Unit: Sensory Deficit Genetics - INSERM (U587)

Director: Christine PETIT

Research in the laboratory of Génétique des Déficits Sensoriels aims at (1) elucidating the molecular bases of hereditary auditory defects in humans, and (2) unravelling the molecular mechanisms underlying the development and functioning of the cochlea, the mammalian auditory organ. Special emphasis is put on the molecular bases of the functioning of the sensory cells' hair bundle, the organelle devoted to auditory mechano-electrical transduction.

Understanding the mechanisms which underlie the function of sensory systems is an objective to which the study of hereditary dysfunctions of these systems can contribute, following the development of tools for genetic and genomic analysis.

At the beginning of the 1990s, we chose to study hereditary forms of human deafness for two reasons: (1) at the time they constituted an unexplored domain of hereditary sensory pathology, and (2) they should lead to an approach to the molecular basis of the development and functioning of the cochlea (the hearing receptor organ), which was then totally unknown.

Since 1995, we have identified the genes responsible for three forms of type I Usher syndrome (USH1B and USH1C, USH1G), in which there is an association of profound sensorineural deafness and progressive retinitis pigmentosa leading to blindness. We have also identified the genes affected in eight forms of isolated recessive deafness (DFNB2, DFNB9, DFNB16, DFNB18, DFNB21, DFNB22, DFNB31 and DFNB59), two forms of isolated dominant dearness (DFNA2 and DFNA3), the branchio-oto-renal and the branchio-oto syndromes, and collaborated to the identification of a few other deafness genes.

Using complementary experimental approaches, we have obtained an ensemble of results concerning the function of the proteins encoded by these genes. Most of these proteins participate in the following processes: (1) the structure of the tectorial membrane, an acellular membrane which covers the auditory sensory epithelium and which participates in the transmission of the energy of the sound wave to the hair bundle of the sensory cells, (2) the development of the hair bundle, the receptor structure to sound, which is composed of a group of rigid microvilli, the stereocilia, and which contains the machinery for mechanotransduction, (3) the function of the synapse of the sensory cells ("ribbon" synapse), and (4) the communication between cells via gap junctions.

In addition, we have undertaken a genetic study of presbycusis, a sensorineural form of deafness that predominates on high frequencies. Presbycusis may affect more than a half of the population over 70 years old. Its pathogeny is poorly understood, and the role played by genetic factors remains to be determined. With the aim of identifying the genes most commonly involved, we are currently collecting a large number of affected families.

Development of the hair bundle

Four components of the hair bundle have been identified: myosin VIIa, whirlin (see picture), harmonin and stereocilin. Mutations of the genes coding for myosin VIIa and harmonin are responsible for type I Usher syndrome (USH1B and USH1C forms), and more rarely for an isolated form of deafness. Harmonin and whirlin are two molecules with PDZ domains, submembrane proteins which organise protein complexes. The tails of unconventional myosins, such as myosin VIIa, are linked to the proteins on which the motor force of myosin is directed. The structures with which these proteins are associated are thus placed under tension, and may even move under certain circumstances. With the aim of understanding the role of myosin VIIa, a study of its ligands has been undertaken. This has allowed us to identify a new ubiquitous transmembrane molecule present in intercellular adhesion junctions, vezatin, which belongs to the same complex as E-cadherin, and thus we could propose a role for myosin VIIa in cell adhesion. Another ligand for this myosin binds to rab27, which is present at the surface of the melanosomes, thus explaining the abnormal position of melanosomes in the cells of the pigmented epithelium of the retina in patients with USH1B syndrome. Finally, we have shown that the b isoforms of harmonin, which are present in the developing hair bundle, both bind to actin filaments and induce their clustering into bundles, and also interact with cadherin-23 and protocadherin-15, two proteins also involved in Usher syndrome type I (USH1D and USH1F), which form transitory interstereociliar links. Thus, four proteins which are defective in four forms of type I Usher syndrome work together in a network of molecular interactions which contributes to the cohesion of the developing hair bundle and stabilises the stereocilia by anchoring the links which connect them to the actin filaments which form the cytoskeleton of each stereocilium. An additional protein, SANS, defective in USH1G, was subsequently included in the molecular interaction network. SANS interacts with harmonin and myosin VIIa. SANS is located beneath the hair bundle, in an apical region of the hair cell that contains many vesicles. This result may indicate that SANS controls the trafficking of myosin VIIa and harmonin towards the hair bundle. We have shown that usherin, a transmembrane protein with a large ectodomain, which underlies a genetic form of Usher syndrome type II (USH2A), contributes to a subset of interstereociliar links (ankle links) during hair bundle development too. In addition, we have shown that whirlin, responsible for the DFNB31 form of deafness, binds to and is colocalised with myosin XV in the most apical part of the growing stereocilia (see picture). Finally, we have identified a novel transmembrane protein of the hair cells, PHR1, which binds both to myosin VIIa and myosin 1c, and could thus play a role in the mechanotransduction adaptation process.

Synapses of the sensory cells

In a collaborative study, we have demonstrated that the DFNA2 form of deafness is due to a defect in the KCNQ4 potassium channel, which is expressed mainly by the outer hair cells (for which the essential function is the amplification of the sound stimulus), and by the neurons of the central auditory pathway. Especially interesting, the identification of the gene responsible for another form of recessive deafness, DFNB9, has led to the discovery of otoferlin, a transmembrane protein from the ferlin family, which is associated to the synaptic vesicles in inner hair cells (the genuine auditory sensory cells). We have produced and analysed mutant mice lacking otoferlin, and have shown that the protein plays a crucial role in neurotransmitter release at the inner hair cell synapse.

Gap junctions

Connexin26 is defective in a form of recessive deafness, DFNB1, which, we have shown, accounts for more than one-third of the cases of severe to profound deafness in children. The two cochlear cellular networks formed by the gap junctions, the epithelial network and the fibrocyte network, express connexin26 and connexin30, for which a deficit also leads to deafness. Because of the high frequency for the DFNB1 form of deafness, we have undertaken a study of its pathogenicity. Ubiquitous inactivation of the gene which codes for connexin26 (Cx26) is lethal in the mouse. We therefore performed a conditional inactivation of Cx26 in the epithelial network, and analysed the phenotype of the mutant mice, as well as that of mice in which the gene which encodes connexin30 (Cx30) was inactivated ubiquitously. In both cases, death by apoptosis was observed in cells of the sensory auditory epithelium beginning in the 3rd week postnatally, i.e. a little after wild-type mice begin to hear. Furthermore, the endocochlear potential, a transepithelial potential difference between endolymphatic and perilymphatic compartments, which normally appears in the 2nd week of life, is absent in Cx30-/- mice. This finding led us to conclude that connexin30 plays an essential role in the stria vascularis, the site of production of the endocochlear potential.

These results have been obtained because of the synergy in the activities of the various members of our laboratory. The subtracted cDNA libraries which were generated by some have also helped others in isolating deafness genes; they have been the source of promoters utilised by several scientists to generate conditional inactivations of genes in the ear. Because of the complex structure of the inner ear, the knowledge of histology and embryology of some has been essential to permit the transition from the isolation of genes responsible for deafness to the study of the pathogenicity of the corresponding forms. A real transfer of knowledge between specialists in molecular biology, embryologists and ENT physicians has taken place very rapidly. In addition, we are now able to carry out protein structure analysis and electrophysiological studies of the mechanoelectrical transduction process that takes place in the hair bundles of mouse auditory sensory cells. A dialog between those who are looking for new genes implicated in deafness and those who are trying to define the networks of molecular interactions in which the products of already-identified genes participate has been a source of ideas and discoveries for both. A continuous articulation with the clinic has made it possible to establish the frequency of defects in the connexin26 gene, to provide the first clinical description of a genetic form of isolated deafness, and above all to evaluate the medical implications of our studies. Genetic counselling for families with deaf individuals has been considerably improved.

Photo :

Whirlin, a protein containing PDZ domains which is responsible for the DFNB31 form of recessive deafness, is revealed by a specific antibody in the auditory sensory cells of a post natal day 12 mouse (red staining). The protein is present in the very apical part of the stereocilia during their differentiation and plays a key role in hair bundle growth. Stereocilia are stained in green by using fluorescent phalloidin, which specifically binds to the actin filaments.

Keywords: Audition, Deafness, Sensory physiology, Sensorineural defects, Human genetics, Cell biology, Electrophysiology


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