Abstract
Purpose :
The daily degradation of phagocytosed photoreceptor outer segments (POS) puts a large burden on lysosomes of RPE cells; patching lysosomal membranes by members of the Endosomal Sorting Complex Required for Transport (ESCRT)-pathway after moderate damage, or lysosomal renewal through lysophagy following more severe insults, both ensure lysosomes can meet the degradative needs of aging cells. This study examined the time course of ESCRT-protein and lysophagy component recruitment to RPE cells following permeation of the lysosomal membrane.
Methods :
Lysosomal damage was induced in human iPS-RPE cells or ARPE-19 cells using L-Leucyl-L-Leucine methyl ester (LLOMe). Chloroquine (CQ) was added on occasion. qPCR identified ESCRT and lysophagy components in RPE cells, while the Live/Dead assay quantified death. Live cell imaging using spinning-disk confocal microscopy, immunohistochemistry, and ImageJ analysis quantified involvement of key proteins.
Results :
In dose-response studies, LLOMe killed RPE cells at 3 mM and 1 mM, but not 300µM or 100µM up to 24 hrs; 300 µM LLOMe was used for subsequent studies. RPE cells expressed mRNA for lysosome repair and lysophagy components including ALIX, p62, CHMP2, CHM4b, Gal3, and TRIM16; only Gal3 mRNA levels increase after exposure to LLOMe or POS while TRIM16 rose after CQ treatment. After 15 min LLOMe exposure, punctate staining of ESCRT-protein CHMP4b was detected; increased LAMP1 staining was observed at 30 min; LAMP1 and CHMP4b were closely associated, with CHMP4b often surrounding the punctate LAMP1 staining. Preliminary data suggest this early response was reduced by Ca2+-chelator BAPTA, suggesting a role for Ca2+. Lysophagy was detected at later time points, with punctate staining for p62 found 24 hrs after LLOMe ± CQ exposure but not earlier. Staining for p62 and LAMP1 showed significant colocalization, consistent with lysophagy. Large LAMP1 condensates ~ 1µm in diameter were frequently seen after 24 hrs LLOMe exposure; these “megalysosomes” were associated with p62 and surrounded by TRIM16.
Conclusions :
This preliminary study suggests ESCRT-protein CHMP4b is rapidly recruited to damaged lysosomes of RPE cells through a Ca2+ dependent process, while prolonged damage leads to recruitment of p62 to lysosomes for organelle renewal through lysophagy. We hypothesize the balance between repair and renewal is distorted in disease.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.