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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.

International audience; 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.

International audience; 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.

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.

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.

Deafness, the most frequent sensory deficit in humans, is extremely heterogenous with hundreds of genes probably 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 15.2 Mb locus on chromosome 4p15.32p15.1 containing a missense pathogenic 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 mutation 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.

International audience; 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.

International audience; 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.

Usher syndrome type 1 (USH1) causes combined hearing and sight defects, but USH1 protein function in the retina is unclear. Schietroma et al. use Xenopus to model the deficiency in two USH1 proteins—protocadherin-15 and cadherin-23—and identify crucial roles for these molecules in shaping the photoreceptor outer segment., Usher syndrome type 1 (USH1) causes combined hearing and sight defects, but how mutations in USH1 genes lead to retinal dystrophy in patients remains elusive. The USH1 protein complex is associated with calyceal processes, which are microvilli of unknown function surrounding the base of the photoreceptor outer segment. We show that in Xenopus tropicalis, these processes are connected to the outer-segment membrane by links composed of protocadherin-15 (USH1F protein). Protocadherin-15 deficiency, obtained by a knockdown approach, leads to impaired photoreceptor function and abnormally shaped photoreceptor outer segments. Rod basal outer disks displayed excessive outgrowth, and cone outer segments were curved, with lamellae of heterogeneous sizes, defects also observed upon knockdown of Cdh23, encoding cadherin-23 (USH1D protein). The calyceal processes were virtually absent in cones and displayed markedly reduced F-actin content in rods, suggesting that protocadherin-15–containing links are essential for their development and/or maintenance. We propose that calyceal processes, together with their associated links, control the sizing of rod disks and cone lamellae throughout their daily renewal.

International audience; 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 Clrn2 clarinet/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 indepth 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.

Determining the genetic bases of age-related disease remains a major challenge requiring a spectrum of approaches from human and clinical genetics to the utilization of model organism studies. Here we report a large-scale genetic screen in mice employing a phenotype-driven discovery platform to identify mutations resulting in age-related disease, both late-onset and progressive. We have utilized N-ethyl-N-nitrosourea mutagenesis to generate pedigrees of mutagenized mice that were subject to recurrent screens for mutant phenotypes as the mice aged. In total, we identify 105 distinct mutant lines from 157 pedigrees analysed, out of which 27 are late-onset phenotypes across a range of physiological systems. Using whole-genome sequencing we uncover the underlying genes for 44 of these mutant phenotypes, including 12 late-onset phenotypes. These genes reveal a number of novel pathways involved with age-related disease. We illustrate our findings by the recovery and characterization of a novel mouse model of age-related hearing loss., Random mutagenesis can uncover novel genes involved in phenotypic traits. Here the authors perform a large-scale phenotypic screen on over 100 mouse strains generated by ENU mutagenesis to identify mice with age-related diseases, which they attribute to specific mutations revealed by whole-genome sequencing.

Clarin-1, a tetraspan-like membrane protein defective in Usher syndrome type IIIA (USH3A), is essential for hair bundle morphogenesis in auditory hair cells. We report a new synaptic role for clarin-1 in mouse auditory hair cells elucidated by characterization of Clrn1 total (Clrn1(ex4-/-)) and postnatal hair cell-specific conditional (Clrn1(ex4fl/fl) Myo15-Cre(+/-)) knockout mice. Clrn1(ex4-/-) mice were profoundly deaf, whereas Clrn1(ex4fl/fl) Myo15-Cre(+/-) mice displayed progressive increases in hearing thresholds, with, initially, normal otoacoustic emissions and hair bundle morphology. Inner hair cell (IHC) patch-clamp recordings for the 2 mutant mice revealed defective exocytosis and a disorganization of synaptic F-actin and Ca(V)1.3 Ca2+ channels, indicative of a synaptopathy. Postsynaptic defects were also observed, with an abnormally broad distribution of AMPA receptors associated with a loss of afferent dendrites and defective electrically evoked auditory brainstem responses. Protein-protein interaction assays revealed interactions between clarin-1 and the synaptic Ca(V)1.3 Ca2+ channel complex via the Ca-V beta(2) auxiliary subunit and the PDZ domain-containing protein harmonin (defective in Usher syndrome type IC). Cochlear gene therapy in vivo, through adeno-associated virus-mediated Clrn1 transfer into hair cells, prevented the synaptic defects and durably improved hearing in Clrn1(ex4fl/fl) Myo15-Cre(+/-) mice. Our results identify clarin-1 as a key organizer of IHC ribbon synapses, and suggest new treatment possibilities for USH3A patients.

International audience; The genetic, mutational and phenotypic spectrum of deafness-causing genes shows great diversity and pleiotropy. The best examples are the group of genes, which when mutated can either cause non-syndromic hearing loss (NSHL) or the most common dual sensory impairment, Usher syndrome (USH). Variants in the CIB2 gene have been previously reported to cause hearing loss at the DFNB48 locus and deaf-blindness at the USH1J locus. In this study, we characterize the phenotypic spectrum in a multiethnic cohort with autosomal recessive non-syndromic hearing loss (ARNSHL) due to variants in the CIB2 gene. Of the 6 families we ascertained, 3 segregated novel loss-of-function (LOF) variants, 2 families segregated missense variants (1 novel) and 1 family segregated a previously reported pathogenic variant in trans with a frameshift variant. This report is the first to show that biallelic LOF variants in CIB2 cause ARNSHL and not USH. In the era of precision medicine, providing the correct diagnosis (NSHL vs USH) is essential for patient care as it impacts potential intervention and prevention options for patients. Here, we provide evidence disqualifying CIB2 as an USH-causing gene.

International audience; 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.

International audience; Usher syndrome (USH), the most prevalent cause of hereditary deafness-blindness, is an autosomal recessive and genetically heterogeneous disorder. Three clinical subtypes (USH1-3) are distinguishable based on the severity of the sensorineural hearing impairment, the presence or absence of vestibular dysfunction, and the age of onset of the retinitis pigmentosa. A total of 10 causal genes, 6 for USH1, 3 for USH2, and 1 for USH3, and an USH2 modifier gene, have been identified. A robust molecular diagnosis is required not only to improve genetic counseling, but also to advance gene therapy in USH patients. Here, we present an improved diagnostic strategy that is both cost- and time-effective. It relies on the sequential use of three different techniques to analyze selected genomic regions: targeted exome sequencing, comparative genome hybridization, and quantitative exon amplification. We screened a large cohort of 427 patients (139 USH1, 282 USH2, and six of undefined clinical subtype) from various European medical centers for mutations in all USH genes and the modifier gene. We identified a total of 421 different sequence variants predicted to be pathogenic, about half of which had not been previously reported. Remarkably, we detected large genomic rearrangements, most of which were novel and unique, in 9% of the patients. Thus, our strategy led to the identification of biallelic and monoallelic mutations in 92.7% and 5.8% of the USH patients, respectively. With an overall 98.5% mutation characterization rate, the diagnosis efficiency was substantially improved compared with previously reported methods.

International audience; The precise architecture of hair bundles, the arrays of mechanosensitive microvilli-like stereocilia crowning the auditory hair cells, is essential to hearing. Myosin IIIa, defective in the late-onset deafness form DFNB30, has been proposed to transport espin-1 to the tips of stereocilia, thereby promoting their elongation. We show that Myo3a(-/-)Myo3b(-/-) mice lacking myosin IIIa and myosin IIIb are profoundly deaf, whereas Myo3a-cKO Myo3b(-/-) mice lacking myosin IIIb and losing myosin IIIa postnatally have normal hearing. Myo3a(-/-)Myo3b(-/-) cochlear hair bundles display robust mechanoelectrical transduction currents with normal kinetics but show severe embryonic abnormalities whose features rapidly change. These include abnormally tall and numerous microvilli or stereocilia, ungraded stereocilia bundles, and bundle rounding and closure. Surprisingly, espin-1 is properly targeted to Myo3a(-/-)Myo3b(-/-) stereocilia tips. Our results uncover the critical role that class III myosins play redundantly in hair-bundle morphogenesis; they unexpectedly limit the elongation of stereocilia and of subsequently regressing microvilli, thus contributing to the early hair bundle shaping.

International audience; The exquisite sensitivity and frequency tuning of hearing depend on the correct structure and functioning of the auditory sensory hair cells, the neighbouring supporting cells, and the homeostasis of their ionic environment. The increasing number of adhesion proteins identified as causing hearing impairment in humans and mice when defective is consistent with a critical role for cellcell junctions between neighbouring epithelial cells of the cochlea, and of fibrous links within the hair bundle, the sensory hair cell structure responsible for sound reception. Classical cadherins and/or associated adherens-junction proteins, such as p120-catenin or nectin 3, have been shown to be essential for establishment of the regular mosaic cellular pattern of the auditory sensory epithelium. Two cadherinrelated proteins, protocadherin-15 and cadherin-23, are key components of both lateral links and tip-links in hair bundles; they are essential components of the mechanoelectrical transduction machinery. Studies of the role of these adhesion proteins and of the pathogenesis of the forms of deafness caused by defects of these proteins have provided considerable insight into the development and functioning of the auditory sensory epithelium, and of the hair cells in particular.

Mice are a poor model for retinal defects caused by type I Usher syndrome (USH1) because their photoreceptors have almost no calyceal processes, the structures in which all USH1 proteins are detected in other vertebrates., The mechanisms underlying retinal dystrophy in Usher syndrome type I (USH1) remain unknown because mutant mice lacking any of the USH1 proteins—myosin VIIa, harmonin, cadherin-23, protocadherin-15, sans—do not display retinal degeneration. We found here that, in macaque photoreceptor cells, all USH1 proteins colocalized at membrane interfaces (i) between the inner and outer segments in rods and (ii) between the microvillus-like calyceal processes and the outer segment basolateral region in rods and cones. This pattern, conserved in humans and frogs, was mediated by the formation of an USH1 protein network, which was associated with the calyceal processes from the early embryonic stages of outer segment growth onwards. By contrast, mouse photoreceptors lacked calyceal processes and had no USH1 proteins at the inner–outer segment interface. We suggest that USH1 proteins form an adhesion belt around the basolateral region of the photoreceptor outer segment in humans, and that defects in this structure cause the retinal degeneration in USH1 patients.

International audience; 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.

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.

Cell-cell interactions play a major role during preimplantation development of the mouse embryo. The formation of adherens junctions is a major feature of compaction, the first morphogenetic event that takes place at the 8-cell stage. Then, during the following two cell cycles, tight junctions form, and the outer layer of cells differentiate into a functional epithelium, leading to the formation of the blastocoel cavity. Until now, E-cadherin was the only transmembrane molecule localized in adherens junctions and required for early development. Vezatin is a transmembrane protein of adherens junctions, interacting with the E-cadherin-catenins complex. Here, we show that vezatin is expressed very early during mouse preimplantation development. It co-localizes with E-cadherin throughout development, being found all around the cell cortex before compaction and basolaterally in adherens junctions thereafter. In addition, vezatin is also detected in nuclei during most of the cell cycle. Finally, using a morpholino-oligonucleotide approach to inhibit vezatin function during preimplantation development, we observed that inhibition of vezatin synthesis leads to a cell cycle arrest with limited cell-cell interactions. This phenotype can be rescued when mRNAs coding for vezatin missing the 5'UTR are co-injected with the anti-vezatin morpholino-oligonucleotide. Cells derived from blastomeres injected with morpholino-oligonucleotide had a reduced amount of vezatin concomitantly with a decrease in the quantity of E-cadherin and beta-catenin localized in the areas of intercellular contact. Shift in E-cadherin cortical distribution was correlated with a strong decrease in E-cadherin mRNA and protein contents. Altogether, these observations demonstrate that vezatin is required for morphogenesis of the preimplantation mouse embryo.

International audience; Defects in myosin VIIa, the PDZ-domain-containing protein harmonin, cadherin 23 and protocadherin 15 (two cadherins with large extracellular regions), and the putative scaffolding protein Sans underlie five genetic forms of Usher syndrome type I (USH1), the most frequent cause of hereditary deafness-blindness in humans. All USH1 proteins are localised within growing stereocilia and/or the kinocilium that make up the developing auditory hair bundle, the mechanosensitive structure receptive to sound stimulation. Cadherin 23 has been shown to be a component of fibrous links interconnecting the growing stereocilia as well as the kinocilium and the nearest tall stereocilia. A similar function is anticipated for protocadherin 15. Multiple direct interactions between USH1 proteins have been demonstrated. In particular, harmonin b can bind to the cytoplasmic regions of cadherin 23 and protocadherin 15, and to F-actin, and thus probably anchors these cadherins to the actin filaments filling the stereocilia. Myosin VIIa and Sans are both involved in the sorting and/or targeting of harmonin b to the stereocilia. Together, this suggests that the disorganisation of the hair bundles observed in mice mutants lacking orthologues of USH1 proteins may result from a defect of hair-bundle-link-mediated adhesion forces. Moreover, several recent evidences suggest that some genes defective in Usher type II syndrome also encode interstereocilia links, thus bridging the pathogenic pathways of USH1 and USH2 hearing impairment. Additional functions of USH1 proteins in the inner ear and the retina are evident from other phenotypic abnormalities observed in these mice. In particular, myosin VIIa could act at the interface between microtubule- and actin-based transport.

International audience; Defects in myosin VIIa, harmonin (a PDZ domain protein), cadherin 23, protocadherin 15 and sans (a putative scaffolding protein), underlie five forms of Usher syndrome type I (USH1). Mouse mutants for all these proteins exhibit disorganization of their hair bundle, which is the mechanotransduction receptive structure of the inner ear sensory cells, the cochlear and vestibular hair cells. We have previously demonstrated that harmonin interacts with cadherin 23 and myosin VIIa. Here we address the extent of interactions between the five known USH1 proteins. We establish the previously suggested sans-harmonin interaction and find that sans also binds to myosin VIIa. We show that sans can form homomeric structures and that harmonin b can interact with all harmonin isoforms. We reveal that harmonin also binds to protocadherin 15. Molecular characterization of these interactions indicates that through its binding to four of the five USH1 proteins, the first PDZ domain (PDZ1) of harmonin plays a central role in this network. We localize sans in the apical region of cochlear and vestibular hair cell bodies underneath the cuticular plate. In contrast to the other four known USH1 proteins, no sans labeling was detected within the stereocilia. We propose that via its binding to myosin VIIa and/or harmonin, sans controls the hair bundle cohesion and proper development by regulating the traffic of USH1 proteins en route to the stereocilia.

International audience; Defects in myosin XVa and the PDZ domain-containing protein, whirlin, underlie deafness in humans and mice. Hair bundles of mutant mice defective for either protein have abnormally short stereocilia. Here, we show that whirlin, like myosin XVa, is present at the very tip of each stereocilium in the developing and mature hair bundles of the cochlear and vestibular system. We found that myosin XVa SH3-MyTH4 region binds to the short isoform of whirlin (PR-PDZ3) that can rescue the stereocilia growth defect in whirlin defective mice. Moreover, the C-terminal MyTH4-FERM region of myosin XVa binds to the PDZ1 and PDZ2 domains of the long whirlin isoform. We conclude that a direct myosin XVa-whirlin interaction at the stereocilia tip is likely to control the elongation of stereocilia. Whirlin, unlike myosin XVa, is also transiently localized in the basal region of developing stereocilia in rat vestibular and cochlear hair cells until P4 and P12, respectively. Notably, whirlin also interacts with myosin VIIa that is present along the entire length of the stereocilia. Finally, we show that the transmembrane netrin-G1 ligand (NGL-1) binds to the PDZ1 and PDZ2 domains of whirlin and has an extracellular region that homophilically self-interacts in a Ca 21-dependent manner. The interaction between whirlin and NGL-1 might be involved in the stabilization of interstereociliar links.

Usher syndrome type I (USH1) is the most frequent cause of hereditary deaf-blindness in humans. Seven genetic loci (USH1A-G) have been implicated in this disease to date, and four of the corresponding genes have been identified: USH1B, C, D and F. We carried out fine mapping of USH1G (chromosome 17q24-25), restricting the location of this gene to an interval of 2.6 Mb and then screened genes present within this interval for mutations. The genes screened included the orthologue of the Sans gene, which is defective in the Jackson shaker deaf mutant and maps to the syntenic region in mice. In two consanguineous USH1G-affected families, we detected two different frameshift mutations in the SANS gene. Two brothers from a German family affected with USH1G were found to be compound heterozygotes for a frameshift and a missense mutation. These results demonstrate that SANS underlies USH1G. The SANS protein contains three ankyrin domains and a sterile alpha motif, and its C-terminal tripeptide presents a class I PDZ-binding motif. We showed, by means of co-transfection experiments, that SANS associates with harmonin, a PDZ domain-containing protein responsible for USH1C. In Jackson shaker mice the hair bundles, the mechanoreceptive structures of inner ear sensory cells, are disorganized. Based on the known interaction between USH1B (myosin VIIa), USH1C (harmonin) and USH1D (cadherin 23) proteins and the results obtained in this study, we suggest that a functional network formed by the USH1B, C, D and G proteins is responsible for the correct cohesion of the hair bundle.

Deaf-blindness in three distinct genetic forms of Usher type I syndrome (USH1) is caused by defects in myosin VIIa, harmonin and cadherin 23. Despite being critical for hearing, the functions of these proteins in the inner ear remain elusive. Here we show that harmonin, a PDZ domain-containing protein, and cadherin 23 are both present in the growing stereocilia and that they bind to each other. Moreover, we demonstrate that harmonin b is an F-actin-bundling protein, which is thus likely to anchor cadherin 23 to the stereocilia microfilaments, thereby identifying a novel anchorage mode of the cadherins to the actin cytoskeleton. Moreover, harmonin b interacts directly with myosin VIIa, and is absent from the disorganized hair bundles of myosin VIIa mutant mice, suggesting that myosin VIIa conveys harmonin b along the actin core of the developing stereocilia. We propose that the shaping of the hair bundle relies on a functional unit composed of myosin VIIa, harmonin b and cadherin 23 that is essential to ensure the cohesion of the stereocilia.

Defects of the myosin VIIa motor protein cause deafness and retinal anomalies in humans and mice. We report on the identification of a novel myosin-VIIa-interacting protein that we have named MyRIP (myosin-VIIa- and Rab-interacting protein), since it also binds to Rab27A in a GTP-dependent manner. In the retinal pigment epithelium cells, MyRIP, myosin VIIa and Rab27A are associated with melanosomes. In transfected PC12 cells, overexpression of MyRIP was shown to interfere with the myosin VIIa tail localization. We propose that a molecular complex composed of Rab27A, MyRIP and myosin VIIa bridges retinal melanosomes to the actin cytoskeleton and thereby mediates the local trafficking of these organelles. The defect of this molecular complex is likely to account for the perinuclear mislocalization of the melanosomes observed in the retinal pigment epithelium cells of myosinVIIa-defective mice.

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.

International audience; The Usher syndrome (USH) is the most prevalent cause of inherited deaf-blindness. Three clinical subtypes, USH1–3, have been defined, and ten USH genes identified. The hearing impairment due to USH gene defects has been shown to result from improper organisation of the hair bundle, the sound receptive structure of sensory hair cells. In contrast, the cellular basis of the visual defect is less well understood as this phenotype is absent in almost all the USH mouse models that faithfully mimic the human hearing impairment. Structural and molecular interspecies discrepancies regarding photoreceptor calyceal processes and the association with the distribution of USH1 proteins have recently been unravelled, and have led to the conclusion that a defect in the USH1 protein complex-mediated connection between the photoreceptor outer segment and the surrounding calyceal processes (in both rods and cones), and the inner segment (in rods only), probably causes the USH1 retinal dystrophy in humans.; Le syndrome de Usher (USH) constitue la première cause de cécité-surdité héréditaire. Trois sous-types cliniques, USH1–3, ont été́ définis, et dix gènes USH ont été identifiés. La déficience auditive due aux défauts des gènes USH résulte d’une désorganisation de la touffe ciliaire des cellules sensorielles. À l’inverse, la base cellulaire du défaut visuel est beaucoup moins bien comprise, car il manque dans presque tous les modèles murins de USH, qui reproduisent pourtant fidèlement la déficience auditive. Les différences inter-espèces structurelles concernant les processus caliciels de la cellule photoréceptrice et leur association avec la localisation des protéines USH1 ont récemment été mises en évidence. Elles ont conduit à la conclusion qu’un défaut dans la connexion médiée par les protéines USH1 entre le segment externe du photorécepteur et, d’une part, les processus caliciels (à la fois dans les bâtonnets et les cônes) et, d’autre part, le segment interne (dans les bâtonnets seulement) est probablement à l’origine de la dystrophie rétinienne USH1 chez l’homme.

Using a PCR-based subtractive method on cDNA from 2-day-old mouse cochlea, we identified a gene encoding otogelin, Otog, an inner ear specific glycoprotein expressed in all acellular structures. Here, we provide evidence that otogelin is detected as early as embryonic day 10 in the otic vesicle. At this stage, otogelin is detected in the epithelial cells which do not overlap with the myosin VIIA-expressing cells, namely the precursors of the hair cells, thus arguing for an early commitment of the two cell populations. Analysis of otogelin spatiotemporal cell distribution allows a molecular tracing for the contribution of the cochlear and vestibular inner ear supporting cells to the formation of the acellular structures. Throughout embryonic and adult life, the expression of the otogelin gene as monitored by LacZ inserted into Otog, and the abundance of the protein are greater in the vestibule than in the cochlea. In adult, otogelin is still produced by the vestibular supporting cells, which argues for a continuous process of otogelin renewal in the otoconial membranes and cupulae. In contrast, in the tectorial membrane, otogelin should be a long-lasting protein since both the otogelin gene and protein were almost undetectable in adult cochlear cells. The data are consistent with the requirement for otogelin in the attachment of the otoconial membranes and cupulae to their corresponding sensory epithelia as revealed in Otog -/- mice.

To gain an insight into the cellular function of the unconventional myosin VIIA, we sought proteins interacting with its tail region, using the yeast two-hybrid system. Here we report on one of the five candidate interactors we identified, namely the type I alpha regulatory subunit (RI alpha) of protein kinase A. The interaction of RI alpha with myosin VIIA tail was demonstrated by coimmunoprecipitation from transfected HEK293 cells. Analysis of deleted constructs in the yeast two-hybrid system showed that the interaction of myosin VIIA with RI alpha involves the dimerization domain of RI alpha. In vitro binding assays identified the C-terminal "4.1, ezrin, radixin, moesin" (FERM)-like domain of myosin VIIA as the interacting domain. In humans and mice, mutations in the myosin VIIA gene underlie hereditary hearing loss, which may or may not be associated with visual deficiency. Immunohistofluorescence revealed that myosin VIIA and RI alpha are coexpressed in the outer hair cells of the cochlea and rod photoreceptor cells of the retina. Our results strongly suggest that myosin VIIA is a novel protein kinase A-anchoring protein that targets protein kinase A to definite subcellular sites of these sensory cells.

Genes specifically expressed in the inner ear are candidates to underlie hereditary nonsyndromic deafness. The gene Otog has been isolated from a mouse subtractive cDNA cochlear library. It encodes otogelin, an N-glycosylated protein that is present in the acellular membranes covering the six sensory epithelial patches of the inner ear: in the cochlea (the auditory sensory organ), the tectorial membrane (TM) over the organ of Corti; and in the vestibule (the balance sensory organ), the otoconial membranes over the utricular and saccular maculae as well as the cupulae over the cristae ampullares of the three semi-circular canals. These membranes are involved in the mechanotransduction process. Their movement, which is induced by sound in the cochlea or acceleration in the vestibule, results in the deflection of the stereocilia bundle at the apex of the sensory hair cells, which in turn opens the mechanotransduction channels located at the tip of the stereo-cilia. We sought to elucidate the role of otogelin in the auditory and vestibular functions by generating mice with a targeted disruption of Otog. In Otog-/- mice, both the vestibular and the auditory functions were impaired. Histological analysis of these mutants demonstrated that in the vestibule, otogelin is required for the anchoring of the otoconial membranes and cupulae to the neuroepithelia. In the cochlea, ultrastructural analysis of the TM indicated that otogelin is involved in the organization of its fibrillar network. Otogelin is likely to have a role in the resistance of this membrane to sound stimulation. These results support OTOG as a possible candidate gene for a human nonsyndromic form of deafness.

International audience; Using a candidate gene approach, we identified a novel human gene, OTOF, underlying an autosomal recessive, nonsyndromic prelingual deafness, DFNB9. The same nonsense mutation was detected in four unrelated affected families of Lebanese origin. OTOF is the second member of a mammalian gene family related to Caenorhabditis elegans fer-1. It encodes a predicted cytosolic protein (of 1,230 aa) with three C2 domains and a single carboxy-terminal transmembrane domain. The sequence homologies and predicted structure of otoferlin, the protein encoded by OTOF, suggest its involvement in vesicle membrane fusion. In the inner ear, the expression of the orthologous mouse gene, mainly in the sensory hair cells, indicates that such a role could apply to synaptic vesicles.

International audience; Potassium channels regulate electrical signaling and the ionic composition of biological fluids. Mutations in the three known genes of the KCNQ branch of the K channel gene family underlie inherited cardiac ar-rhythmias (in some cases associated with deafness) and neonatal epilepsy. We have now cloned KCNQ4, a novel member of this branch. It maps to the DFNA2 locus for a form of nonsyndromic dominant deafness. In the cochlea, it is expressed in sensory outer hair cells. A mutation in this gene in a DFNA2 pedigree changes a residue in the KCNQ4 pore region. It abol-ishes the potassium currents of wild-type KCNQ4 on which it exerts a strong dominant-negative effect. Whereas mutations in KCNQ1 cause deafness by af-fecting endolymph secretion, the mechanism leading to KCNQ4-related hearing loss is intrinsic to outer hair cells.

Classically, it was thought that the adenohypophyseal gland originated from the oral ectoderm. Its development has been the object of numerous studies over many years. However, several questions are still raised about its origin, differentiation, and commitment. The adenohypophyseal gland could originate from the anterior ridge of the neural plate. Glandular adenohypophyseal cells are committed very early in embryonic life. Interactions between adenohypophyseal presumptive territory and neighboring tissues can exist very soon, as early as at the open neural stage. The expression of a given phenotype by the committed cells seems to be controlled by a number of differentiation and/or transcription factors. In view of all these studies, performed with the use of different in vivo and in vitro models, classical concepts of the embryology of the adenohypophyseal gland need to be reevaluated. Indeed, many questions remain unanswered concerning the molecular mechanisms of known and unknown factors controlling development of the adenohypophyseal gland. Microsc. Res. Tech. 39:98–113, 1997. © 1997 Wiley-Liss, Inc.

Several lines of evidence indicate a crucial role for unconventional myosins in the function of the sensory hair cells of the inner ear. We report here the characterization of the cDNAs encoding two unconventional type I myosins from a mouse cochlear cDNA library. The first cDNA encodes a putative protein named Myo1c, which is likely to be the murine orthologue of the bullfrog myosin I{beta} and which may be involved in the gating of the mechanotransduction channel of the sensory hair cells. This myosin belongs to the group of short-tailed myosins I, with its tail ending shortly after a polybasic, TH-1-like domain. The second cDNA encodes a novel type I myosin Myo1f which displays three regions: a head domain with the conserved ATP- and actin-binding sites, a neck domain with a single IQ motif, and a tail domain with the tripartite structure initially described in protozoan myosins I. The tail of Myo1f includes (1) a TH-1 region rich in basic residues, which may interact with anionic membrane phospholipids; (2) a TH-2 proline-rich region, expected to contain an ATP-insensitive actin-binding site; and (3) an SH-3 domain found in a variety of cytoskeletal and signaling proteins. Northern blot analysis indicated that themore » genes encoding Myo1c and Myo1f display a widespread tissue expression in the adult mouse. Myo1c and Myo1f were mapped by in situ hybridization to the chromosomal regions 11D-11E and 17B-17C, respectively. The human orthologuous genes MYO1C and MYO1F were also characterized, and mapped to the human chromosomal regions 17p13 and 19p13.2- 19p1.3.3, respectively. 45 refs., 5 figs., 2 tabs.« less

International audience; 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 bV, the mammalian b-heavy spectrin, as a myosin VIIa-and rhodopsin-interacting partner in photoreceptor cells. Spectrin bV displays a polarized distribution from the Golgi apparatus to the base of the outer segment, which, unlike that of other b spectrins, matches the trafficking route of opsin and other phototransduction proteins. Formation of spectrin bV-rhodopsin complex could be detected in the differentiating photoreceptors as soon as their outer segment emerges. A failure of the spectrin bV-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 bV also associates with two USH1 proteins, sans (USH1G) and harmonin (USH1C). Spectrins are supposed to function as heteromers of a and b subunits, but fluorescence resonance energy transfer and in vitro binding experiments indicated that spectrin bV can also form homodimers, which likely supports its aII-independent bV functions. Finally, consistent with its distribution along the connecting cilia axonemes, spectrin bV binds to several subunits of the microtubule-based motor proteins, kinesin II and the dynein complex. We therefore suggest that spectrin bV 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.

The tight control of cell-cell connectivity mediated by cadherins is a key issue in human health and disease. The human genome contains over 115 genes encoding cadherins and cadherin-like proteins. Defects in about 21 of these proteins (8 classical, 5 desmosomal, 8 atypical cadherins) have been linked to inherited disorders in humans, including skin and hair disorders, cardiomyopathies, sensory defects associated with deafness and blindness, and psychiatric disorders. With the advent of exome and genome sequencing techniques, we can anticipate the discovery of yet more evidence for the involvement of additional cadherins. Elucidation of the related physiopathological mechanisms underlying these conditions should help to clarify the roles played by these cadherins in tissues and the ways in which defects in different cadherins cause such a wide spectrum of associated phenotypes. These disorders also constitute disparate model systems for investigations of the relative contributions of mechanical adhesive strength and intracellular signaling pathways to the pathogenic process for a given cadherin.

The tight control of cell–cell connectivity mediated by cadherins is a key issue in human health and disease. The human genome contains over 115 genes encoding cadherins and cadherin-like proteins. Defects in about 21 of these proteins (8 classical, 5 desmosomal, 8 atypical cadherins) have been linked to inherited disorders in humans, including skin and hair disorders, cardiomyopathies, sensory defects associated with deafness and blindness, and psychiatric disorders. With the advent of exome and genome sequencing techniques, we can anticipate the discovery of yet more evidence for the involvement of additional cadherins. Elucidation of the related physiopathological mechanisms underlying these conditions should help to clarify the roles played by these cadherins in tissues and the ways in which defects in different cadherins cause such a wide spectrum of associated phenotypes. These disorders also constitute disparate model systems for investigations of the relative contributions of mechanical adhesive strength and intracellular signaling pathways to the pathogenic process for a given cadherin.

Les dystrophies retiniennes hereditaires constituent une cause frequente de cecite chez l'homme. Leur prevalence a ete estimee a 1/3000. Les retinites pigmentaires representent a elles seules 66% de ces atteintes. A ce jour, seuls quatre genes responsables ont ete identifies chez l'homme, qui ne rendent compte que de 15% des atteintes. L'etude de la fonction et de la regulation de l'ensemble des genes responsables de retinite pigmentaire dans differents modeles animaux devrait, un jour, permettre de comprendre la physiopathologie de ces dystrophies retiniennes pour lesquelles il n'existe encore aucun traitement. Il s'agit d'animaux porteurs de mutations spontanees, ou d'animaux transgeniques ou chimeriques.

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.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.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.