The integrity of cochlear hair cells is established and maintained through the localization of Dia1 at apical junctional complexes and stereocilia.

Dia1, which belongs to the diaphanous-related formin family, influences a variety of cellular processes through straight actin elongation activity. Recently, novel DIA1 mutants such as p.R1213X (p.R1204X) and p.A265S, have been reported to cause an autosomal dominant sensorineural hearing loss (DFNA1). Additionally, active DIA1 mutants induce progressive hearing loss in a gain-of-function manner. However, the subcellular localization and pathological function of DIA1(R1213X/R1204X) remains unknown. In the present study, we demonstrated the localization of endogenous Dia1 and the constitutively active DIA1 mutant in the cochlea, using transgenic mice expressing FLAG-tagged DIA1(R1204X) (DIA1-TG). Endogenous Dia1 and the DIA1 mutant were regionally expressed at the organ of Corti and the spiral ganglion from early life; alongside cochlear maturation, they became localized at the apical junctional complexes (AJCs) between hair cells (HCs) and supporting cells (SCs). To investigate HC vulnerability in the DIA1-TG mice, we exposed 4-week-old mice to moderate noise, which induced temporary threshold shifts with cochlear synaptopathy and ultrastructural changes in stereocilia 4 weeks post noise exposure. Furthermore, we established a knock-in (KI) mouse line expressing AcGFP-tagged DIA1(R1213X) (DIA1-KI) and confirmed mutant localization at AJCs and the tips of stereocilia in HCs. In MDCK cells with stable expression of AcGFP-DIA1(R1213X), AcGFP-DIA1(R1213X) revealed marked localization at microvilli on the apical surface of cells and decreased localization at cell-cell junctions. The DIA1-TG mice demonstrated hazy and ruffled circumferential actin belts at AJCs and abnormal stereocilia accompanied with HC loss at 5 months of age. In conclusion, Dia1 plays a pivotal role in the development and maintenance of AJCs and stereocilia, ensuring cochlear and HC integrity. Subclinical/latent vulnerability of HCs may be the cause of progressive hearing loss in DFNA1 patients, thus suggesting new therapeutic targets for preventing HC degeneration and progressive hearing loss associated with DFNA1.