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Radouil T. Tzekov, Lei Lei, Huapeng Li, J Hugh McDowell, Guangping Gao, W. Clay Smith, Shibo Tang, Shalesh Kaushal; Analysis Of The Components And Clearance Of Retinal Lipofuscin. Invest. Ophthalmol. Vis. Sci. 2012;53(14):4272.
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To evaluate several aspects of lipofuscin-like material (LLM) accumulation in retinal pigment epithelial (RPE) cells, including time course, natural clearance, effects of phagocytosis rate, effects of rod outer segment (ROS) oxidation, contribution of different ROS components and pharmacological manipulation of autophagy.
Post-confluent ARPE-19 cells were cultured in 24 well plates and fed with different types of bovine ROS for 7 to 14 days. Additionally, several ROS components were added to the RPE cells separately: 11-cis retinal, all-trans retinal, 9-cis retinal, opsin, liposomes containing phosphatidylethanolamine and phosphatidylcholine. Lipofuscin-like autofluorescence (LLAF) was determined at two wavelengths (530 and 585 nm) by FACS. Cells were also treated with lysosomal (chloroquine, NH4Cl) or autophagy inhibitors (3-MA), or with autophagy inducers (Ku-0063794, PI-103, PIK-90) to evaluate the effect on LLAF. Additionally, rapamycin (10 and 20 μM) was added over a 2-day period or as a single application for live cell imaging. Autophagy inhibition was also performed by siRNA and shRNA knockdown of Atg5 and Atg7.
Application of ROS indicates that the rate of LLM accumulation is linear over a 14-day period with higher rate of increase for the first 7 days. The rate of lipofuscin accumulation was proportional to the rate of phagocytosis of the ROS; LLM appears relatively stable once it accumulates in the RPE cell with only a limited loss (10-15%) over a 7-day period. Feeding with oxidatively modified ROS, and individual retinoids such as 11-cis and all-trans retinal all enhanced LLAF, with a more pronounced increase at 585 nm. Application of lysosomal or autophagy inhibitors and suppression of Atg5 or Atg7 increased LLAF, while application of autophagy inducers decreased LLAF. Rapamycin decreased LLAF in a dose-dependent way and most of the decrease occurred within the first 30 min after application.
These results emphasize the role of retinoids, lipids and oxidation in LLM accumulation in RPE cells. Furthermore, it confirms the possibility for a pharmacological clearance of RPE LLM by small molecules modulating the mTOR/autophagy pathway, thus opening new avenues for the treatment of dry ARMD and other lipofuscinopathies.
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