TRPML1 channels activation facilitates lysosomal exocytosis and reduces autofluorescent lysosomal accumulations, opsin retention and lipid peroxidation in RPE cells compromised by chloroquine

Abstract

Purpose : Excessive lysosomal accumulations are associated with multiple neurodegenerations including age-related macular degeneration. Treatment to reduce these accumulations is of broad interest. Lysosomes store calcium, and the regulated efflux through TRPML1 channels contributes to the fusion of lysosomal membranes with the plasma membrane during lysosomal exocytosis. We previously identified functional TRPML1 channels on RPE lysosomes. Here we ask whether TRPML1 activation can facilitate lysosomal exocytosis and clearance of accumulations in RPE cells

Methods : Experiments were performed on confluent aged ARPE-19 cells. Autofluorescence (AF) at 488 ex/560 em was determined in a Fluoroskan plate reader and microscopically. Extracellular acid phosphatase (AP) was measured with a standard colorimetric kit. Cells were fed with bovine photoreceptor outer segments (POS) using a pulse chase protocol to minimize interference in phagocytosis; cells were fed 1x10⁶ POS/ml for 2 h (pulse), medium alone for 2 h (chase), then 20 h in control or chloroquine (CHQ) medium. Opsin levels were determined with immunoblots. Lipid peroxidation were determined from measurements of Bodipy-C11

Results : Cellular accumulations were induced by exposing ARPE-19 cells to CHQ for 7 days followed by 24 hr exposure to U18666A (U18). CHQ/U18 treatment increased AF accumulations. Exposure of these cells to TRPML1 agonist ML-SA1 reduced AF levels. To determine whether ML-SA1 increased lysosomal exocytosis, extracellular levels of lysosomal AP were determined. ML-SA1 increased extracellular levels of AP in both control and CHQ-treated cells, suggesting ML-SA1 triggered lysosomal exocytosis. Furthermore, the effect of TRPML1 on opsin turnover was investigated. RPE treated with CHQ and loaded with POS showed a significant rise in opsin on immunoblots. Critically, ML-SA1 decreased retention of opsin in CHQ-treated cells.

Conclusions : Activation of TRPML1 in compromised RPE cells increased lysosomal exocytosis and decreased lysosomal autofluorescence. In cells loaded with POS, CHQ treatment increased opsin retention and lipid peroxidation, while TRPML1 activation decreased both. Further investigation is needed to identify how TRPML1 activation enhances lysosomal clearance in compromised RPE cells

This is a 2021 ARVO Annual Meeting abstract.