Analyzing the Functional Role of Calycleal Processes in CDHR1-dependent Cone Rod Dystrophy

Retinal congenital disease is a major contributor to blindness disorders, affecting up to 4.5 million people worldwide. Cone-rod dystrophies are a group of inherited retinal diseases that first affect cone photoreceptors, then rod photoreceptors, or in some cases affect rods and cones simultaneously. Generally, photoreceptors begin to degenerate, causing progressive loss in visual acuity, color and central vision, and light sensitivity. To develop therapeutics for cone-rod dystrophies, understanding how each of the over 30 genes implicated in cone-rod dystrophy contribute to the onset and progression of this disease is imperative. To do this, most studies have aimed to elucidate the role of these genes in retinal development and maintenance in vertebrate models such as mouse and zebrafish. A well-established candidate gene associated with cone-rod dystrophy which has yet to be explored in a developmental context is photoreceptor specific cadherin CDHR1. Several clinical studies have described mutations in CDHR1 associated with inherited cone-rod dystrophy. Conserved among vertebrates, CDHR1 belongs to the cadherin superfamily of calcium-dependent cell adhesion molecules but is exclusively expressed in photoreceptors of zebrafish, chickens, mice, and humans. CDHR1 encodes an intracellular domain, a transmembrane domain in addition to six cadherin repeats. Previous studies in mice using tomography, electron microscopy and immunohistochemistry have unequivocally defined CDHR1 localization to the base of outer segment (OS) of photoreceptor cells and proposed it plays a role during rod OS disk release. Despite a mouse model for CDHR1 loss of function, we still lack an detailed understanding of CHDR1’s molecular function and how it relates to our understanding of OS homeostasis. Recent findings form our group have discovered a novel connection between CDHR1 and the often-overlooked calyceal processes (CP). CPs are actin rich membrane extensions that surround the OS that are hypothesized to play roles in OS assembly, and structural integrity, yet have not been examined in the context of macular degeneration. Using super resolution microscopy, we have observed that the zebrafish homologue, Cdhr1a, localizes juxtaposed to CP resident protein Pcdh15b, another retinal cadherin, suggesting a potential interaction bridging the OS and the CPs. Cross species examination, including non-human primates, confirms this localization pattern as evolutionarily conserved. Furthermore, co-immunoprecipitation confirms an interaction between Cdhr1a and Pcdh15b. Additionally, a newly generated cdhr1a loss of function zebrafish mutant line from our lab exhibits cone-rod retinal dystrophy and disorganized CPs. Taken together, we hypothesize that Cdhr1a plays a crucial role during assembly of nascent rod photoreceptor outer segments and maintains cone OS stability by facilitating a direct connection between forming disks and calyceal processes. Determining the molecular function of Cdhr1a, is key to understanding its connection to cone-rod dystrophies and retinal degeneration in general. We believe zebrafish offers a unique opportunity to not only study the molecular function of Cdhr1a but also model and assay CDHR1-associated cone-rod dystrophy and the functional contribution of CPs. To achieve these goals we propose: AIM 1: Model cone-rod dystrophy in a Cdhr1a loss of function mutant. The zebrafish cone-rich retina offers a highly relatable model for human cone-rod dystrophy. Using a newly generated Cdhr1a loss of function mutant line we aim to comprehensively characterize the progression and catalogue molecular signatures associated with cone and rod degeneration from early development and into adulthood. AIM 2: Investigate the molecular function of Cdhr1a in photoreceptors. We propose that a physical interaction between Cdhr1a and Pcdh15b facilitates connections between newly forming OS disks and the CPs which facilitates proper OS assembly to establish and maintain OS structural integrity. AIM 3: Assay photoreceptor homeostatic function of Cdhr1a. examine UPS interaction with cdhr1a VS residence/clearance from the OS. Examine protein interactome network linking OS and CP – proximity ligation approach.