Mouse Model of Retinitis Pigmentosa 2 has a Photoreceptor Nuclear Migration Defect

Abstract

Purpose : Arl3 is a small GTPase protein that regulates the ciliary enrichment of lipidated proteins. Human mutations in ARL3 gene have been linked to autosomal dominant and recessive forms of retinal degeneration. Our lab recently showed that dominant human mutations in the ARL3 elevate Arl3-GTP activity levels in the cell and result in a developmental phenotype whereby photoreceptor nuclei are mislocalized from the outer nuclear layer (ONL) to the inner nuclear layer (INL). In a follow-up study, we investigate whether defects in Arl3 binding to RP2, the GTPase activating protein that facilitates Arl3 hydrolysis of GTP to GDP, affect photoreceptor nuclear migration. Understanding whether RP2 defects could also lead to mislocalized photoreceptor nuclei is important as human mutations in the RP2 gene are associated with X-linked retinitis pigmentosa.

Methods : Dissected eyes were collected and hard fixed in 4% Paraformaldehyde and sliced into 100µM agarose retinal sections on Vibratome. Sections were blocked in 5% donkey serum in 1X PBS with 0.05% TritonX for 1 hour, then incubated in primary and secondary antibody solutions. We also collected dissected eyes and hard fixed in 2% Paraformaldehyde/2% Glutaraldehyde and sliced into thin plastic retinal sections that were stained with Toluidine-blue. Images were collected and analyzed on a Zeiss LSM confocal microscope.

Results : To assess whether loss of RP2 results in a nuclear migration phenotype, we collected retinal cross-sections at P21 from WT, Rp2^het, and Rp2^null mice and stained for phosducin, a photoreceptor-specific protein found in the cell body. Phosducin-positive nuclei were found within the INL of retinal sections in Rp2^het, and Rp2^null mice, but not WT controls. We also found that displaced photoreceptor nuclei contained ectopic synapses that could possibly explain why ERG dysfunction precedes photoreceptor cell loss as was previously reported in the Rp2^null mice.

Conclusions : Identifying that the same photoreceptor pathobiology underlies both autosomal dominant mutations in ARL3 and XLRP mutations in RP2 suggests that a single treatment option could benefit both patient groups. Future investigation into the developmental process of photoreceptor nuclear migration in both healthy and diseased retinas will help elucidate potential therapeutic targets.

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