IFT88 Mediates Cilia-Associated Wound Repair in Retinal Pigment Epithelium

Abstract

Purpose : Primary cilia are microtubule-based organelles that transduce extracellular cues into intracellular signals critical for diverse processes including proliferation and tissue repair. Defective ciliary function results in multisystemic diseases known as ciliopathies in humans. Many ciliopathies in the eye, such as Bardet-Biedl Syndrome, share a common feature of atrophy of the retinal pigment epithelium (RPE). Mutation in genes involved in cilia formation, including IFT88 or BBS4 which cause Bardet-Biedl Syndrome, result in loss of or shortened cilia. However, the roles for RPE cilia in vivo remain unknown. This study investigates the role of primary cilia in the proliferative and reparative responses of mouse RPE after targeted laser-injury.

Methods : RPE flatmount from Bbs4^-/- mutant mice and wildtype controls from pups on the day of birth (P0), were obtained and immunostained for cilia markers. Transgenic mice were generated with RPE-selective loss of primary cilia (RPE-cKO mice) by crossing IFT88^flox/flox mice and BEST1-cre mice. Using a laser-induced injury model in vivo, primary cilia formation was assessed by ciliary markers on RPE flatmounts in response to injury using fluorescence microscopy. Anti-Ki67 antibody was used as a proliferative cell marker. The re-epithelialization was analyzed for wound size measurement with anti ZO-1 antibody and phalloidin. Statistical analyses were performed using Student’s t-test or One-way ANOVA, p<0.05 was considered statistically significant.

Results : Ciliation in Bbs4 mutant RPE cells is disrupted early during eye development. RPE cilium were reassembled after laser injury, followed by rapid disassembly when repair is completed (n=4 mice). Consequently, faster repair is observed with loss of cilia in RPE-cKO mice than control eyes. Additionally, the removal of cilia by RPE-cKO mice improved wound healing by enhancing cellular proliferation in mouse RPE (n=6-10 eyes, Student’s t-test, P<0.05).

Conclusions : This study supports a novel role for primary cilia in RPE repair and provides insights into potential cilia-based therapeutic targets for RPE degenerative diseases.

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