Mouse Prpf3, 8 and 31 mutants show altered rhythms of retinal phagocytosis and adhesion

Abstract

Purpose: Mutations in the Pre-mRNA Processing Factors 3, 8 and 31 (PRPF3, 8 and 31) cause autosomal dominant retinitis pigmentosa (adRP). We previously showed that gene-targeted mice for these 3 factors display late-onset morphological changes in the retinal pigment epithelium (RPE). Since the RPE is critically important for the overall health of the retina, we wanted to determine which abnormalities precede the morphological changes observed in these mice. We have focused our investigation on two of the most important RPE functions that follow a daily rhythm: phagocytosis of photoreceptor outer segment (POS) and retinal adhesion.

Methods: Phagocytosis was analyzed in vitro and in vivo on Prpf-mutant and wild-type mice. In vitro phagocytosis assays were performed on primary RPE cell cultures from 9-10 day-old mice fed with FITC-labeled POS. Ratios of bound and ingested phagosomes versus cell numbers were assessed on a fluorescent microscope. For in vivo phagocytosis assays, mice were euthanized at different times of the day, eyes processed for electron microscopy or paraffin sectioning and phagosome numbers were counted on electron micrographs or paraffin section lengths labeled with an anti-rhodopsin antibody. Strength of retinal adhesion was evaluated by quantification of RPE-specific melanin and RPE65 proteins present on peeled retinas. Expression of phagocytosis receptors and ligands were analyzed by immunohistological labelings.

Results: Primary Prpf-mutants RPE cells show a 40% decrease in total phagocytosis. We found that POS binding is reduced by around 53% in Prpf31-mutant cells, whereas POS internalization remained similar to wild-type cells. In vivo, we observed the normal daily phagocytosis peak 2 hours after light-onset in wild-type controls, while it was greatly reduced in all three mutants. Similarly, retinal adhesion was reduced at the peak adhesion time in mutants, while being normal at other times. Some of the main phagocytic proteins showed altered localization in RPE cells and the POS/interphotoreceptor matrix area.

Conclusions: Our results suggest that early-onset defects in the synchronized rhythmicity of retinal phagocytosis and adhesion could explain the morphological changes observed in aging mutant RPE cells. Studies are underway to identify the exact cellular modifications created by altered splicing in these animals.

Keywords: 701 retinal pigment epithelium • 645 phagocytosis and killing • 695 retinal degenerations: cell biology