Pathogenic mechanism of the PLS1 gene variant in hearing loss and functional validation in a zebrafish model.

PLS1, encoding an actin-bundling protein Plastin 1, is one of the pathogenic genes responsible for autosomal dominant non-syndromic hearing loss. The cellular damage caused by mutant plastin proteins may arise from loss of function and/or dominant-negative effects. In this study, we investigated the pathogenic mechanism of PLS1 mutations by establishing both in vitro cellular models and in vivo zebrafish models. Immunoprecipitation demonstrated that the ΔExon8 mutation disrupts the intramolecular interaction between the ABD1 and ABD2 domains, while immunofluorescence colocalization revealed that the mutation alters the binding pattern of PLS1 to actin bundles and disturbs cytoskeletal organization. Through zebrafish FM1-43 uptake assays, we showed that the mutant protein impaired the functional opening and permeability of the stereociliary mechanoelectrical transduction (MET) channel. Although both PLS1-knockout zebrafish and zebrafish expressing ΔExon8-PLS1 exhibited behavioral signs of hearing impairment, defects in MET channel uptake were observed only in the mutant-expressing fish. Notably, neither PLS1 deficiency nor re-expression of human wild-type PLS1 in knockout zebrafish reproduces this MET defect. Thus, the mutation not only reduces native PLS1 function but also interferes with normal MET channel activity. In summary, our findings indicate that the DFNA76 phenotype caused by the PLS1ΔExon8 mutation is likely due to a dominant-negative effect with partial loss-of-function.