Abstract
Purpose :
Recessive Stargardt disease (STGD1) is a juvenile maculopathy caused by mutations in the ABCA4 gene without a suitable treatment. STGD1 manifests with accumulation of auto-fluorescent lipofuscin containing bisretinoids, lipid, and protein aggregates within the retinal pigment epithelium (RPE). Protein degradation in the RPE is primarily mediated by the lysosomal protease Cathepsin D (CatD). The functional activity and maturation of CatD is dependent on a narrow range of acidic pH within the endo-lysosomes. We previously reported elevated lysosomal pH and reduced CatD activity using iPSC-derived RPE cells from three STGD1 patients (H, J, and S). Here, we tested for CatD functional rescue using the P2Y12 antagonist, Ticagrelor, previously shown to reduce lysosomal acidity in Abca4-/- mice.
Methods :
CatD protein levels were evaluated by immunoblotting and immunostaining in RPE cells from STGD1 patients (confirmed ABCA4 mutations) and unaffected control (no ABCA4 mutations) at 3-months (mo) in-culture. Accumulation of α-synuclein, the physiological substrate for CatD, was assessed by immunocytochemistry. Lysosomal pH was measured using LysoSensor Yellow/Blue DND-160 ratiometric probe. CatD activity determined by fluorometric assay. For phenotypic rescue studies, RPE cells were treated with 50µM Ticagrelor in media for one week. Statistical significance was determined by student’s t-test.
Results :
At 3-mo, STGD1 RPE cells from H- and J-patients exhibit elevated immature/intermediate CatD levels versus normal, that inversely correlated with levels of mature CatD. At 6-mo, all three STGD1 RPE cells (H, J, and S) display aggregation of α-synuclein that was not present in normal. Lysosomal pH was reduced in Ticagrelor-treated STGD1 cells compared to untreated STGD1. Elevated levels of immature CatD in STGD1 RPE cells were reduced to near normal with Ticagrelor treatment. Additionally, functional activity of CatD was restored to normal levels in treated STGD1 RPE versus untreated.
Conclusions :
These studies provide further evidence of RPE endo-lysosomal dysfunction in STGD1, which is in-part mediated by CatD deficiencies. Reduction in CatD protein maturation and functional activity further contribute to the degrative burden in STGD1 RPE cells. CatD deficiencies can be rescued by Ticagrelor treatment, suggesting the therapeutic potential for targeting CatD in STGD1.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.