Beyond PDZ binding: The USH2 complex in zebrafish photoreceptors requires interactions among multiple protein domains

Abstract

Purpose : Usher syndrome (USH), hearing loss combined with retinitis pigmentosa, is clinically and genetically complex. USH proteins form complexes mediated by PDZ binding, and mutations that block these interactions presumably underlie clinical symptoms. We used zebrafish mutants to analyze changes in protein interactions and retinal degeneration resulting from dysfunctional USH2 complex components.

Methods : We obtained mutants by CRISPR/Cas9 gene editing or from ZIRC and used antibodies to visualize USH2 proteins in cryosectioned retinas. Active Caspase-3 antibody was used to identify apoptotic cells. We analyzed confocal signal intensity with LSM5 and ImageJ software.

Results : We targeted mutations in ush2a^b1245 and adgrv1^b1333 to preserve the extracellular and transmembrane domains but delete intracellular PDZ-binding domains. whirlin is duplicated in zebrafish, and both encode proteins localized to the periciliary region (PCR) of photoreceptors. Whirlin A is variably reduced at the PCR in ush2a^b1245 and ush2a^sa1881 mutants and is partially dependent on light adaptation; retinas collected late in the day show more reduced Whirlin A than those dark adapted or collected earlier. In contrast, Whirlin A is minimally reduced in adgrv1 mutants, and does not appear light dependent. Whirlin B is unaffected in ush2a^b1245, variably reduced in ush2a^sa1881 (an early truncating mutation), and diminished in both early and late truncating adgrv1 mutants. Whirlin A localization is comparable in adgrv1^sa10539;ush2a^b1245 double and single mutants, but Whirlin B is undetectable in double mutants. Photoreceptor degeneration is enhanced in adgrv1;ush2a double mutants compared to either mutant alone.

Conclusions : We conclude that although PDZ binding by Usherin and Adgrv1 is indispensable for localization of Whirlin at the PCR, additional stability of the USH2 complex relies on interactions mediated by the Usherin extracellular domain. Increased photoreceptor death when Usherin and Adgrv1 functions are both impaired correlates with loss of Whirlin B localization from the PCR, suggesting that complex instability is related to severity of USH2 retinal pathology. Complete understanding of how various functional domains and interactions among USH2 proteins relate to the molecular pathology of the disease is critical for optimizing emerging treatments for USH2 patients with a broad range of genetic lesions.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.