Abstract
Purpose: :
A2E is a component of lipofuscin that is toxic to RPE cells in vitro. A2E has been implicated in several retinal diseases, e.g. Stargardt1. Lemieux et. al (Analyt. Biochem. 327:247) reported that lysosomotropes, e.g. chlorpromazine (CPZ), can remove lysotracker from lysosomes of cells. We tested the ability of CPZ and other lysosomotropes for clearance of A2E from RPE cells, as a novel approach to alleviate A2E-mediated cytotoxicity.
Methods: :
ARPE-19 cells were incubated overnite with 8 uM A2E, or 30 min with 0.3 uM lysotracker. After a brief (1-4 hrs) incubation with the lysosomotrope, cells were analyzed for fluorescence of lysotracker or A2E by high content imaging on an Opera or ViewLux microplate imager. Retinas from Abca4-/- mice were characterized by lipofuscin autofluorescence and by immunofluorescenct staining with an acrolein antibody as a marker for oxidative stress.
Results: :
A2E above 25 uM became cytotoxic, but 8 uM A2E did not affect viability. Exposure of ARPE-19 cells to blue light caused extensive cytotoxocity with A2E-laden cells but not naive cells. Abca4-/- mice showed age-related accumulation in ocular A2E, lipofuscin accumulation, and elevated oxidative stress in the retinas, particularly in the RPE layer. In vitro experiments showed that brief treatment with 100 uM CPZ reduced lysotracker fluorescence in ARPE-19 cells by 45% +/- 4% and A2E fluorescence by 62% +/- 5 compared to the respective controls. Loss of A2E fluorescence was dose-dependent and not due to quenching by CPZ or cytotoxicity. Other lysosomotropes also showed reduction of A2E fluorescence, although to various degrees; some were ineffective. Studies with Abca4-/- mice are underway to evaluate the in vivo efficacy of A2E clearance by lysosomotropes.
Conclusions: :
Accumulation of A2E and similar lipofuscin components in the RPE may initiate or exacerbate retinal pathogenic mechanisms (e.g. oxidative stress, complement activation, etc.) Our in vitro data suggest that A2E, and possibly other toxins that sequester in lysosomes, can be removed by chemical clearance using select lysosomotropes. Since accumulation of A2E is a relatively slow process, even partial clearance may help delay any A2E-mediated vision loss. It remains to be seen whether this approach is safe and efficacious for clinical use.
Keywords: ipofuscin • retinal pigment epithelium • neuroprotection