Impaired Cathepsin D evidenced in iPSC-derived RPE cells of STGD1 patients

Abstract

Purpose : Recessive Stargardt disease (STGD1) is an inherited maculopathy caused predominantly by mutations in the ABCA4 gene. The buildup of auto-fluorescent lipofuscin within the retinal pigment epithelium (RPE) is a pathological hallmark of STGD1. Daily phagocytosis of outer-segments membranes requires a functional endolysosomal system to prevent additional stress on the RPE. Cathepsin D (CatD) is a primary lysosomal protease responsible for protein degradation in the RPE. Both CatD activity and protein maturation are dependent on acidic pH in the endolysosomal compartments. We previously reported impaired maturation and reduced activity of CatD in the STGD1 mouse model (Abca4^-/-). Here, we sought to investigate CatD activity using induced pluripotent stem cell (iPSC)-derived RPE cells from three STGD1 patients.

Methods : Immature CatD protein was evaluated by immunostaining in iPSC-RPE cells from STGD1 patients with confirmed ABCA4 mutations, and unaffected control (no ABCA4 mutations) at 1-and 2-mo in-culture. Distribution of immature CatD with endolysosomal markers was assessed in iPSC-RPE cells by immunocytochemistry using super-resolution and confocal microscopy. Lysosomal pH was measured using LysoSensor Yellow/Blue DND-160 ratiometric probe (n=3), and CatD functional activity by fluorometric assay (n=4-7) in iPSC-RPE cells. Statistical significance was determined by ANOVA testing with Tukey-Kramer post-hoc analysis.

Results : At 1- and 2-mo, STGD1 iPSC-RPE cells exhibit aggregation of immature CatD. Interestingly, immature CatD in STGD1 iPSC-RPE cells co-localizes with both Rab5 and Lamp1 endo-lysosomal markers respectively. In contrast, immature CatD only co-localizes with the early endosome marker Rab5 in control iPSC-RPE cells. Lysosomal pH was elevated in all three STGD1 patients’ iPSC-RPE cells vs control (p<0.05). Notably, CatD functional activity was significantly reduced (p<0.05) in two of the donor STGD1 iPSC-RPE cell lines compared to control.

Conclusions : Consistent with studies in the STGD1 mouse model, we provide further evidence for endo-lysosomal dysfunction in STGD1 patient-derived iPSC-RPE cells, mediated by CatD deficiency. Improper localization of immature CatD coupled with reduced functional activity indicates an amplified degrative burden in STGD1 RPE cells. These findings support the contribution of CatD in developing a STGD1 phenotype and suggest its potential as a therapeutic target.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.