Wennan Lu
| Wennan Lu |
Wennan Lu, Nestor Mas Gomez, Claire H Mitchell
Basic & Translational Science, University of Pennsylvania, School of Dental Medicine
Introduction
Understanding how injured retinal pigment epithelial(RPE) cells communicate trouble to neighboring cells is central. RPE cells are particularly susceptible to lysosomal compromise as they degrade photoreceptor outer segments released each day, and as they are post-mitotic cells, allowing chronic accumulation of toxins in the lysosomes. Given the pathways connecting lysosomes and multivesicular bodies (MVBs), and the role of MVBs in exosome release, we hypothesized that lysosomal compromise alteres exosome release from RPE cells.
Methods
Lysosomal compromise in iPS-RPE or ARPE-19 cells was induced by exposing cells to 10µM chloroquine for 7 days. Immunoblots were performed on vesicles collected from supernatant using ExoQuick. Vesicle size and quantity were analyzed with a Spectradyne system and exosome marker analysis performed with an ExoView chip system. C57BL6J mice received intraperitoneal chloroquine injections 50 mg/kg; 3x/wk for 6 weeks.
Results
Immunoblots showed a rise in exosome marker CD63 in vesicular material from the supernatents of iPS-RPE cells exposed to chloroquine. A similar rise in CD63 was observed in immunoblots from the supernatant of ARPE-19 cells exposed to chloroquine. Quantification of extracellular vesicle size confirmed these vesicles to be exosomes, with a mean diameter of 79.7 ±1.4 nm in control; particle size was not changed after exposure to chloroquine. However, Spectradyne analysis confirmed exosome number was increased by chloroquine. ExoView analysis showed chloroquine induced a differential increase in the number of CD63+ and CD81+ vesicles, with a smaller rise in CD9+ vesicles. Initial trials suggest supernatant from chloroquine-treated ARPE-19 cells reduced viable 661w cells, although chloroquine by itself did not. Increased staining for CD63 was found in RPE cells from mice, and in cultured RPE cells, treated with chloroquine.
Conclusion
These findings suggest lysosomal compromise increases exosome release from RPE cells. Links between exosome release and photoreceptor health require confirmation. Exosome markers CD63 and CD81 were previously identified in drusen from AMD patients; whether reducing lysosomal compromise can reduce these markers in drusen remains to be determined.