Disfunción peroxisomal altera el metabolismo de los segmentos exteriores fotorreceptores en el epitelio pigmentario...

Peroxisomes are ubiquitous organelles that compartmentalize metabolic reactions including lipid catabolism and cellular detoxification. Biallelic loss-of-function variants in genes responsible for peroxisome assembly and function cause peroxisome biogenesis disorders (PBDs). Approximately three-quarters of PBDs are due to pathogenic variants in PEX1 or PEX6 and result in multisystem disease, including retinal degeneration and blindness. Despite retinal pigment epithelial (RPE) dysfunction and retinal degeneration occurring frequently in PBDs, precisely how impaired peroxisome activity disrupts retinal function remains to be fully explored. To address this, we differentiated PEX1 knockout ( PEX1 ^-/- ), PEX6 knockout ( PEX6 ^-/- ), and wildtype human induced pluripotent stem cells (iPSCs) into RPE to study the consequences of peroxisome dysfunction in this disease-relevant cell type.

CRISPR/Cas9-mediated genome editing was used to generate PEX1 ^-/- and PEX6 ^-/- in human iPSCs. The knockouts and isogenic wildtype iPSCs were differentiated into RPE (iRPE) and characterized by morphology, pigmentation, transepithelial electrical resistance (TEER), and expression of proteins associated with differentiated RPE using immunofluorescence microscopy and flow cytometry. Immunoblot analysis of whole iRPE lysates was used to evaluate the proteolytic processing of peroxisome enzymes, a measure of the integrity of peroxisome matrix protein import. Immunofluorescence detection of peroxisome membrane proteins was used to determine the abundance of peroxisomes across iRPE lines. Targeted lipidomics by gas and liquid chromatography with mass spectrometry were used to quantitatively compare the profiles of wildtype, PEX1 ^-/- , and PEX6 ^-/- iRPE. Accumulation of intracellular neutral lipid, and more specifically lipid droplets, in iRPE was measured using flow cytometry and perilipin-2 immunoblotting, respectively. Phagocytosis of photoreceptor outer segments (POS) by iRPE was evaluated using rhodopsin immunoblotting.

PEX1 ^-/- , PEX6 ^-/- , and wildtype iRPE all had comparable hexagonal morphology and integrity of tight junctions, developed pigment, and similarly expressed proteins characteristic of RPE. Immunoblot analysis demonstrated aberrant processing of ACOX1, MFP2, and ACAA1 in PEX1 ^-/- and PEX6 ^-/- iRPE, suggesting impaired peroxisome matrix protein import. Targeted lipid profiling, including total fatty acid (FA) lipid profile analysis, revealed that in comparison to wildtype, PEX1 ^-/- and PEX6 ^-/- iRPE had significantly reduced docosahexaenoic acid (C22:6ω3) ( p <0.001) and phosphoethanolamine plasmalogens ( p <0.0001 for C18:0(plasm)-Total-PE), while very long-chain fatty acids (VLCFA) ( p <0.001 for C26:0 and p <0.01 for C26:1ω9) and branched-chain FAs ( p <0.01 for both pristanic acid and phytanic acid) were significantly elevated. Following a POS challenge, wildtype iRPE had significantly elevated eicosapentaenoic acid (C20:5ω3) ( p =9.3E-5) and arachidonic acid (C20:4ω6) ( p =0.013), both retroconversion products from the two most abundant polyunsaturated FAs in POS: C22:6ω3 and docosapentaenoic acid (C22:5ω6). PEX1 ^-/- and PEX6 ^-/- iRPE accumulated twice the amount of C22:5ω6 ( p <0.05) relative to wildtype iRPE following a POS challenge, without a significant increase in C20:5ω3 or C20:4ω6, suggesting impaired ω3 and ω6 retroconversion pathways. Finally, PEX1 ^-/- and PEX6 ^-/- iRPE demonstrated delayed degradation of internalized POS based on rhodopsin immunoblotting, increased intracellular lipid droplets, and reduced TEER following a POS challenge.

Our work has established the first human iRPE models to study the effects of peroxisome dysfunction in the RPE. Impaired peroxisome activity resulted in significant lipid profile changes and defective phagocytosis in iRPE. Dysregulated POS metabolism in the RPE is a potential mechanism driving retinal degeneration in patients with PBDs. Fully understanding the role of peroxisomes in the RPE will facilitate the development of novel therapies for degenerative retinal disorders.