Rev-Erb\u03b1 and Photoreceptor Outer Segments modulate the Circadian Clock in Retinal Pigment Epithelial Cells

Nemanja Milićević,Jorge Mendoza,Arthur Bergen,Nadia Mazzaro,Marie-Paule Felder-Schmittbuhl,Anneloor Ten Asbroek,Jacoline Ten Brink,Esmée Wils,Ivanka De Bruin

doi:10.1038/s41598-019-48203-3

Abstract

Retinal photoreceptor outer segments (POS) are renewed daily through phagocytosis by the adjacent retinal pigment epithelial (RPE) monolayer. Phagocytosis is mainly driven by the RPE circadian clock but the underlying molecular mechanisms remain elusive. Using ARPE-19 (human RPE cell-line) dispersed and monolayer cell cultures, we investigated the influence of cellular organization on the RPE clock and phagocytosis genes. PCR analysis revealed rhythmic expression of clock and phagocytosis genes in all ARPE-19 cultures. Monolayers had a tendency for higher amplitudes of clock gene oscillations. In all conditions ARNTL, CRY1, PER1-2, REV-ERBα, ITGB5, LAMP1 and PROS1 were rhythmically expressed with REV-ERBα being among the clock genes whose expression showed most robust rhythms in ARPE-19 cells. Using RPE-choroid explant preparations of the mPer2Luc knock-in mice we found that Rev-Erbα deficiency induced significantly longer periods and earlier phases of PER2-bioluminescence oscillations. Furthermore, early phagocytosis factors β5-Integrin and FAK and the lysosomal marker LAMP1 protein levels are rhythmic. Finally, POS incubation affects clock and clock-controlled phagocytosis gene expression in RPE monolayers in a time-dependent manner suggesting that POS can reset the RPE clock. These results shed some light on the complex interplay between POS, the RPE clock and clock-controlled phagocytosis machinery which is modulated by Rev-Erbα.

Highlights

The photoreceptors and the retinal pigment epithelial (RPE) initiate rhythmic phagocytosis[7]
We found that the ARPE-19 monolayers show rhythms in the photoreceptor outer segments (POS) phagocytosis machinery, and that POS might in turn entrain the RPE clock
Results of non-linear regression analysis showed that the dispersed cells exhibited rhythmic expression of the following clock genes: ARNTL, CRY1, CRY2, PER1, PER2, REV-ERBα, and the phagocytosis genes: ITGB5, lysosomal associated membrane protein 1 (LAMP1), www.nature.com/scientificreports

Summary

Introduction

The photoreceptors and the RPE initiate rhythmic phagocytosis[7]. Recent evidence indicates that RPE possesses a circadian oscillator[10,11], but its potential role in regulating the phagocytosis machinery remains unknown. The removal of damaged POS is performed by RPE apical microvilli that project into the interphotoreceptor matrix. The daily clearance of POS makes the RPE one of the most phagocytically active cells in the human body[17]. This imposes a high metabolic burden in the RPE, in particular at the beginning of the light phase of the light/dark (LD) cycle, during peak phagocytic activity. We found that the ARPE-19 monolayers show rhythms in the POS phagocytosis machinery, and that POS might in turn entrain the RPE clock

Methods

Results

Conclusion