Usherin defects lead to early-onset retinal dysfunction in zebrafish

Abstract

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-technology, we introduced protein-truncating germline lesions into the zebrafish ush2a gene (ush2a: c.2337_2342delinsAC; p.Cys780GlnfsTer32 and ush2a: c.15520_15523delinsTG; p.Ala5174fsTer). 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, but presence of the ectodomain of usherin at the periciliary membrane of ush2a-derived photoreceptors. Furthermore, defects of usherin led to a reduction in localization of USH2 complex members, whirlin and Adgrv1, at the photoreceptor periciliary membrane of both mutants. Significantly elevated levels of apoptotic photoreceptors could be observed 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 as well as ush2a 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.

Modeling ZNF408-Associated FEVR in Zebrafish Results in Abnormal Retinal Vasculature

“…33,34 The loss of function znf408 zebrafish models (as in znf408 rmc103/rmc103 and znf408 rmc104/rmc104 ) showed defective retinal vasculature development and only mild visual impairment in visuomotor assay, particularly if compared with the visual impairment observed in retinitis pigmentosa zebrafish models. [35][36][37] One explanation for this is that the visuomotor assay we used here is not sensitive enough, and alternatives (e.g., electroretinogram recordings, optokinetic response measurements) would be needed. Thus, it is yet to be determined how (predicted) loss of function of znf408 (as in znf408 rmc103/rmc103 and znf408 rmc104/rmc104 ) showed a retinal vasculature phenotype instead of retinitis pigmentosa-like symptoms in zebrafish.…”

Usher Syndrome: Genetics and Molecular Links of Hearing Loss and Directions for Therapy

“…These Ush2a mutant mice models have provided information on the role of usherin in the auditory system, but limited data on the visual system. Usherin protein appears to be functional in zebrafish despite them lacking a full-length protein (Dona et al, 2018). In addition, ush2a knockout zebrafish have been generated successfully and these exhibit auditory abnormalities and retinal degeneration, replicating the disease phenotypes of human USH2A patients (Han et al, 2018).…”

“…Usherin is a large protein comprised of a number of domains ( Figure 3A; Eudy et al, 1998;Weston et al, 2000;Sheng and Sala, 2001;Dona et al, 2018). USH2A has two isoforms, A and B, with isoform B being the predominant form in retina ( Figure 3A; Liu et al, 2007;Toms et al, 2015).…”

“…Two of the most frequent mutations in USH2A occur on exon 13, namely c.2276C>T (p.C759F) and c.2299delG (p.E767fs), with the latter accounting for over 30% of USH2A mutations in an USH cohort (Le Quesne Stabej et al, 2012;Fuster-Garcia et al, 2017). Due to the high frequency of mutations on exon 13, antisense oligonucleotide treatments targeted at skipping this region of the gene are under development (Dona et al, 2018;van Wijk, 2019).…”

“…Two designed ASO successfully targeted a pseudo-exoncausing USH2A mutation, increasing the amount of correctly spliced transcript in USH2A patient-derived fibroblasts (Slijkerman et al, 2016). A novel drug, QR-421a (ProQR Therapeutics) demonstrated a successful "exon skip" in an Ush2a mutant zebrafish model, leading to restored Usherin expression and electroretinogram (ERG) recordings (Dona et al, 2018;van Diepen et al, 2019). QR-421a is currently in clinical trial (Stellar; NCT03780257), with preliminary results reported in April 2020.…”