Lack of PDZD7 long isoform disrupts ankle‐link complex and causes hearing loss in mice

Abstract

Usher syndrome (USH) is the most frequent form of combined hereditary deafnessblindness, characterized by hearing loss and retinitis pigmentosa, with or without vestibular dysfunction. PDZD7 is a PDZ domain-containing scaffold protein that was suggested to be a USH modifier and a contributor to digenic USH. In the inner ear hair cells, PDZD7 localizes at the ankle region of the stereocilia and constitutes the so-called ankle-link complex together with three other USH proteins Usherin, WHRN, and ADGRV1. PDZD7 gene is subjected to alternative splicing, which gives rise to two types of PDZD7 isoforms, namely the long and short isoforms. At present, little is known which specific isoform is involved in ankle-link formation and stereocilia development. In this work, we showed that PDZD7 long isoform, but not short isoforms, localizes at the ankle region of the stereocilia. Moreover, we established Pdzd7 mutant mice by introducing deletions into exon 14 of the Pdzd7 gene, which causes potential premature translational stop in the long isoform but leaves short isoforms unaffected. We found that lack of PDZD7 long isoform affects the localization of other ankle-link complex components in the stereocilia. Consequently, Pdzd7 mutant mice showed stereocilia development deficits and hearing loss as well as reduced mechanotransduction (MET) currents, suggesting that PDZD7 long isoform is indispensable for hair cells. Furthermore, by performing yeast two-hybrid screening, we identified a PDZD7 long isoform-specific binding partner PIP5K1C, which has been shown to play important roles in hearing and might participate in the function and/or transportation of PDZD7. F I G U R E 1 PDZD7 long isoform, but not short isoform, localizes in the stereocilia of auditory hair cells. A, Schematic drawing of Pdzd7 genomic structure and the domain architecture of PDZD7 protein. Mouse Pdzd7 gene contains 16 exons, and deletions were introduced into exon 14 that is unique for PDZD7 long isoform. The coding sequences were indicated by black boxes, whereas the uncoding sequences were indicated by grey boxes. The various domains and the antigens recognized by different antibodies were indicated. B and C, Cochlear whole mounts from Pdzd7 +/△PDZ3 and Pdzd7 △PDZ3/△PDZ3 mice at P4 were stained with PDZD7_C antibody (B) or PDZD7_N antibody (C). Phalloidin staining was also performed to indicate the stereocilia. Images were taken from the middle turn of the cochlea. D-E', Cochlear explants from wild-type mice at P3 were injectoporated with expression vectors to express HA-tagged PDZD7 long isoform (D and D') or short isoform (E and E') in hair cells. GFP fluorescence indicated successfully injectoporated hair cells. Immunostaining with anti-HA antibody was performed to locate HA-PDZD7. Phalloidin staining was performed to indicate the stereocilia. Scale bars: 2 μm in (B) and (C), 20 μm in (D) and (E), and 5 μm in (D') and (E')

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