Defective lysosomal calcium signaling in retinal pigmented epithelium cells from ABCA4^-/- mice

Abstract

Purpose : Lysosomes are recognized as a major store of intracellular Ca²⁺, and lysosomal Ca²⁺ signaling is implicated in exocytosis, vesicle fusion, and transcriptional control of autophagy. The TRPML1 cation channel is a major conduit for lysosomal Ca²⁺ efflux and its activity can be compromised by lysosomal lipid accumulation. The lysosomes of RPE cells process the shed tips of photoreceptor outer segments and their own autophagic material, and undegraded material can accumulate as lipofuscin. RPE cells from the ABCA4^-/- mouse model of Stargardt’s disease are characterized by large deposit of lipofuscin. Here we asked whether lysosomal Ca²⁺ signaling in the ABCA4^-/- mouse was defective

Methods : Mice were treated in accordance with ARVO guidelines. Ca²⁺ levels from isolated RPE cells were determined by Ca²⁺ imaging with dye fura-2; cellular lipofuscin did not affect the dye signal. TRPML1 mRNA was quantified by qPCR. Total lysosomal Ca²⁺ was determined from levels released into the cytoplasm following lysosomal rupture by glycyl-L-phenylalanine-beta-naphthylamide (GPN).

Results : The TRPML1 Ca²⁺ channel was expressed in mouse RPE cells. Stimulation of RPE cells from control mice with channel agonist MLSA1 induced a robust rise in cytoplasmic Ca²⁺. This Ca²⁺ rise was not dependent on extracellular Ca²⁺ or disrupted by thapsigargin, consistent with its lysosomal source. RPE cells from TRPML1^-/- mice did not respond to MLSA1, confirming receptor identification. Finally, ABCA4^-/- RPE cells were stimulated with MLSA1 and Ca²⁺ release was decreased by 85% compared to control RPE cells. TRPML1 expression did not differ between RPE cells from control and ABCA4^-/- mice. Total lysosomal Ca²⁺ were the same in RPE cells from control and ABCA4^-/- mice, as determined with GPN

Conclusions : The release of Ca²⁺ from lysosomes in mouse RPE cells is substantial, and is mediated, at least in part, by the TRPML1 channel. The impaired release of lysosomal Ca²⁺ in RPE cells of the ABCA4^-/- mouse may reflect impaired channel activity, as TRPML1 expression and lysosomal Ca²⁺ levels were unchanged. Whether this impaired lysosomal signaling contributes to the pathology in Stargardt’s remains to be determined

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.