A new mutation of Sgms1 causes gradual hearing loss associated with a reduced endocochlear potential.

Sgms1 encodes sphingomyelin synthase 1, an enzyme in the sphingosine-1-phosphate signalling pathway, and was previously reported to underlie hearing impairment in the mouse. A new mouse allele, Sgms1tm1a, unexpectedly showed normal Auditory Brainstem Response thresholds. We found that the Sgms1tm1a mutation led to incomplete knockdown of transcript to 20 % of normal values, which was enough to support normal hearing. The Sgms1tm1b allele was generated by knocking out exon 7, leading to a complete lack of detectable transcript in the inner ear. Sgms1tm1b homozygotes showed largely normal auditory brainstem response thresholds at first, followed by progressive loss of sensitivity until they showed severe impairment at 6 months old. The endocochlear potential was consistently reduced in Sgms1tm1b mutants at 3, 4 and 8 weeks old, to around 80 mV compared with around 120 mV in control littermates. The stria vascularis showed a characteristic irregularity of marginal cell surfaces and patchy loss of Kcnq1 expression at their apical membrane, and expression analysis of the lateral wall suggested that marginal cells were the most likely initial site of dysfunction in the mutants. Finally, significant association of auditory thresholds with DNA markers within and close to the human SGMS1 gene were found in the 1958 Birth Cohort, suggesting that SGMS1 variants may play a role in the range of hearing abilities in the human population.

A genome-wide association study reveals a polygenic architecture of speech-in-noise deficits in individuals with self-reported normal hearing.

Speech-in-noise (SIN) perception is a primary complaint of individuals with audiometric hearing loss. SIN performance varies drastically, even among individuals with normal hearing. The present genome-wide association study (GWAS) investigated the genetic basis of SIN deficits in individuals with self-reported normal hearing in quiet situations. GWAS was performed on 279,911 individuals from the UB Biobank cohort, with 58,847 reporting SIN deficits despite reporting normal hearing in quiet. GWAS identified 996 single nucleotide polymorphisms (SNPs), achieving significance (p < 5*10-8) across four genomic loci. 720 SNPs across 21 loci achieved suggestive significance (p < 10-6). GWAS signals were enriched in brain tissues, such as the anterior cingulate cortex, dorsolateral prefrontal cortex, entorhinal cortex, frontal cortex, hippocampus, and inferior temporal cortex. Cochlear cell types revealed no significant association with SIN deficits. SIN deficits were associated with various health traits, including neuropsychiatric, sensory, cognitive, metabolic, cardiovascular, and inflammatory conditions. A replication analysis was conducted on 242 healthy young adults. Self-reported speech perception, hearing thresholds (0.25-16 kHz), and distortion product otoacoustic emissions (1-16 kHz) were utilized for the replication analysis. 73 SNPs were replicated with a self-reported speech perception measure. 211 SNPs were replicated with at least one and 66 with at least two audiological measures. 12 SNPs near or within MAPT, GRM3, and HLA-DQA1 were replicated for all audiological measures. The present study highlighted a polygenic architecture underlying SIN deficits in individuals with self-reported normal hearing.

A burden shared: The evolutionary case for studying human deafness in Drosophila.

Hearing impairment is the most prevalent sensory disease in humans and can have dramatic effects on the development, and preservation, of our cognitive abilities and social interactions. Currently 20 % of the world's population suffer from a form of hearing impairment; this is predicted to rise to 25 % by 2050. Despite this staggering disease load, and the vast damage it inflicts on the social, medical and economic fabric of humankind, our ability to predict, or prevent, the loss of hearing is very poor indeed. We here make the case for a paradigm shift in our approach to studying deafness. By exploiting more forcefully the molecular-genetic conservation between human hearing and hearing in morphologically distinct models, such as the fruit fly Drosophila melanogaster, we believe, a deeper understanding of hearing and deafness can be achieved. An understanding that moves beyond the surface of the 'deafness genes' to probe the underlying bedrock of hearing, which is shared across taxa, and partly shared across modalities. When it comes to understanding the workings (and failings) of human sensory function, a simple fruit fly has a lot to offer and a fly eye might sometimes be a powerful model for a human ear. Particularly the use of fly avatars, in which specific molecular (genetic or proteomic) states of humans (e.g. specific patients) are experimentally reproduced, in order to study the corresponding molecular mechanisms (e.g. specific diseases) in a controlled yet naturalistic environment, is a tool that promises multiple unprecedented insights. The use of the fly - and fly avatars - would benefit humans and will help enhance the power of other scientific models, such as the mouse.

miR-130b-3p involved in the pathogenesis of age-related hearing loss via targeting PPARγ and autophagy.

The study focuses on the underlying regulatory mechanism of age-related hearing loss (ARHL), which results from autophagy dysregulation mediated by miR-130b-3p targeting PPARγ. We constructed miR-130b-3p knockout (antagomir) and PPARγ over-expression (OE-PPARγ) mice model by injecting mmu-miR-130b-3p antagomir and HBAAV2/Anc80-m-Pparg-T2A-mCHerry into the right ear' round window of each mouse, respectively. In vitro, we introduced oxidative stress within HEI-OC1 cells by H2O2 and exogenously changed the miR-130b-3p and PPARγ levels. MiRNA level was detected by RT-qPCR, proteins by western blotting and immunohistochemistry. Morphology of autophagosomes was observed by electron microscopy. In vivo, the cochlea of aged mice showed higher miR-130b-3p expression and lower PPARγ expression, while exogenous inhibition of miR-130b-3p up-regulated PPARγ expression. Autophagy-related biomarkers expression (ATG5, Beclin-1 and LC3B II/I) decreased in aged mice, which reversely increased after the inhibition of miR-130b-3p. The elevation of PPARγ demonstrated similar effects. Contrarily, exogenous overexpression of miR-130b-3p resulted in the decrease of ATG5, Beclin-1 and LC3B II/I. We created oxidative stress within HEI-OC1 by H2O2, subsequently observed the formation of autophagosomes under electron microscope, so as the elevated cell apoptosis rate and weakened cell viability. MiR-130b-3p/PPARγ contributed to the premature senescence of these H2O2-induced HEI-OC1 cells. MiR-130b-3p regulated HEI-OC1 cell growth by targeting PPARγ, thus leading to ARHL.

Molecular adaptations underlying high-frequency hearing in the brain of CF bats species.

BACKGROUND: The majority of bat species have developed remarkable echolocation ability, especially for the laryngeally echolocating bats along with high-frequency hearing. Adaptive evolution has been widely detected for the cochleae in the laryngeally echolocating bats, however, limited understanding for the brain which is the central to echolocation signal processing in the auditory perception system, the laryngeally echolocating bats brain may also undergo adaptive changes. RESULT: In order to uncover the molecular adaptations related with high-frequency hearing in the brain of laryngeally echolocating bats, the genes expressed in the brain of Rhinolophus ferrumequinum (CF bat) and Myotis pilosus (FM bat) were both detected and also compared. A total of 346,891 genes were detected and the signal transduction mechanisms were annotated by the most abundant genes, followed by the transcription. In hence, there were 3,088 DEGs were found between the two bat brains, with 1,426 highly expressed in the brain of R. ferrumequinum, which were significantly enriched in the neuron and neurodevelopmental processes. Moreover, we found a key candidate hearing gene, ADCY1, playing an important role in the R. ferrumequinum brain and undergoing adaptive evolution in CF bats. CONCLUSIONS: Our study provides a new insight to the molecular bases of high-frequency hearing in two laryngeally echolocating bats brain and revealed different nervous system activities during auditory perception in the brain of CF bats.

Comparative exploration of mammalian deafness gene homologues in the Drosophila auditory organ shows genetic correlation between insect and vertebrate hearing.

Johnston's organ, the Drosophila auditory organ, is anatomically very different from the mammalian organ of Corti. However, recent evidence indicates significant cellular and molecular similarities exist between vertebrate and invertebrate hearing, suggesting that Drosophila may be a useful platform to determine the function of the many mammalian deafness genes whose underlying biological mechanisms are poorly characterized. Our goal was a comprehensive screen of all known orthologues of mammalian deafness genes in the fruit fly to better understand conservation of hearing mechanisms between the insect and the fly and ultimately gain insight into human hereditary deafness. We used bioinformatic comparisons to screen previously reported human and mouse deafness genes and found that 156 of them have orthologues in Drosophila melanogaster. We used fluorescent imaging of T2A-GAL4 gene trap and GFP or YFP fluorescent protein trap lines for 54 of the Drosophila genes and found 38 to be expressed in different cell types in Johnston's organ. We phenotypically characterized the function of strong loss-of-function mutants in three genes expressed in Johnston's organ (Cad99C, Msp-300, and Koi) using a courtship assay and electrophysiological recordings of sound-evoked potentials. Cad99C and Koi were found to have significant courtship defects. However, when we tested these genes for electrophysiological defects in hearing response, we did not see a significant difference suggesting the courtship defects were not caused by hearing deficiencies. Furthermore, we used a UAS/RNAi approach to test the function of seven genes and found two additional genes, CG5921 and Myo10a, that gave a statistically significant delay in courtship but not in sound-evoked potentials. Our results suggest that many mammalian deafness genes have Drosophila homologues expressed in the Johnston's organ, but that their requirement for hearing may not necessarily be the same as in mammals.

Interaural and sex differences in the natural evolution of hearing levels in pre-symptomatic and symptomatic carriers of the p.Pro51Ser variant in the COCH gene.

Hearing impairment constitutes a significant health problem in developed countries. If hearing loss is slowly progressive, the first signs may not be noticed in time, or remain untreated until the moment the auditory dysfunction becomes more apparent. The present study will focus on DFNA9, an autosomal dominant disorder caused by pathogenic variants in the COCH gene. Although several cross-sectional studies on this topic have been conducted, a crucial need for longitudinal research has been reported by many authors. Longitudinal trajectories of individual hearing thresholds were established as function of age and superimposed lowess curves were generated for 101 female and male carriers of the p.Pro51Ser variant. The average number of times patients have been tested was 2.49 years with a minimum of 1 year and a maximum of 4 years. In addition, interaural and sex differences were studied, as they could modify the natural evolution of the hearing function. The current study demonstrates that, both in female carriers and male carriers, the first signs of hearing decline, i.e. hearing thresholds of 20 dB HL, become apparent as early as the 3rd decade in the highest frequencies. In addition, a rapid progression of SNHL occurs between 40 and 50 years of age. Differences between male and female carriers in the progression of hearing loss are most obvious between the age of 50 and 65 years. Furthermore, interaural discrepancies also manifest from the age of 50 years onwards. High-quality prospective data on the long-term natural evolution of hearing levels offer the opportunity to identify different disease stages in each cochlea and different types of evolution. This will provide more insights in the window of opportunity for future therapeutic intervention trials.

The cochlear matrisome: Importance in hearing and deafness.

The extracellular matrix (ECM) consists in a complex meshwork of collagens, glycoproteins, and proteoglycans, which serves a scaffolding function and provides viscoelastic properties to the tissues. ECM acts as a biomechanical support, and actively participates in cell signaling to induce tissular changes in response to environmental forces and soluble cues. Given the remarkable complexity of the inner ear architecture, its exquisite structure-function relationship, and the importance of vibration-induced stimulation of its sensory cells, ECM is instrumental to hearing. Many factors of the matrisome are involved in cochlea development, function and maintenance, as evidenced by the variety of ECM proteins associated with hereditary deafness. This review describes the structural and functional ECM components in the auditory organ and how they are modulated over time and following injury.

Critical role of TPRN rings in the stereocilia for hearing.

Inner ear hair cells detect sound vibration through the deflection of mechanosensory stereocilia. Cytoplasmic protein TPRN has been shown to localize at the taper region of the stereocilia, and mutations in TPRN cause hereditary hearing loss through an unknown mechanism. Here, using biochemistry and dual stimulated emission depletion microscopy imaging, we show that the TPRN, together with its binding proteins CLIC5 and PTPRQ, forms concentric rings in the taper region of stereocilia. The disruption of TPRN rings, triggered by the competitive inhibition of the interaction of TPRN and CLIC5 or exogenous TPRN overexpression, leads to stereocilia degeneration and severe hearing loss. Most importantly, restoration of the TPRN rings can rescue the damaged auditory function of Tprn knockout mice by exogenously expressing TPRN at an appropriate level in HCs via promoter recombinant adeno-associated virus (AAV). In summary, our results reveal highly structured TPRN rings near the taper region of stereocilia that are crucial for stereocilia function and hearing. Also, TPRN ring restoration in stereocilia by AAV-Tprn effectively repairs damaged hearing, which lays the foundation for the clinical application of AAV-mediated gene therapy in patients with TPRN mutation.

A nonneural miRNA cluster mediates hearing via repression of two neural targets.

We show here that mir-279/996 are absolutely essential for development and function of Johnston's organ (JO), the primary proprioceptive and auditory organ in Drosophila Their deletion results in highly aberrant cell fate determination, including loss of scolopale cells and ectopic neurons, and mutants are electrophysiologically deaf. In vivo activity sensors and mosaic analyses indicate that these seed-related miRNAs function autonomously to suppress neural fate in nonneuronal cells. Finally, genetic interactions pinpoint two neural targets (elav and insensible) that underlie miRNA mutant JO phenotypes. This work uncovers how critical post-transcriptional regulation of specific miRNA targets governs cell specification and function of the auditory system.

Convergence in hearing-related genes between echolocating birds and mammals.

Echolocation, the detection of objects by means of sound waves, has evolved independently in diverse animals. Echolocators include not only mammals such as toothed whales and yangochiropteran and rhinolophoid bats but also Rousettus fruit bats, as well as two bird lineages, oilbirds and swiftlets. In whales and yangochiropteran and rhinolophoid bats, positive selection and molecular convergence has been documented in key hearing-related genes, such as prestin (SLC26A5), but few studies have examined these loci in other echolocators. Here, we examine patterns of selection and convergence in echolocation-related genes in echolocating birds and Rousettus bats. Fewer of these loci were under selection in Rousettus or birds compared with classically recognized echolocators, and elevated convergence (compared to outgroups) was not evident across this gene set. In certain genes, however, we detected convergent substitutions with potential functional relevance, including convergence between Rousettus and classic echolocators in prestin at a site known to affect hair cell electromotility. We also detected convergence between Yangochiroptera, Rhinolophidea, and oilbirds in TMC1, an important mechanosensory transduction channel in vertebrate hair cells, and observed an amino acid change at the same site within the pore domain. Our results suggest that although most proteins implicated in echolocation in specialized mammals may not have been recruited in birds or Rousettus fruit bats, certain hearing-related loci may have undergone convergent functional changes. Investigating adaptations in diverse echolocators will deepen our understanding of this unusual sensory modality.

Rescue of hearing by adenine base editing in a humanized mouse model of Usher syndrome type 1F.

Usher syndrome type 1F (USH1F), characterized by congenital lack of hearing and balance and progressive loss of vision, is caused by mutations in the PCDH15 gene. In the Ashkenazi population, a recessive truncation mutation accounts for a large proportion of USH1F cases. The truncation is caused by a single C→T mutation, which converts an arginine codon to a stop (R245X). To test the potential for base editors to revert this mutation, we developed a humanized Pcdh15R245X mouse model for USH1F. Mice homozygous for the R245X mutation were deaf and exhibited profound balance deficits, while heterozygous mice were unaffected. Here we show that an adenine base editor (ABE) is capable of reversing the R245X mutation to restore the PCDH15 sequence and function. We packaged a split-intein ABE into dual adeno-associated virus (AAV) vectors and delivered them into cochleas of neonatal USH1F mice. Hearing was not restored in a Pcdh15 constitutive null mouse despite base editing, perhaps because of early disorganization of cochlear hair cells. However, injection of vectors encoding the split ABE into a late-deletion conditional Pcdh15 knockout rescued hearing. This study demonstrates the ability of an ABE to correct the PCDH15 R245X mutation in the cochlea and restore hearing.

Deafness: from genetic architecture to gene therapy.

Progress in deciphering the genetic architecture of human sensorineural hearing impairment (SNHI) or loss, and multidisciplinary studies of mouse models, have led to the elucidation of the molecular mechanisms underlying auditory system function, primarily in the cochlea, the mammalian hearing organ. These studies have provided unparalleled insights into the pathophysiological processes involved in SNHI, paving the way for the development of inner-ear gene therapy based on gene replacement, gene augmentation or gene editing. The application of these approaches in preclinical studies over the past decade has highlighted key translational opportunities and challenges for achieving effective, safe and sustained inner-ear gene therapy to prevent or cure monogenic forms of SNHI and associated balance disorders.

Mini-PCDH15 gene therapy rescues hearing in a mouse model of Usher syndrome type 1F.

Usher syndrome type 1 F (USH1F), caused by mutations in the protocadherin-15 gene (PCDH15), is characterized by congenital deafness, lack of balance, and progressive blindness. In hair cells, the receptor cells of the inner ear, PCDH15 is a component of tip links, fine filaments which pull open mechanosensory transduction channels. A simple gene addition therapy for USH1F is challenging because the PCDH15 coding sequence is too large for adeno-associated virus (AAV) vectors. We use rational, structure-based design to engineer mini-PCDH15s in which 3-5 of the 11 extracellular cadherin repeats are deleted, but which still bind a partner protein. Some mini-PCDH15s can fit in an AAV. An AAV encoding one of these, injected into the inner ears of mouse models of USH1F, produces a mini-PCDH15 which properly forms tip links, prevents the degeneration of hair cell bundles, and rescues hearing. Mini-PCDH15s may be a useful therapy for the deafness of USH1F.

Correlation between genotype and phenotype with special attention to hearing in 14 Japanese cases of NF2-related schwannomatosis.

NF2-related schwannomatosis (NF2) is an autosomal dominant genetic disorder caused by variants in the NF2 gene. Approximately 50% of NF2 patients inherit pathogenic variants, and the remainder acquire de novo variants. NF2 is characterized by development of bilateral vestibular schwannomas. The genetic background of Japanese NF2 cases has not been fully investigated, and the present report performed a genetic analysis of 14 Japanese NF2 cases and examined genotype-phenotype correlations. DNA samples collected from peripheral blood were analyzed by next-generation sequencing, multiplex ligation-dependent probe amplification analysis, and in vitro electrophoresis. Ten cases had pathogenic or likely pathogenic variants in the NF2 gene, with seven truncating variants and three non-truncating variants. The age of onset in all seven cases with truncating variants was < 20 years. The age of onset significantly differed among cases with truncating NF2 variants, non-truncating NF2 variants, and no NF2 variants. However, the clinical course of tumor growth and hearing deterioration were not predicted only by germline pathogenic NF2 variants. The rate of truncating variants was higher in the present study than that of previous reports. Genotype-phenotype correlations in the age of onset were present in the analyzed Japanese NF2 cases.

The Expression Levels of MicroRNAs Differentially Expressed in Sudden Sensorineural Hearing Loss Patients' Serum Are Unchanged for up to 12 Months after Hearing Loss Onset.

Sudden sensorineural hearing loss (SSNHL) is an acquired idiopathic hearing loss. Serum levels of small, non-coding RNAs and microRNAs (miRNAs) miR-195-5p/-132-3p/-30a-3p/-128-3p/-140-3p/-186-5p/-375-3p/-590-5p are differentially expressed in SSNHL patients within 28 days of hearing loss onset. This study determines if these changes persist by comparing the serum miRNA expression profile of SSNHL patients within 1 month of hearing loss onset with that of patients 3-12 months after hearing loss onset. We collected serum from consenting adult SSNHL patients at presentation or during clinic follow-up. We matched patient samples drawn 3-12 months after hearing loss onset (delayed group, n = 9 patients) by age and sex to samples drawn from patients within 28 days of hearing loss onset (immediate group, n = 14 patients). We compared the real-time PCR-determined expression levels of the target miRNAs between the two groups. We calculated the air conduction pure-tone-averaged (PTA) audiometric thresholds in affected ears at the initial and final follow-up visits. We undertook inter-group comparisons of hearing outcome status and initial and final PTA audiometric thresholds. There was no significant inter-group difference in miRNA expression level, hearing recovery status and initial and final affected ear PTA audiometric thresholds.

Experience-dependent flexibility in a molecularly diverse central-to-peripheral auditory feedback system.

Brainstem olivocochlear neurons (OCNs) modulate the earliest stages of auditory processing through feedback projections to the cochlea and have been shown to influence hearing and protect the ear from sound-induced damage. Here, we used single-nucleus sequencing, anatomical reconstructions, and electrophysiology to characterize murine OCNs during postnatal development, in mature animals, and after sound exposure. We identified markers for known medial (MOC) and lateral (LOC) OCN subtypes, and show that they express distinct cohorts of physiologically relevant genes that change over development. In addition, we discovered a neuropeptide-enriched LOC subtype that produces Neuropeptide Y along with other neurotransmitters. Throughout the cochlea, both LOC subtypes extend arborizations over wide frequency domains. Moreover, LOC neuropeptide expression is strongly upregulated days after acoustic trauma, potentially providing a sustained protective signal to the cochlea. OCNs are therefore poised to have diffuse, dynamic effects on early auditory processing over timescales ranging from milliseconds to days.

Just as our pupils dilate or shrink depending on the amount of light available to our eyes, our ears adjust their sensitivity based on the sound environment we encounter. Evidence suggests that a group of cells known as olivocochlear neurons (OCNs for short) may be involved in this process. These cells are located in the brainstem but project into the cochlea, the inner ear structure that converts sound waves into the electrical impulses relayed to the brain. OCNs may mediate how sounds are detected and encoded "at the source." Historically, OCNs have been divided into two groups (medial or lateral OCNs) based on different morphologies and roles in hearing. For instance, medial OCNs are thought to protect our ears against loud sounds by sending molecular signals to the inner ear cells that amplify certain auditory signals. However, it remains difficult to disentangle the precise function of the different types of OCNs, in part because scientists still lack markers that would allow them to distinguish between medial and lateral cells simply based on genetic activity. Frank et al. aimed to eliminate this bottleneck by identifying which genes were switched on and to what degree in individual mouse medial and lateral OCNs; this was done throughout development and after exposure to loud noises. The experiments uncovered a range of genetic markers for medial and lateral OCNs, showing that these cells switch on different sets of genes relevant to their role over development. This gene expression data also revealed that two distinct groups of lateral OCNs exist, one of which is characterised by the production of large amounts of neuropeptides, a type of chemical messenger that can modulate neural circuit activity. Further work in both developing and adult mice showed that this production is shaped by the activity of the cells, with the neuropeptide levels increasing when the animals are exposed to damaging levels of noise. This change lasts for several days, suggesting that such an experience can have long-lasting effects on how the brain provides feedback to the ear. Overall, the results by Frank et al. will help to better identify and characterize the different types of OCNs and the role that they have in hearing. By uncovering the chemical messengers that mediate the response to loud noises, this research may contribute to a better understanding of how to prevent or reduce hearing loss.

Cx26 heterozygous mutations cause hyperacusis-like hearing oversensitivity and increase susceptibility to noise.

Gap junction gene GJB2 (Cx26) mutations cause >50% of nonsyndromic hearing loss. Its recessive hetero-mutation carriers, who have no deafness, occupy ~10 to 20% of the general population. Here, we report an unexpected finding that these heterozygote carriers have hearing oversensitivity, and active cochlear amplification increased. Mouse models show that Cx26 hetero-deletion reduced endocochlear potential generation in the cochlear lateral wall and caused outer hair cell electromotor protein prestin compensatively up-regulated to increase active cochlear amplification and hearing sensitivity. The increase of active cochlear amplification also increased sensitivity to noise; exposure to daily-level noise could cause Cx26+/- mice permanent hearing threshold shift, leading to hearing loss. This study demonstrates that Cx26 recessive heterozygous mutations are not "harmless" for hearing as previously considered and can cause hyperacusis-like hearing oversensitivity. The data also indicate that GJB2 hetero-mutation carriers are vulnerable to noise and should avoid noise exposure in daily life.

Syntaxin 4 is essential for hearing in human and zebrafish.

Congenital hearing impairment (HI) is a genetically highly heterogeneous disorder in which prompt recognition and intervention are crucial to optimize outcomes. In this study, we used exome sequencing to investigate a large consanguineous Pakistani family with eight affected individuals showing bilateral severe-to-profound HI. This identified a homozygous splice region variant in STX4 (c.232 + 6T>C), which causes exon skipping and a frameshift, that segregated with HI (two-point logarithm of odds (LOD) score = 5.9). STX4, a member of the syntaxin family, is a component of the SNARE machinery involved in several vesicle transport and recycling pathways. In silico analysis showed that murine orthologue Stx4a is highly and widespread expressed in the developing and adult inner ear. Immunofluorescent imaging revealed localization of STX4A in the cell body, cell membrane and stereocilia of inner and outer hair cells. Furthermore, a morpholino-based knockdown of stx4 in zebrafish showed an abnormal startle response, morphological and developmental defects, and a disrupted mechanotransduction function in neuromast hair cells measured via FM1-43 uptake. Our findings indicate that STX4 dysfunction leads to HI in humans and zebrafish and supports the evolutionary conserved role of STX4 in inner ear development and hair cell functioning.