Abstract
Purpose :
In the mammalian retina, daily renewal of light-sensitive photoreceptor outer segments involves coordinated shedding of distal outer segment tips and their engulfment by adjacent retinal pigment epithelial (RPE) cells. Exposure of the anionic membrane lipid phosphatidylserine (PS) at the outer leaflet of photoreceptor outer segment tips promotes shedding and subsequent phagocytosis by RPE cells. The mechanisms underlying this PS exposure remain to be understood. Anoctamin-6 (also known as TMEM16F) is a calcium-activated lipid scramblase that can promote PS exposure. Here we investigated whether anoctamin-6 has a role in PS exposure by photoreceptor outer segments in mouse retina in vivo.
Methods :
ano6+/- mice were extensively backcrossed to 129T2/SvEmsJ (WT129) and then bred to yield ano6-/- mice. For experiments, ano6-/- and control WT129 mice were housed in strict 12 h light/dark cycles and sacrificed at 4-5 months of age at time points relative to light onset. Retinas were immediately dissected and incubated photoreceptor side up with PS detection reagent pSIVA (polarity-sensitive indicator of viability and apoptosis). X-y image stacks were obtained using a Leica TSP8 laser scanning confocal microscopy system. Maximum projections were analyzed using ImageJ. Data sets of n≥4 were analyzed using two-tailed Student’s t-test.
Results :
Anoctamin-6 knockout mice (ano6-/-) are not viable in C57BL6/J genetic background. We found that ano6-/- mice in 129 background are viable and have no obvious gross abnormalities up to 6 months of age. Moreover, ano6-/- mice breed with fecundity similar to WT129 mice. Ano6 protein was detected in WT129 but not ano6-/- retina. ano6-/- retina behaved similarly to WT129 tissue in dissections. In WT129 mice, outer segment PS tips elongate and change in frequency in a characteristic diurnal rhythm. Here, we found that at light onset, when outer segment PS tip length in WT129 peaks, PS tip length in ano6-/- was significantly less than in WT129 (p<0.05). One hour after light onset, the peak of frequency of outer segment PS tips in WT129 retina, PS tip frequency in ano6-/- was significantly less than in WT129 (p<0.05).
Conclusions :
Lack of anoctamin-6 leads to loss of rhythmic outer segment PS tip elongation and change in frequency. To our knowledge, anoctamin-6 is the first scramblase to be linked to PS exposure of photoreceptor outer segment tips.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.