Core Circadian Clock Genes Per1 and Per2 drive the Rhythm in Photoreceptor Outer Segment Phagocytosis and Photoreceptor DNA repair

Abstract

Purpose : Retinal 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. In this study, we tested the hypothesis that, within photoreceptors, the outputs of circadian clock genes Per1 and Per2 initiate the peak of POS phagocytosis.

Methods : QPCR gene expression analysis was performed on whole retinas obtained at 3h intervals from wild-type (WT) and Per1, Per2 double mutant (Per1^-/-Per2^Brdm1) mice kept either in LD (12h light: 12h dark) or DD (24h dark) conditions. By immunohistochemistry and microscopy, POS were quantified from WT and Per1^-/-Per2^Brdm1 mouse retinas obtained under LD and DD at 3h intervals. RNA-sequencing was performed on selectively laser-capture microdissected photoreceptors from WT and Per1^-/-Per2^Brdm1 mouse eyes obtained under DD, prior, during and after the (expected) peak in phagocytosis. Gene ontology and pathway analysis was performed using g:Profiler.

Results : Under DD, Per1^-/-Per2^Brdm1 mice lack a daily peak in POS phagocytosis. We found that this deficit is partially rescued by light. By qPCR analysis we found that core clock genes were rhythmic in both WT and Per1^-/-Per2^Brdm1 whole retinas. More precise transcriptomics analysis of laser-capture microdissected photoreceptors showed that 2.372 genes (=10.45% total) showed temporal variation in WT mice. In contrast, the same analysis of Per1^-/-Per2^Brdm1 mice photoreceptors yielded only 2 time-affected genes. Unexpectedly, in WT photoreceptors, there were no differentially expressed genes between time-points preceding and during the peak of POS phagocytosis. However, we found a significant gene expression response characterized by Gene Ontology terms and pathways related to DNA repair at the end of the subjective day-time, which was absent in clock-mutant photoreceptors.

Conclusions : Our findings indicate that Per1 and Per2 are necessary clock components for driving POS phagocytosis and DNA repair in photoreceptors. Our data cast doubt on the widely-held view that photoreceptors themselves initiate POS phagocytosis and reveal a transcriptional link between clock genes and DNA repair.

This is a 2020 ARVO Annual Meeting abstract.