Abstract
Purpose :
Peroxisomes are ubiquitous organelles that house metabolic reactions including lipid catabolism and cellular detoxification. Peroxisomal biogenesis disorders (PBDs) frequently cause retinal degeneration and retinal pigment epithelial (RPE) dysfunction, and other systemic abnormalities due to biallelic loss-of-function mutations in genes responsible for peroxisome assembly and function. Precisely how impaired peroxisome function causes retinal degeneration remains to be fully explored. We hypothesize that peroxisome-deficient RPE develops lipid accumulation and increased oxidative stress due to an inability to process and metabolize phagocytosed photoreceptor outer segments (POS).
Methods :
Previously generated PEX1 and PEX6 knockout human induced pluripotent stem cells (iPSCs) were differentiated into RPE, along with the parental control line. The resulting iPSC-RPE were validated via flow cytometry by determining the expression of TYRP1, PAX6, PMEL17, BEST1, and MITF. Immunofluorescence microscopy and immunoblotting for the peroxisomal proteins PMP70, catalase and thiolase were used to assess peroxisomal number and matrix-protein import. Mass spectrometry was used to quantitatively compare the lipid profile of the peroxisome-deficient knockouts and parental line. Lastly, quantitative flow cytometry was used to measure lipid accumulation (BODIPY) in the iPSC-RPE lines before and after a POS challenge. A similar experiment is underway to evaluate oxidative stress (CellROX).
Results :
The peroxisome-deficient and parental iPSC-RPE lines developed tight junctions and expressed markers consistent with mature RPE. The PEX1 and PEX6 knockout iPSC-RPE had similar peroxisomes abundance, but impaired matrix protein import compared to control. Following a POS challenge, peroxisome-deficient iPSC-RPE accumulated more neutral lipids compared to the control line.
Conclusions :
Peroxisome-deficient iPSC-RPE models accumulate lipid when challenged with POS loads, shedding light on important roles of peroxisomes in the RPE. Our iPSC-RPE models will be used to further study the contribution of peroxisomes to RPE health and disease.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.