Removal of Dopamine 2 Receptor abolishes the daily rhythm of phagocytosis in the mouse RPE.

Abstract

Purpose : A burst in phagocytosis of photoreceptor outer segment discs by retinal pigmented epithelium (RPE) is a rhythmic process that occurs daily 1-2 hours after the onset of light. This phenomenon has been considered crucial for the health and physiology of retina and RPE. Previously we have reported that dopamine via dopamine 2 receptor (D₂R) shifts the circadian rhythm in cultured RPE. Here, we investigated the impact of the removal of D₂R receptor on the phagocytic activity of RPE.

Methods : Control and D₂R knock-out (D₂R^-/-) mice (3 months) were sacrificed one hour before (ZT23), one hour after (ZT1) and three hours after (ZT3) the onset of light. Immunohistochemistry was used to label and count the phagosomes in the eye sections. Phosphorylation of focal adhesion kinase (pFAK, a key regulator in the daily burst of phagocytic activity) was analyzed by immunoblotting. Changes in gene expression between the 3 different time-points and genotypes were investigated by RNA-seq. Structure and function of retina and RPE were assessed by SD-OCT, Zo-1 immunohistochemistry and ERG in both genotypes at 3 and 12 months.

Results : D₂R^-/- mice did not show a daily burst of phagocytic activity after the onset of light and FAK phosphorylation did not increase in the D₂R^-/- mice at ZT1. RNA-seq revealed a total of 235 differentially expressed genes (DEGs) between ZT23 and ZT1 in the control mice, whereas D₂R KO had signifcantly higher 485 DEGs. Pathway analysis of the gene expression data indicated that at ZT1 integrin signaling was down-regulated in D₂R^-/- mice at ZT1. No differences in retinal and RPE morphology or ERG responses – as well as retinal functioning - were observed between control and D₂R^-/- at 3 and 12 months of age.

Conclusions : Our data indicate that removal of D₂R prevents the daily burst in phagocytosis and the related increase in FAK phosphorylation. Our RNA-seq data suggest a putative role of D₂R signaling in controlling the integrin pathway which is known to play an important role in the control of the daily burst of phagocytosis by the RPE. Surprisingly the lack in the daily burst in phagocytic activity did not affect retinal or RPE structure and function.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.