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

Abstract

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 MDCKAcGFP-DIA1(R1213X) 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.

Nox3-Derived Superoxide in Cochleae Induces Sensorineural Hearing Loss

“…To achieve a statistical power of 0.8 with a 50% effect size with means and standard deviations, sample sizes were calculated using a power analysis based on variances of the ABR threshold and hair cell and presynaptic ribbon counts reported previously (Ueyama et al, 2016;Ninoyu et al, 2020). As several experiments had considerably large effect sizes, sample sizes were adjusted accordingly.…”

Variable autoinhibition among deafness-associated variants of Diaphanous 1 (DIAPH1)

“…Mouse models have been invaluable in understanding human deafness variants, and a transgenic mouse expressing DIAPH1-1213x shows progressive hearing loss with loss of ≈10 and 40% of inner and outer hair cells, respectively, between 10- and 25-weeks (Ueyama et al , 2016). Surprisingly, 8-week-old 1213x-expressing mice show greater hair cell damage following sound exposure (specifically loss of ribbon synapses and hair bundle abnormalities), but these changes were not correlated with permanent threshold shifts (Ninoyu et al , 2020). To date, no mouse model has been reported for the other pathogenic DFNA1 variants.…”

“…Through a systematic analysis of five variants, we report here highly disparate levels of autoinhibition, indicating that these variants could have distinct activation levels in vivo . Recently reported mouse models of DFNA1 will hopefully uncover the cellular mechanisms of DIAPH1 -associated pathology in the inner ear (Ninoyu et al , 2020). Thus far, those efforts have been focused on 1213X, which appears to date to be the most commonly occurring variant in the human population.…”

“…However, endogenous formin localization to the hair bundle has not been reported to date. In a knock-in mouse, expressing a fluorescently tagged DIAPH1-R1213x, localization of mutant DIAPH1 was observed in a subset of inner hair cell stereocilia tips (Ninoyu et al , 2020). In the same study, endogenous Dia1 was localized most strikingly to apical junction complexes in inner hair cells, as well as in outer hair cells, Deiters’ cells, inner pillar cells, and spiral ganglion in both WT and transgenic mice at P5 (Ninoyu et al , 2020).…”

“…In a knock-in mouse, expressing a fluorescently tagged DIAPH1-R1213x, localization of mutant DIAPH1 was observed in a subset of inner hair cell stereocilia tips (Ninoyu et al , 2020). In the same study, endogenous Dia1 was localized most strikingly to apical junction complexes in inner hair cells, as well as in outer hair cells, Deiters’ cells, inner pillar cells, and spiral ganglion in both WT and transgenic mice at P5 (Ninoyu et al , 2020). This study also revealed that WT mDia1 and mutant Dia1 (R1213X) were first observed in apical junction complexes of inner and outer hair cells at the basal turn which spread up to middle and apical turns as the Organ of Corti matures (from P5 to P14).…”