Clarin‐2 is essential for hearing by maintaining stereocilia integrity and function

Abstract Hearing relies on mechanically gated ion channels present in the actin‐rich stereocilia bundles at the apical surface of cochlear hair cells. Our knowledge of the mechanisms underlying the formation and maintenance of the sound‐receptive structure is limited. Utilizing a large‐scale forward genetic screen in mice, genome mapping and gene complementation tests, we identified Clrn2 as a new deafness gene. The Clrn2 clarinet/clarinet mice (p.Trp4* mutation) exhibit a progressive, early‐onset hearing loss, with no overt retinal deficits. Utilizing data from the UK Biobank study, we could show that CLRN2 is involved in human non‐syndromic progressive hearing loss. Our in‐depth morphological, molecular and functional investigations establish that while it is not required for initial formation of cochlear sensory hair cell stereocilia bundles, clarin‐2 is critical for maintaining normal bundle integrity and functioning. In the differentiating hair bundles, lack of clarin‐2 leads to loss of mechano‐electrical transduction, followed by selective progressive loss of the transducing stereocilia. Together, our findings demonstrate a key role for clarin‐2 in mammalian hearing, providing insights into the interplay between mechano‐electrical transduction and stereocilia maintenance.

A-D Representative retinal cryosections of Clrn2 clarinet/+ and Clrn2 clarinet/clarinet mice at 7 months. Phalloidin and DAPI staining show the retina have a normal thickness and architecture, with a normal photoreceptor cells layer organization (A). Focusing on photoreceptor cells, we found no difference in the distribution of cone opsin or rhodopsin, which were confined to the outer segments of cones and rods, respectively, in Clrn2 clarinet/+ and Clrn2 clarinet/clarinet mice (B). The absence of morphological or functional abnormalities in Clrn2 clarinet/clarinet mice is consistent with absence of TUNEL-positive nuclei (C) and normal distribution of Iba-1, a gliosis marker (D). IS/OS, inner segment and outer segment; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Scale bars, 20 lm. Outer hair cell stereocilia Figure EV3. Median Z-projections of IHC and OHC stereocilia tips from the second tallest row.
A pseudo-stack comprising of ≥ 80 images of individual stereocilia tips extracted from scanning electron micrographs prepared from P8 wild-type and clarinet mutant mice. The Adobe Photoshop "Auto tone" function was used to increase the contrast of each median projection, followed by pseudo-colouring to highlight the edges of the projected "averaged" stereocilia tips. These image adjustments allow an accurate outline of each median projection to be drawn. For both inner and outer hair cells, the tips of the stereocilia from the second tallest row of Clrn2 clarinet/clarinet mice appear more rounded than those of their wild-type (Clrn2 +/+ ) littermates.

A B
C D E Figure EV4. Age-dependent differential expression of Clrn2 in wild-type cochlea, and tests of anti-Clrn2 antibodies.
A RT-PCR analysis. To assess the temporal expression of Clrn2 in the cochlea, cochlear RNA was extracted from wild-type mice at several perinatal and early postnatal timepoints and utilized for qRT-PCR studies to determine the abundance of Clrn2 transcripts. Values were calculated relative to expression level at P4. Expression is constant during embryonic and early postnatal timepoints (E17.5 to P12), then increases~3-fold between P12 and P16. RQ: relative quantification (arbitrary units). For each timepoint, the data shown are mean AE SD of 5 biological replicates. *P < 0.05, **P < 0.01, one-way ANOVA. B-D Clarin-2 expression and test of anti-clarin-2 antibodies. Transfected HeLa cells producing FLAG-or GFP-tagged clarin-2 (green) were labelled by the anti-clarin-2 antibodies (green): homemade (B), commercial Proteintech (23994-1-AP) (C) or Atlas (HPA042407) (D). Only the homemade antibody clearly labelled the two overexpressed clarin-2 fusion proteins (overlapping immunostaining in yellow-some highlighted by arrows, B). Conversely, none of the commercial antibodies could detect the GFP-tagged clarin-2, as visualized by the lack of yellow staining in the left panels in (C) (Proteintech) and (D) (Atlas). E Immunostaining using anti-clarin-2 homemade antibody showed no specific staining in the F-actin-labelled hair cells (asterisks). The white arrows indicate the presence of non-specific immunostaining over the supporting cells.
A B C D Figure EV5. Distribution of hair bundle proteins in clarinet mice.
A-D Confocal images of whole-mount preparations of cochlear sensory epithelia from Clrn2 clarinet/+ and Clrn2 clarinet/clarinet P6 mice immunostained for the Usher 1B protein myosin VIIa (green in A) and actin (red), PDZD7 (green in B) and actin (red). The PDZD7 immunostaining is normally restricted to the base of stereocilia in both Clrn2 clarinet/+ and Clrn2 clarinet/clarinet mice. (C) Examples of IHCs from Clrn2 clarinet/+ and Clrn2 clarinet/clarinet P6 mice, showing the change in harmonin-b localization in the absence of Clrn2. (D) EPS8 immunostaining (green) illustrating the protein enrichment at the tips of actin-labelled stereocilia (red) in both Clrn2 clarinet/+ and Clrn2 clarinet/clarinet IHCs. Scale bars, 2 lm.