Novel association to retinal homeostasis identified for a gene involved in ubiquitination and proteasomal degradation.

Abstract

Purpose : Our goal is to identify and study genes essential to retinal development and homeostasis using an unbiased forward genetics approach. Here we characterize a novel association involving a gene with a known role in the ubiquitin–proteasome system (UPS).

Methods : We used a forward genetics screening pipeline (N-ethyl-N- nitrosourea mutagenesis in C57BL/6J mice) to identify, in an unbiased manner, non-redundant genes leading to the accumulation of fundus spots. All G3 mice used in our screening have been pre-genotyped at all mutant loci, making it possible to instantly determine the causative mutation of a given phenotype. We identified a mutation that led to a phenotype of increased fundus spots, that we termed aegean, and used CRISPR/Cas9 mutagenesis to generate mice deficient in the affected gene. We then characterized the retina in these mice.

Results : The aegean phenotype was recapitulated in the CRISPR-generated KO mouse line. This line showed progressive accumulation of fundus spots with age and a statistically significant thinning of the outer retina. We then used serial fundus photos, optical coherence tomography (OCT), immunohistochemistry (of both retinal sections and flat mounts), and electron microscopy to characterize the retinal phenotype. Functional retinal testing with electrophysiology will also be shown.

Conclusions : A forward genetics screening protocol using a semiquantitative fundus spot scale allowed us to identify a novel gene-retinal phenotype association involving a gene important for the ubiquitin proteasome system. This pathway plays a key role in cell cycle control, signal transduction, and removal of toxic/damaged proteins from the cell. Further studies will be needed to further characterize the specific role of this gene in retinal homeostasis.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.