Usherin defects lead to early-onset retinal dysfunction in zebrafish

Mutations in USH2A are the most frequent cause of Usher syndrome and autosomal recessive nonsyndromic retinitis pigmentosa. To unravel the pathogenic mechanisms underlying USH2A-associated retinal degeneration and to evaluate future therapeutic strategies that could potentially halt the progression of this devastating disorder, an animal model is needed. The available Ush2a knock-out mouse model does not mimic the human phenotype, because it presents with only a mild and late-onset retinal degeneration. Using CRISPR/Cas9-tech-nology, we introduced protein-truncating germline lesions into the zebrafish ush2a gene (ush2a(rmc1): c.2337_2342delinsAC; p.Cys780GlnfsTer32 and ush2a(b1245): c.15520_15523delinsTG; p.A1a5174fsTer). Homozygous mutants were viable and displayed no obvious morphological or developmental defects. Immunohistochemical analyses with antibodies recognizing the N- or C-terminal region of the ush2a-encoded protein, usherin, demonstrated complete absence of usherin in photoreceptors of ush2a(rmc1), but presence of the ectodomain of usherin at the periciliary membrane of ush2a(b1245)-derived photoreceptors. Furthermore, defects of usherin led to a reduction in localization of USH2 complex members, whirlin and Adgrvl, at the photoreceptor periciliary membrane of both mutants. Significantly elevated levels of apoptotic photoreceptors could be ob-served in both mutants when kept under constant bright illumination for three days. Electroretinogram (ERG) recordings revealed a significant and similar decrease in both a- and b-wave amplitudes in ush2a(rmc1) as well as ush2a(b1245) larvae as compared to strain- and age-matched wild-type larvae. In conclusion, this study shows that mutant ush2a zebrafish models present with early-onset retinal dysfunction that is exacerbated by light exposure. These models provide a better understanding of the pathophysiology underlying USH2A-associated RP and a unique opportunity to evaluate future therapeutic strategies.