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• W2119942550 abstract "Report17 June 2014Open Access The CD2 isoform of protocadherin-15 is an essential component of the tip-link complex in mature auditory hair cells Elise Pepermans Elise Pepermans Unité de Génétique et Physiologie de l'Audition, Institut Pasteur, Paris, France UMRS 1120, Institut National de la Santé et de la Recherche Médicale (INSERM), Paris, France Université Pierre et Marie Curie (Paris VI), Paris, France Search for more papers by this author Vincent Michel Vincent Michel Unité de Génétique et Physiologie de l'Audition, Institut Pasteur, Paris, France UMRS 1120, Institut National de la Santé et de la Recherche Médicale (INSERM), Paris, France Université Pierre et Marie Curie (Paris VI), Paris, France Search for more papers by this author Richard Goodyear Richard Goodyear School of Life Sciences, University of Sussex, Brighton, UK Search for more papers by this author Crystel Bonnet Crystel Bonnet UMRS 1120, Institut National de la Santé et de la Recherche Médicale (INSERM), Paris, France Université Pierre et Marie Curie (Paris VI), Paris, France Syndrome de Usher et autres Atteintes Rétino-Cochléaires, Institut de la vision, Paris, France Search for more papers by this author Samia Abdi Samia Abdi Centre Hospitalier universitaire de Blida, Université Saad Dahleb, Blida, Algérie Search for more papers by this author Typhaine Dupont Typhaine Dupont Unité de Génétique et Physiologie de l'Audition, Institut Pasteur, Paris, France UMRS 1120, Institut National de la Santé et de la Recherche Médicale (INSERM), Paris, France Université Pierre et Marie Curie (Paris VI), Paris, France Search for more papers by this author Souad Gherbi Souad Gherbi Centre de référence des Surdités Génétiques, Hôpital Necker, Paris, France Search for more papers by this author Muriel Holder Muriel Holder Service de Génétique Clinique, Hôpital Jeanne-de-Flandre, Lille, France Search for more papers by this author Mohamed Makrelouf Mohamed Makrelouf Laboratoire de Biochimie Génétique, Université d'Alger 1, Alger, Algérie Search for more papers by this author Jean-Pierre Hardelin Jean-Pierre Hardelin Unité de Génétique et Physiologie de l'Audition, Institut Pasteur, Paris, France UMRS 1120, Institut National de la Santé et de la Recherche Médicale (INSERM), Paris, France Université Pierre et Marie Curie (Paris VI), Paris, France Search for more papers by this author Sandrine Marlin Sandrine Marlin Centre de référence des Surdités Génétiques, Hôpital Necker, Paris, France Search for more papers by this author Akila Zenati Akila Zenati Laboratoire de Biochimie Génétique, Université d'Alger 1, Alger, Algérie Search for more papers by this author Guy Richardson Guy Richardson School of Life Sciences, University of Sussex, Brighton, UK Search for more papers by this author Paul Avan Paul Avan Laboratoire de Biophysique Sensorielle, Université d'Auvergne, Clermont-Ferrand, France UMR 1107, Institut National de la Santé et de la Recherche Médicale (INSERM), Clermont-Ferrand, France Centre Jean Perrin, Clermont-Ferrand Cedex 01, France Search for more papers by this author Amel Bahloul Amel Bahloul Unité de Génétique et Physiologie de l'Audition, Institut Pasteur, Paris, France UMRS 1120, Institut National de la Santé et de la Recherche Médicale (INSERM), Paris, France Université Pierre et Marie Curie (Paris VI), Paris, France Search for more papers by this author Christine Petit Corresponding Author Christine Petit Unité de Génétique et Physiologie de l'Audition, Institut Pasteur, Paris, France UMRS 1120, Institut National de la Santé et de la Recherche Médicale (INSERM), Paris, France Université Pierre et Marie Curie (Paris VI), Paris, France Syndrome de Usher et autres Atteintes Rétino-Cochléaires, Institut de la vision, Paris, France Collège de France, Paris, France Search for more papers by this author Elise Pepermans Elise Pepermans Unité de Génétique et Physiologie de l'Audition, Institut Pasteur, Paris, France UMRS 1120, Institut National de la Santé et de la Recherche Médicale (INSERM), Paris, France Université Pierre et Marie Curie (Paris VI), Paris, France Search for more papers by this author Vincent Michel Vincent Michel Unité de Génétique et Physiologie de l'Audition, Institut Pasteur, Paris, France UMRS 1120, Institut National de la Santé et de la Recherche Médicale (INSERM), Paris, France Université Pierre et Marie Curie (Paris VI), Paris, France Search for more papers by this author Richard Goodyear Richard Goodyear School of Life Sciences, University of Sussex, Brighton, UK Search for more papers by this author Crystel Bonnet Crystel Bonnet UMRS 1120, Institut National de la Santé et de la Recherche Médicale (INSERM), Paris, France Université Pierre et Marie Curie (Paris VI), Paris, France Syndrome de Usher et autres Atteintes Rétino-Cochléaires, Institut de la vision, Paris, France Search for more papers by this author Samia Abdi Samia Abdi Centre Hospitalier universitaire de Blida, Université Saad Dahleb, Blida, Algérie Search for more papers by this author Typhaine Dupont Typhaine Dupont Unité de Génétique et Physiologie de l'Audition, Institut Pasteur, Paris, France UMRS 1120, Institut National de la Santé et de la Recherche Médicale (INSERM), Paris, France Université Pierre et Marie Curie (Paris VI), Paris, France Search for more papers by this author Souad Gherbi Souad Gherbi Centre de référence des Surdités Génétiques, Hôpital Necker, Paris, France Search for more papers by this author Muriel Holder Muriel Holder Service de Génétique Clinique, Hôpital Jeanne-de-Flandre, Lille, France Search for more papers by this author Mohamed Makrelouf Mohamed Makrelouf Laboratoire de Biochimie Génétique, Université d'Alger 1, Alger, Algérie Search for more papers by this author Jean-Pierre Hardelin Jean-Pierre Hardelin Unité de Génétique et Physiologie de l'Audition, Institut Pasteur, Paris, France UMRS 1120, Institut National de la Santé et de la Recherche Médicale (INSERM), Paris, France Université Pierre et Marie Curie (Paris VI), Paris, France Search for more papers by this author Sandrine Marlin Sandrine Marlin Centre de référence des Surdités Génétiques, Hôpital Necker, Paris, France Search for more papers by this author Akila Zenati Akila Zenati Laboratoire de Biochimie Génétique, Université d'Alger 1, Alger, Algérie Search for more papers by this author Guy Richardson Guy Richardson School of Life Sciences, University of Sussex, Brighton, UK Search for more papers by this author Paul Avan Paul Avan Laboratoire de Biophysique Sensorielle, Université d'Auvergne, Clermont-Ferrand, France UMR 1107, Institut National de la Santé et de la Recherche Médicale (INSERM), Clermont-Ferrand, France Centre Jean Perrin, Clermont-Ferrand Cedex 01, France Search for more papers by this author Amel Bahloul Amel Bahloul Unité de Génétique et Physiologie de l'Audition, Institut Pasteur, Paris, France UMRS 1120, Institut National de la Santé et de la Recherche Médicale (INSERM), Paris, France Université Pierre et Marie Curie (Paris VI), Paris, France Search for more papers by this author Christine Petit Corresponding Author Christine Petit Unité de Génétique et Physiologie de l'Audition, Institut Pasteur, Paris, France UMRS 1120, Institut National de la Santé et de la Recherche Médicale (INSERM), Paris, France Université Pierre et Marie Curie (Paris VI), Paris, France Syndrome de Usher et autres Atteintes Rétino-Cochléaires, Institut de la vision, Paris, France Collège de France, Paris, France Search for more papers by this author Author Information Elise Pepermans1,2,3, Vincent Michel1,2,3, Richard Goodyear4, Crystel Bonnet2,3,5, Samia Abdi6, Typhaine Dupont1,2,3, Souad Gherbi7, Muriel Holder8, Mohamed Makrelouf9, Jean-Pierre Hardelin1,2,3, Sandrine Marlin7, Akila Zenati9, Guy Richardson4, Paul Avan10,11,12, Amel Bahloul1,2,3 and Christine Petit 1,2,3,5,13 1Unité de Génétique et Physiologie de l'Audition, Institut Pasteur, Paris, France 2UMRS 1120, Institut National de la Santé et de la Recherche Médicale (INSERM), Paris, France 3Université Pierre et Marie Curie (Paris VI), Paris, France 4School of Life Sciences, University of Sussex, Brighton, UK 5Syndrome de Usher et autres Atteintes Rétino-Cochléaires, Institut de la vision, Paris, France 6Centre Hospitalier universitaire de Blida, Université Saad Dahleb, Blida, Algérie 7Centre de référence des Surdités Génétiques, Hôpital Necker, Paris, France 8Service de Génétique Clinique, Hôpital Jeanne-de-Flandre, Lille, France 9Laboratoire de Biochimie Génétique, Université d'Alger 1, Alger, Algérie 10Laboratoire de Biophysique Sensorielle, Université d'Auvergne, Clermont-Ferrand, France 11UMR 1107, Institut National de la Santé et de la Recherche Médicale (INSERM), Clermont-Ferrand, France 12Centre Jean Perrin, Clermont-Ferrand Cedex 01, France 13Collège de France, Paris, France *Corresponding author. Tel: +33 145688890; E-mail: [email protected] EMBO Mol Med (2014)6:984-992https://doi.org/10.15252/emmm.201403976 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Protocadherin-15 (Pcdh15) is a component of the tip-links, the extracellular filaments that gate hair cell mechano-electrical transduction channels in the inner ear. There are three Pcdh15 splice isoforms (CD1, CD2 and CD3), which only differ by their cytoplasmic domains; they are thought to function redundantly in mechano-electrical transduction during hair-bundle development, but whether any of these isoforms composes the tip-link in mature hair cells remains unknown. By immunolabelling and both morphological and electrophysiological analyses of post-natal hair cell-specific conditional knockout mice (Pcdh15ex38-fl/ex38-fl Myo15-cre+/−) that lose only this isoform after normal hair-bundle development, we show that Pcdh15-CD2 is an essential component of tip-links in mature auditory hair cells. The finding, in the homozygous or compound heterozygous state, of a PCDH15 frameshift mutation (p.P1515Tfs*4) that affects only Pcdh15-CD2, in profoundly deaf children from two unrelated families, extends this conclusion to humans. These results provide key information for identification of new components of the mature auditory mechano-electrical transduction machinery. This will also serve as a basis for the development of gene therapy for deafness caused by PCDH15 defects. Synopsis Pcdh15-CD2 isoform is shown essential for auditory function in mature hair cells both in humans and mice but not for visual function and make the lower part of the tip-link in inner ears. This is a major advance for the development of gene therapy for deafness caused by PCDH15 defects. In immature mouse cochlear hair cells, the three protocadherin-15 splice isoforms (CD1, CD2 and CD3), which differ only by their cytoplasmic region, are redundant to form the lower part of the tip-links, fibrous link that gate the mechano-electrical transduction channels. In contrast, the CD2 isoform is an indispensable tip-link component in mature mouse cochlear hair cells. The CD2 isoform of protocadherin-15 is also mandatory for hearing in humans as revealed by the identification of mutations only affecting this isoform in profoundly deaf patients. Introduction Three transmembrane protocadherin-15 (Pcdh15) splice isoforms (Pcdh15-CD1, Pcdh15-CD2 and Pcdh15-CD3) differing only in the C-terminal part of their cytoplasmic domains (Fig 1A) are present in the hair bundles of developing cochlear hair cells (Ahmed et al, 2006). The inner and outer hair cells (IHCs and OHCs) of the cochlea have distinct roles (signal transmission for IHCs; frequency dependent mechanical amplification for OHCs), but both perform mechano-electrical transduction (MET). MET takes place in the hair bundle, an apical ensemble of stiff microvilli (stereocilia) organized in three rows of increasing height, the short, middle and tall rows. The oblique tip-link connects the tip of a stereocilium in one row to the side of an adjacent taller stereocilium. This link controls the open probability of the MET channels located at its lower insertion point, namely at the tips of short- and middle-row stereocilia (Howard & Hudspeth, 1988; Beurg et al, 2009). Pcdh15 and cadherin-23 form the lower and upper parts of this link, respectively (Kazmierczak et al, 2007). Kinociliary links, also composed of these cadherins (Goodyear et al, 2010), connect the stereocilia to the kinocilium, a structure that regresses before the onset of hearing but is necessary for the correct planar polarization of the hair bundle during the early stages of development. Figure 1. Immunolabelling of Pcdh15-CD2 Schematic of Pcdh15 isoforms. The position of the CD2 fragment used to produce the anti-Pcdh15-CD2 antibody is indicated (138 C-terminal amino acids). (TM: transmembrane domain, * PBM: PDZ-binding motif). Confocal images of hair cells (OHC above, IHC below) stained for Pcdh15-CD2 (green) and actin (red) at P5 (immature hair cells) and P16 (mature hair cells). Scale bars: 2 μm. Transmission electron micrographs of a Pcdh15-CD2 immunoreactive IHC hair bundle at P15 (left panel), enlargements of boxed region: two adjacent sections of the same hair bundle (central panels) and a tip-link profile from an OHC (right panel). Arrowheads indicate gold particles. Note that the presence of the gold particles is restricted to the tips of the short- and middle-row stereocilia, consistent with Pcdh15-CD2 being a component of the lower part of the tip-link, and to the apico-lateral region of the tallest stereocilia, suggesting that Pcdh15-CD2 could also be a component of the lateral links between stereocilia of the tallest row. Scale bars: 200 nm. Download figure Download PowerPoint Auditory defects are not detected in mice lacking Pcdh15-CD1 or Pcdh15-CD3, whereas mice lacking Pcdh15-CD2 (PCDH15-ΔCD2) are profoundly deaf. However, tip-links are observed and MET currents can be recorded in immature hair cells of PCDH15-ΔCD2 mice, and consequently, it has been suggested that the three Pcdh15 isoforms function redundantly in the tip-link (Webb et al, 2011). The deafness of PCDH15-ΔCD2 mice has been attributed to defects in hair-bundle polarity, a phenotype consistent with Pcdh15-CD2 being an essential component of kinociliary links (Ahmed et al, 2006; Webb et al, 2011). Considering that reported mouse mutants with misoriented hair bundles display only moderate hearing impairments (Curtin et al, 2003; Jagger et al, 2011; Copley et al, 2013), we investigated whether Pcdh15-CD2 could play a previously unrecognized but critical role in mature auditory hair cells. Results and Discussion Pcdh15-CD2 is located at the stereociliary tips in auditory hair bundles Previous immunofluorescence studies have not demonstrated that Pcdh15-CD2 is present in mature hair bundles (Ahmed et al, 2006). We therefore generated a new polyclonal antibody specific to this isoform (Supplementary Fig S1): from post-natal day 5 (P5) onwards, Pcdh15-CD2 was detected at the tip of every stereocilium, in both IHCs and OHCs (Fig 1B). In mature IHCs, Pcdh15-CD2 antibody labelling was conspicuous at the tips of the three rows of stereocilia, in particular at the lower tip-link insertion points. Transmission electron microscopy of immunogold-labelled longitudinal sections of hair bundles showed that almost all gold particles were at the apices of the three stereociliary rows in mature OHCs and IHCs (Fig 1C). The high concentration of gold particles at the extreme apex of a small row IHC stereocilium in one section (Fig 1C, second panel) and at the extreme apex of a neighbouring, middle-row stereocilium in an adjacent section (Fig 1C, third panel) exemplifies the restricted distribution of Pcdh15-CD2. A typical tip-link profile from an OHC hair bundle showing immunogold labelling at the tip-link lower insertion point is also shown in the last panel of Fig 1C. These observations suggest that Pcdh15-CD2 is a lower tip-link component in mature auditory hair cells (see also Supplementary Figs S2 and S3). Absence of Pcdh15-CD2 results in the loss of tip-links in mature auditory hair cells To probe the role of Pcdh15-CD2 in mature hair bundles, a post-natal hair cell-specific conditional knockout mouse model, Pcdh15ex38-fl/ex38-flMyo15-cre+/− mice, was generated. Conditional post-natal deletion of exon 38, specific to the Pcdh15-CD2 isoform, circumvented the early morphogenetic defects caused by the absence of this isoform during hair-bundle development (Webb et al, 2011; see Methods and Supplementary Fig S4). The auditory function of these mutant mice was probed by in vivo audiometric tests, which explore the activities of IHCs and OHCs. At the onset of hearing, on P15, auditory function, measured as auditory brainstem responses (ABRs), was identical in Pcdh15ex38-fl/ex38-flMyo15-cre+/−mice (referred to as conditional Pcdh15Δ'CD2 mice) and their Pcdh15ex38-fl/ex38-fl littermate controls. By P17, ABR thresholds in the mutants started to increase, and by P30, they were above 90 dB SPL across the frequency spectrum tested (5–40 kHz). By P45, the conditional Pcdh15Δ'CD2 mice lacked any identifiable ABR response to loud sound stimulation (115 dB SPL), indicating complete hearing loss and fully defective IHCs. Distortion-product otoacoustic emissions (DPOAEs), which involve OHC MET channel function (Avan et al, 2013), increased in threshold and decreased in amplitude from P24 onwards, had almost disappeared by P30 and were completely absent on P45. Cochlear microphonic (CM) potentials, phasic extracellular potentials reflecting MET currents in the OHCs of the basal region of the cochlea (Patuzzi et al, 1989), had an amplitude reduced to 4% of that in controls by P30, indicating a loss of MET in OHCs. As ABR thresholds were 40 dB higher on P21 despite normal DPOAEs, this indicates that IHC function was already impaired at this age. Consistent with this, the amplitude of compound action potentials [representing synchronous firing of afferent neurons innervating the IHCs (Spoendlin & Baumgartner, 1977)] in response to loud sound stimuli (105 dB SPL, the processing of which relies only on IHC function) on P30 was only 3% of that in controls (Fig 2A–C and Supplementary Fig S5). Thus, MET, whilst initially normal in both IHCs and OHCs in conditional Pcdh15Δ'CD2 mice, is totally abolished by P45. In contrast, conditional Pcdh15Δ'CD2 mice explored by behavioural tests (see Methods) did not show vestibular dysfunction, as is the case for PCDH15-ΔCD2 mice (Webb et al, 2011). Figure 2. Auditory testing and morphological analysis of hair bundles in conditional Pcdh15Δ'CD2 mice A. ABR thresholds across the 5-40 kHz frequency spectrum on P30 and P45. Blue and red curves (mean ± SEM) correspond to Pcdh15ex38-fl/ex38-fl (control) and conditional Pcdh15-Δ'CD2 mice, respectively. In P45 conditional Pcdh15Δ'CD2 mice, ABR waves could not be detected even at 115 dB SPL (P30: conditional Pcdh15Δ'CD2 mice n = 7, control n = 15, P45: conditional Pcdh15Δ'CD2 mice n = 6, control n = 15). B, C. CM and CAP responses to a 10 kHz, 105 dB SPL tone burst in a Pcdh15ex38-fl/ex38-fl P30 control mouse (blue) and a P30 conditional Pcdh15Δ'CD2 mouse (red). D. Scanning electron micrographs showing that the orientation of OHC hair bundles is normal in P30 conditional Pcdh15Δ'CD2 mice in which Pcdh15-CD2 is lost after hair-bundle development, in contrast to KO Pcdh15Δ'CD2 mice that lack Pcdh15-CD2 throughout development. Scale bars: 2 μm. E. Scanning electron micrographs showing IHC and OHC hair bundles of Pcdh15 ex38-fl/ex38-fl (control) and conditional Pcdh15Δ'CD2 mice on P30 and P45. White arrowheads indicate stereocilia with prolate-shaped tips, and arrows show regression of small row stereocilia. Tip-links (black arrowheads) are visible in controls on P30 and P45 but not in conditional Pcdh15ΔCD2 mice at either age. Scale bars: 500 nm. Download figure Download PowerPoint Auditory hair bundles were analysed morphologically by scanning electron microscopy (Fig 2D and E). In conditional Pcdh15Δ'CD2 mice, hair bundles were correctly oriented, unlike those of Pcdh15ex38-fl/ex38-fl PGK-cre+/−mice (referred to as KO Pcdh15Δ'CD2 mice) that lack Pcdh15-CD2 throughout development (Fig 2D; see Materials and Methods and Supplementary Figs S4 and S6). In conditional Pcdh15Δ'CD2 mice, very few tip-links were observed on OHCs on P30, and none were detected by P45; these links were consistently observed in littermate controls at the same ages. In P30 IHCs of conditional Pcdh15Δ'CD2 mice, most middle-row stereocilia had lost their distal prolate shape, indicating the loss of tip-link tension (Tilney et al, 1988). By P45, many middle-row stereocilia of IHCs had reduced lengths and most, if not all, short-row stereocilia had regressed entirely (Fig 2E). Some OHC short-row stereocilia were also missing. These anomalies are reminiscent of those reported in post-natal conditional knockout mice lacking other proteins of the tip-link complex (Caberlotto et al, 2011) and consequently are consistent with the existence of a functional connection between the tip-link and F-actin polymerization in the stereocilia. Thus, in conditional Pcdh15Δ'CD2 mice, when Pcdh15-CD2 is no longer expressed in auditory hair bundles, tip-links are lost, both from IHCs and OHCs: this explains the loss of MET. These results demonstrate that Pcdh15-CD2 is an essential component of tip-links in mature auditory hair cells, which accounts for the profound deafness of mutant mice lacking this isoform. Patients lacking PCDH15-CD2 are profoundly deaf In humans, biallelic loss-of-function mutations in PCDH15 result in Usher syndrome of type 1 (Usher 1), a dual sensory disorder combining severe to profound congenital deafness, vestibular disorders and prepubertal onset retinitis pigmentosa eventually leading to blindness. To date, no Usher 1 patient carrying a mutation specifically affecting only one of the three Pcdh15 isoforms has been reported, although more than 45 different PCDH15 mutations have been detected in the about 400 patients analysed (https://grenada.lumc.nl/LOVD2/Usher_montpellier/USHbases.html; Bonnet et al, 2011; and Crystel Bonnet, unpublished results). We therefore extended our search for specific Pcdh15 isoform defects to patients affected by isolated (nonsyndromic) deafness. Sanger sequencing was used to analyse PCDH15 in 60 unrelated individuals with congenital profound sensorineural deafness, for whom common pathogenic mutations in the most prevalent deafness genes (GJB2, MYO15A and OTOF) had been excluded. In three patients from two independent families (patient IV.2 from family CPID4744 and patients IV.4 and IV.5 from family CPIDS6-10), we found a frameshift mutation specific to the CD2 isoform: c.4542dup (p.P1515Tfs*4), in exon 38 of PCDH15. This mutation was not found in the Exome Variant Server database (http://evs.gs.washington.edu/EVS/). It is predicted to lead either to a truncated form (lacking the 273 C-terminal amino acids) or to the absence of the CD2 isoform due to nonsense-mediated mRNA decay (Maquat, 1995). The patient from family CPID4744 carried this mutation in the homozygous state, and the two patients from family CPIDS6-10 carried it in the heterozygous state, in association with a nonsense mutation, c.400C>T (p.R134*), located in PCDH15 exon 6, which is common to the three Pcdh15 isoforms. Segregation analysis of these mutations (see Methods) showed that each parent had transmitted one mutation to the affected children, who are thus compound heterozygotes. Five siblings of the two families (patient IV.1 from family CPID4744 and patients IV.1, IV.2, IV.3 and IV.6 from family CPIDS6-10) with normal hearing did not carry the c.4542dup (p.P1515Tfs*4) mutation in the homozygous state (family CPID4744) or in the compound heterozygous state (family CPIDS6-10). Whole exome sequencing was performed in the three patients and did not detect any mutations with predicted pathogenicity in either the homozygous or the compound heterozygous state in known deafness genes or in other genes. These three patients only show hearing impairment and no clinical signs of vestibular dysfunction (no delay in walking and no video-nystagmography abnormalities) or retinal defects (no vision difficulties in reduced illumination, and no abnormalities in any of fundus autofluorescence, optical coherence tomography, or scotopic or photopic electroretinogram; Fig 3). Abnormalities of the electroretinogram are systematically detected earlier in Usher 1 patients. This therefore suggests that the absence of the Pcdh15-CD2 isoform is responsible for an isolated (nonsyndromic) form of profound deafness. Figure 3. Profound deafness in patients carrying mutations affecting PCDH15-CD2 Segregation of the PCDH15 mutations in the two families. Air-conduction pure-tone audiometric curves for patients IV.2 (family CPID4744) and IV.4 (family CPIDS6-10) at the age of 3 and 5 years, respectively. Hearing thresholds at all sound frequencies tested (0.5, 1, 2, 4 and 8 kHz) were above 120 dB HL, the largest intensity tested, for both ears in both patients, indicating bilateral profound deafness. Scotopic and photopic electroretinogram in patient IV.2 (family CPID4744) at the age of 7 years, showing normal a- and b-waves in both traces. Download figure Download PowerPoint Conclusion The functional and morphological defects of conditional Pcdh15Δ'CD2 mice show that Pcdh15-CD2, an isoform with a specific 284-amino acid C-terminal region, is an essential component of the tip-link in mature auditory hair cells. Knockout mice for Pcdh15-CD1 or Pcdh15-CD3 are not hearing-impaired (Webb et al, 2011), and consequently, we can conclude that the Pcdh15-CD2 isoform is the only Pcdh15 isoform required for the maintenance and/or function of the MET machinery in the two types of mature auditory sensory cells, the IHCs and OHCs. Pcdh15-CD2 is therefore essential both for hair-bundle morphogenesis, as a component of the kinociliary links, and later in mature auditory hair cells, for MET, as a component of the tip-links. Our work thus demonstrates that the auditory MET machinery undergoes a molecular maturation process, switching from a developmental form in which the Pcdh15 isoforms are functionally redundant to a fully mature form in which Pcdh15-CD2 is critical. Identifying which physiological features of the MET machinery are modified by this molecular maturation would require ex vivo MET current recording which, however, has not yet been successfully implemented at mature stages. Our results should lead to a shift of focus in the search for new components of the lower part of the MET machinery. Because of the apparent functional redundancy observed between the various Pcdh15 isoforms, this search has so far been concentrated on proteins that can interact within the stereocilia with the sequences common to the three Pcdh15 isoforms, that is, the transmembrane and the juxtamembrane sequences (a total of 81 amino acids). Our findings indicate that the identification of the ligands of the Pcdh15-CD2 cytoplasmic C-terminal region may be particularly pertinent. The absence of Pcdh15-CD2 affects mature auditory transduction but not vestibular transduction, both in patients and in the conditional knockout mice, showing that the function of Pcdh15-CD2 in the inner ear is conserved between mice and humans. By demonstrating the requirement for the Pcdh15-CD2 isoform for auditory function in humans, this constitutes a major step towards the development of gene therapy strategies for deafness caused by PCDH15 defects. Materials and Methods Animals Animals were housed in the Institut Pasteur animal facilities accredited by the French Ministry of Agriculture to perform experiments on live mice (accreditation 75-15-01, issued on 6 September 2013 in appliance of the French and European regulations on care and protection of the Laboratory Animals (EC Directive 2010/63, French Law 2013-118, 6 February 2013). The corresponding author confirms that protocols were approved by the veterinary staff of the Institut Pasteur animal facility and were performed in compliance with the NIH Animal Welfare Insurance #A5476-01 issued on 31 July 2012. Pcdh15av3J/av3J mice Pcdh15av3J/av3J mice were obtained from Jackson Laboratories (Bar Arbor, ME). Pcdh15 ex38-fl/ex38-fl mice A targeting vector was designed in which loxP sites were introduced upstream and downstream from Pcdh15 exon 38, and a neomycin resistance (neo) cassette flanked with Frt sites as selectable marker was introduced downstream of exon 38. The targeting construct was introduced by electroporation into embryonic stem (ES) cells from the 129S1/SvlmJ mouse strain, and positive ES cells were selected by their resistance to G418. Stem cells carrying the intended construct were injected into blastocysts from C57BL/6J mice to obtain chimeric mice. After germline transmission, mice were crossed with C57BL/6J mice producing Flp recombinase to remove the neo cassette. The Pcdh15ex38-fl/ex38-fl mice (MGI: 5566900) lack the neo cassette and behave like wild-type (Pcdh15+/+) mice. Pcdh15 ex38-fl/ex38-fl mice were crossed with PGK-cre transgenic mice carrying the cre recombinase gene driven by the early and ubiquitously active phosphoglycerate kinase-1 gene promoter (Lallemand et al, 1998; mutant offspring referred to as KO Pcdh15Δ'CD2 mice); they were also crossed with Myo15-cre recombinant mice carrying the cre recombinase gene driven by the myosin-15 gene promoter which, in the" @default.
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• W2119942550 title "The <scp>CD</scp> 2 isoform of protocadherin‐15 is an essential component of the tip‐link complex in mature auditory hair cells" @default.
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