Abstract
Purpose :
CLN3 disease is a lysosomal storage disorder that leads to retina degeneration and vision loss. Previous studies from our laboratory utilizing pluripotent stem cell derived-RPE (hPSC-RPE) from CLN3 disease patients has shown reduced microvilli density and decreased phagocytosis of photoreceptor outer segments (POS) in CLN3 disease. Notably, dysregulated sphingolipid metabolism, a hallmark of CLN3 disease, is known to regulate microvilli biogenesis in other cells. Therefore, the purpose of this study was to determine whether altered sphingolipid metabolism contributes to observed microvilli defects and consequently impaired POS phagocytosis by CLN3 disease hPSC-RPE cells.
Methods :
hPSC-RPE from control and CLN3 disease (harboring the common disease-associated biallelic deletion of exons 7 and 8) were cultured on transwells. An untargeted proteomic analysis and a targeted lipidomic analyses (Targeted Sphingolipids Analysis Panel; Creative Proteomics) in conjunction with Western blotting and immunocytochemistry (ICC) were performed to identify and validate alterations in sphingolipid metabolism in CLN3 disease hPSC-RPE cells. POS phagocytosis assay was performed on control and CLN3-hPSC RPE by feeding RPE cells with unlabeled or FITC-labeled POS (~20 POS/RPE) for 30 min and then measuring levels of i) rhodopsin (RHO; a POS protein) and/or ii) FITC-POS by Western blotting and ICC. To evaluate a specific role of acid ceramidase1 (ASAH1) in POS phagocytosis, parallel cultures of unsupplemented CLN3-hESC RPE cells and recombinant ASAH1 (30µg/ml) supplemented CLN3 hPSC-RPE cells were subjected to POS phagocytosis assay.
Results :
Compared to control hPSC-RPE cells, CLN3 disease hPSC-RPE cells showed decreased i) POS phagocytosis, ii) ASAH1 levels, iv) sphingosine 1 phosphate (S1P) levels and iv) Ezrin (EZR) activation. Directly linking ASAH1-SP1-EZR axis to impaired POS phagocytosis by CLN3 disease hPSC RPE cells, supplementation of recombinant ASAH1 increased i) S1P levels and ii) EZR activation and iii) POS phagocytosis in CLN3-hPSC RPE cells.
Conclusions :
Our data on the CLN3 hPSC-RPE model suggests that perturbation of ASAH1-S1P-EZR axis contributes to POS phagocytosis defect in CLN3 disease.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.