A mouse model for nonsyndromic (DFNB12) links loss to defects in tip links of mechanosensory hair cells

Martin Schwandera,b, Wei Xionga,b, Joshua Tokitac, Andrea Lellid, Heather M. Elledgea,b, Piotr Kazmierczaka,b, Anna Sczanieckaa,b, Anand Kolatkara, Tim Wiltshiree,1, Peter Kuhna, Jeffrey R. Holtd, Bechara Kacharc, Lisa Tarantinoe,1, and Ulrich Mu¨ llera,b,2

aDepartment of Cell Biology and bInstitute of Childhood and Neglected Disease, The Scripps Research Institute, La Jolla, CA 92037; cLaboratory of Cellular Biology, National Institute of Deafness and Other Communication Disorders, National Institutes of Health, Bethesda, MD 20892; dDepartment of Neuroscience, University of Virginia School of Medicine, Charlottesville, VA 22908; and eGenomics Institute of the Novartis Research Foundation, San Diego, CA 92121

Communicated by Bruce A. Beutler, The Scripps Research Institute, La Jolla, CA, January 26, 2009 (received for review January 9, 2009)

Deafness is the most common form of sensory impairment in mechanism by which such mutations cause disease is unclear. humans and is frequently caused by single gene mutations. Inter- In an N-ethyl-N-nitrosourea (ENU) mutagenesis screen (35), estingly, different mutations in a gene can cause syndromic and we have now identified salsa mice, which suffer from progres- nonsyndromic forms of deafness, as well as progressive and sive and carry a Cdh23 missense mutation that is age-related hearing loss. We provide here an explanation for the predicted to affect Ca2ϩ binding by the extracellular CDH23 phenotypic variability associated with mutations in the cadherin 23 domain. Similar mutations in the human CDH23 gene cause gene (CDH23). CDH23 null alleles cause deaf-blindness (Usher DFNB12 (1, 5, 6, 10, 13, 36). Unlike in mice with predicted syndrome type 1D; USH1D), whereas missense mutations cause Cdh23 null alleles, hair bundle development appears unaf- nonsyndromic deafness (DFNB12). In a forward genetic screen, we fected in salsa mice. Instead, tip links are progressively lost, have identified salsa mice, which suffer from hearing loss due to a suggesting that similar mutations in DFNB12 patients lead to Cdh23 missense mutation modeling DFNB12. In contrast to waltzer deafness by affecting tip links. mice, which carry a CDH23 null allele mimicking USH1D, hair cell development is unaffected in salsa mice. Instead, tip links, which Results are thought to gate mechanotransduction channels in hair cells, are Progressive Hearing Loss in salsa Mice. In an ENU mutagenesis progressively lost. Our findings suggest that DFNB12 belongs to a screen, we identified salsa mice, which show no auditory startle new class of disorder that is caused by defects in tip links. We response (35). Recordings of the auditory brainstem response propose that mutations in other genes that cause USH1 and (ABR) revealed that salsa mice suffer from progressive hearing nonsyndromic deafness may also have distinct effects on hair cell loss. Wild-type mice at 3 weeks and 2 months of age had ABR development and function. thresholds to click stimuli at 20 Ϯ 5 dB. Thresholds in salsa mice were by 3 weeks at 78 Ϯ 15 dB and by 2 months at 100 Ϯ 5dB cadherin 23 ͉ Cdh23 ͉ ͉ progressive hearing loss (Fig. 1 C and D). salsa mice were hearing-impaired across all frequencies (Fig. 1E). No defect was observed in heterozygous ramatic progress has been made in the identification of gene salsa mice, demonstrating recessive mode of inheritance (35). Dmutations that cause hearing loss, but we know compara- Distortion product otoacoustic emissions were not detected in tively little about the mechanisms by which the mutations lead to salsa mice (Fig. S1), indicating that outer hair cell function was disease. Interestingly, different mutations in a gene can cause perturbed. Movement and the ability to swim were unaffected distinct disease outcomes. The cadherin 23 gene (CDH23) (Fig. 1F) (35), indicating that the vestibular system of salsa mice provides a striking example. Predicted CDH23 null mutations was intact. lead to deaf-blindness (USH1D), whereas missense mutations cause nonsyndromic deafness (DFNB12) (1–13). A polymor- salsa Mice Carry a Cdh23 Point Mutation. salsa mice were derived on phism in Cdh23 is linked to age-related hearing loss (14). a C57BL/6J background (35). To identify the affected gene, we Similarly, mutations in the genes for VIIa (MYO7A) and crossed salsa mice to 129S1/SvImJ mice. Offspring were inter- protocadherin 15 (PCDH15) cause USH1 and nonsyndromic crossed to obtain F2 mice for ABR measurements and DNA deafness (http://webh01.ua.ac.be/hhh/). preparation. By using single-nucleotide polymorphisms (SNPs), Recent studies in mice suggest that USH1 is caused by defects the affected genomic locus was mapped to a 4-MB interval on in hair cell development. Each developing hair cell contains at containing Cdh23 (Fig. S2A). Sequencing re- the apical surface a single kinocilium and rows of stereocilia, vealed a single point mutation, A2210T, in exon 22 of Cdh23 which form the mechanically sensitive organelle of a hair cell (Fig. 1A). Extracellular filaments connect the stereocilia and kinocilium of a developing hair cell (15). CDH23 and PCDH15 Author contributions: M.S., W.X., A.L., H.M.E., P. Kazmierczak, A.S., T.W., J.R.H., B.K., L.T., and U.M. designed research; M.S., W.X., J.T., A.L., H.M.E., P. Kazmierczak, A.S., B.K., and L.T. are components of transient lateral links, kinociliary links, and performed research; A.K. and P. Kuhn contributed new reagents/analytic tools; M.S., W.X., tip links (Fig. 1A) (16–20), and their cytoplasmic domains bind J.T., A.L., H.M.E., P. Kazmierczak, A.S., A.K., T.W., P. Kuhn, J.R.H., B.K., L.T., and U.M. to protein complexes containing harmonin, MYO7A, and sans analyzed data; and M.S. and U.M. wrote the paper. (21–25). Predicted null mutations in murine USH1 genes cause The authors declare no conflict of interest. defects in hair bundle development (24, 26–34), suggesting that See Commentary on page 4959. USH1 proteins form transmembrane complexes that regulate 1Present address: Department of Psychiatry, University of North Carolina, Chapel Hill, NC hair bundle morphogenesis. Failure of these complexes likely 27516. causes USH1. 2To whom correspondence should be addressed. E-mail: [email protected]. So far, there are no animal models for nonsyndromic This article contains supporting information online at www.pnas.org/cgi/content/full/ deafness caused by mutations in any USH1 gene, and the 0900691106/DCSupplemental.

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Fig. 1. Analysis of auditory function. (A) Diagram of a developing hair bundle. Stereocilia are connected to each other and to the kinocilium by various linkages. Linkages that contain PCDH15 and CDH23 are indicated. (B) The diagram shows the localization of CDH23 and PCDH15 at tip links. (C) 2ϩ Click-evoked ABR for a 2-month-old wild-type (black lines) and a salsa mouse Fig. 2. The mutation in salsa mice maps to a Ca -binding motif in CDH23. (red line) at different sound intensities (dB). ABR waves I–IV are indicated. (D) (A) Sequence chromatograph from wild-type and salsa mice reveals an A-to-T Average auditory thresholds for 3-week-old and 2-month-old mice [wild-type mutation in exon 22 of Cdh23.(B) The C-terminal part of EC7 of CDH23 from n ϭ 4 for 3 weeks (white) and 2 months (gray); salsa n ϭ 5 for 3 weeks (orange) different species is shown. The Glu737Val substitution in salsa affects a con- 2ϩ and 2 months (red)]. The mean Ϯ SD is indicated; a Student’s t test was served Ca -binding motif (yellow boxes). CDH1 and CDH2 are shown for performed. (E) Auditory thresholds in 3-week-old (triangles) and 2-month-old comparison. (C) CDH23 EC7/8 wild-type (blue) and salsa mutant (red) se- (circles) mice as determined by pure tone ABR recordings. In contrast to quences threaded onto the E-cadherin EC1/2 by using the automodel class and wild-type (gray and black lines) salsa mutants (orange and red lines) showed a sequence alignment produced by T-Coffee. Energy-minimized model shows 2ϩ progressive hearing loss. (F) Analysis of movement in the open field. salsa mice that the Glu737Val mutation affects Ca coordination. (D) Domain structure (red) showed normal numbers of small-diameter rotations (Ͼ2.75-cm radius) of CDH23 indicating the 27 extracellular cadherin repeats (blue). Missense 2ϩ and were not hyperactive. As a positive control, the sirtaki mouse line (gray) mutations in Ca -binding motifs in the CDH23 extracellular domain cause that has vestibular defects (35) is shown. Values are mean Ϯ SD. A Student t DFNB12 in humans (purple shaded box). Nonsense and splice site mutations have been identified in waltzer mice and USH1D (gray shaded box). SS test was performed. **, P Ͻ 0.01; ***, P Ͼ 0.001. indicates signal sequence; TM, transmembrane domain.

(Fig. 2A). Compound heterozygous mice carrying 1 salsa allele and 1 previously reported mutant Cdh23 allele (waltzerv2J) (27) Staining with CDH23 antibodies revealed that CDH23 was targeted to the stereocilia of hair cells in salsa mice by P5 and were deaf (Fig. S2B), confirming that the salsa mutation causes P10 (Fig. 4 A–F and K), but expression was only occasionally deafness. The salsa mutation leads to a Glu737Val substitution detectable by P30 (Fig. 4 G–K). However, no defects in CDH23 within an LDRE motif in the seventh cadherin repeat of CDH23 cell surface transport were observed (Fig. S3). Because (Fig. 2B), which is conserved in CDH23 across species and in CDH23 is a tip-link component (16, 20), tip-link maintenance cadherin repeats of other cadherins (Fig. 2B), and is required for 2ϩ may be affected. Tips of stereocilia show characteristic tenting Ca chelation (Fig. 2C) (37, 38). Several mutations that cause that is thought to be the consequence of tip-link-mediated 2ϩ DFNB12 resemble the salsa mutation and affect Ca -binding tension (39–42). Tenting can therefore be used to quantify the motifs (LDRE, DXND, DXD; Fig. 2D and Table S1) (1, 5–7, 10, presence of tip links. Tenting was observed in wild-type hair 13, 36). salsa mice are therefore a model for some forms of cells at P5, P21, and P60 (Fig. 5 A, C, and H and Figs. S4 and DFNB12. Mutations that lead to predicted CDH23 null alleles S5). In salsa mice, tenting was observed throughout the cause USH1D (Fig. 2D) (1–5, 7–12), which is modeled by cochlear duct at P5 (Fig. 5 B and H and Fig. S4); by P21, tenting mutations in waltzer mice (Fig. 2D) (27, 33, 34). was no longer observed in the basal turn of the , and was absent in all hair cells by P60 (Fig. 5D and Fig. S5). salsa Progressive Tip-Link Loss in salsa Mice. Although hair bundles are mice showed no signs of vestibular dysfunction (Fig. 1F), and disrupted in waltzer mice at early postnatal ages (Fig. 3F) (27, tenting of stereociliary tips and tip links were maintained in

34), hair bundles were unaffected in salsa mice at postnatal day vestibular hair cells (Fig. 5 F and G). Finally, by P90, the organ GENETICS 5 (P5) and P28 (Fig. 3). This suggests that CDH23 plays an of Corti and spiral ganglion degenerated in salsa mice, whereas important role in steps beyond hair bundle development. the vestibule was unaffected (Fig. S6). Staining for activated

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Fig. 3. Preserved hair bundle morphology in salsa mice. (A–F) Scanning electron microscopy micrographs of cochlear whole mounts from P5 wild-type mice and salsa and waltzer mutants. (A and B) The organ of Corti in salsa mice was patterned normally in 3 rows of outer and 1 row of inner hair cells. (C–F) Hair bundles in salsa displayed the characteristic polarized morphology with a single kinocilium. Hair bundles in waltzer mice were fragmented. (G–L)At Fig. 4. Progressive loss of CDH23 expression in salsa mice. (A–J) Cochlear P28, hair bundle morphology was indistinguishable in wild-type and salsa whole mounts of wild-type and salsa mice were stained with an antibody mice. [Scale bars: A and B (5 ␮m), C–F (2 ␮m), G and H (10 ␮m), and I–L (2 ␮m).] against the CDH23 cytoplasmic domain (green) and with phalloidin (red). (A and B) Levels of CDH23 expression in homozygous salsa and wild-type mice at P5 were similar. (C and D) Higher-magnification view of CDH23 expression in caspase 3 showed that cochlear hair cells and spiral ganglion hair bundles at P5. (E and F) At P10, CDH23 staining was maintained at neurons died by apoptosis (Fig. S6). stereociliary tips. (G–J) At P30, CDH23 staining was barely detectable in salsa. Arrowheads point to CDH23 in wild types and residual staining in salsa.(K) Quantification of CDH23 staining in hair cells at P10 and P30. salsa mice (black) Defects in Hair Cell Function by P7/8. To test whether hair cell showed reduced numbers of CDH23 puncta in hair bundles at P30. Values are function was affected before detectable morphological changes, mean Ϯ SD. A Student’s t test was performed. ***, P Ͻ 0.001. [Scale bar: A and we recorded mechanotransduction currents from P7/8 cochlear B (8 ␮m), C–J (2 ␮m).] hair cells in response to 5-ms bundle deflections ranging from Ϫ400 to 1000 nm. Current-displacement [I(X)] relationship plots did not reveal a difference between wild-type and salsa mice dependent interactions between PCDH15-His and CDH23salsa- (Fig. 6 A and B). Peak currents at maximal deflection were Fc, but interactions were diminished (Fig. 7B). To determine similar in controls (419 Ϯ 40 pA) and mutants (427 Ϯ 35 pA). whether mutations in CDH23 that cause DFNB12 reduce inter- We next analyzed the adaptation kinetics of mechanotransduc- actions with PCDH15, we engineered 2 human mutations into tion currents in outer hair cells stimulated with 100-ms deflec- CDH23-Fc (Fig. 7A) (1). Both mutations reduced interactions of tions of 100–800 nm. Normalization of each current trace to its CDH23 with PCDH15 (Fig. 7B). peak current showed faster transduction current decline in salsa compared with wild type, which was only observed at deflections Discussion above 300 nm (Fig. 6 C and D). We conclude that hair cell Previous studies have shown that predicted Cdh23 null alleles function was mildly affected by P7/8. perturb hair bundle development, likely as a consequence of defects in the filaments that connect the stereocilia and kinoci- The salsa Mutation Affects CDH23 Adhesive Function. CDH23 inter- lium of a developing hair cell. In contrast, we show here that a acts with PCDH15 to form tip links (16). To test the effects of Cdh23 missense mutation leads to progressive tip-link loss and, the salsa mutation on CDH23 properties, we incubated the ultimately, to hair cell death. In salsa mice, a small defect in purified extracellular domains of CDH23 with or without the mechanotransduction by cochlear hair cells was apparent at salsa mutation fused to Fc tags (referred to as CDH23wt-Fc and P7/P8, indicating that hair cell function was already affected. At CDH23salsa-Fc, respectively) with the purified His-tagged ex- subsequent ages, tip links were lost, as revealed by diminished tracellular domain of protocadherin 15 (PCDH15-His; Fig. 7A). levels of CDH23 protein in stereocilia and loss of tenting of Protein complexes were analyzed by Western blotting. As re- stereociliary tips. Because predicted CDH23 null alleles are ported, PCDH15-His bound to CDH23wt-Fc in a Ca2ϩ- associated with USH1D, whereas missense mutations similar to dependent manner (Fig. 7B) (16). We also observed Ca2ϩ- the one observed in salsa mice cause DFNB12, we propose that

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Fig. 5. Progressive loss of tenting at stereociliary tips. (A and B) Scanning electron microscopy analysis in P5 wild-type and salsa mice revealed tip tenting in stereocilia of cochlear hair cells (arrowheads). (C and D) Defects in tenting in salsa mice at P60 (arrowheads in C indicate tenting in wild-type). (E–G) Tips in vestibular hair cells display normal tenting. Tip links (arrowhead in G) were preserved. (H) Quantification of tip tenting at P5 and P21. Tenting was reduced in hair cells from the medial and basal cochlear turns at P21. *, P Ͻ 0.05. [Scale bars: A–D (300 nm), E and F (500 nm), and G (150 nm).]

USH1D is caused by developmental defects in hair bundles and tip-link function without effects on hair cell development. The DFNB12 by tip-link defects. duality of USH1 protein function for hair cell development and Our biochemical studies show that the salsa mutation affects mechanotransduction provides a plausible explanation for the interactions of CDH23 and PCDH15, even though the muta- different disease phenotypes that are associated with distinct tion affects an amino acid outside the ligand-binding domain mutations in USH1 genes (http://webh01.ua.ac.be/hhh/). in EC1 (16). Hair cell function was also maintained for some Based on our findings, we predict that some missense muta- time in salsa mice, and tip links sustained substantial forces in tions in USH1 genes, which cause nonsyndromic deafness, electrophysiological experiments. These findings seem contra- affect the mechanotransduction machinery of hair cells with- dictory at first glance, but the study of classical cadherins out effects on hair bundle development. Null mutations in- ϩ provides clues that likely explain the results. Ca2 binding stead affect hair cell morphogenesis. It has been proposed that rigidifies the cadherin extracellular domain. Disruption of USH1 genes also participate in photoreceptor morphogenesis ϩ Ca2 binding influences protein structure, often perturbing (46), a process that is likely affected by USH1 null alleles but adhesive function (37, 38). In analogy to classical cadherins, it not by missense mutations that affect mechanotransduction. is likely that defects in ligand binding caused by the salsa Strikingly, tip links in vestibular hair cells and vestibular mutation report changes in CDH23 structure that are propa- function are maintained in salsa mice and DFNB12 patients, gated over a considerable distance within the extracellular which may be a consequence of the different mechanical domain. Structural changes might predispose tip links to properties of these low-frequency mechanoreceptors. Consis- breakage, necessitating frequent regeneration that cannot be tent with this model, loss of tip links in salsa mice was first sustained. CDH23 and PCDH15 at tip links might also carry observed in the basal part of the cochlea, which contains hair additional posttranslational modifications, which stabilize cells that respond to the highest frequencies. their interaction but were not present in the recombinant proteins. An interesting implication of our findings is that Materials and Methods some CDH23 mutations may predispose individuals to hearing An extended section is provided as SI Materials and Methods. loss caused by mechanical insults. In fact, polymorphisms in CDH23 are associated with noise-induced hearing loss (43), ENU Mutagenesis, Functional Studies, Positional Cloning, Histology, and Bio- and mice heterozygous for the Cdh23 waltzer allele show chemistry. ENU mutagenesis, analysis of ABRs and distortion product oto- increased noise susceptibility (44). Finally, a polymorphism in acoustic emissions, test of vestibular function, staining of sections and whole Cdh23 is associated with age-related hearing loss (14), which mounts, electron microscopy, positional cloning, the expression and purifica- may be related to instability of tip links. tion of recombinant CDH23 and PCDH15, and protein interaction studies have All USH1 proteins control hair bundle development (15). been described previously (16, 35, 47, 48). Tip tenting was quantified by CDH23, PCDH15, and MYO7A are also expressed in mature counting tented stereocilia in the medium row in inner hair cell bundles (in 2–6 hair cells per time point and genotype). For quantification of tip staining, hair cells (the mature expression pattern for harmonin and the number of CDH23 fluorescent puncta in bundles was determined (Ͼ27 sans has yet to be determined). As tip-link proteins, CDH23 hair cells per time point and genotype). and PCDH15 are thought to gate transduction channels,

whereas MYO7A contributes to channel adaptation (45). Molecular Modeling. Using Modeler version 9v4 (49), cadherin repeat 7 and 8 GENETICS Missense mutations in the genes for MYO7A and PCDH15, of CDH23 were threaded onto cadherin repeat 1 and 2 of CDH1 using the similar to the salsa mutation in CDH23, might also affect automodel class and the sequence alignment produced by T-Coffee (50). The

Schwander et al. PNAS ͉ March 31, 2009 ͉ vol. 106 ͉ no. 13 ͉ 5255 Downloaded by guest on September 30, 2021 Fig. 7. salsa and DFNB12 mutations affect interactions between CDH23 and PCDH15. (A) Diagram of CDH23 and PCDH15 constructs. The extracellular domains were fused to a His or Fc tag. (B) CDH23Fc and the mutant derivatives were incubated with PCDH15-His in the presence of 1 mM EDTA or in the presence of 10 ␮M and 100 ␮MCa2ϩ. Protein complexes were purified and analyzed by Western blotting. Complex formation was observed in the pres- ence but not absence of Ca2ϩ. The salsa and DFNB12 mutations weakened interactions between CDH23 and PCDH15. (Right) Controls for input amounts of CDH23Fc and mutant derivatives.

model was energy-minimized to remove bad contacts. The minimized, threaded coordinates were used to mutate Glu-66 to Val to produce a mutant model. Minimization consisted of 20 cycles of conjugate gradient minimiza- tion followed by 50 cycles of molecular dynamics optimization using the Verlet algorithm and finished with 20 cycles of conjugate gradient minimization. Fig. 6. Mechanotransduction currents. Data from wild type are represented in blue and from salsa in red. (A) Examples of transduction currents in cochlear Outer hair cells in the apical/middle turn were outer hair cells at P7 in response to 5-ms mechanical stimulation. (B) Current- Mechanotransduction Currents. displacement [I(X)] relationships revealed no obvious difference between wild recorded. Cells were whole-cell-patched, and hair bundles were deflected type and salsa.(C) Examples of transduction currents in cochlear outer hair with a stiff glass probe. cells at P7 in response to 100-ms mechanical stimulation. (D) Averaged trans- duction currents for deflections between 100 and 800 nm expressed as a ACKNOWLEDGMENTS. We thank T. Ricci for advice with electrophysiology percentage of peak currents at 800 nm. Current amplitude was lower in and K. Spencer for help with microscopy. This work was funded by National homozygous salsa mice between 300-nm and 800-nm deflection. Data are Institutes of Health Grants DC005969 and DC007704 (to U.M.), the Skaggs shown as mean Ϯ SEM. Student’s 2-tailed unpaired t test was performed (*, Institute for Chemical Biology (U.M.), and the Bruce Ford and Anne Smith P Ͻ 0.05; **, P Ͻ 0.01). Bundy Foundation (W.X.).

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