Core Circadian Clock GenesPer1andPer2regulate the Rhythm in Photoreceptor Outer Segment Phagocytosis
Authors: Milićević Nemanja , Hakkari Ouafa Ait-Hmyed , Bagchi Udita , Sandu Cristina , Jongejan Aldo , Moerland Perry D. , ten Brink Jacoline B. , Hicks David , Bergen Arthur A. , Felder-Schmittbuhl Marie-Paule 
Published: December 2020

AbstractRetinal photoreceptors undergo daily renewal of their distal outer segments, a process indispensable for maintaining retinal health. Photoreceptor Outer Segment (POS) phagocytosis occurs as a daily peak, roughly about one hour after light onset. However, the underlying cellular and molecular mechanisms which initiate this process are still unknown. Here we show that, under constant darkness, mice deficient for core circadian clock genes (Per1andPer2), lack a daily peak in POS phagocytosis. By qPCR analysis we found that core clock genes were rhythmic over 24h in both WT andPer1, Per2double mutant whole retinas. More precise transcriptomics analysis of laser capture microdissected WT photoreceptors revealed no differentially, expressed genes between time-points preceding and during the peak of POS phagocytosis. By contrast, we found that microdissected WT retinal pigment epithelium (RPE) had a number of genes that were differentially expressed at the peak phagocytic time-point compared to adjacent ones. We also found a number of differentially expressed genes inPer1, Per2double mutant RPE compared to WT ones at the peak phagocytic time-point. Finally, based on STRING analysis we found a group of interacting genes which potentially drive POS phagocytosis in the RPE. This potential pathway consists of genes such as:Pacsin1,Syp,Camk2bandCamk2damong others. Our findings indicate thatPer1andPer2are necessary clock components for driving POS phagocytosis and suggest that this process is transcriptionally driven by the RPE.DeclarationsFundingThis project has been funded with support from the NeuroTime Erasmus+ grant (European Union), Rotterdamse Stichting Blindenbelangen (Netherlands), Nelly Reef fund (Netherlands), Stichting voor Ooglijders (Netherlands), Stichting tot Verbetering van het Lot der Blinden (Netherlands) and Retina France (France).Conflicts of interest/Competing interestsThe authors declare no competing interests.Availability of data and materialData supporting the conclusions of this article are included within the article and are available from the corresponding authors on reasonable request.Code availabilityThe R code for analysis is available from the corresponding authors on reasonable request.Ethics approvalAll experimental procedures were performed in accordance with the Association for Research in Vision and Ophthalmology Statement on Use of Animals in Ophthalmic and Vision Research, as well as with the European Union Directive (2010/63/EU).Consent to participateNot applicableConsent for publicationAll authors read and approve of the contents of this manuscript.Author contributionsN.M. performed experiments, analysis, prepared figures, wrote the manuscript and obtained funding. O.A.-H.H. performed experiments, data analysis, prepared figures and obtained funding. P.D.M. and A.J. performed bioinformatics analysis and edited the manuscript. U.B., J.B.t.B. and C.S. provided technical assistance, performed experiments, prepared figures and edited the manuscript. D.H., A.A.B. and M.-P.F.-S. conceptualized and directed the project, obtained funding, provided resources, performed analysis and edited the manuscript.