Abstract
Purpose :
Routine renewal of photoreceptor outer segments follows a circadian rhythm with a burst of phosphatidylserine (PS) externalization and RPE phagocytosis of spent outer segment tips following light onset. In wild-type mice lack of expected light onset results in attenuated outer segment PS exposure and RPE phagocytosis. Here, we investigate if the photo-sensitive protein melanopsin plays a role in regulating outer segment turnover activities upon light onset.
Methods :
3-4 month-old wild-type (WT) and melanopsin-deficient (Opn4-/-) mice of either sex were entrained to 12h:12h dark light cycles. Animals were sacrificed and eyes dissected at precise times relative to entrained light onset either in room light or under infrared illumination using night vision goggles. Exposure of externalized PS by outer segments was tested with PS biosensor live imaging of dissected retina. RPE phagocytosis was quantified using immunofluorescence microscopy of RPE flatmounts with rhodopsin-positive inclusions of 0.5 to 1 μm diameter counted as phagosomes. Cryosection immunofluorescence microscopy and lysate immunoblotting of marker proteins served to assess retinal and RPE tissue health. A minimum of 4 independent samples for each genotype and condition were quantified, and only differences with P<0.05 were considered significant.
Results :
Phagosome content in Opn4-/- RPE was reduced by about half 1.5 h after light compared to phagosome content of WT RPE. WT mice sacrificed in the dark after missing a single light onset showed significantly attenuated RPE phagosome content (P<0.01), while Opn4-/- mouse RPE phagosome content was identical regardless of whether light onset occurred. Similar to photoreceptors in vivo, outer segments in isolated WT eyecups extend PS exposure if exposed to light ex vivo. Comparing ex vivo PS exposure by outer segments in eyecups with and without melanopsin antagonist, we found that the increase induced by illumination was abolished by melanopsin inhibition (P<0.01).
Conclusions :
Outer segment renewal in mice lacking melanopsin is attenuated and non-responsive to expected dark-light transition. Moreover, acute pharmacological inhibition of melanopsin prevents extension of PS exposure by outer segment tips in response to light. These results imply that the direct and acute effect of light onset on the diurnal burst of photoreceptor outer segment renewal is mediated by melanopsin.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.