Michel Leibovici

A human homologue of the Drosophila eyes absent gene underlies branchio-oto-renal (BOR) syndrome and identifies a novel gene family.

A candidate gene for Branchio-Oto-Renal (BOR) syndrome was identified at chromosome 8q13.3 by positional cloning and shown to underlie the disease. This gene is a human homologue of the Drosophila eyes absent gene (eya), and was therefore called EYA1. A highly conserved 271-amino acid C-terminal region was also found in the products of two other human genes (EYA2 and EYA3), demonstrating the existence of a novel gene family. The expression pattern of the murine EYA1 orthologue, Eya1, suggests a role in the development of all components of the inner ear, from the emergence of the otic placode. In the developing kidney, the expression pattern is indicative of a role for Eya1 in the metanephric cells surrounding the ‘just-divided’ ureteric branches.

A defect in harmonin, a PDZ domain-containing protein expressed in the inner ear sensory hair cells, underlies Usher syndrome type 1C

Usher syndrome type 1 (USH1) is an autosomal recessive sensory defect involving congenital profound sensorineural deafness, vestibular dysfunction and blindness (due to progressive retinitis pigmentosa). Six different USH1 loci have been reported. So far, only MYO7A (USH1B), encoding myosin VIIA (ref. 2), has been identified as a gene whose mutation causes the disease. Here, we report a gene underlying USH1C (MIM 276904), a USH1 subtype described in a population of Acadian descendants from Louisiana and in a Lebanese family. We identified this gene (USH1C), encoding a PDZ-domain–containing protein, harmonin, in a subtracted mouse cDNA library derived from inner ear sensory areas. In patients we found a splice-site mutation, a frameshift mutation and the expansion of an intronic variable number of tandem repeat (VNTR). We showed that, in the mouse inner ear, only the sensory hair cells express harmonin. The inner ear Ush1c transcripts predicted several harmonin isoforms, some containing an additional coiled-coil domain and a proline- and serine-rich region. As several of these transcripts were absent from the eye, we propose that USH1C also underlies the DFNB18 form of isolated deafness.

Mutations in the gene encoding pejvakin, a newly identified protein of the afferent auditory pathway, cause DFNB59 auditory neuropathy

Auditory neuropathy is a particular type of hearing impairment in which neural transmission of the auditory signal is impaired, while cochlear outer hair cells remain functional. Here we report on DFNB59, a newly identified gene on chromosome 2q31.1–q31.3 mutated in four families segregating autosomal recessive auditory neuropathy. DFNB59 encodes pejvakin, a 352-residue protein. Pejvakin is a paralog of DFNA5, a protein of unknown function also involved in deafness. By immunohistofluorescence, pejvakin is detected in the cell bodies of neurons of the afferent auditory pathway. Furthermore, Dfnb59 knock-in mice, homozygous for the R183W variant identified in one DFNB59 family, show abnormal auditory brainstem responses indicative of neuronal dysfunction along the auditory pathway. Unlike previously described sensorineural deafness genes, all of which underlie cochlear cell pathologies, DFNB59 is the first human gene implicated in nonsyndromic deafness due to a neuronal defect.

Otoancorin, an inner ear protein restricted to the interface between the apical surface of sensory epithelia and their overlying acellular gels, is defective in autosomal recessive deafness DFNB22

A 3,673-bp murine cDNA predicted to encode a glycosylphosphatidylinositol-anchored protein of 1,088 amino acids was isolated during a study aimed at identifying transcripts specifically expressed in the inner ear. This inner ear-specific protein, otoancorin, shares weak homology with megakaryocyte potentiating factor/mesothelin precursor. Otoancorin is located at the interface between the apical surface of the inner ear sensory epithelia and their overlying acellular gels. In the cochlea, otoancorin is detected at two attachment zones of the tectorial membrane, a permanent one along the top of the spiral limbus and a transient one on the surface of the developing greater epithelial ridge. In the vestibule, otoancorin is present on the apical surface of nonsensory cells, where they contact the otoconial membranes and cupulae. The identification of the mutation (IVS12+2T>C) in the corresponding gene OTOA in one consanguineous Palestinian family affected by nonsyndromic recessive deafness DFNB22 assigns an essential function to otoancorin. We propose that otoancorin ensures the attachment of the inner ear acellular gels to the apical surface of the underlying nonsensory cells.

Mutations in a new gene encoding a protein of the hair bundle cause non-syndromic deafness at the DFNB16 locus.

Hearing impairment affects about 1 in 1,000 children at birth. Approximately 70 loci implicated in non-syndromic forms of deafness have been reported in humans and 24 causative genes have been identified (see also http://www.uia.ac.be/dnalab/hhh). We report a mouse transcript, isolated by a candidate deafness gene approach, that is expressed almost exclusively in the inner ear. Genomic analysis shows that the human ortholog STRC (so called owing to the name we have given its protein—stereocilin), which is located on chromosome 15q15, contains 29 exons encompassing approximately 19 kb. STRC is tandemly duplicated, with the coding sequence of the second copy interrupted by a stop codon in exon 20. We have identified two frameshift mutations and a large deletion in the copy containing 29 coding exons in two families affected by autosomal recessive non-syndromal sensorineural deafness linked to the DFNB16 locus. Stereocilin is made up of 1,809 amino acids, and contains a putative signal petide and several hydrophobic segments. Using immunohistolabeling, we demonstrate that, in the mouse inner ear, stereocilin is expressed only in the sensory hair cells and is associated with the stereocilia, the stiff microvilli forming the structure for mechanoreception of sound stimulation.

Chapter 8 Mouse Models for Human Hereditary Deafness

Hearing impairment is a frequent condition in humans. Identification of the causative genes for the early onset forms of isolated deafness began 15 years ago and has been very fruitful. To date, approximately 50 causative genes have been identified. Yet, limited information regarding the underlying pathogenic mechanisms can be derived from hearing tests in deaf patients. This chapter describes the success of mouse models in the elucidation of some pathophysiological processes in the auditory sensory organ, the cochlea. These models have revealed a variety of defective structures and functions at the origin of deafness genetic forms. This is illustrated by three different examples: (1) the DFNB9 deafness form, a synaptopathy of the cochlear sensory cells where otoferlin is defective; (2) the Usher syndrome, in which deafness is related to abnormal development of the hair bundle, the mechanoreceptive structure of the sensory cells to sound; (3) the DFNB1 deafness form, which is the most common form of inherited deafness in Caucasian populations, mainly caused by connexin-26 defects that alter gap junction communication between nonsensory cochlear cells.

Stereocilin connects outer hair cell stereocilia to one another and to the tectorial membrane

Stereocilin is defective in a recessive form of deafness, DFNB16. We studied the distribution of stereocilin in the developing and mature mouse inner ear and analyzed the consequences of its absence in stereocilin‐null (Strc−/−) mice that suffer hearing loss starting at postnatal day 15 (P15) and progressing until P60. Using immunofluorescence and immunogold electron microscopy, stereocilin was detected in association with two cell surface specializations specific to outer hair cells (OHCs) in the mature cochlea: the horizontal top connectors that join the apical regions of adjacent stereocilia within the hair bundle, and the attachment links that attach the tallest stereocilia to the overlying tectorial membrane. Stereocilin was also detected around the kinocilium of vestibular hair cells and immature OHCs. In Strc−/− mice the OHC hair bundle was structurally and functionally normal until P9. Top connectors, however, did not form and the cohesiveness of the OHC hair bundle progressively deteriorated from P10. The stereocilia were still interconnected by tip links at P14, but these progressively disappeared from P15. By P60 the stereocilia, still arranged in a V‐shaped bundle, were fully disconnected from each other. Stereocilia imprints on the lower surface of the tectorial membrane were also not observed in Strc−/− mice, thus indicating that the tips of the tallest stereocilia failed to be embedded in this gel. We conclude that stereocilin is essential to the formation of horizontal top connectors. We propose that these links, which maintain the cohesiveness of the mature OHC hair bundle, are required for tip‐link turnover. J. Comp. Neurol. 519:194‐210, 2011.

Characterization and developmental expression of Xenopus proliferating cell nuclear antigen (PCNA).

The coding sequence of the proliferating cell nuclear antigen (PCNA) was characterized in the amphibian Xenopus laevis. The deduced protein sequence shares an extensive homology (89%) with the mammalian PCNA coding sequences. Xenopus PCNA is expressed beginning in early oogenesis and reaches a level of 3 X 10(7) transcripts per mature oocyte, whereas proliferative somatic cells contain 3 X 10(2) PCNA transcripts per cell. Most of the PCNA protein is expressed during late oogenesis and one single stage VI oocyte contains the amount of PCNA protein present in 4 X 10(5) somatic cells in culture. Thus most, if not all, of the PCNA required for early development is stored as a maternal gene product. Part of the mRNA stockpile is degraded during the cleavage stage and then new PCNA zygotic expression at the neurula stage maintains a constitutive value of 30 transcripts per cell until the tailbud stage. The maternal protein is maintained at a constant level during embryonic development at least until the swimming tadpole stage. The protein is localized in the nuclei at all stages of oogenesis and development that were examined.

Cochlear outer hair cells undergo an apical circumference remodeling constrained by the hair bundle shape.

Epithelial cells acquire diverse shapes relating to their different functions. This is particularly relevant for the cochlear outer hair cells (OHCs), whose apical and basolateral shapes accommodate the functioning of these cells as mechano-electrical and electromechanical transducers, respectively. We uncovered a circumferential shape transition of the apical junctional complex (AJC) of OHCs, which occurs during the early postnatal period in the mouse, prior to hearing onset. Geometric analysis of the OHC apical circumference using immunostaining of the AJC protein ZO1 and Fourier-interpolated contour detection characterizes this transition as a switch from a rounded-hexagon to a non-convex circumference delineating two lateral lobes at the neural side of the cell, with a negative curvature in between. This shape tightly correlates with the ‘V’-configuration of the OHC hair bundle, the apical mechanosensitive organelle that converts sound-evoked vibrations into variations in cell membrane potential. The OHC apical circumference remodeling failed or was incomplete in all the mouse mutants affected in hair bundle morphogenesis that we tested. During the normal shape transition, myosin VIIa and myosin II (A and B isoforms) displayed polarized redistributions into and out of the developing lobes, respectively, while Shroom2 and F-actin transiently accumulated in the lobes. Defects in these redistributions were observed in the mutants, paralleling their apical circumference abnormalities. Our results point to a pivotal role for actomyosin cytoskeleton tensions in the reshaping of the OHC apical circumference. We propose that this remodeling contributes to optimize the mechanical coupling between the basal and apical poles of mature OHCs.

Initial characterization of kinocilin, a protein of the hair cell kinocilium

A subtracted library prepared from vestibular sensory areas [Nat. Genet. 26 (2000) 51] was used to identify a 960bp murine transcript preferentially expressed in the inner ear and testis. The cDNA predicts a basic 124aa protein that does not share any significant sequence homology with known proteins. Immunofluorescence and immunoelectron microscopy revealed that the protein is located mainly in the kinocilium of sensory cells in the inner ear. The protein was thus named kinocilin. In the mouse, kinocilin is first detected in the kinocilia of vestibular and auditory hair cells at embryonic days 14.5, and 18.5, respectively. In the mature vestibular hair cells, kinocilin is still present in the kinocilium. As the auditory hair cells begin to lose the kinocilium during postnatal development, kinocilin becomes distributed in an annular pattern at the apex of these cells, where it co-localizes with the tubulin belt [Hear. Res. 42 (1989) 1]. In mature auditory hair cells, kinocilin is also present at the level of the cuticular plate, at the base of each stereocilium. In addition, as the kinocilium regresses from developing auditory hair cells, kinocilin begins to be expressed by the pillar cells and Deiters cells, that both contain prominent transcellular and apical bundles of microtubules. By contrast, kinocilin was not detected in the supporting cells in the vestibular end organs. The protein is also present in the manchette of the spermatids, a transient structure enriched in interconnected microtubules. We propose that kinocilin has a role in stabilizing dense microtubular networks or in vesicular trafficking.