Abstract
Purpose :
Photoreceptor outer segment renewal maintains photoreceptor integrity for life. During renewal, spent photoreceptor outer segments expose phosphatidylserine (PS) at their distal tips triggering phagocytosis by the adjacent retinal pigment epithelium (RPE). Earlier studies determined the circadian rhythmicity of RPE phagocytosis by housing animals in darkness for several days. We tested the effect of acute lack of morning light onset or shifted light onset on outer segment PS exposure and RPE phagocytosis in mice.
Methods :
Age-matched 129Sv male and female mice were housed in a 12 h light/dark cycle. Light onset occurred normally at ZT0 (LD), was omitted (DD) or shifted to 1 hour earlier or later. For detection of externalized PS, dissected retinas were incubated with PS biosensor and imaged live. Immunofluorescence microscopy of RPE flatmounts was used to quantify rhodopsin-positive RPE phagosomes.
Results :
In wild-type retina, PS tip exposure was attenuated in DD both in terms of individual PS tip length and frequency of PS tips: in DD at ZT0, when PS tips are longest in LD, PS tips had the same length as at ZT1 (P>0.05), and PS tips were shorter than in LD (P<0.05). Similarly, PS tip frequency at ZT1, when density peaks, was less in DD than in LD (P<0.05). When light onset was shifted 1 hour earlier or later, PS tips did not elongate at the new time of light onset. However, when eyes were enucleated and partially dissected in the dark, then exposed to light at entrained time of light onset, PS tips elongated as in LD. Moreover, peak phagosome content in wild-type RPE was decreased in DD compared to LD (P<0.05). In contrast, RPE phagosome content at the same time point was the same in DD and LD in itgb5-/- mice, which lack the diurnal rhythm of outer segment renewal.
Conclusions :
Acute lack of light onset reduces outer segment PS exposure and RPE phagocytosis, while shifted light onset cannot induce PS exposure to the same extent as light onset at the entrained time. These results imply that outer segment renewal is not purely circadian but requires additional light input. Enucleated eyes respond to light by elongating outer segment PS tips indicating the relevant light response is intrinsic to the eye. Lack of effect of light onset on itgb5-/- RPE phagocytosis suggests a molecular link to altered RPE signaling. Altogether, our results show that morning light onset has a critical role in outer segment renewal.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.