Cdhr1a connects the outer segment to calyceal processes via interaction with pcdh15b, implications for pathogenesis of cone rod dystrophy.

Abstract

Purpose : Mutations in CDHR1, a photoreceptor specific cadherin lead to cone rod dystrophy. Mice and xenopus Cdhr1 mutant models exhibit abnormal outer segment (OS) morphology and photoreceptor (PRC) degeneration. However, the function of CDHR1 underlying pathogenesis remains unknown. By using a cone dominant zebrafish model, we explored how loss of Cdhr1a function leads to cone rod dystrophy and also discovered that Cdhr1a interacts with Pcdh15b thus physically linking the outer segment with calyceal processes (CP).

Methods : To establish a zebrafish model of cone rod dystrophy we generated a CRISPR knock-out line for Cdhr1a (Cdhr1a^D173) and verified it to be null using Immunofluorescence (IF). Cdhr1a KO maternal zygotes at 5 days post fertilization (dpf), 15dpf, 30dpf, 90dpf, and 180dpf were analyzed using confocal microscopy. Prph2 staining was used to demarcate OS morphology while fluorescent peanut and wheat germ agglutinins were used to distinguish between cones and rods. To examine subcellular localization, IF imaging for Cdhr1a and Pcdh15 was visualized using structured illumination microscopy (SIM). Finally, biochemical interactions between Cdhr1a and Pcdh15b were tested by coimmunoprecipitation and cell aggregation assay using HEK293T and K562 cells.

Results : Cdhr1a mutants exhibit minor gross cone OS morphology defects starting at 15dpf and severe OS disruption by 3 months while rod OS defects appeared at 3 months. Using SIM we observed juxtaposition of Cdhr1a at the leading edge of OS with Pcdh15b localizing to the CP of inner segments in zebrafish, human, macaque, xenopus, mouse, and gerbil PRCs. Additionally, coimmunoprecipitation in HEK293T cells confirmed cadherin-based interactions between Cdhr1a and Pcdh15b while co-culturing Cdhr1a and Pcdh15b transfected K562 cells led to cell aggregation indicating functional extracellular interactions. Finally, using SIM, we correlated the disruption of CP attachments to cone OS degeneration in the Cdhr1a^D173 line.

Conclusions : Phenotypes observed in the Cdhr1a^D173 phenocopy cone-rod dystrophy observed in patients. Our discovery of the interaction between Cdhr1a and Pcdh15b as being required for OS-CP interactions suggests a novel mechanism for pathogenesis of cone rod dystrophy.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.

 

Figure 1. Evolutionarily conserved Cdhr1 and Pcdh15 juxtaposition in vertebrate photoreceptors