The acquisition of mechano-electrical transducer current adaptation in auditory hair cells requires myosin VI

Mutations in Myo6, the gene encoding the (F-actin) minus end-directed unconventional myosin, myosin VI, cause hereditary deafness in mice (Snell's waltzer) and humans. In the sensory hair cells of the cochlea, myosin VI is expressed in the cell bodies and along the stereocilia that project from the cells’ apical surface. It is required for maintaining the structural integrity of the mechanosensitive hair bundles formed by the stereocilia. In this study we investigate whether myosin VI contributes to mechano-electrical transduction. We report that Ca²+-dependent adaptation of the mechano-electrical transducer (MET) current, which serves to keep the transduction apparatus operating within its most sensitive range, is absent in outer and inner hair cells from homozygous Snell's waltzer mutant mice, which fail to express myosin VI. The operating range of the MET channels is also abnormal in the mutants, resulting in the absence of a resting MET current. We found that cadherin 23, a component of the hair bundle's transient lateral links, fails to be downregulated along the length of the stereocilia in maturing Myo6 mutant mice. MET currents of heterozygous littermates appear normal. We propose that myosin VI, by removing key molecules from developing hair bundles, is required for the development of the MET apparatus and its Ca²+-dependent adaptation

Lhx3, a LIM domain transcription factor, is regulated by Pou4f3 in the auditory but not in the vestibular system

A dominant mutation of the gene encoding the POU4F3 transcription factor underlies human non-syndromic progressive hearing loss DFNA15. Using oligonucleotide microarrays to generate expression profiles of inner ears of Pou4f3 ddl/ ddl mutant and wild-type mice, we have identified and validated Lhx3, a LIM domain transcription factor, as an in vivo target gene regulated by Pou4f3. Lhx3 is a hair cell-specific gene expressed in all hair cells of the auditory and vestibular system as early as embryonic day 16. The level of Lhx3 mRNA is greatly reduced in the inner ears of embryonic Pou4f3 mutant mice. Our data also show that the expression of Lhx3 is regulated differently in auditory and vestibular hair cells. This is the first example of a hair cell-specific gene expressed both in auditory and in vestibular hair cells, with differential regulation of expression in these two closely related systems.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/72019/1/j.1460-9568.2007.05332.x.pd

CLRN1 Is Nonessential in the Mouse Retina but Is Required for Cochlear Hair Cell Development

Mutations in the CLRN1 gene cause Usher syndrome type 3 (USH3), a human disease characterized by progressive blindness and deafness. Clarin 1, the protein product of CLRN1, is a four-transmembrane protein predicted to be associated with ribbon synapses of photoreceptors and cochlear hair cells, and recently demonstrated to be associated with the cytoskeleton. To study Clrn1, we created a Clrn1 knockout (KO) mouse and characterized the histological and functional consequences of Clrn1 deletion in the retina and cochlea. Clrn1 KO mice do not develop a retinal degeneration phenotype, but exhibit progressive loss of sensory hair cells in the cochlea and deterioration of the organ of Corti by 4 months. Hair cell stereocilia in KO animals were longer and disorganized by 4 months, and some Clrn1 KO mice exhibited circling behavior by 5–6 months of age. Clrn1 mRNA expression was localized in the retina using in situ hybridization (ISH), laser capture microdissection (LCM), and RT–PCR. Retinal Clrn1 transcripts were found throughout development and adulthood by RT–PCR, although expression peaked at P7 and declined to undetectable levels in adult retina by ISH. LCM localized Clrn1 transcripts to the retinas inner nuclear layer, and WT levels of retinal Clrn1 expression were observed in photoreceptor-less retinas. Examination of Clrn1 KO mice suggests that CLRN1 is unnecessary in the murine retina but essential for normal cochlear development and function. This may reflect a redundancy in the mouse retina not present in human retina. In contrast to mouse KO models of USH1 and USH2, our data indicate that Clrn1 expression in the retina is restricted to the Müller glia. This is a novel finding, as most retinal degeneration associated proteins are expressed in photoreceptors, not in glia. If CLRN1 expression in humans is comparable to the expression pattern observed in mice, this is the first report of an inner retinal protein that, when mutated, causes retinal degeneration

USH3A transcripts encode clarin-1, a four-transmembrane-domain protein with a possible role in sensory synapses

[EN] Usher syndrome type 3 (USH3) is an autosomal recessive disorder characterised by the association of post-lingual progressive hearing loss, progressive visual loss due to retinitis pigmentosa and variable presence of vestibular dysfunction. Because the previously defined transcripts do not account for all USH3 cases, we performed further analysis and revealed the presence of additional exons embedded in longer human and mouse USH3A transcripts and three novel USH3A mutations. Expression of Ush3a transcripts was localised by whole mount in situ hybridisation to cochlear hair cells and spiral ganglion cells. The full length USH3A transcript encodes clarin-1, a four-transmembrane-domain protein, which defines a novel vertebrate-specific family of three paralogues. Limited sequence homology to stargazin, a cerebellar synapse four-transmembrane-domain protein, suggests a role for clarin-1 in hair cell and photoreceptor cell synapses, as well as a common pathophysiological pathway for different Usher syndromes.We are grateful to all patients and their family members who participated in this study. We would also like to thank Ronna Hertzano for the preparation of the mouse inner ear cDNA. This work was funded by an Infrastructure grant of the Israeli Ministry of Science Culture and Sports, the Crown Human Genome Center at The Weizmann Institute of Science, the Alfried Krupp Foundation and by the Finnish Eye and Tissue Bank Foundation, the Finnish Eye Foundation, the Maud Kuistila Memorial Foundation, the Oskar Oflund Foundation, Finnish State grant TYH9235, the European Commission (QLG2-CT-1999-00988) (KB Araham) and by the Foundation Fighting Blindness. JS Beckman holds the, Hermann Mayer professorial chair and D Lancet holds the Ralf and Lois Silver professorial chair.Adato, A.; Vreugde, S.; Joensuu, T.; Avidan, N.; Hamalainen, R.; Belenkiy, O.; Olender, T.... (2002). USH3A transcripts encode clarin-1, a four-transmembrane-domain protein with a possible role in sensory synapses. European Journal of Human Genetics. 10(6):339-350. https://doi.org/10.1038/sj.ejhg.520083133935010

A Myo6 Mutation Destroys Coordination between the Myosin Heads, Revealing New Functions of Myosin VI in the Stereocilia of Mammalian Inner Ear Hair Cells

Myosin VI, found in organisms from Caenorhabditis elegans to humans, is essential for auditory and vestibular function in mammals, since genetic mutations lead to hearing impairment and vestibular dysfunction in both humans and mice. Here, we show that a missense mutation in this molecular motor in an ENU-generated mouse model, Tailchaser, disrupts myosin VI function. Structural changes in the Tailchaser hair bundles include mislocalization of the kinocilia and branching of stereocilia. Transfection of GFP-labeled myosin VI into epithelial cells and delivery of endocytic vesicles to the early endosome revealed that the mutant phenotype displays disrupted motor function. The actin-activated ATPase rates measured for the D179Y mutation are decreased, and indicate loss of coordination of the myosin VI heads or ‘gating’ in the dimer form. Proper coordination is required for walking processively along, or anchoring to, actin filaments, and is apparently destroyed by the proximity of the mutation to the nucleotide-binding pocket. This loss of myosin VI function may not allow myosin VI to transport its cargoes appropriately at the base and within the stereocilia, or to anchor the membrane of stereocilia to actin filaments via its cargos, both of which lead to structural changes in the stereocilia of myosin VI–impaired hair cells, and ultimately leading to deafness

MuD: an interactive web server for the prediction of non-neutral substitutions using protein structural data

The discrimination between functionally neutral amino acid substitutions and non-neutral mutations, affecting protein function, is very important for our understanding of diseases. The rapidly growing amounts of experimental data enable the development of computational tools to facilitate the annotation of these substitutions. Here, we describe a Random Forests-based classifier, named Mutation Detector (MuD) that utilizes structural and sequence-derived features to assess the impact of a given substitution on the protein function. In its automatic mode, MuD is comparable to alternative tools in performance. However, the uniqueness of MuD is that user-reported protein-specific structural and functional information can be added at run-time, thereby enhancing the prediction accuracy further. The MuD server, available at http://mud.tau.ac.il, assigns a reliability score to every prediction, thus offering a useful tool for the prioritization of substitutions in proteins with an available 3D structure

HIF–VEGF Pathways Are Critical for Chronic Otitis Media in Junbo and Jeff Mouse Mutants

Otitis media with effusion (OME) is the commonest cause of hearing loss in children, yet the underlying genetic pathways and mechanisms involved are incompletely understood. Ventilation of the middle ear with tympanostomy tubes is the commonest surgical procedure in children and the best treatment for chronic OME, but the mechanism by which they work remains uncertain. As hypoxia is a common feature of inflamed microenvironments, moderation of hypoxia may be a significant contributory mechanism. We have investigated the occurrence of hypoxia and hypoxia-inducible factor (HIF) mediated responses in Junbo and Jeff mouse mutant models, which develop spontaneous chronic otitis media. We found that Jeff and Junbo mice labeled in vivo with pimonidazole showed cellular hypoxia in inflammatory cells in the bulla lumen, and in Junbo the middle ear mucosa was also hypoxic. The bulla fluid inflammatory cell numbers were greater and the upregulation of inflammatory gene networks were more pronounced in Junbo than Jeff. Hif-1a gene expression was elevated in bulla fluid inflammatory cells, and there was upregulation of its target genes including Vegfa in Junbo and Jeff. We therefore investigated the effects in Junbo of small-molecule inhibitors of VEGFR signaling (PTK787, SU-11248, and BAY 43-9006) and destabilizing HIF by inhibiting its chaperone HSP90 with 17-DMAG. We found that both classes of inhibitor significantly reduced hearing loss and the occurrence of bulla fluid and that VEGFR inhibitors moderated angiogenesis and lymphangiogenesis in the inflamed middle ear mucosa. The effectiveness of HSP90 and VEGFR signaling inhibitors in suppressing OM in the Junbo model implicates HIF–mediated VEGF as playing a pivotal role in OM pathogenesis. Our analysis of the Junbo and Jeff mutants highlights the role of hypoxia and HIF–mediated pathways, and we conclude that targeting molecules in HIF–VEGF signaling pathways has therapeutic potential in the treatment of chronic OM

Consensus interpretation of the p.Met34Thr and p.Val37Ile variants in GJB2 by the ClinGen Hearing Loss Expert Panel

Purpose: Pathogenic variants in GJB2 are the most common cause of autosomal recessive sensorineural hearing loss. The classification of c.101T>C/p.Met34Thr and c.109G>A/p.Val37Ile in GJB2 are controversial. Therefore, an expert consensus is required for the interpretation of these two variants. Methods: The ClinGen Hearing Loss Expert Panel collected published data and shared unpublished information from contributing laboratories and clinics regarding the two variants. Functional, computational, allelic, and segregation data were also obtained. Case-control statistical analyses were performed. Results: The panel reviewed the synthesized information, and classified the p.Met34Thr and p.Val37Ile variants utilizing professional variant interpretation guidelines and professional judgment. We found that p.Met34Thr and p.Val37Ile are significantly overrepresented in hearing loss patients, compared with population controls. Individuals homozygous or compound heterozygous for p.Met34Thr or p.Val37Ile typically manifest mild to moderate hearing loss. Several other types of evidence also support pathogenic roles for these two variants. Conclusion: Resolving controversies in variant classification requires coordinated effort among a panel of international multi-institutional experts to share data, standardize classification guidelines, review evidence, and reach a consensus. We concluded that p.Met34Thr and p.Val37Ile variants in GJB2 are pathogenic for autosomal recessive nonsyndromic hearing loss with variable expressivity and incomplete penetrance