Abstract
Purpose :
In Choroideremia (CHM), progressive degeneration of photoreceptors (PR), retinal pigment epithelium (RPE) and choroid is caused by mutations of the CHM gene encoding Rab escort protein 1 (REP1). REP1 is required for prenylation of Rab GTPases, and its loss leads to Rab mislocalization and impaired vesicle trafficking. Phagocytic uptake and disposal of shed PR outer segments (OS) are among the myriad RPE functions critical to retinal health. Phagosome maturation requires extensive Rab-mediated membrane remodeling. Metabolites such as βHB, derived from fatty acid oxidation (FAO) of OS lipids in RPE, are recycled back to PR as the ketone fuel βHB. Our goal is to determine if loss of REP1 expression leads to compromised RPE degradative capacity resulting in nutrient deprivation in PR and RPE dysfunction, which can be ameliorated by therapeutic intervention. Here we establish an in vitro cell culture model to access lipid homeostasis in RPE from CHM patients.
Methods :
Human RPE lines were induced from pluripotent stem cells (iRPEs) derived from 4 CHM patients and 4 unaffected controls (Ctrl) with no history of vision impairments. Cells were grown on permeable transwells and fed outer segments (OS) for 3h. After OS removal, samples were collected after 30 and 120-minute chases. In such OS uptake studies, we defined the relationship between phagosome maturation and FAO using confocal imaging and metabolic analyses, respectively. The levels of key nutrient transporters, GLUT1, MCT3, and MCT1 as well as metabolic enzymes, HMGCS2 and BDH were determined by Western blot. Transporter localization was assessed with immunohistochemistry and confocal imaging. Secreted βHB was measured with a colorimetric assay.
Results :
Expression of nutrient transporters and lipid metabolic enzymes necessary for ketone body formation were comparable in Ctrl and CHM iRPE. After a 30-minute chase following OS uptake, levels of rhodopsin were high in both control and CHM iRPE. After 120 min of chase, opsin levels declined in Ctrl cells but were elevated in CHM iRPE. Coincident with delayed phagosome maturation in the CHM iRPE, there was an increase in intracellular lipid and decreased βHB secretion.
Conclusions :
Loss of REP1 activity in CHM patient-derived iRPE contributes to metabolic dysregulation manifest as loss of ketone body synthesis and intracellular lipid accumulation, collectively contributing to a pro-inflammatory microenvironment.
This is a 2020 ARVO Annual Meeting abstract.