Hearing loss in audio-1 mice. A, Diagram of the ENU-induced forward genetics screen in C57BL/6J mice. G3 animals were tested for hearing impairment. Red asterisks indicate mutation originating from the G0 male, the blue asterisks from the G0 females. Animals with putative homozygous phenotype are represented with a black-filled square or circle, for male and female, respectively. B, Representative ABR traces to click stimuli in the indicated control and mutant mice at 8 weeks of age. C, Statistic results of ABR thresholds to click stimuli at P21 and P60 of wild-type (control) and homozygous audio-1 mutant mice. n = 15 mice for each group. D, ABR thresholds to pure tones at P21 and P60 of wild-type (control) and homozygous audio-1 mutant mice. n = 15 mice for each group.

Positional cloning of the audio-1 mutation. A, Mapping of the audio-1 locus. The diagram shows the results from fine mapping of the region of chromosome 9 (Chr 9) linked to the mutant phenotype. Audio-1 mice (generated on a C57BL/6J background) were crossed to 129S1/SvlmJ mice. The F1 offspring were intercrossed to obtain F2 progeny for ABR phenotyping. DNA from affected and unaffected F2 mice and control mice was analyzed with SNP markers that distinguish between C57BL/6J and 129S1/SvlmJ alleles to detect linkage of a genomic region to the audio-1 phenotype. The locations of SNP markers on chromosome 9 are listed in the first column (the kilobase position 56,586,960–77,090,797 is indicated). The mouse identifiers and phenotypes are shown in the two top rows. The genotype of each mouse for a given SNP marker is represented as follows: B, homozygous C57BL/6J; C, homozygous 129S1/SvlmJ; H, heterozygous. The chromosomal interval that segregates with the mutant phenotype is highlighted with a red box. Two affected animals selected for the exome sequencing are indicated with an asterisk. B, Homozygous mutation found in the two animals subjected to exome sequencing (A24289 and A28196). The chromosomal position, the reference nucleotide (Ref), the nucleotide variation identified (Var), and the gene concerned are indicated. C, Verification of the nucleotide variation on a large mouse cohort for the two homozygous mutations lying in the identified locus. The mouse identifier (ID), the hearing ability (af, affected; un, unaffected), and the nucleotide variation observed are indicated. Outliers are indicated with an asterisk. D, Exon structure of the NPTN gene. Exons are shown as black squares. Coding exons are numbered. Sequence chromatograms of wild-type (WT) and homozygous audio-1 mice reveal a T-to-A transversion in exon 3 (box). E, Schematic representation of the two splicing isoform of Nptn. Np65 contains three Ig domains. Np55 contains only two. The missense mutation, indicted in red, is found in the amino acid #122 for Np65 and amino acid #6 for Np55. It leads to the replacement of an isoleucine by an asparagine. SP, Signal peptide.

Nptn knock-out mice are deaf. A, Diagram of the Nptn allele used in the current study. The “knock-out-first” allele (Nptn^null) contains an SA:lacZ trapping cassette and a floxed promoter-driven neo cassette inserted into the intron the Nptn gene, disrupting gene function. B, PCR analysis of wild-type (+/+), heterozygous (+/−), and homozygous mutant (−/−) mice obtained after crossing the Nptn^null mice. C, Nptn^−/− mice show reduced size during development when compared with Nptn^+/− littermates. D, Immunoblots of brain extracts from Nptn^+/+, Nptn^+/−, and Nptn^−/− mice probed with either Np55/65 or Np65-specific antibody. Immunoreactive bands of the expected molecular mass are detected in Nptn^+/+ and Nptn^+/− extracts, but not in Nptn^−/− extracts. As a control, anti-α tubulin antibody was used to confirm equal loading of total protein lysate between groups. E, Immunoblots of brain extracts from +/+ and homozygous audio-1 mutant mice probed with Np55/65 antibody. Immunoreactive bands of the expected molecular mass are detected in +/+ but not in homozygous audio-1 mutant extracts. As a control, anti-α tubulin antibody was used to confirm equal loading of total protein lysate between groups. F, Statistic results of ABR thresholds to click stimuli at P21 and P60 of wild-type (control) and Nptn^−/− mice. n = 15 mice for each group. G, ABR thresholds to pure tones at P21 and P60 of wild-type (control) and Nptn^−/− mice. n = 15 mice for each group.

Nptn transcripts are expressed in the inner ear. A, Graphical representation of cDNA/protein composition of the Nptn isoforms. The position of the primer used in RT-PCR and in situ hybridization probes to amplify the sequence of each isoform is indicated. SP, Signal peptide; TM, Transmembrane domain; Cyto, cytoplasmic domain. B, Isoform-specific RT-PCR on cerebral cortex and cochlea from P7, P14, and P28. C–V, Analysis of Nptn expression by in situ hybridization on P4 inner ear sections. Comparable probes specific for both isoform (Nptn 55/65) and for the long isoform (Nptn 65) were used. A probe specific for hair cell expression (Loxhd1) was also used as positive control. AS, Antisense; S, sense. C–G, Sections of the inner ear showing three turns and expression of Np55 and Np65 in the SGNs (black arrowheads). H–L, Expression of Np55 in the organ of Corti and in hair cells (yellow dotted lines). M–Q, hair cells close up. R–V, Np55 is also expressed in the vestibular hair cells (white arrowheads). R'–V', Close-up of the vestibular sensory epithelium. Note that the Nptn 55/65 probe revealed expression in hair cells and support cells (R'). Scale bars: C–G, 200 μm; H–L, 50 μm; M–Q, 20 μm; R–V, 100 μm; R'–V', 50 μm.

Np55 is expressed in SGNs and in the stereocilia of OHCs but not IHCs. A, Sagittal inner ear sections of SGNs from wild-type (WT) mice at P7 were stained for Np55/65 (red) and Tuj1 (green). Note the localization of Np55/65 at the cell surface of SGN cell bodies. B, Cochlear whole mounts from C57BL/6 mice at P7 were stained for Np55/65 (red) and phalloidin (green). Note the localization of Np55/65 only in stereocilia OHCs but not IHCs. C, Higher-magnification view of hair cells showing Np55/65 immunoreactivity near the distal ends of stereocilia (top). No staining was observed in homozygous audio-1 mutant OHCs (middle) and IHCs of WT (bottom). D, Expression of Nptn in vestibular hair cells. E, F, Immunogold localization of Nptn in stereocilia at P7 and P60. Nptn was distributed along the length of stereocilia but with higher density toward their upper part and less staining in their lower part. Note IHC shows negative Nptn immune-reactivity. Scale bars: A, 10 μm; B, 2 μm; C, D, 10 μm; E, 500 nm.

NPTN in purified vestibular hair bundles. A, Relative molar abundance of NPTN (summed isoforms) from purified hair bundles and whole epithelium from mouse utricles. NPTN is highly enriched in bundles. B, Protein immunoblotting of purified bundles using Np55/Np65 antibody. BUN, 18 ear-equivalents of P21–P24 C57BL/6 mouse utricle hair bundles; Agar, equivalent amount of agarose in saline; UTR, two P22 mouse utricles; Brain, 2 μg of C57BL/6 mouse brain extract. While the Np55 and Np65 isoforms are clearly identified in brain, whole mouse utricle has a complex isoform pattern. Bundles predominantly contain an NPTN isoform that migrates between Np55 and Np65. C, D, Peptide mapping of NPTN isoforms from utricle bundles and epithelium, using pooled P5 and P23 peptide identifications. The frequency of peptide identification is plotted as a function of the position of the peptide in the amino acid sequence. The exon structures of the Np55 and Np65 isoforms are indicated. No peptides in the Np65-specific exon (#2) were detected in bundles (C) or epithelium (D), despite frequent detection in other experiments.

Trafficking of Np55 to hair cell stereocilia of OHCs but not IHCs. A, Graphical representation of the injectoporation setup used for the transfection of hair cells of the organ of Corti. The HA-tagged Np55 plasmid for delivering (left), as well as the intercellular space for plasmid injection (right), are indicated. B, Organs of Corti from P3 mice were injectoporated with Np55-HA constructs and stained with phalloidin (Phal, F-actin) and anti-HA antibody after 24 h in vitro. C–E, Higher-magnification view of hair cells overexpressed with Np55-WT or audio-1 mutant (Np55-mut). Note the expression of Np55-HA in stereocilia of OHCs (D), not in IHCs (E). Np55-HA construct carrying the audio-1 mutation failed to be transported to the stereocilia of OHCs (D). Scale bars: B, 10 μm; C–E, 5 μm. Number of transfected cells: C, n = 36; D, n = 16; E, n = 12.

Hair cell survival and mechanotransduction. A, B, Scanning electron microscopy analysis of hair bundles from wild-type (control) and homozygous audio-1 mutant mice in the midapical cochlea at P14 and P40. Hair bundles from OHCs at higher magnification are shown. C, Severe degeneration of the OHCs was evident at P120 in homozygous audio-1 mutant mice. D, The average number of OHCs per sample of the entire organ of Corti was calculated at P60 and P120. Cell losses in each group are marked as red. Number of cell loss/survival from each group: 4/730 (P60, Control), 15/730 (P60, audio-1), 14/706 (P120, Control), and 167/545 (P120, audio-1). E, Traces of transduction currents in OHCs from wild-type (control) and Nptn^−/− mice at P7 in response to a set of 10 ms hair bundle deflections ranging from −400 to 1000 nm (100 nm steps) at a holding potential of −70 mV. F, Current displacement plots obtained from similar data as shown in E. All values are mean ± SEM by Student's t test.

VOCTV reveals absence of cochlear amplification. A, B, In vivo cochlear cross-sectional images from the apical cochlear turn of control (Nptn^+/+) and Nptn^−/− mice revealed no gross anatomical differences in the TM and its relationship to the BM and the intervening hair cell epithelium. Also, Reissner's membrane (RM) appeared normal in Nptn^−/− mice. C, D, Sound-evoked BM and TM vibrations in representative control and Nptn^−/− mice. Thicker lines were used to represent the response to higher stimulus intensities. E, BM sensitivity was calculated by normalizing BM displacement to middle ear displacement. In control mice, BM sensitivity increased and tuning became sharper with decreasing stimulus level, a hallmark of cochlear amplification. For Nptn^−/− mice, BM sensitivity was broad and did not change with stimulus level, indicating a lack of cochlear amplification. F, Measurements of cochlear gain at both the BM and TM were normal in control mice but absent in Nptn^−/− mice (p = 0.02 for both comparisons). G, The phase of the TM relative to the BM was not significantly different between Nptn^−/− and control mice (p > 0.7 at both 5 and 9 kHz, the frequencies of largest vibration to low-intensity stimuli in Nptn^−/− and control mice; gray arrows). All values are mean ± SEM.

OHC amplification, electromotility, and coupling of OHC stereocilia to the TM. A, Representative DPOAE response spectra from wild-type (control) and homozygous audio-1 mutant mice at a single stimulus condition. Note the 2f1–f2 peak (gray arrow), which is absent in mutant mice. B, DPOAE thresholds at different frequencies at P21 and P60 of wild-type (control) and homozygous audio-1 mutant mice. n = 15 mice for each group. C, DPOAE thresholds at different frequencies at P21 and P60 of wild-type (control) and Nptn^−/− mice. n = 15 mice for each group. D, NLC recorded from wild-type (control) and Nptn^−/− mice at P21. E, Parameters of NLC fitting. Normalized NLC peak (Norm. NLCmax) was performed using the peak value of NLC normalized with the base line. Clin, Linear capacitance. F, Scanning electron micrographs of V-shaped TM imprints from wild-type (control) and Nptn^−/− mice of different ages. These imprints (arrows) are formed by the notches remaining after the tallest row of OHC stereocilia was pulled away from the TM. Such imprints are not found or are disorganized in the TM of Nptn^−/− mice. Scale bars, 2 μm. All values are mean ± SEM. ***p < 0.001, by two-way ANOVA in B–D and Student's t test in E. G–I, CM. Representative CM recordings measured from a P28 Nptn^−/− mouse (in red) and a WT littermate (in black) in response to a 6 kHz stimulus at 80, 90, and 100 dB SPL. The peak-to-peak amplitude of the CM versus sound intensity measured in P28 Nptn^−/− mice (n = 2) and WT littermates (n = 2) in H, and measured in P30 audio-1 mice (n = 3) and littermate WT (n = 4) in I.