Han, J. Y. S. | Search bioRxiv

bioRxiv2026

Peroxisome dysfunction alters metabolism of photoreceptor outer segments in human retinal pigment epithelium

Peroxisomes are ubiquitous organelles that compartmentalize metabolic reactions including lipid catabolism and cellular detoxification. Pathogenic variants in PEX1 and PEX6 disrupt essential peroxisome functions and cause profound neurodegenerative diseases called peroxisome biogenesis disorders (PBDs). Despite retinal degeneration and blindness occurring frequently in PBDs, precisely how impaired peroxisome activity disrupts retinal function remains to be fully explored. To address this, we differentiated PEX1-/-, PEX6-/-, and wildtype human induced pluripotent stem cells into retinal pigment epithelium (iRPE) to study the consequences of peroxisome dysfunction in this disease-relevant cell type. Despite exhibiting impaired peroxisome matrix protein import, PEX1-/- and PEX6-/-iRPE had comparable morphology, tight junctions, and expression of proteins characteristic of RPE compared to wildtype iRPE. Targeted lipid profiling revealed reduced docosahexaenoic acid, a polyunsaturated fatty acid (PUFA) essential for retinal function, and elevated lipid species exclusively metabolized by peroxisomes in PEX1-/- and PEX6-/- iRPE. Following a photoreceptor outer segment (POS) challenge, PEX1-/- and PEX6-/- iRPE demonstrated disrupted PUFA retroconversion and lipid droplet accumulation. Additionally, PEX1-/- and PEX6-/-iRPE had impaired rhodopsin degradation, lysosomal dysfunction, and reduced transepithelial electrical resistance. These findings suggest that dysregulated POS metabolism in the RPE is a potential mechanism driving retinal degeneration in patients with PBDs. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/701576v2_ufig1.gif" ALT="Figure 1"> View larger version (99K): org.highwire.dtl.DTLVardef@bf2389org.highwire.dtl.DTLVardef@b62e1forg.highwire.dtl.DTLVardef@8e0b21org.highwire.dtl.DTLVardef@17cb332_HPS_FORMAT_FIGEXP M_FIG C_FIG Schematic summarizing the consequences of PEX1 and PEX6 knockout on iRPE biology, including the presence of import-incompetent peroxisomes, impaired {omega}3 and {omega}6 fatty acid retroconversion, lipid droplet accumulation, and defective photoreceptor outer segment phagocytosis.

cell biology↗

bioRxiv2024

Reassessing Retinal Pigment Epithelial Ketogenesis: Enzymatic Assays for Ketone Body Levels Provide Inaccurate Results

The retinal pigment epithelium (RPE) is omnivorous and can utilize a wide range of substrates for oxidative phosphorylation. Certain tissues with high mitochondrial metabolic load are capable of ketogenesis, a biochemical pathway that consolidates acetyl-CoA into ketone bodies. Earlier work demonstrated that the RPE expresses the rate-limiting enzyme for ketogenesis, 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2), and that the RPE indeed produces ketone bodies, including beta-hydroxybutyrate ({beta}-HB). Prior work, based on detecting {beta}-HB via enzymatic assays, suggested that differentiated cultures of primary RPE preferentially export {beta}-HB across the apical membrane. Here, we compare the accuracy of measuring {beta}-HB by enzymatic assay kits to mass spectrometry analysis. We found that commercial kits lack the sensitivity to accurately measure the levels of {beta}-HB in RPE cultures and are prone to artifact. Using mass spectrometry, we found that while RPE cultures secrete {beta}-HB, they do so equally to both apical and basal sides. We also find RPE is capable of consuming {beta}-HB as levels rise. Using isotopically labeled glucose, amino acid, and fatty acid tracers, we found that carbons from both fatty acids and ketogenic amino acids, but not from glucose, produce {beta}-HB. Altogether, we substantiate {beta}-HB secretion in RPE but find that the secretion is equal apically and basally, RPE {beta}-HB can derive from ketogenic amino acids or fatty acids, and accurate {beta}-HB assessment requires mass spectrometric analysis.

cell biology↗