Abstract
Purpose :
Accumulation of A2E in the lysosomes of retinal pigment epithelium (RPE) is a primary etiological agent for recessive Stargardt disease and ABCA4-related forms of cone-rod dystrophy and retinitis pigmentosa and a risk factor for AMD. Currently, there is no FDA-approved treatment for any of these diseases. Hence, drugs that remove A2E could fulfill this therapeutic void. We have shown that β-cyclodextrins (βCD) bind and reduce A2E from RPE cells (Nociari et al., PNAS, 2014). However, to be therapeutically useful, βCDs require modifications that enhance their clearance strength. In order to develop a high throughput quantitative A2E-removal assay that guided βCDs optimization process, we prepared a novel, A2E surrogate whose fluorescent signal intensity depended on the amount of material present and no on the polarity of its environment.
Methods :
We synthesized a highly fluorescent A2E derivative (FIG.1) by replacing Nitrobenzoxadiazole (NBD) for ethanolamine during synthesis (Parish et al., PNAS, 1998). The purity and identity of NBD-A2E was assessed by HPLC-MSMS and NMR.
Results :
NBD-A2E displayed fluorescence excitation peaks at 350 nm and 450 nm and an emission maximum at 500 nm, and was 600% more fluorescent than A2E both in water and in methanol. In contrast with A2E, which drastically increases its fluorescence upon encapsulation by βCD, NBD-A2E fluorescence properties were minimally affected by βCD. Photo-oxidative decay was very similar for both bisretinoids. Surprisingly, whereas A2E displays dose-dependent toxicity for ARPE-19 cells, NBD-A2E was not toxic. Pulse-chase fluorescence microscopy studies revealed that although both bisretinoids entered ARPE-19 cells, A2E, but not NBD-A2E was retained in the lysosomal compartment. Separate experiments indicated that the toxicity and propensity of bisretinoids (such as A2E and all trans retinal dimer (ATRD)) to accumulate in lysosomes correlates with their abilities to aggregate in water and to cause lysosomal membrane permeabilization. In contrast, NBD-A2E neither forms aggregates, nor causes lysosomal membrane permeabilization.
Conclusions :
In addition to their potential utility to facilitate the screening for more potent pharmacological agents that remove A2E, A2E derivatives provide excellent tools to explore current hypotheses on mechanisms of bisretinoid toxicity.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.