Potential role of modulating autophagy levels in sensorineural hearing loss.

In recent years, extensive research has been conducted on the pathogenesis of sensorineural hearing loss (SNHL). Apoptosis and necrosis have been identified to play important roles in hearing loss, but they cannot account for all hearing loss. Autophagy, a cellular process responsible for cell self-degradation and reutilization, has emerged as a significant factor contributing to hearing loss, particularly in cases of autophagy deficiency. Autophagy plays a crucial role in maintaining cell health by exerting cytoprotective and metabolically homeostatic effects in organisms. Consequently, modulating autophagy levels can profoundly impact the survival, death, and regeneration of cells in the inner ear, including hair cells (HCs) and spiral ganglion neurons (SGNs). Abnormal mitochondrial autophagy has been demonstrated in animal models of SNHL. These findings indicate the profound significance of comprehending autophagy while suggesting that our perspective on this cellular process holds promise for advancing the treatment of SNHL. Thus, this review aims to clarify the pathogenic mechanisms of SNHL and the role of autophagy in the developmental processes of various cochlear structures, including the greater epithelial ridge (GER), SGNs, and the ribbon synapse. The pathogenic mechanisms of age-related hearing loss (ARHL), also known as presbycusis, and the latest research on autophagy are also discussed. Furthermore, we underscore recent findings on the modulation of autophagy in SNHL induced by ototoxic drugs. Additionally, we suggest further research that might illuminate the complete potential of autophagy in addressing SNHL, ultimately leading to the formulation of pioneering therapeutic strategies and approaches for the treatment of deafness.

Generation of the human induced pluripotent stem cell line PUMCi005-A from a patient with Perrault syndrome.

We generated PUMCi005-A, an induced pluripotent stem cell (iPSC) line, from dermal fibroblasts of a 32-year-old female Perrault syndrome patient with double heterozygous (794 G > A and 1181 G > A) mutations in the TWNK gene using Sendai viral delivery of OCT4, SOX2, KLF4, and c-MYC. The PUMCi005-A iPSC line carried the TWNK mutations, displayed typical iPSC morphology, expressed pluripotent stem cell markers, did not have integration of Sendai virus, and exhibited a normal karyotype and differentiation into three germ layers.

Pharmacokinetics of monoclonal antibodies locally-applied into the middle ear of guinea pigs.

Countless therapeutic antibodies are currently available for the treatment of a broad range of diseases. Some target molecules of therapeutic antibodies are involved in the pathogenesis of sensorineural hearing loss (SNHL), suggesting that SNHL may be a novel target for monoclonal antibody (mAb) therapy. When considering mAb therapy for SNHL, understanding of the pharmacokinetics of mAbs after local application into the middle ear is crucial. To reveal the fundamental characteristics of mAb pharmacokinetics following local application into the middle ear of guinea pigs, we performed pharmacokinetic analyses of mouse monoclonal antibodies to FLAG-tag (FLAG-mAbs), which have no specific binding sites in the middle and inner ear. FLAG-mAbs were rapidly transferred from the middle ear to the cochlear fluid, indicating high permeability of the round window membrane to mAbs. FLAG-mAbs were eliminated from the cochlear fluid 3 h after application, similar to small molecules. Whole-body autoradiography and quantitative assessments of cerebrospinal fluid and serum demonstrated that the biodistribution of FLAG-mAbs was limited to the middle and inner ear. Altogether, the pharmacokinetics of mAbs are similar to those of small molecules when locally applied into the middle ear, suggesting the necessity of drug delivery systems for appropriate mAb delivery to the cochlear fluid after local application into the middle ear.

Chromodomain helicase DNA binding protein 4 in cell fate decisions.

Loss of spiral ganglion neurons (SGNs) in the cochlea causes hearing loss. Understanding the mechanisms of cell fate transition accelerates efforts that employ directed differentiation and lineage conversion to repopulate lost SGNs. Proposed strategies to regenerate SGNs rely on altering cell fate by activating transcriptional regulatory networks, but repressing networks for alternative cell lineages is also essential. Epigenomic changes during cell fate transitions suggest that CHD4 represses gene expression by altering the chromatin status. Despite limited direct investigations, human genetic studies implicate CHD4 function in the inner ear. The possibility of CHD4 in suppressing alternative cell fates to promote inner ear regeneration is discussed.

Early Steps towards Hearing: Placodes and Sensory Development.

Sensorineural hearing loss is the most prevalent sensory deficit in humans. Most cases of hearing loss are due to the degeneration of key structures of the sensory pathway in the cochlea, such as the sensory hair cells, the primary auditory neurons, and their synaptic connection to the hair cells. Different cell-based strategies to replace damaged inner ear neurosensory tissue aiming at the restoration of regeneration or functional recovery are currently the subject of intensive research. Most of these cell-based treatment approaches require experimental in vitro models that rely on a fine understanding of the earliest morphogenetic steps that underlie the in vivo development of the inner ear since its initial induction from a common otic-epibranchial territory. This knowledge will be applied to various proposed experimental cell replacement strategies to either address the feasibility or identify novel therapeutic options for sensorineural hearing loss. In this review, we describe how ear and epibranchial placode development can be recapitulated by focusing on the cellular transformations that occur as the inner ear is converted from a thickening of the surface ectoderm next to the hindbrain known as the otic placode to an otocyst embedded in the head mesenchyme. Finally, we will highlight otic and epibranchial placode development and morphogenetic events towards progenitors of the inner ear and their neurosensory cell derivatives.

The NLRP3 inflammasome as a target for sensorineural hearing loss.

Sensorineural hearing loss is the most common type of hearing loss in adults and occurs due to damage of the inner ear caused by a range of factors including ageing, excessive noise, toxins, and cancer. Auto-inflammatory disease is also a cause of hearing loss and there is evidence that inflammation could contribute to hearing loss in other conditions. Within the inner ear there are resident macrophage cells that respond to insults and whose activation correlates with damage. The NLRP3 inflammasome is a multi-molecular pro-inflammatory protein complex that forms in activated macrophages and may contribute to hearing loss. The aim of this article is to discuss the evidence for the NLRP3 inflammasome and associated cytokines as potential therapeutic targets for sensorineural hearing loss in conditions ranging from auto-inflammatory disease to tumour-induced hearing loss in vestibular schwannoma.

Regenerative Therapy Approaches and Encountered Problems in Sensorineural Hearing Loss.

Hearing loss is one of the most important public health matters worldwide, severely affecting people's social, psychological, and cognitive development. The perception of sound, movement, and balance in vertebrates depends on a special sensory organ called the cochlea, which contains hair cells and supporting cells in the inner ear. Genetic factors, epigenetics, the use of ototoxic drugs (some antibiotics and chemotherapeutics), noise, infections, or even aging can cause loss of hair cells and their related primary neurons, leading to sensorineural hearing loss. Although a sensorineural hearing loss, also known as permanent hearing loss, is treated with hearing aids and cochlear implants, treatment methods are limited. Since even the best implant cannot exhibit the characteristics of the original ear, the permanent sensory deficit will be permanent. For this reason, it has become important to develop regenerative treatment methods to regenerate and replace lost or damaged hair cells and neurons. Developments in stem cell technology have led to promising studies in regenerating damaged/lost hair cells or neurons with endogenous or exogenous cell-based therapies. Epigenetic mechanisms can turn hearing-related genes on and off and determine which proteins to copy. In addition, due to gene silencing, gene replacement, and CRISPR/CAS9 technology, gene therapy methods have accelerated, and studies have been carried out to treat dominant and recessive mutations that cause genetic-induced hearing loss or increase hair cell regeneration. In this paper, potential gene therapy and stem cell applications in the acquisition of cochlear function, which causes sensorineural hearing loss, and the difficulties encountered in these applications are compiled from a bioengineering perspective.

Current development of patient-specific induced pluripotent stem cells harbouring mitochondrial gene mutations and their applications in the treatment of sensorineural hearing loss.

Of all the human body's sensory systems, the auditory system is perhaps its most intricate. Hearing loss can result from even modest damage or cell death in the inner ear, and is the most common form of sensory loss. Human hearing is made possible by the sensory epithelium, the lateral wall, and auditory nerves. The most prominent functional cells in the sensory epithelium are outer hair cells (OHCs), inner hair cells (IHCs), and supporting cells. Different sound frequencies are processed by OHCs and IHCs in different cochlear regions, with those in the apex responsible for low frequencies and those in the basal region responsible for high frequencies. Hair cells can be damaged or destroyed by loud noise, aging process, genetic mutations, ototoxicity, infection, and illness. As such, they are a primary target for treating sensorineural hearing loss. Other areas known to affect hearing include spiral ganglion neurons (SGNs) in the auditory nerve. Age-related degradation of HCs and SGNs can also cause hearing loss. The aim of this review is to introduce the roles of mitochondria in human auditory system and the inner ear's main cell types and cellular functions, before going on to detail the likely health benefits of iPSC technology. We posit that patient-specific iPSCs with mitochondrial gene mutations will be an important aspect of regenerative medicine and will lead to significant progress in the treatment of SNHL.

Aucubin protects mouse cochlear hair cells from cisplatin-induced ototoxicity via activation of the PI3K/AKT/STAT3 pathway.

Cisplatin is commonly used to treat cancers and is associated with a significant risk of irreversible sensorineural hearing loss. However, no effective preventive strategies are available for cisplatin-induced HL. Therefore, significant efforts have been made to discover new drugs protecting cochlear hair cells from cisplatin-induced damage. We found that a new phytochemical, aucubin, attenuated cisplatin-induced apoptosis, the production of reactive oxygen species, and mitochondrial dysfunction in House Ear Institute Organ of Corti 1 cells and cochlear hair cells. Moreover, aucubin attenuated cisplatin-induced sensorineural hearing loss and hair cells loss in vivo. Furthermore, RNA sequencing analysis revealed that the otoprotective effects of aucubin were mainly mediated by increased STAT3 phosphorylation via the PI3K/AKT pathway. Inhibition of the STAT3 signaling pathway with the inhibitor S3I-201 or siRNA disrupted the protective effects of aucubin on cisplatin-induced apoptosis. In conclusion, we identified an otoprotective effect of aucubin. Therefore, aucubin could be used to prevent cisplatin-induced ototoxicity.

Intratympanic steroid treatment can reduce ROS and immune response in human perilymph investigated by in-depth proteome analysis.

Intratympanic (IT) steroid treatment is one of the most widely used and effective treatments for inner ear disorders such as sudden sensorineural hearing loss (SNHL). However, a clear mechanism of IT steroids in inner ear recovery has not yet been revealed. Therefore, we investigated proteome changes in extracted human perilymph after steroid treatment. In this study, we applied a tandem mass spectrometry (MS/MS)-based proteomics approach to discover global proteome changes by comparing human perilymph after steroid treatment with non-treated perilymph group. Using liquid chromatography-MS/MS analysis, we selected 156 differentially expressed proteins (DEPs) that were statistically significant according to Student's t-test. Functional annotation analysis showed that upregulated proteins after steroid treatment are related to apoptosis signaling, as well as reactive oxygen species (ROS) and immune responses. The protein-protein interaction (PPI) clusters the proteins associated with these processes and attempts to observe signaling circuitry, which mediates cellular response after IT steroid treatments. Moreover, we also considered the interactome analysis of DEPs and observed that those with high interaction scores were categorized as having equivalent molecular functions (MFs). Collectively, we suggest that DEPs and interacting proteins in human perilymph after steroid treatment would inhibit the apoptotic and adaptive immune processes that may lead to anti-inflammatory effects.

Murine cytomegalovirus employs the mixed lineage kinases family to regulate the spiral ganglion neuron cell death and hearing loss.

Cytomegalovirus (CMV)-induced sensorineural hearing loss (SNHL) is a worldwide epidemic. Recent studies have shown that the degree of spiral ganglion neuron (SGN) loss is correlated with hearing loss after CMV infection. We aimed to better understand the pathological mechanisms of CMV-related SGN death and to search for intervention measures. We found that both apoptosis and pyroptosis are involved in CMV-induced SGN death, which may be caused by the simultaneous activation of the p53/JNK and NLRP3/caspase-1 signaling pathways, respectively. Moreover, considering that mixed lineage kinase family (MLK1/2/3) are host restriction factors against viral infection and upstream regulators of the p53/JNK and inflammatory (including NLRP3-caspase1) signaling pathways, we further demonstrated that the MLKs inhibitor URMC-099 exhibited a protective effect against CMV-induced SGN death and hearing loss. These results indicate that MLKs signaling may be a key regulator and promising novel target for preventing apoptosis and even pyroptosis during the CMV infection of SGN cells and for treating hearing loss.

PIN1 protects auditory hair cells from senescence via autophagy.

Background: Age-related hearing loss is an increasing sensorineural hearing loss. But the pathogenesis of ARHL has not been clarified. Herein, we studied the role and significance of PIN1 in regulating autophagy activity in senescence HEI-OC1cells and HCs. Methods and Results: C57BL/6 mice and HEI-OC1 cells were contained in our research. Transfection of plasmids and juglone were used to upregulate or inhibit the PIN 1 expression. Immunofluorescence and Western blot were used to detect the expression of PIN1, LC3, p62, p21 and p16 protein levels in the hair cells of C57BL/6 mice cochleae and HEI-OC1 cells. Senescence-associated ß-galactosidase (SA-ß-gal) staining was used to investigate the senescent level.The results of this study showed that the level of autophagy increased in the senescent auditory hair cells. When inhibited the autophagy level with 3-MA, the senescent HEI-OC1 cells were alleviated. The autophagy activity in senescent HEI-OC1 cells also could be reduced by overexpressing PIN1 protein. On the contrary, inhibiting PIN1 could increase the autophagy level of senescent cells and cochlear hair cells. Conclusion: PIN1 might regulate autophagy activity to induce the senescent of HEI-OC1cells and HCs, which will provide a theoretical support for the prevention and treatment of age-related hearing loss.

Approaches to Mitigate Mitochondrial Dysfunction in Sensorineural Hearing Loss.

Mitochondria are highly dynamic multifaceted organelles with various functions including cellular energy metabolism, reactive oxygen species (ROS) generation, calcium homeostasis, and apoptosis. Because of these diverse functions, mitochondria are key regulators of cell survival and death, and their dysfunction is implicated in numerous diseases, particularly neurodegenerative disorders such as Alzheimer's Disease, Parkinson's Disease, and Huntington's Disease. One of the most common neurodegenerative disorders is sensorineural hearing loss (SNHL). SNHL primarily originates from the degenerative changes in the cochlea, which is the auditory portion of the inner ear. Many cochlear cells contain an abundance of mitochondria and are metabolically highly active, rendering them susceptible to mitochondrial dysfunction. Indeed, the causal role of mitochondrial dysfunction in SNHL progression is well established, and therefore, targeted for treatment. In this review, we aim to compile the emerging findings in the literature indicating the role of mitochondrial dysfunction in the progression of sensorineural hearing loss and highlight potential therapeutics targeting mitochondrial dysfunction for hearing loss treatment.

Gene therapy with a synthetic adeno-associated viral vector improves audiovestibular phenotypes in Pjvk-mutant mice.

Recessive PJVK mutations that cause a deficiency of pejvakin, a protein expressed in both sensory hair cells and first-order neurons of the inner ear, are an important cause of hereditary hearing impairment. Patients with PJVK mutations garner limited benefits from cochlear implantation; thus, alternative biological therapies may be required to address this clinical difficulty. The synthetic adeno-associated viral vector Anc80L65, with its wide tropism and high transduction efficiency in various inner ear cells, may provide a solution. We delivered the PJVK transgene to the inner ear of Pjvk mutant mice using the synthetic Anc80L65 vector. We observed robust exogenous pejvakin expression in the hair cells and neurons of the cochlea and vestibular organs. Subsequent morphologic and audiologic studies demonstrated significant restoration of spiral ganglion neuron density and hair cells in the cochlea, along with partial recovery of sensorineural hearing impairment. In addition, we observed a recovery of vestibular ganglion neurons and balance function to WT levels. Our study demonstrates the utility of Anc80L65-mediated gene delivery in Pjvk mutant mice and provides insights into the potential of gene therapy for PJVK-related inner ear deficits.

Head and neck radiotherapy causes significant disruptions of cochlear ribbon synapses and consequent sensorineural hearing loss.

PURPOSE: To investigate the possible effects of head and neck radiotherapy on hearing function in mice. METHOD: Adult C57BL/6J mice were irradiated to the head and neck once with cobalt-60 rays at doses of 10 Gy or 20 Gy. Hearing function was estimated by the detection of auditory brainstem response (ABR) thresholds and the suprathreshold function of cochlear was indicated by the peak amplitudes and latencies of wave I. The mice were tested on days 1, 7, 14, and 21 after radiation treatment, and untreated mice in littermates served as controls. The cochlear pre-synaptic ribbons were labeled using an anti-RIBEYE/CtBP2 antibody, and the synaptic vesicle membrane was traced using anti-vesicular glutamate transporter 3 (VGLUT-3) antibody. The number and size of the pre-synaptic ribbons were counted along the cochlear axis from the apex to the base. The expression of VGLUT-3 was measured by the intensity of immunofluorescence. Hematoxylin and eosin (H&E) staining was also performed to evaluate the structural changes in the cochlea. RESULTS: Compared with the controls, mice treated with 10 Gy and 20 Gy doses on days 1, 7, 14, and 21 were found to have significant disruptions in ABR thresholds and amplitudes (p < 0.05). Moreover, mice in the 20 Gy group, compared with the 10 Gy group, showed greater hearing loss and suprathreshold deficits (p < 0.05). Quantitative analysis revealed a decrease in the number and size of CtBP2-positive puncta in both the 10 Gy and 20 Gy groups compared with the controls (p < 0.05); in the 20 Gy group, the number and size of CtBP2-positive puncta were less than those in the 10 Gy group (p < 0.05). We observed a significant disruption in the expression of VGLUT-3 in the group treated with 20 Gy. However, compared with the control group, both immunofluorescence and H&E staining revealed no significant changes in the number of hair cells or the array for the 10 or 20 Gy treatments (p > 0.05). CONCLUSION: Radiation therapy targeting the head and neck can cause sensorineural hearing loss via disruption specific to the cochlear ribbon synapses. To our knowledge, this is the first study to demonstrate that cochlear ribbon synapses may be a subcellular target of radiation-induced hearing loss.

Activation of Rictor/mTORC2 signaling acts as a pivotal strategy to protect against sensorineural hearing loss.

The Food and Drug Administration­approved drug sirolimus, which inhibits mechanistic target of rapamycin (mTOR), is the leading candidate for targeting aging in rodents and humans. We previously demonstrated that sirolimus could treat ARHL in mice. In this study, we further demonstrate that sirolimus protects mice against cocaine-induced hearing loss. However, using efficacy and safety tests, we discovered that mice developed substantial hearing loss when administered high doses of sirolimus. Using pharmacological and genetic interventions in murine models, we demonstrate that the inactivation of mTORC2 is the major driver underlying hearing loss. Mechanistically, mTORC2 exerts its effects primarily through phosphorylating in the AKT/PKB signaling pathway, and ablation of P53 activity greatly attenuated the severity of the hearing phenotype in mTORC2-deficient mice. We also found that the selective activation of mTORC2 could protect mice from acoustic trauma and cisplatin-induced ototoxicity. Thus, in this study, we discover a function of mTORC2 and suggest that its therapeutic activation could represent a potentially effective and promising strategy to prevent sensorineural hearing loss. More importantly, we elucidate the side effects of sirolimus and provide an evaluation criterion for the rational use of this drug in a clinical setting.

Perilymph metabolomic and proteomic MALDI-ToF profiling with porous silicon chips: A proof-of-concept study.

INTRODUCTION: Sensorineural hearing losses (SNHLs) are a significant public health issue, and the hearing loss field is desperately in need of effective therapy. Pathophysiological mechanisms are not yet clearly understood in the absence of validated methods to assess the inner ear content. Proteomic and metabolomic analysis of perilymph is opening new research perspectives for SNHLs. We aimed to demonstrate the feasibility of an innovative mass spectrometry (MS) strategy using porous silicon chips (PSCs) to investigate the low molecular weight (LMW) protein and metabolite content of human perilymph. Our second objective was to stratify perilymph samples according to their MS profiles and compare these results with clinical data. MATERIAL AND METHODS: Perilymph samples obtained during cochlear implant surgery from patients with SNHLs were retrieved from a validated biobank. To focus on LMW entities, we used a PSC enrichment protocol before MALDI-ToF MS analysis. PSCs were used as a LMW molecular preanalytical stabilizer and amplifier. Patients' clinical data and SNHL characteristics were retrieved retrospectively from medical charts. RESULTS: We successfully acquired and compared 59 exploitable MS profiles out of 71 perilymph samples. There was a good correlation between duplicates. Comparing both ears from the same patient, we found good reproducibility even when there was a one-year interval between samplings. We identified three distinct groups when comparing the samples' metabolomic profiles and four homogeneous groups comparing their LMW proteome profiles. Clinical data analysis suggested that some groups shared clinical or preanalytical characteristics. CONCLUSION: This proof-of-concept study confirms that LMW proteome and metabolome content of perilymph can be analyzed with PSCs. Based on protein profiles, we managed to stratify perilymp samples according to their molecular composition. These results must be confirmed with a larger population, and sampling methods require improvement, but this approach seems promising. In the future, this approach may pave the way for companion test strategies to precisely diagnose and define potential molecular targets for audioprotective therapies.

Mechanism of NLRX1 Mediating JNK Apoptosis Pathway to Participate in Functional Impairment of Cochlear Hair Cells in Mice with Senile Deafness.

To explore the mechanism of cochlear hair cell damage and study the prevention and treatment of sensorineural hearing loss, the effect of NLRX1 gene expression on the functional damage of cochlear hair cells in presbycusis was comprehensively analyzed. In the in vivo detection, C57BL/6 mice of different ages were used as experimental subjects. Cochlear tissues were taken after the hearing test of mice, and the number of cells and protein changes in NLRX1 immunofluorescence staining were detected. In the in vitro detection, the cochlear hair cell HEI-OE1 was used as the experimental object, and the cell proliferation activity was detected after overexpression or silencing of NLRX1.In the in vivo and in vitro experiments, the expression of JNK pathway-related proteins was simultaneously detected. The results of in vivo experiments showed that the hearing threshold of 270d -old mice was substantially greater than that of 15d-, 30d-, and 90d-old mice (P <0.05). I addition, with increasing age, the expression of p-JNK, Bcl-2, Bax, and Caspase-3 in the mouse cochlea gradually increased (P<0.05).In vitro experimental results showed that cell proliferation activity decreased after overexpression of NLRX1, and the expression of p-JNK, Bcl-2, Bax, and Caspase-3 was substantially decreased (P<0.05). Silencing NLRX1 can inhibit the above phenomenon, indicating that NLRX1 can inhibit the proliferation of hair cells in old mice through the activation of the JNK apoptosis pathway, thereby promoting the occurrence of sensorineural hearing loss.

Cubilin-, megalin-, and Dab2-dependent transcription revealed by CRISPR/Cas9 knockout in kidney proximal tubule cells.

The multiligand receptors megalin (Lrp2) and cubilin (Cubn) and their endocytic adaptor protein Dab2 (Dab2) play essential roles in maintaining the integrity of the apical endocytic pathway of proximal tubule (PT) cells and have complex and poorly understood roles in the development of chronic kidney disease. Here, we used RNA-sequencing and CRISPR/Cas9 knockout (KO) technology in a well-differentiated cell culture model to identify PT-specific transcriptional changes that are directly consequent to the loss of megalin, cubilin, or Dab2 expression. KO of Lrp2 had the greatest transcriptional effect, and nearly all genes whose expression was affected in Cubn KO and Dab2 KO cells were also changed in Lrp2 KO cells. Pathway analysis and more granular inspection of the altered gene profiles suggested changes in pathways with immunomodulatory functions that might trigger the pathological changes observed in KO mice and patients with Donnai-Barrow syndrome. In addition, differences in transcription patterns between Lrp2 and Dab2 KO cells suggested the possibility that altered spatial signaling by aberrantly localized receptors contributes to transcriptional changes upon the disruption of PT endocytic function. A reduction in transcripts encoding sodium-glucose cotransporter isoform 2 was confirmed in Lrp2 KO mouse kidney lysates by quantitative PCR analysis. Our results highlight the role of megalin as a master regulator and coordinator of ion transport, metabolism, and endocytosis in the PT. Compared with the studies in animal models, this approach provides a means to identify PT-specific transcriptional changes that are directly consequent to the loss of these target genes.NEW & NOTEWORTHY Megalin and cubilin receptors together with their adaptor protein Dab2 represent major components of the endocytic machinery responsible for efficient uptake of filtered proteins by the proximal tubule (PT). Dab2 and megalin expression have been implicated as both positive and negative modulators of kidney disease. We used RNA sequencing to knock out CRISPR/Cas9 cubilin, megalin, and Dab2 in highly differentiated PT cells to identify PT-specific changes that are directly consequent to knockout of each component.

Human deafness-associated variants alter the dynamics of key molecules in hair cell stereocilia F-actin cores.

Stereocilia protrude up to 100 µm from the apical surface of vertebrate inner ear hair cells and are packed with cross-linked filamentous actin (F-actin). They function as mechanical switches to convert sound vibration into electrochemical neuronal signals transmitted to the brain. Several genes encode molecular components of stereocilia including actin monomers, actin regulatory and bundling proteins, motor proteins and the proteins of the mechanotransduction complex. A stereocilium F-actin core is a dynamic system, which is continuously being remodeled while maintaining an outwardly stable architecture under the regulation of F-actin barbed-end cappers, severing proteins and crosslinkers. The F-actin cores of stereocilia also provide a pathway for motor proteins to transport cargos including components of tip-link densities, scaffolding proteins and actin regulatory proteins. Deficiencies and mutations of stereocilia components that disturb this "dynamic equilibrium" in stereocilia can induce morphological changes and disrupt mechanotransduction causing sensorineural hearing loss, best studied in mouse and zebrafish models. Currently, at least 23 genes, associated with human syndromic and nonsyndromic hearing loss, encode proteins involved in the development and maintenance of stereocilia F-actin cores. However, it is challenging to predict how variants associated with sensorineural hearing loss segregating in families affect protein function. Here, we review the functions of several molecular components of stereocilia F-actin cores and provide new data from our experimental approach to directly evaluate the pathogenicity and functional impact of reported and novel variants of DIAPH1 in autosomal-dominant DFNA1 hearing loss using single-molecule fluorescence microscopy.