The Role of Peroxins 1 and 6 in the Retinal Pigment Epithelium
Creators: Mouzaaber, Constantin
Publisher: name: University of Alberta Library
Year: 2024

<p>Purpose. Peroxisomes are ubiquitous organelles that compartmentalize metabolic reactions including lipid catabolism and cellular detoxification. Enzymes flagged by peroxisomal targeting sequences are delivered into peroxisomes by the cumulative action of some peroxins (PEX), including PEX5 which shuttles the enzymes. Peroxisome biogenesis disorders (PBDs) are recessive conditions caused by biallelic loss-of-function mutations in genes responsible for peroxisome assembly and function. Approximately two-thirds of PBDs are caused by mutations in PEX1 or PEX6, which encode PEX1 and 6, respectively. Together, PEX1 and PEX6 form an ATPase Associated with diverse cellular Activities (AAA ATPase) exportomer that facilitates the release of monoubiquitinated PEX5 from the peroxisomal membrane to accommodate further shuttling of peroxisome enzymes. The RPE is a specialized monolayer of cells that performs several critical roles in the retina, including the daily phagocytosis of light-sensitive photoreceptor outer segments (POS), facilitating their renewal. PBDs are frequently associated with retinal pigment epithelial (RPE) dysfunction and retinal degeneration, but precisely how impaired peroxisome activity perturbs retinal function remains to be fully explored. Effective degradation of the lipid-rich POS ingested by the RPE is hypothesized to depend on intact and functional peroxisomes. This project aims to study the effects of peroxisome dysfunction in the RPE and highlight the importance of intact peroxisomes in maintaining normal vision.</p>

<p>Methods. To study the effects of a severe PBD genotype, PEX1 and PEX6 knockout (KO) induced pluripotent stem cells (iPSCs), along with the isogenic wild-type iPSCs, were differentiated into RPE cells (iRPE) in culture. Immunoblot analysis of whole iRPE cell lysates was performed, and the processing of ACAA1, a peroxisome matrix protein, was evaluated to assess the fidelity of peroxisome matrix protein import in the PEX1 KO and PEX6 KO iRPE cells relative to wild-type. Immunofluorescence detection of an antibody against PMP70, a peroxisomal membrane protein, in combination with image analysis were used to determine the abundance of peroxisomes across iRPE cell lines. Gas chromatography with flame-ionization detection was used to quantitatively compare the fatty acid lipid profiles of the wild-type and PEX6 KO iRPE. POS isolated from bovine eyes were added to iRPE cell culture media to examine the effect of PEX1 and PEX6 mutation on POS phagocytosis in iRPE. Immunofluorescence experiments focused upon lipid droplets were performed to interrogate lipid accumulation in iRPE. Flow cytometry experiments were performed to assess the heterogeneity of the differentiated iRPE populations and quantify the intracellular neutral lipid content of the cells before and after addition of isolated POS.</p>

<p>Results. Fewer PMP70-positive puncta were observed in PEX1 KO and PEX6 KO iRPE, but no statistical significance was reached (P= 0.29). Immunoblot analysis demonstrated disrupted processing of matrix-destined ACAA1, likely due to impaired peroxisome exportomer machinery in PEX1 KO and PEX6 KO iRPE cells. PEX6 KO iRPE had significantly reduced levels of docosahexaenoic acid (DHA) (P&lt;0.0001), a long-chainiv polyunsaturated fatty acid that can be generated by peroxisomes but is largely acquired from diet. Additionally, PEX6 KO iRPE significantly accumulated DHA-precursor fatty acids (P &lt;0.0001). At baseline, PEX1 KO and PEX6 KO iRPE cells exhibited an increased number of lipid droplets, and contained significantly more intracellular neutral lipids relative to wild-type iRPE (P=0.0005 and P&lt;0.0001, respectively). After challenging the cells with POS, PEX1 KO and PEX6 KO iRPE exhibited an increased size and number of lipid droplets, and demonstrated significantly elevated binding of dyes for intracellular neutral lipids relative to wild-type iRPE (P&lt;0.0001).</p>

<p>Conclusions. This work established the first human iRPE culture model to study the effects of peroxisome dysfunction. PEX1 KO and PEX6 KO iRPE displayed impaired peroxisomal matrix protein import. Peroxisome dysfunction induced significant lipid profile changes in iRPE cells, including elevated very long-chain fatty acids and reduced DHA. PEX1 KO and PEX6 KO iRPE accumulated intracellular lipid droplets, which is a biomarker of aged and diseased RPE. Challenging iRPE with POS aggravated an already significant lipid handling defect in the PEX1 KO and PEX6 KO iRPE. A better understanding of the role of peroxisomes in the RPE will facilitate the development of novel therapeutic approaches for patients with peroxisomal disorders and retinal degeneration. </p>