Clarin-2 gene supplementation durably preserves hearing in a model of progressive hearing loss.

Hearing loss is a major health concern affecting millions of people worldwide with currently limited treatment options. In clarin-2-deficient Clrn2-/- mice, used here as a model of progressive hearing loss, we report synaptic auditory abnormalities in addition to the previously demonstrated defects of hair bundle structure and mechanoelectrical transduction. We sought an in-depth evaluation of viral-mediated gene delivery as a therapy for these hearing-impaired mice. Supplementation with either the murine Clrn2 or human CLRN2 genes preserved normal hearing in treated Clrn2-/- mice. Conversely, mutated forms of CLRN2, identified in patients with post-lingual moderate to severe hearing loss, failed to prevent hearing loss. The ectopic expression of clarin-2 successfully prevented the loss of stereocilia, maintained normal mechanoelectrical transduction, preserved inner hair cell synaptic function, and ensured near-normal hearing thresholds over time. Maximal hearing preservation was observed when Clrn2 was delivered prior to the loss of transducing stereocilia. Our findings demonstrate that gene therapy is effective for the treatment of post-lingual hearing impairment and age-related deafness associated with CLRN2 patient mutations.

The genetic and phenotypic landscapes of Usher syndrome: from disease mechanisms to a new classification.

Usher syndrome (USH) is the most common cause of deaf-blindness in humans, with a prevalence of about 1/10,000 (~ 400,000 people worldwide). Cochlear implants are currently used to reduce the burden of hearing loss in severe-to-profoundly deaf patients, but many promising treatments including gene, cell, and drug therapies to restore the native function of the inner ear and retinal sensory cells are under investigation. The traditional clinical classification of Usher syndrome defines three major subtypes-USH1, 2 and 3-according to hearing loss severity and onset, the presence or absence of vestibular dysfunction, and age at onset of retinitis pigmentosa. Pathogenic variants of nine USH genes have been initially reported: MYO7A, USH1C, PCDH15, CDH23, and USH1G for USH1, USH2A, ADGRV1, and WHRN for USH2, and CLRN1 for USH3. Based on the co-occurrence of hearing and vision deficits, the list of USH genes has been extended to few other genes, but with limited supporting information. A consensus on combined criteria for Usher syndrome is crucial for the development of accurate diagnosis and to improve patient management. In recent years, a wealth of information has been obtained concerning the properties of the Usher proteins, related molecular networks, potential genotype-phenotype correlations, and the pathogenic mechanisms underlying the impairment or loss of hearing, balance and vision. The advent of precision medicine calls for a clear and more precise diagnosis of Usher syndrome, exploiting all the existing data to develop a combined clinical/genetic/network/functional classification for Usher syndrome.

Review of Genotype-Phenotype Correlations in Usher Syndrome.

Usher syndrome (USH) encompasses a group of clinically and genetically heterogenous disorders defined by the triad of sensorineural hearing loss (SNHL), vestibular dysfunction, and vision loss. USH is the most common cause of deaf blindness. USH is divided clinically into three subtypes-USH1, USH2, and USH3-based on symptom severity, progression, and age of onset. The underlying genetics of these USH forms are, however, significantly more complex, with over a dozen genes linked to the three primary clinical subtypes and other atypical USH phenotypes. Several of these genes are associated with other deaf-blindness syndromes that share significant clinical overlap with USH, pointing to the limits of a clinically based classification system. The genotype-phenotype relationships among USH forms also may vary significantly based on the location and type of mutation in the gene of interest. Understanding these genotype-phenotype relationships and associated natural disease histories is necessary for the successful development and application of gene-based therapies and precision medicine approaches to USH. Currently, the state of knowledge varies widely depending on the gene of interest. Recent studies utilizing next-generation sequencing technology have expanded the list of known pathogenic mutations in USH genes, identified new genes associated with USH-like phenotypes, and proposed algorithms to predict the phenotypic effects of specific categories of allelic variants. Further work is required to validate USH gene causality, and better define USH genotype-phenotype relationships and disease natural histories-particularly for rare mutations-to lay the groundwork for the future of USH treatment.

A biallelic variant in CLRN2 causes non-syndromic hearing loss in humans.

Deafness, the most frequent sensory deficit in humans, is extremely heterogeneous with hundreds of genes involved. Clinical and genetic analyses of an extended consanguineous family with pre-lingual, moderate-to-profound autosomal recessive sensorineural hearing loss, allowed us to identify CLRN2, encoding a tetraspan protein, as a new deafness gene. Homozygosity mapping followed by exome sequencing identified a 14.96 Mb locus on chromosome 4p15.32p15.1 containing a likely pathogenic missense variant in CLRN2 (c.494C > A, NM_001079827.2) segregating with the disease. Using in vitro RNA splicing analysis, we show that the CLRN2 c.494C > A variant leads to two events: (1) the substitution of a highly conserved threonine (uncharged amino acid) to lysine (charged amino acid) at position 165, p.(Thr165Lys), and (2) aberrant splicing, with the retention of intron 2 resulting in a stop codon after 26 additional amino acids, p.(Gly146Lysfs*26). Expression studies and phenotyping of newly produced zebrafish and mouse models deficient for clarin 2 further confirm that clarin 2, expressed in the inner ear hair cells, is essential for normal organization and maintenance of the auditory hair bundles, and for hearing function. Together, our findings identify CLRN2 as a new deafness gene, which will impact future diagnosis and treatment for deaf patients.

The auditory hair cell ribbon synapse: from assembly to function.

Cochlear inner hair cells (IHCs), the mammalian auditory sensory cells, encode acoustic signals with high fidelity by Graded variations of their membrane potential trigger rapid and sustained vesicle exocytosis at their ribbon synapses. The kinetics of glutamate release allows proper transfer of sound information to the primary afferent auditory neurons. Understanding the physiological properties and underlying molecular mechanisms of the IHC synaptic machinery, and especially its high temporal acuity, which is pivotal to speech perception, is a central issue of auditory science. During the past decade, substantial progress in high-resolution imaging and electrophysiological recordings, as well as the development of genetic approaches both in humans and in mice, has produced major insights regarding the morphological, physiological, and molecular characteristics of this synapse. Here we review this recent knowledge and discuss how it enlightens the way the IHC ribbon synapse develops and functions.

Identification of a CDH12 potential candidate genetic variant for an autosomal dominant form of transgrediens and progrediens palmoplantar keratoderma in a Tunisian family.

Molecular diagnosis of rare inherited palmoplantar keratoderma (PPK) is still challenging. We investigated at the clinical and genetic level a consanguineous Tunisian family presenting an autosomal dominant atypical form of transgrediens and progrediens PPK to better characterize this ultrarare disease and to identify its molecular etiology. Whole-exome sequencing (WES), filtering strategies, and bioinformatics analysis have been achieved. Clinical investigation and follow up over 13 years of this Tunisian family with three siblings formerly diagnosed as an autosomal recessive form of Mal de Melela-like conducted us to reconsider its initial phenotype. Indeed, the three patients presented clinical features that overlap both Mal de Meleda and progressive symmetric erythrokeratoderma (PSEK). The mode of inheritance was also reconsidered, since the mother, initially classified as unaffected, exhibited a similar expression of the disease. WES analysis showed the absence of potentially functional rare variants in known PPKs or PSEK-related genes. Results revealed a novel heterozygous nonsynonymous variant in cadherin-12 gene (CDH12, NM_004061, c.1655C > A, p.Thr552Asn) in all affected family members. This variant is absent in dbSNP and in 50 in-house control exomes. In addition, in silico analysis of the mutated 3D domain structure predicted that this variant would result in cadherin-12 protein destabilization and thermal instability. Functional annotation and biological network construction data provide further supporting evidence for the potential role of CDH12 in the maintenance of skin integrity. Taken together, these results suggest that CDH12 gene is a potential candidate gene for an atypical presentation of an autosomal dominant form of transgrediens and progrediens PPK.

Spectrin ßV adaptive mutations and changes in subcellular location correlate with emergence of hair cell electromotility in mammalians.

The remarkable hearing capacities of mammals arise from various evolutionary innovations. These include the cochlear outer hair cells and their singular feature, somatic electromotility, i.e., the ability of their cylindrical cell body to shorten and elongate upon cell depolarization and hyperpolarization, respectively. To shed light on the processes underlying the emergence of electromotility, we focused on the ßV giant spectrin, a major component of the outer hair cells' cortical cytoskeleton. We identified strong signatures of adaptive evolution at multiple sites along the spectrin-ßV amino acid sequence in the lineage leading to mammals, together with substantial differences in the subcellular location of this protein between the frog and the mouse inner ear hair cells. In frog hair cells, spectrin ßV was invariably detected near the apical junctional complex and above the cuticular plate, a dense F-actin meshwork located underneath the apical plasma membrane. In the mouse, the protein had a broad punctate cytoplasmic distribution in the vestibular hair cells, whereas it was detected in the entire lateral wall of cochlear outer hair cells and had an intermediary distribution (both cytoplasmic and cortical, but restricted to the cell apical region) in cochlear inner hair cells. Our results support a scenario where the singular organization of the outer hair cells' cortical cytoskeleton may have emerged from molecular networks initially involved in membrane trafficking, which were present near the apical junctional complex in the hair cells of mammalian ancestors and would have subsequently expanded to the entire lateral wall in outer hair cells.

An unusually powerful mode of low-frequency sound interference due to defective hair bundles of the auditory outer hair cells.

A detrimental perceptive consequence of damaged auditory sensory hair cells consists in a pronounced masking effect exerted by low-frequency sounds, thought to occur when auditory threshold elevation substantially exceeds 40 dB. Here, we identified the submembrane scaffold protein Nherf1 as a hair-bundle component of the differentiating outer hair cells (OHCs). Nherf1(-/-) mice displayed OHC hair-bundle shape anomalies in the mid and basal cochlea, normally tuned to mid- and high-frequency tones, and mild (22-35 dB) hearing-threshold elevations restricted to midhigh sound frequencies. This mild decrease in hearing sensitivity was, however, discordant with almost nonresponding OHCs at the cochlear base as assessed by distortion-product otoacoustic emissions and cochlear microphonic potentials. Moreover, unlike wild-type mice, responses of Nherf1(-/-) mice to high-frequency (20-40 kHz) test tones were not masked by tones of neighboring frequencies. Instead, efficient maskers were characterized by their frequencies up to two octaves below the probe-tone frequency, unusually low intensities up to 25 dB below probe-tone level, and growth-of-masker slope (2.2 dB/dB) reflecting their compressive amplification. Together, these properties do not fit the current acknowledged features of a hypersensitivity of the basal cochlea to lower frequencies, but rather suggest a previously unidentified mechanism. Low-frequency maskers, we propose, may interact within the unaffected cochlear apical region with midhigh frequency sounds propagated there via a mode possibly using the persistent contact of misshaped OHC hair bundles with the tectorial membrane. Our findings thus reveal a source of misleading interpretations of hearing thresholds and of hypervulnerability to low-frequency sound interference.

The giant spectrin ßV couples the molecular motors to phototransduction and Usher syndrome type I proteins along their trafficking route.

Mutations in the myosin VIIa gene cause Usher syndrome type IB (USH1B), characterized by deaf-blindness. A delay of opsin trafficking has been observed in the retinal photoreceptor cells of myosin VIIa-deficient mice. We identified spectrin ßV, the mammalian ß-heavy spectrin, as a myosin VIIa- and rhodopsin-interacting partner in photoreceptor cells. Spectrin ßV displays a polarized distribution from the Golgi apparatus to the base of the outer segment, which, unlike that of other ß spectrins, matches the trafficking route of opsin and other phototransduction proteins. Formation of spectrin ßV-rhodopsin complex could be detected in the differentiating photoreceptors as soon as their outer segment emerges. A failure of the spectrin ßV-mediated coupling between myosin VIIa and opsin molecules thus probably accounts for the opsin transport delay in myosin VIIa-deficient mice. We showed that spectrin ßV also associates with two USH1 proteins, sans (USH1G) and harmonin (USH1C). Spectrins are supposed to function as heteromers of α and ß subunits, but fluorescence resonance energy transfer and in vitro binding experiments indicated that spectrin ßV can also form homodimers, which likely supports its αII-independent ßV functions. Finally, consistent with its distribution along the connecting cilia axonemes, spectrin ßV binds to several subunits of the microtubule-based motor proteins, kinesin II and the dynein complex. We therefore suggest that spectrin ßV homomers couple some USH1 proteins, opsin and other phototransduction proteins to both actin- and microtubule-based motors, thereby contributing to their transport towards the photoreceptor outer disks.

Successful gene therapy in the RPGRIP1-deficient dog: a large model of cone-rod dystrophy.

For the development of new therapies, proof-of-concept studies in large animal models that share clinical features with their human counterparts represent a pivotal step. For inherited retinal dystrophies primarily involving photoreceptor cells, the efficacy of gene therapy has been demonstrated in canine models of stationary cone dystrophies and progressive rod-cone dystrophies but not in large models of progressive cone-rod dystrophies, another important cause of blindness. To address the last issue, we evaluated gene therapy in the retinitis pigmentosa GTPase regulator interacting protein 1 (RPGRIP1)-deficient dog, a model exhibiting a severe cone-rod dystrophy similar to that seen in humans. Subretinal injection of AAV5 (n = 5) or AAV8 (n = 2) encoding the canine Rpgrip1 improved photoreceptor survival in transduced areas of treated retinas. Cone function was significantly and stably rescued in all treated eyes (18-72% of those recorded in normal eyes) up to 24 months postinjection. Rod function was also preserved (22-29% of baseline function) in four of the five treated dogs up to 24 months postinjection. No detectable rod function remained in untreated contralateral eyes. More importantly, treatment preserved bright- and dim-light vision. Efficacy of gene therapy in this large animal model of cone-rod dystrophy provides great promise for human treatment.

Defects in whirlin, a PDZ domain molecule involved in stereocilia elongation, cause deafness in the whirler mouse and families with DFNB31.

The whirler mouse mutant (wi) does not respond to sound stimuli, and detailed ultrastructural analysis of sensory hair cells in the organ of Corti of the inner ear indicates that the whirler gene encodes a protein involved in the elongation and maintenance of stereocilia in both inner hair cells (IHCs) and outer hair cells (OHCs). BAC-mediated transgene correction of the mouse phenotype and mutation analysis identified the causative gene as encoding a novel PDZ protein called whirlin. The gene encoding whirlin also underlies the human autosomal recessive deafness locus DFNB31. In the mouse cochlea, whirlin is expressed in the sensory IHC and OHC stereocilia. Our findings suggest that this novel PDZ domain-containing molecule acts as an organizer of submembranous molecular complexes that control the coordinated actin polymerization and membrane growth of stereocilia.

Usher syndrome (sensorineural deafness and retinitis pigmentosa): pathogenesis, molecular diagnosis and therapeutic approaches.

PURPOSE OF REVIEW: Usher syndrome (USH) is the most prevalent cause of hereditary deafness-blindness in humans. In this review, we pinpoint new insights regarding the molecular mechanisms defective in this syndrome, its molecular diagnosis and prospective therapies. RECENT FINDINGS: Animal models wherein USH proteins were targeted at different maturation stages of the auditory hair cells have been engineered, shedding new light on the development and functioning of the hair bundle, the sound receptive structure. Improved protocols and guidelines for early molecular diagnosis of USH (USH genotyping microarrays, otochips and complete Sanger sequencing of the 366 coding exons of identified USH genes) have been developed. Approaches to alleviate or cure hearing and visual impairments have been initiated, leading to various degrees of functional rescuing. SUMMARY: Whereas the mechanisms underlying hearing impairment in USH patients are being unraveled, showing in particular that USH1 proteins are involved in the shaping of the hair bundle and the functioning of the mechanoelectrical transduction machinery, the mechanisms underlying the retinal defects are still unclear. Efforts to improve clinical diagnosis have been successful. Yet, despite some encouraging results, further development of therapeutic approaches is necessary to ultimately treat this dual sensory defect.

Vestibular Deficits in Deafness: Clinical Presentation, Animal Modeling, and Treatment Solutions.

The inner ear is responsible for both hearing and balance. These functions are dependent on the correct functioning of mechanosensitive hair cells, which convert sound- and motion-induced stimuli into electrical signals conveyed to the brain. During evolution of the inner ear, the major changes occurred in the hearing organ, whereas the structure of the vestibular organs remained constant in all vertebrates over the same period. Vestibular deficits are highly prevalent in humans, due to multiple intersecting causes: genetics, environmental factors, ototoxic drugs, infections and aging. Studies of deafness genes associated with balance deficits and their corresponding animal models have shed light on the development and function of these two sensory systems. Bilateral vestibular deficits often impair individual postural control, gaze stabilization, locomotion and spatial orientation. The resulting dizziness, vertigo, and/or falls (frequent in elderly populations) greatly affect patient quality of life. In the absence of treatment, prosthetic devices, such as vestibular implants, providing information about the direction, amplitude and velocity of body movements, are being developed and have given promising results in animal models and humans. Novel methods and techniques have led to major progress in gene therapies targeting the inner ear (gene supplementation and gene editing), 3D inner ear organoids and reprograming protocols for generating hair cell-like cells. These rapid advances in multiscale approaches covering basic research, clinical diagnostics and therapies are fostering interdisciplinary research to develop personalized treatments for vestibular disorders.

Progress in Gene Editing Tools and Their Potential for Correcting Mutations Underlying Hearing and Vision Loss.

Blindness and deafness are the most frequent sensory disorders in humans. Whatever their cause - genetic, environmental, or due to toxic agents, or aging - the deterioration of these senses is often linked to irreversible damage to the light-sensing photoreceptor cells (blindness) and/or the mechanosensitive hair cells (deafness). Efforts are increasingly focused on preventing disease progression by correcting or replacing the blindness and deafness-causal pathogenic alleles. In recent years, gene replacement therapies for rare monogenic disorders of the retina have given positive results, leading to the marketing of the first gene therapy product for a form of childhood hereditary blindness. Promising results, with a partial restoration of auditory function, have also been reported in preclinical models of human deafness. Silencing approaches, including antisense oligonucleotides, adeno-associated virus (AAV)-mediated microRNA delivery, and genome-editing approaches have also been applied to various genetic forms of blindness and deafness The discovery of new DNA- and RNA-based CRISPR/Cas nucleases, and the new generations of base, prime, and RNA editors offers new possibilities for directly repairing point mutations and therapeutically restoring gene function. Thanks to easy access and immune-privilege status of self-contained compartments, the eye and the ear continue to be at the forefront of developing therapies for genetic diseases. Here, we review the ongoing applications and achievements of this new class of emerging therapeutics in the sensory organs of vision and hearing, highlighting the challenges ahead and the solutions to be overcome for their successful therapeutic application in vivo.

Stem Cells and Gene Therapy in Progressive Hearing Loss: the State of the Art.

Progressive non-syndromic sensorineural hearing loss (PNSHL) is the most common cause of sensory impairment, affecting more than a third of individuals over the age of 65. PNSHL includes noise-induced hearing loss (NIHL) and inherited forms of deafness, among which is delayed-onset autosomal dominant hearing loss (AD PNSHL). PNSHL is a prime candidate for genetic therapies due to the fact that PNSHL has been studied extensively, and there is a potentially wide window between identification of the disorder and the onset of hearing loss. Several gene therapy strategies exist that show potential for targeting PNSHL, including viral and non-viral approaches, and gene editing versus gene-modulating approaches. To fully explore the potential of these therapy strategies, a faithful in vitro model of the human inner ear is needed. Such models may come from induced pluripotent stem cells (iPSCs). The development of new treatment modalities by combining iPSC modeling with novel and innovative gene therapy approaches will pave the way for future applications leading to improved quality of life for many affected individuals and their families.

[Stem cell therapy in the inner ear: recent achievements and prospects]. / Thérapie cellulaire dans l'oreille interne : nouveaux développements et perspectives.

Because of its high prevalence and social impact, hearing impairment is a major public health problem. Whatever the cause--heredity, acoustic trauma, aminoglycoside antibiotics, noise exposure or aging--the hearing impairment is often caused by an irreversible loss of sensory hair cells. So far, hearing aids and cochlear implants are the only possibility to "treat" profound deafness. With the advent of regenerative medicine, extensive studies aimed to repair, regenerate or replace lost hair cells have been initiated. Recently, Stefan Heller and colleagues described a guidance protocol to induce mouse embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC) to differentiate into mechanosensitive hair cells. The resulting hair cells hold promise as a tool for hair cell molecular physiology and physiopathology, drug discovery, and possibly also hair cell replacement. The next challenges, alternative strategies, their limitations and prospects are also discussed.

Inner Ear Gene Therapies Take Off: Current Promises and Future Challenges.

Hearing impairment is the most frequent sensory deficit in humans of all age groups, from children (1/500) to the elderly (more than 50% of the over-75 s). Over 50% of congenital deafness are hereditary in nature. The other major causes of deafness, which also may have genetic predisposition, are aging, acoustic trauma, ototoxic drugs such as aminoglycosides, and noise exposure. Over the last two decades, the study of inherited deafness forms and related animal models has been instrumental in deciphering the molecular, cellular, and physiological mechanisms of disease. However, there is still no curative treatment for sensorineural deafness. Hearing loss is currently palliated by rehabilitation methods: conventional hearing aids, and for more severe forms, cochlear implants. Efforts are continuing to improve these devices to help users to understand speech in noisy environments and to appreciate music. However, neither approach can mediate a full recovery of hearing sensitivity and/or restoration of the native inner ear sensory epithelia. New therapeutic approaches based on gene transfer and gene editing tools are being developed in animal models. In this review, we focus on the successful restoration of auditory and vestibular functions in certain inner ear conditions, paving the way for future clinical applications.

Disease mechanisms and gene therapy for Usher syndrome.

Usher syndrome (USH) is a major cause of deaf-blindness in humans, affecting ∼400 000 patients worldwide. Three clinical subtypes, USH1-3, have been defined, with 10 USH genes identified so far. In recent years, in addition to identification of new Usher genes and diagnostic tools, major progress has been made in understanding the role of Usher proteins and how they cooperate through interaction networks to ensure proper development, architecture and function of the stereociliary bundle at the apex of sensory hair cells in the inner ear. Several Usher mouse models of known human Usher genes have been characterized. These mice faithfully reproduce the auditory phenotype associated with Usher syndrome and the vestibular phenotype associated with some mutations in USH genes, particularly USH1. Interestingly, very few mouse models of Usher syndrome recapitulate the retinal phenotype associated with the disease in human. Usher patients can benefit from hearing aids or cochlear implants, which partially alleviate auditory sensory deprivation. However, there are currently no biological treatments available for auditory or visual dysfunction in Usher patients. Development of novel therapies for Usher syndrome has sprouted over the past decade, building on recent progress in gene transfer and new gene editing tools. Promising success demonstrating recovery of hearing and balance functions have been obtained via distinct therapeutic strategies in animal models. Clinical translation to Usher patients, however, calls for further improvements and concerted efforts to overcome the challenges ahead.

Rab27A and its effector MyRIP link secretory granules to F-actin and control their motion towards release sites.

The GTPase Rab27A interacts with myosin-VIIa and myosin-Va via MyRIP or melanophilin and mediates melanosome binding to actin. Here we show that Rab27A and MyRIP are associated with secretory granules (SGs) in adrenal chromaffin cells and PC12 cells. Overexpression of Rab27A, GTPase-deficient Rab27A-Q78L, or MyRIP reduced secretory responses of PC12 cells. Amperometric recordings of single adrenal chromaffin cells revealed that Rab27A-Q78L and MyRIP reduced the sustained component of release. Moreover, these effects on secretion were partly suppressed by the actin-depolymerizing drug latrunculin but strengthened by jasplakinolide, which stabilizes the actin cortex. Finally, MyRIP and Rab27A-Q78L restricted the motion of SGs in the subplasmalemmal region of PC12 cells, as measured by evanescent-wave fluorescence microscopy. In contrast, the Rab27A-binding domain of MyRIP and a MyRIP construct that interacts with myosin-Va but not with actin increased the mobility of SGs. We propose that Rab27A and MyRIP link SGs to F-actin and control their motion toward release sites through the actin cortex.

Clarin-2 is essential for hearing by maintaining stereocilia integrity and function.

Hearing relies on mechanically gated ion channels present in the actin-rich stereocilia bundles at the apical surface of cochlear hair cells. Our knowledge of the mechanisms underlying the formation and maintenance of the sound-receptive structure is limited. Utilizing a large-scale forward genetic screen in mice, genome mapping and gene complementation tests, we identified Clrn2 as a new deafness gene. The Clrn2clarinet/clarinet mice (p.Trp4* mutation) exhibit a progressive, early-onset hearing loss, with no overt retinal deficits. Utilizing data from the UK Biobank study, we could show that CLRN2 is involved in human non-syndromic progressive hearing loss. Our in-depth morphological, molecular and functional investigations establish that while it is not required for initial formation of cochlear sensory hair cell stereocilia bundles, clarin-2 is critical for maintaining normal bundle integrity and functioning. In the differentiating hair bundles, lack of clarin-2 leads to loss of mechano-electrical transduction, followed by selective progressive loss of the transducing stereocilia. Together, our findings demonstrate a key role for clarin-2 in mammalian hearing, providing insights into the interplay between mechano-electrical transduction and stereocilia maintenance.