Nestor Mas Gomez
| Nestor Mas Gomez |
Nestor Mas Gomez, Wennan Lu, Claire, H Mitchel
Basic and Translational Sciences, University of Pennsylvania, School of Dental Medicine
Introduction
Compromised lysosomal integrity can lead to neurodegenerations like Alzheimer disease and age-related macular degeneration. Lysosomes of retinal pigmented epithelial (RPE) cells are particularly vulnerable to compromise given their substantial processing of oxidized lipids. Recent findings suggest moderate lysosomal damage is repaired via the endosomal sorting complexes required for transport (ESCRT) machinery, that more extensive damage requires organelle turnover through lysophagy, and that extreme lysosomal strain can lead to cell death. This preliminary study examined the involvement of ESCRT proteins, lysophagy and cell death in response to increasing levels of lysosomal damage
Methods
Experiments were performed in ARPE19 and iPS-RPE derived cells, with lysosomal membrane permeabilization induced by exposure to L-Leucyl L-Leucine methyl ester (LLoMe). Lysosomal alkalinization was induced by chloroquine (CQ). Identification of relevant proteins was determined using standard immunohistochemical procedures. Cell survival was determined using the LIVE/DEAD assay, while mRNA levels were measured using qPCR
Results
Initial experiments exposed cells to 300µM-LLoMe for short durations to examine repair. Staining for ESCRT protein CHMP4b was increased 30min after treatment with LLoMe, with a rise in the size of stained clusters. qPCR indicated no change in expression of CHMP4b mRNA after 1hr exposure to LLoMe. Levels of mRNA for lysophagy marker galectin 3 (Gal3) were increased after 1hr of LLoMe, although Gal3 was not detected in control or LLoMe-treated cells using two different antibodies. Exposure to LLoMe for 24hrs did not change levels of autophagy marker p62 on its own, although simultaneous exposure to CQ did increase p62 staining. Concurrent treatment with both lysosomal insults also increased colocalization of p62 with lysosomal marker LAMP1, consistent with induction of lysophagy. To understand whether these attempts at cell repair or turnover were successful, cell survival was examined. No change in cell death was detected 24hr after exposure 300µM-LLoMe, but was found with higher concentrations
Conclusion
This initial study has identified several parameters involved in lysosomal repair and lysophagy. The increase in CHMP4b staining after 30min exposure to LLoMe is consistent with membrane repair. The colocalization of p62 and LAMP1 following longer exposure to LLoMe in the presence of CQ suggests induction of lysophagy. While the lack of cell death induced by moderate LLoMe levels suggests these attempts to repair lysosomes were successful, future studies will expand our understanding