Abstract
Purpose :
Bisretinoids, the precursors of lipofuscin, cannot be degraded by enzymes within the RPE. Degradation of these compounds is however possible after light exposure and there is no accumulation in young individuals although the load of bisretinoids is not age-dependent. During phagocytosis of outer segments containing bisretinoid, superoxide, hydroxyl radical and H2O2 are formed by NADPH oxidase within the phagosomes, which is similar to the respiratory burst by which macrophages kill bacteria1.Here we investigated whether the superoxide generator riboflavin can remove lipofuscin from RPE.
Methods :
Nine month old Abca4(-/-) mice were daily treated orally with 1µg riboflavin-5-phosphate ester over 4 weeks. The eyes were fixed and embedded for electron microscopy. The trainable Weka Segmentation plugin for Fiji software, a machine learning tool, was used to determine the area of lipofuscin in about 30 electron micrographs/eye from 7 eyes (4 treated/3 untreated) as described2.
Results :
Free and melanin associated lipofuscin was significantly reduced by treatment. Without treatment 13.3 + 4.5 % and after riboflavin treatment 9.2 + 3.4 % of the cell cytoplasm was occupied by lipofuscin (t-test p < 0.001).
Conclusions :
Bisretinoid cracking occurs shortly after phagocytosis within phagosomes of the RPE by superoxide and other radicals. For this reason radical generators (NADPH oxidase, Riboflavin) are specifically present in phagosomes and melanosomes of the RPE. If the amount of bisretinoids is too large as in Stargardt's disease, they cannot be degraded completely and fuse with melanosomes to melanolipofuscin. Melanin has unusual redox properties and generates radicals in order to continue lipofuscin degradation within these spezialized lysosomes. It is known that melanin binds, interacts and surprisingly does not quench riboflavin fluorescence3.If melanin becomes exhausted, lipofuscin degradation is slowed down and melanolipofuscin (Fig) accumulates. Here we show that substitution with the radical generator riboflavin, which is naturally present in phagosomes 4 and melanosomes, can remove lipofuscin from the RPE.
1 Miceli et al (1994) Exp Cell Res 214(1): 242-249.
2. Taubitz et al (2018). PeerJ 6: e5215.
3 Kozik et al (1990) Biophys Chem 38(1-2): 39-48.
4 Said et al (2005). J Physiol 566(Pt 2): 369-377.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.