Hair Cell Function Research Articles

Therapeutic Effects of GDF6-Overexpressing Mesenchymal Stem Cells through Upregulation of the GDF15/SIRT1 Axis in Age-Related Hearing Loss.

It has been reported the therapeutic effects of mesenchymal stem cells (MSCs) on hearing loss. This study explored the therapeutic effects of growth differentiation factor 6 (GDF6) overexpression-induced MSCs (MSCs-GDF6) on age-related hearing loss (ARHL) and its underlying mechanisms. Reverse transcription-quantitative PCR and western blotting were used to evaluate gene expression. Flow cytometry and immunofluorescence assays were performed for the detection of apoptosis and autophagy, respectively. Hearing function and loss of outer hair cells (HCs) in ARHL rats were measured using the auditory brainstem response and cochlear silver nitrate staining, respectively. MSC proliferation was evaluated with the Cell Counting Kit-8 assay. Growth differentiation factor 15 (GDF15) and sirtuin 1 (SIRT1) expression was significantly decreased in hydrogen peroxide (H2O2)-induced House Ear Institute-Organ of Corti 1 (HEI-OC1) cells and the cochlea of ARHL rats. Elevated apoptosis and blocked autophagic flux were uncovered in H2O2-induced HEI-OC1 cells and ARHL rats. GDF15 overexpression inhibited apoptosis and restored autophagic flux in vitro and in vivo. Meanwhile, GDF15 positively regulated SIRT1 protein expression. MSCs-GDF6 not only upregulated GDF15 and SIRT1 expression but also suppressed apoptosis and restored autophagic flux to reduce loss of HCs and hearing loss in ARHL rats. MSCs-GDF6 prevented loss of HCs to relieve ARHL by inhibiting apoptosis and restoring autophagic flux, likely in association with upregulation of the GDF15/SIRT1 axis.

MYO7A is required for the functional integrity of the mechanoelectrical transduction complex in hair cells of the adult cochlea

Myosin-VIIA (MYO7A) is an unconventional myosin responsible for syndromic (Usher 1B) or nonsyndromic forms of deafness in humans when mutated. In the cochlea, MYO7A is expressed in hair cells, where it is believed to act as the motor protein tensioning the mechanoelectrical transducer (MET) channels, thus setting their resting open probability (Po). However, direct evidence for this unique role for an unconventional myosin in mature hair cells is lacking. Here, we show that MYO7A has a distinct role in hair cells, being crucial for the structural integrity of hair bundles. Postnatal deletion of Myo7a leads to 87 to 96% reduction in MYO7A from hair cells by postnatal day 20 (P20), without affecting hearing function. During the following week, mice showed progressive decline in both hearing function and MET current amplitude in hair cells without affecting the resting Po and calcium sensitivity of the MET channel. Hair-bundle stiffness was normal at P20 but halved at P30, despite it having a normal staircase morphology and tip links. The reduction of MYO7A in the stereocilia (>87%) increased their vulnerability to sound-induced damage, with significantly more hearing loss and hair bundle deterioration than in control mice. RNA-sequencing identified a downregulation of several stereociliary genes in the Myo7a-deficient cochlea, indicating the presence of indirect compensatory mechanisms. This study reveals that mature hair cells seem to use a MYO7A-independent mechanism to maintain the resting Po of the MET channels. Instead, MYO7A is essential for maintaining the structural and functional integrity of the hair bundles.

Bridging the gap between presynaptic hair cell function and neural sound encoding

Bridging the gap between presynaptic hair cell function and neural sound encoding

Conditional Tnfaip6-Knockout in Inner Ear Hair Cells Does not Alter Auditory Function.

Noise-induced hearing loss is a worldwide public health issue that is characterized by temporary or permanent changes in hearing sensitivity. This condition is closely linked to inflammatory responses, and interventions targeting the inflammatory gene tumor necrosis factor-alpha (TNFα) are known to mitigate cochlear noise damage. TNFα-induced proteins (TNFAIPs) are a family of translucent acidic proteins, and TNFAIP6 has a notable association with inflammatory responses. To date, there have been few reports on TNFAIP6 levels in the inner ear. To elucidate the precise mechanism, we generated transgenic mouse models with conditional knockout of Tnfaip6 (Tnfaip6 cKO). Evaluation of hair cell morphology and function revealed no significant differences in hair cell numbers or ribbon synapses between Tnfaip6 cKO and wild-type mice. Moreover, there were no notable variations in hair cell numbers or hearing function in noisy environments. Our results indicate that Tnfaip6 does not have a substantial impact on the auditory system.

Assessment of the Peripheral and Central Auditory System in Infants Whose Mothers Tested Positive for COVID-19 During Pregnancy

Introduction: Coronavirus disease 2019 (COVID-19) is an infectious disease caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), a new member of the coronavirus family. While respiratory transmission is the main route, concerns have arisen regarding possible vertical transmission, which refers to the transmission of the virus from mother to fetus through the dissemination of viral particles in the amniotic fluid. Fetal viral infection via the placenta can affect the formation of the auditory system and lead to congenital hearing disorders. The aim of this research was to investigate the effects of vertical exposure to COVID-19 on the auditory system of newborns. Methodology: The study included a study group (SG) and a control group (CG). Selected during neonatal hearing screening, the SG consisted of 23 infants up to 1 year old whose mothers had been infected with SARS-CoV-2. The CG consisted of 15 infants whose mothers became pregnant after the end of the pandemic, had tested negative for COVID-19, and had no respiratory symptoms during pregnancy. The assessments for both groups were brainstem auditory evoked potentials (BAEPs), tympanometry, transient evoked otoacoustic emissions (TEOAEs), and distortion product otoacoustic emissions (DPOAEs). The research was divided into two studies, one cross-sectional and the other longitudinal. Results: All participants exhibited absolute latencies within the normal range for waves I, III, and V, although in the SG, there was a statistically significant increase in the latency of wave I in the left ear. In terms of OAEs, in the SG, there appeared to be a tendency for TEOAEs to be absent at high frequencies. Over several months, there was a general decrease in the amplitude of high-frequency responses in both TEOAEs and DPOAEs. Conclusion: No evidence was found that vertical exposure to COVID-19 causes hearing loss, although there were signs of possible deterioration in hair cell functioning.

Serum Levels of Stereocilin as a Hearing Biomarker.

Noise-induced hearing loss (NIHL) often presents with an insidious onset, resulting from the cumulative effect of chronic, high-level noise exposure regardless of etiology. Stereocilin (STRC) is a protein that supports stereocilia attachment and cochlear hair cell function, 2 common targets of noise trauma. In this study, we explored the relationship between STRC and daily noise exposure in young, healthy adults. We found that higher noise exposure levels were associated with lower serum levels of STRC, as was the case for another inner-ear protein, prestin. There was a statistically significant positive correlation between serum STRC and prestin levels. These results support a biomarker approach for the diagnosis and monitoring of NIHL. The ability to detect and measure STRC in the blood also has implications for targeted gene therapy. STRC mutations are known to be associated with autosomal recessive deafness, a condition that is now amenable to targeted gene therapy.

Regulation of cochlear hair cell function by intracellular calcium stores.

Mammalian hearing depends on the dual mechanosensory and motor functions of cochlear hair cells. Both these functions may be regulated by Ca2+ release from intracellular stores. However, it is still unclear how exactly intracellular Ca2+ release may affect either hair cell mechano-electrical transduction (MET) or prestin-dependent electromotility in outer hair cells (OHCs). Here, we used photo-activatable (caged) compounds to generate fast increases of either Ca2+ or inositol-3-phosphate (IP3) in the cytosol of young postnatal rodent auditory hair cells, thereby stimulating either Ca2+- or IP3- induced releases of Ca2+ from intracellular stores. Fast Ca2+ imaging was used to monitor propagation of Ca2+ signals along the length of a hair cell. To access potential physiological role(s) of intracellular Ca2+ releases, we used whole cell patch clamp to record: i) OHC voltage-dependent capacitance, a known electrical correlate of prestin-based electromotility, and ii) MET currents evoked by stereocilia bundle deflections with fluid-jet. In the latter experiments, changes of mechanical stiffness of the hair bundles were also quantified from video recordings of stereocilia movements. Ca2+ uncaging at the OHC apex initiated Ca2+ wave propagating to the base of the cell with subsequent Ca2+ build-up there. Ca2+ uncaging at the OHC base generated long-lasting and apparently self-sustained Ca2+ responses, further confirming Ca2+-induced Ca2+ release in the OHC basal region. Photoactivated IP3 initiated a slow increase of cytosolic Ca2+ ([Ca2+] i ) throughout the whole OHC, confirming the presence of slow-activated IP3-gated Ca2+ stores in OHCs. Interestingly, Ca2+ uncaging produced no effects on OHC voltage-dependent capacitance. In an OHC, the rise of [Ca2+] i is known to decrease axial stiffness of the cell and may modulate the stiffness of mechanosensory stereocilia bundles. To separate these two phenomena, we explored the potential effects of intracellular Ca2+ release on mechanical properties of stereocilia bundles in cochlear inner hair cells (IHCs). Ca2+ uncaging at the apex of an IHC caused a long-lasting increase in mechanical stiffness of stereocilia bundle without any changes in the amplitude or deflection sensitivity of the MET current. We concluded that the most likely physiological role of IP3-gated Ca2+ release at the apex of the cell is the regulation of hair bundle stiffness. In contrast, Ca2+-induced Ca2+ release at the base of OHCs seems to regulate axial stiffness of the cells and its hyperpolarization in response to efferent stimuli, without direct effects on the OHC prestin-based membrane motors.

Protective Effect of Marine Peptide from Netunea arthritica cumingii Against Gentamicin-Induced Hair Cell Damage in Zebrafish.

Auditory hair cell damage induced by aminoglycoside antibiotics (AmAn) leads to hearing loss, which has a serious effect on people's mental and physical health. This ototoxicity is thought to be related with the excessive accumulation of reactive oxygen species (ROS) in hair cells. However, therapeutic agents that protect hair cells are limited. Marine peptides have been shown to have excellent potential applications in disease prevention and treatment. Therefore, this study investigated the protective effects of an active peptide from Neptunea arthritica cumingii against AmAn-induced hair cell damage using the model of hair cell damage zebrafish. We identified the number, ultrastructure, and function of hair cells using fluorescence probes and scanning electron microscopy. The uptake of AmAn, ROS level, mitochondrial permeability transition pore, and apoptosis in hair cells were also tested by fluorescence labeling and TUNEL assay. The molecular mechanism for hair cell protection exerted by the peptide was detected by a real-time quantitative PCR assay. The results indicated that the peptide suppressed the uptake of AmAn but did not damage the function of hair cells mediating hearing. It also prevented ROS accumulation, decreased the occurrence of apoptosis, and rescued the abnormal opening and expressions of mitochondrial permeability transition pore and genes related to antioxidants. The peptide may be an effective therapeutic agent for AmAn-induced ototoxicity. In the future, we plan to use mammalian models to further investigate the otoprotective effect of the peptide.

A Novel Algorithm to Analyze Multi-Frequency Electrocochleography Measurements to Monitor Electrode Placement During Cochlear Implant Surgery.

During cochlear implant (CI) electrode placement, single low-frequency (e.g., 500 Hz) cochlear microphonics (CM) measurements are used to monitor hair-cell function and provide feedback to avert insertion trauma. However, it can be difficult to differentiate between trauma and the electrode's progression through the cochlea when monitored with a single frequency. Multi-frequency CM measurements, while more complex to analyze, can provide more accurate feedback by measuring CM from various locations along the basilar membrane. A new algorithm was developed to analyze multi-frequency CM tracings by comparing amplitude and phase changes across different test frequencies. The new algorithm was evaluated as to its ability to identify drop-alarm instances with the multi-frequency approach, as compared to single-frequency 500 Hz tracings. The algorithm presented in this manuscript uses the relationship between CM amplitude and phase changes across frequencies to provide real-time feedback during CI electrode placement. The results show that multi-frequency CM tracings raised an alarm only 0.5 times, as compared to 2.8 instances of alarm raised for the single-frequency 500 Hz CM measurements. Multi-frequency CM tracings can help reduce the number of alarms which may be false positives prompting unnecessary electrode manipulations, thereby minimizing the risk of insertion trauma.

Vital Dye Uptake of YO-PRO-1 and DASPEI Depends Upon Mechanoelectrical Transduction Function in Zebrafish Hair Cells.

Vital dyes allow the visualization of cells in vivo without causing tissue damage, making them a useful tool for studying lateral line and inner ear hair cells in living zebrafish and other vertebrates. FM1-43, YO-PRO-1, and DASPEI are three vital dyes commonly used for hair cell visualization. While it has been established that FM1-43 enters hair cells of zebrafish and other organisms through the mechanoelectrical transduction (MET) channel, the mechanism of entry into hair cells for YO-PRO-1 and DASPEI has not been established despite widespread use. We hypothesize that YO-PRO-1 and DASPEI entry into zebrafish hair cells is MET channel uptake dependent similar to FM1-43. To test this hypothesis, we used both genetic and pharmacologic means to block MET channel function. Genetic based MET channel assays were conducted with two different mechanotransduction defective zebrafish lines, specifically the myo7aa-/- loss of function mutant tc320b (p.Y846X) and cdh23-/- loss of function mutant (c.570-571del). Pharmacologic assays were performed with Gadolinium(III) Chloride (Gad(III)), a compound that can temporarily block mechanotransduction activity. Five-day post fertilization (5dpf) myo7aa-/- and cdh23-/- larvae incubated with FM1-43, YO-PRO-1, and DASPEI all showed nearly absent uptake of each vital dye. Treatment of wildtype zebrafish larvae with Gad(III) significantly reduces uptake of FM1-43, YO-PRO-1, and DASPEI vital dyes. These results indicate that YO-PRO-1 and DASPEI entry into zebrafish hair cells is MET channel dependent similar to FM1-43. This knowledge expands the repertoire of vital dyes that can be used to assess mechanotransduction and MET channel function in zebrafish and other vertebrate models of hair cell function.

Kif1a and intact microtubules maintain synaptic-vesicle populations at ribbon synapses in zebrafish hair cells.

Sensory hair cells of the inner ear utilize specialized ribbon synapses to transmit sensory stimuli to the central nervous system. This transmission necessitates rapid and sustained neurotransmitter release, which depends on a large pool of synaptic vesicles at the hair-cell presynapse. While previous work in neurons has shown that kinesin motor proteins traffic synaptic material along microtubules to the presynapse, the mechanisms of this process in hair cells remain unclear. Our study demonstrates that the kinesin motor protein Kif1a, along with an intact microtubule network, is essential for enriching synaptic vesicles at the presynapse in hair cells. Through genetic and pharmacological approaches, we disrupt Kif1a function and impair microtubule networks in hair cells of the zebrafish lateral-line system. These manipulations led to a significant reduction in synaptic-vesicle populations at the presynapse in hair cells. Using electron microscopy, in vivo calcium imaging, and electrophysiology, we show that a diminished supply of synaptic vesicles adversely affects ribbon-synapse function. Kif1aamutants exhibit dramatic reductions in spontaneous vesicle release and evoked postsynaptic calcium responses. Furthermore, kif1aamutants exhibit impaired rheotaxis, a behaviour reliant on the ability of hair cells in the lateral line to respond to sustained flow stimuli. Overall, our results demonstrate that Kif1a-mediated microtubule transport is critical to enrich synaptic vesicles at the active zone, a process that is vital for proper ribbon-synapse function in hair cells. KEY POINTS: Kif1a mRNAs are present in zebrafish hair cells. Loss of Kif1a disrupts the enrichment of synaptic vesicles at ribbon synapses. Disruption of microtubules depletes synaptic vesicles at ribbon synapses. Kif1aa mutants have impaired ribbon-synapse and sensory-system function.

Cingulin-nonmuscle myosin interaction plays a role in epithelial morphogenesis and cingulin nanoscale organization.

Cingulin (CGN) tethers nonmuscle myosin 2B (NM2B; heavy chain encoded by MYH10) to tight junctions (TJs) to modulate junctional and apical cortex mechanics. Here, we studied the role of the CGN-nonmuscle myosin 2 (NM2) interaction in epithelial morphogenesis and nanoscale organization of CGN by expressing wild-type and mutant CGN constructs in CGN-knockout Madin-Darby canine kidney (MDCK) epithelial cells. We show that the NM2-binding region of CGN is required to promote normal cyst morphogenesis of MDCK cells grown in three dimensions and to maintain the C-terminus of CGN in a distal position with respect to the ZO-2 (or TJP2)-containing TJ submembrane region, whereas the N-terminus of CGN is localized more proximal to the TJ membrane. We also show that the CGN mutant protein that causes deafness in human and mouse models is localized at TJs but does not bind to NM2B, resulting in decreased TJ membrane tortuosity. These results indicate that the interaction between CGN and NM2B regulates epithelial tissue morphogenesis and nanoscale organization of CGN and suggest that CGN regulates the auditory function of hair cells by organizing the actomyosin cytoskeleton to modulate the mechanics of the apical and junctional cortex.

Hearing Evaluations in Children With Retinoblastoma Treated With Intra-arterial Carboplatin Chemotherapy: A Single Institution Review.

To determine whether the administration of intra-arterial carboplatin affected the hearing of children with retinoblastoma. Children with retinoblastoma who were treated with intra-arterial carboplatin chemotherapy were included. Hearing tests before chemotherapy including tympanometry, distortion product otoacoustic emissions, and audiogram (if achievable) were performed and repeated 3 to 9 months after concluding intra-arterial therapy. The study was approved by the institutional review board. Patients were identified from the retinoblastoma clinic when the treatment plan included intra-arterial carboplatin chemotherapy. Children were excluded if they had previous intra-arterial carboplatin or preexisting hearing loss but were included if they had systemic carboplatin and dosing was available. Tympanometry was performed to rule out inner ear fluid. All examinations were performed by a certified audiologist with the same equipment, calibrated regularly by a certified technician. Twenty-two children (32 eyes) were evaluable. Because most children are diagnosed at a young age and are unable to participate in an audiogram, distortion product otoacoustic emission measurement was the primary measurement. No child displayed hearing loss. Intra-arterial chemotherapy with carboplatin did not cause ototoxicity in any child by distortion product otoacoustic emission measurement in contrast to systemic chemotherapy where ototoxicity is common. Distortion product otoacoustic emission levels were essentially unchanged from before to after intra-arterial chemotherapy in children with retinoblastoma. These findings suggest that intra-arterial carboplatin does not affect outer hair cell function, and distortion product otoacoustic emission tests can provide useful information when monitoring children at risk of developing carboplatin ototoxicity. [J Pediatr Ophthalmol Strabismus. 20XX;X(X):XXX-XXX.].

A longitudinal study investigating the effects of noise exposure on behavioural, electrophysiological and self-report measures of hearing in musicians with normal audiometric thresholds

Musicians are at risk of hearing loss and tinnitus due to regular exposure to high levels of noise. This level of risk may have been underestimated previously since damage to the auditory system, such as cochlear synaptopathy, may not be easily detectable using standard clinical measures. Most previous research investigating hearing loss in musicians has involved cross-sectional study designs that may capture only a snapshot of hearing health in relation to noise exposure. The aim of this study was to investigate the effects of cumulative noise exposure on behavioural, electrophysiological, and self-report indices of hearing damage in early-career musicians and non-musicians with normal hearing over a 2-year period. Participants completed an annual test battery consisting of pure tone audiometry, extended high-frequency hearing thresholds, distortion product otoacoustic emissions (DPOAEs), speech perception in noise, auditory brainstem responses, and self-report measures of tinnitus, hyperacusis, and hearing in background noise. Participants also completed the Noise Exposure Structured Interview to estimate cumulative noise exposure across the study period. Linear mixed models assessed changes over time. The longitudinal analysis comprised 64 early-career musicians (female n = 34; age range at T0 = 18–26 years) and 30 non-musicians (female n = 20; age range at T0 = 18–27 years). There were few longitudinal changes as a result of musicianship. Small improvements over time in some measures may be attributable to a practice/test-retest effect. Some measures (e.g., DPOAE indices of outer hair cell function) were associated with noise exposure at each time point, but did not show a significant change over time. A small proportion of participants reported a worsening of their tinnitus symptoms, which participants attributed to noise exposure, or not using hearing protection. Future longitudinal studies should attempt to capture the effects of noise exposure over a longer period, taken at several time points, for a precise measure of how hearing changes over time. Hearing conservation programmes for “at risk” individuals should closely monitor DPOAEs to detect early signs of noise-induced hearing loss when audiometric thresholds are clinically normal.

Polymer-Based Nanoparticles for Inner Ear Targeted Trans Differentiation Gene Therapy.

Hearing loss is a significant disability that often goes under recognised, largely due to poor identification, prevention, and treatment. Steps are being made to amend these pitfalls in the investigation of hearing loss, however, the development of a cure to reverse advanced forms remains distant. This review details some current advances in the treatment of hearing loss, with a particular focus on genetic-based nanotechnology and how it may provide a useful avenue for further research. This review presents a broad background on the pathophysiology of hearing loss and some current interventions. We also highlight some potential genes that may be useful in the amelioration of hearing loss. Pathways of cellular differentiation from stem or supporting cell to functional hair cell are covered in detail, as this mechanism represents a key means of regenerating these cell types. Overall, we believe that polymer-based nanotechnology coupled with novel excipients represents a useful area of further research in the treatment of hearing loss, although further studies in this area are required.

Zebrafish myo7aa affects congenital hearing by regulating Rho-GTPase signaling.

myo7aa, the homolog of the human Usher 1B syndrome pathogenic gene, myo7A, plays an important role in stereociliary development and maintenance, therefore, is critical for hearing and balance. However, the molecular mechanisms that myo7aa regulate hearing and balance still need to be studied. In this study, we generated two independent zebrafish myo7aa knockout lines using CRISPR/Cas9 technology. To investigate the effects of myo7aa on hearing, YO-PRO-1 staining and startle response assay were used. To gain insight into the specific molecular mechanisms by which myo7aa affects hearing, transcriptome sequencing and bioinformatics analysis were employed. Our study showed that hair cells of myo7aa-/- zebrafish can not take up YO-PRO-1 fluorescent dye and are insensitive to acoustic stimulation in myo7aa-/- zebrafish compared to wild type. Genes related to the Rho GTPase signaling pathway, such as arhgap33, dab2ip, and arghef40, are significantly down-regulated in myo7aa-/- zebrafish embryos at 3 dpf. GTP and ATP compensation can partially rescue the hair cell defects in myo7aa knockout zebrafish. Our findings suggest that zebrafish myo7aa affects congenital hearing by regulating Rho GTPase signaling, and loss of myo7aa leads to abnormal Rho GTPase signaling and impairs hair cell function. myo7aa, myo7A, arhgap33, dab2ip, arghef40 and myo7aa-/- fonts in the abstract are italicized. -/- is a superscript format.

Metabolic Profiling of Cochlear Organoids Identifies α-Ketoglutarate and NAD+ as Limiting Factors for Hair Cell Reprogramming.

Cochlear hair cells are the sensory cells responsible for transduction of acoustic signals. In mammals, damaged hair cells do not regenerate, resulting in permanent hearing loss. Reprogramming of the surrounding supporting cells to functional hair cells represent a novel strategy to hearing restoration. However, cellular processes governing the efficient and functional hair cell reprogramming are not completely understood. Employing the mouse cochlear organoid system, detailed metabolomic characterizations of the expanding and differentiating organoids are performed. It is found that hair cell differentiation is associated with increased mitochondrial electron transport chain (ETC) activity and reactive oxidative species generation. Transcriptome and metabolome analyses indicate reduced expression of oxidoreductases and tricyclic acid (TCA) cycle metabolites. The metabolic decoupling between ETC and TCA cycle limits the availability of the key metabolic cofactors, α-ketoglutarate (α-KG) and nicotinamide adenine dinucleotide (NAD+). Reduced expression of NAD+ in cochlear supporting cells by PGC1α deficiency further impairs hair cell reprogramming, while supplementation of α-KG and NAD+ promotes hair cell reprogramming both in vitro and in vivo. These findings reveal metabolic rewiring as a central cellular process during hair cell differentiation, and highlight the insufficiency of key metabolites as a metabolic barrier for efficient hair cell reprogramming.

Identification of Druggable Binding Sites and Small Molecules as Modulators of TMC1.

Our ability to hear and maintain balance relies on the proper functioning of inner ear sensory hair cells, which translate mechanical stimuli into electrical signals via mechano-electrical transducer (MET) channels, composed of TMC1/2 proteins. However, the therapeutic use of ototoxic drugs, such as aminoglycosides and cisplatin, which can enter hair cells through MET channels, often leads to profound auditory and vestibular dysfunction. Despite extensive research on otoprotective compounds targeting MET channels, our understanding of how small molecule modulators interact with these channels remains limited, hampering the discovery of novel compounds. Here, we propose a structure-based screening approach, integrating 3D-pharmacophore modeling, molecular simulations, and experimental validation. Our pipeline successfully identified several novel compounds and FDA-approved drugs that reduced dye uptake in cultured cochlear explants, indicating MET modulation activity. Molecular docking and free-energy estimations for binding allowed us to identify three potential drug binding sites within the channel pore, phospholipids, and key amino acids involved in modulator interactions. We also identified shared ligand-binding features between TMC and structurally related TMEM16 protein families, providing novel insights into their distinct inhibition, while potentially guiding the rational design of MET-channel-specific modulators. Our pipeline offers a broad application to discover small molecule modulators for a wide spectrum of mechanosensitive ion channels.

MEK/ERK signaling drives the transdifferentiation of supporting cells into functional hair cells by modulating the Notch pathway.

Loss of cochlear hair cells (HCs) leads to permanent hearing loss in mammals, and regenerative medicine is regarded as an ideal strategy for hearing recovery. Limited genetic and pharmaceutical approaches for HC regeneration have been established, and the existing strategies cannot achieve recovery of auditory function. A promising target to promote HC regeneration is MEK/ERK signaling because dynamic shifts in its activity during the critical stages of inner ear development have been observed. Here, we first showed that MEK/ERK signaling is activated specifically in supporting cells (SCs) after aminoglycoside-induced HC injury. We then selected 4 MEK/ERK signaling inhibitors, and PD0325901 (PD03) was found to induce the transdifferentiation of functional supernumerary HCs from SCs in the neonatal mammalian cochlear epithelium. We next found that PD03 facilitated the generation of HCs in inner ear organoids. Through genome-wide high-throughput RNA sequencing and verification, we found that the Notch pathway is the downstream target of MEK/ERK signaling. Importantly, delivery of PD03 into the inner ear induced mild HC regeneration in vivo. Our study thus reveals the importance of MEK/ERK signaling in cell fate determination and suggests that PD03 might serve as a new approach for HC regeneration.

Correlation of SNR Value on DPOAE Examination with HSP70 Levels in Blood and HSP70 Expression in Cochlea of Noise Model Rattus norvegicus as an Indicator of Inner Ear Damage

Background: Cellular stress caused by noise-induced hearing loss can be observed through the damage of cochlear hair cells, mechanically and metabolically. SNR value on the DPOAE examination assesses the sensory function of cochlear outer hair cells. HSP70 as marker of cellular stress can be identified from its levels in blood and its expression in the cochlea.Methods: Three groups of rats were used for the noise intervention: Group 1 was the control group, Group 2 had a noise exposure of 100 dB, and Group 3 received a noise exposure of 110 dB. DPOAE examination was then conducted, blood samples from rats’ tail were collected after a noise treatment, followed by the measurement of HSP70 levels by ELISA readings. Rats were terminated afterwards and immunohistochemical examination of the cochlear organ of Corti was performed to calculate the expression of HSP70. The data obtained were analyzed using SPSS.Results: A decrease occurred in the values of SNR and an increase occurred in HSP70 levels in blood along with its expression in the cochlea of noise model Rattus norvegicus, and there is a correlation between the three.Conclusion: The decrease in SNR values, increase in HSP70 levels in blood, and increase in HSP70 expression in the cochlear organ of Corti of Rattus norvegicus, proves that noise-induced hearing loss triggers the production of HSP70 as a cytoprotective agent in preventing inner ear damage.Keywords: DPOAE; Rattus norvegicus; NIHL; Heat Shock Protein 70