Impaired lysosomal calcium signaling in RPE cells with an in vitro model of chloroquine retinopathy

Abstract

Purpose : The incidence of chloroquine retinopathy is increasing, with patients experiencing a loss of central vision and photoreceptors. While the initial step in the pathology is widely acknowledged to involve lysosomal alkalinization by chloroquine, the links between alkalinization and photoreceptor death remain unknown. The lysosome is a major source of intracellular Ca²⁺, and release of lysosomal Ca²⁺ through the TRPML1 channel is necessary for signaling and lysosomal exocytosis. In this study we examined the effect of an in vitro model of chloroquine retinopathy on the release of Ca²⁺ through TRPML1 in RPE cells and asked whether lysosomal exocytosis contains protective components

Methods : For Ca²⁺ measurements, RPE cells from C57Bl6J mice and ARPE19 cells were grown on coverslips, loaded with fura-2 and the 340/380 nm signal was recorded using microscope or fluorimeter based readings. Exocytosis was recorded from mouse cells grown on transwell filters. IL-6 was measured with an Elisa kit and acid phosphatase (AP) with a colorimetric assay. TRPML1 mRNA levels were determined with qPCR. The in vitro model of chloroquine retinopathy consisted of ARPE-19 cells treated with 10 µM CHQ for 7 days and 1 µM U18666A for 1 day

Results : The TRPML1 channel agonist ML-SA1 led to reversible and repeatable elevations in cytoplasmic Ca²⁺ in mouse RPE and ARPE-19 cells. The response to ML-SA1 was significantly reduced in the chloroquine model. Treatment increased TRPML1 mRNA levels, while lysosomal Ca²⁺ levels released by glycyl-L-phenylalanine-beta-naphthylamide were not altered; together this suggests the reduced Ca²⁺ efflux in the chloroquine model reflected a reduction in channel function. In polarized mouse RPE cells, ML-SA1 triggered a release of lysosomal enzyme AP across the apical membrane. Importantly, the cytokine IL-6 was also released in the same samples, suggesting lysosomal release of IL-6 following activation of TRPML1

Conclusions : The ability of ML-SA1 to trigger lysosomal Ca²⁺ release and concurrent exocytosis of acid phosphatase and IL-6 implies that lysosomal Ca²⁺ contributes to exocytosis in RPE cells. The reduced response to ML-SA1 in the in vitro chloroquine retinopathy model predicts that IL-6 release will also be reduced. As IL-6 is reported to protect photoreceptors, this reduced signal may be detrimental, although this awaits direct confirmation

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.