalcitriol (Cal) is the most active metabolite of vitamin D and has antioxidant and anti-inflammatory properties. The aim of this study was to investigate the role of Cal in noise-induced hearing loss (NIHL) to further elucidate the mechanism of noise-induced oxidative stress in the mouse cochlea. MethodsC57BL/6 J mice were given six intraperitoneal injections of Cal (500 ng/kg/d). After 14 days of noise exposure, auditory brainstem response (ABR) thresholds, and the cochlear outer hair cell loss rate were analysed to evaluate auditory function. Real-time fluorescence quantitative PCR, immunofluorescence and western blotting were performed in vitro after the treatment of cochlear explants with 100 µM tert-butyl hydroperoxide (TBHP) for 2.5 h and HEI-OC1 cells with 250 µM TBHP for 1.5 h. ResultsIn vivo experiments confirmed that Cal pretreatment mitigated NIHL and outer hair cell death. The in vitro results demonstrated that Cal significantly reduced TBHP-induced cochlear auditory nerve fibre degradation and spiral ganglion neuron damage. Moreover, treatment with Cal inhibited the expression of oxidative stress-related factors (3-NT and 4-HNE) and DNA damage-related factors (γ-H2A.X) and attenuated TBHP-induced apoptosis in cochlear explants and HEI-OC1 cells. A total of 1479 upregulated genes and 1443 downregulated genes were screened in cochlear tissue 1 h after noise exposure. The level of transcription factor 3 (ATF3) was significantly elevated in HEI-OC1 cells after TBHP stimulation. Gene Transcription Regulation Database （GTRD）and Cistrome database analyses revealed that the downstream target gene of ATF3 is dual specificity phosphatase 1 (DUSP1). Cistrome DB Toolkit database results showed that the transcription factor of DUSP1 was ATF3. In addition, the ChIP-PCR results indicated that ATF3 might be a direct transcription factor of DUSP1. ConclusionThe results of our study suggest that Cal attenuates NIHL and inhibits noise-induced apoptosis by regulating the ATF3/DUSP1 signalling pathway.

Effects of Castanopsis echinocarpa on Sensorineural Hearing Loss via Neuronal Gene Regulation.

Sensorineural hearing loss (SNHL), characterized by damage to the inner ear or auditory nerve, is a prevalent auditory disorder. This study explores the potential of Castanopsis echinocarpa (CAE) as a therapeutic agent for SNHL. In vivo experiments were conducted using zebrafish and mouse models. Zebrafish with neomycin-induced ototoxicity were treated with CAE, resulting in otic hair cell protection with an EC50 of 0.49 µg/mL and a therapeutic index of 1020. CAE treatment improved auditory function and protected cochlear sensory cells in a mouse model after noise-induced hearing loss (NIHL). RNA sequencing of NIHL mouse cochleae revealed that CAE up-regulates genes involved in neurotransmitter synthesis, secretion, transport, and neuronal survival. Real-time qPCR validation showed that NIHL decreased the mRNA expression of genes related to neuronal function, such as Gabra1, Gad1, Slc32a1, CaMK2b, CaMKIV, and Slc17a7, while the CAE treatment significantly elevated these levels. In conclusion, our findings provide strong evidence that CAE protects against hearing loss by promoting sensory cell protection and enhancing the expression of genes critical for neuronal function and survival.

Deletion of the Ebf1, a mouse deafness gene, causes a dramatic increase in hair cells and support cells of the organ of Corti.

Following up on our previous observation that early B cell factor (EBF) sites are enriched in open chromatin of the developing sensory epithelium of the mouse cochlea, we investigated the effect of deletion of Ebf1 on inner ear development. We used a Cre driver to delete Ebf1 at the otocyst stage before development of the cochlea. We examined the cochlea at postnatal day (P) 1 and found that the sensory epithelium had doubled in size but the length of the cochlear duct was unaffected. We also found that deletion of Ebf1 led to ectopic sensory patches in the Kölliker's organ. Innervation of the developing organ of Corti was disrupted with no obvious spiral bundles. The ectopic patches were also innervated. All the extra hair cells (HCs) within the sensory epithelium and Kölliker's organ contained mechanoelectrical transduction channels, as indicated by rapid uptake of FM1-43. The excessive numbers of HCs were still present in the adult Ebf1 conditional knockout (cKO) animal. The animals had significantly elevated auditory brainstem response thresholds, suggesting that this gene is essential for hearing development.

Piplartine attenuates aminoglycoside-induced TRPV1 activity and protects from hearing loss in mice.

Hearing loss is a major health concern in our society, affecting more than 400 million people worldwide. Among the causes, aminoglycoside therapy can result in permanent hearing loss in 40% to 60% of patients receiving treatment, and despite these high numbers, no drug for preventing or treating this type of hearing loss has yet been approved by the US Food and Drug Administration. We have previously conducted high-throughput screenings of bioactive compounds, using zebrafish as our discovery platform, and identified piplartine as a potential therapeutic molecule. In the present study, we expanded this work and characterized piplartine's physicochemical and therapeutic properties. We showed that piplartine had a wide therapeutic window and neither induced nephrotoxicity in vivo in zebrafish nor interfered with aminoglycoside antibacterial activity. In addition, a fluorescence-based assay demonstrated that piplartine did not inhibit cytochrome C activity in microsomes. Coadministration of piplartine protected from kanamycin-induced hair cell loss in zebrafish and protected hearing function, outer hair cells, and presynaptic ribbons in a mouse model of kanamycin ototoxicity. Last, we investigated piplartine's mechanism of action by phospho-omics, immunoblotting, immunohistochemistry, and molecular dynamics experiments. We found an up-regulation of AKT1 signaling in the cochleas of mice cotreated with piplartine. Piplartine treatment normalized kanamycin-induced up-regulation of TRPV1 expression and modulated the gating properties of this receptor. Because aminoglycoside entrance to the inner ear is, in part, mediated by TRPV1, these results suggested that by regulating TRPV1 expression, piplartine blocked aminoglycoside's entrance, thereby preventing the long-term deleterious effects of aminoglycoside accumulation in the inner ear compartment.