|Biblioteca NMS|FCM online
Choroideremia (CHM) is a form of retinal degeneration, with an X-linked pattern of inheritance caused by mutations in the CHM gene that codifies the Rab Escort Protein 1 (REP1). Given its genetic component and the closed system of the eye, CHM is an excellent target for gene therapy. However, this type of treatment still presents its challenges, thus it is important to uncover new molecular mechanisms of the disease, with the hopes of finding new therapeutic targets. The retinal pigment epithelium (RPE) consists of a monolayer of cells, localized between the choroid and the photoreceptors. The RPE is functionally diverse, performing tasks such as the daily phagocytosis and degradation of photoreceptor outer segments (POS), secretion of growth factors and transport of nutrients and metabolites. The REP1 protein is responsible for the geranylgeranylation of Rab GTPases, a post-translational modification that facilitates the binding of these small GTPases to membranes and therefore, exert their role in the regulation of membrane traffic. CHM patients have been shown to have RPE dysfunction; accumulation of unprocessed POS accompanied by a reduced degradation capacity and a decrease in phagosomal acidity. With this project we intended to make a full characterization of cellular organelles and pathways, to dissect new uncharacterized cellular defects, with a focus on the endo-lysosomal pathway, as well as the impact of unprocessed POS accumulation in CHM RPE cells. To carry out our objective we used two different in vitro RPE cell models: differentiated RPE from human induced pluripotent stem cells from both healthy and CHM patient donors, hiPSc-RPE, and a commonly used immortalized RPE cell line, ARPE-19 where we used CRISPR/Cas9 to generate a CHM KO line. Characterization of the endo-lysosomal pathway revealed an increase in the number of LAMP1+ and CD63+ vesicles in both hiPSc-RPE and ARPE-19 CHM cell models, suggesting an increase in the number of both lysosomes and multivesicular bodies (MVBs), respectively. Western blot analysis of CHM hiPSc-RPE whole cell lysates revealed an increase in (inactive) procathepsin D and L, suggesting an impairment in the delivery of the immature form of these antibodies cathepsin forms to their proper cellular compartments, pointing to a targeting dysfunction within the endo-lysosomal pathway. Additionally, in ARPE-19 CHM cells, after a single pulse of POS, a significant accumulation of autofluorescent granules (AFGs) was observed, verified at both 24h- and 72h- post feeding resulting from incomplete digestion of POS. Furthermore, hiPSc-RPE CHM cells revealed an increase in cell size as well as a decrease in polarization, as analysed by a decrease in the transepithelial electrical resistance (TER), suggesting a dysfunction in the establishment of a tight monolayer, this evidence should be further analysed. Overall, this work provides novel evidence of cellular dysfunction in CHM cells, specifically on the endo-lysosomal pathway, as well as POS incorporation and degradation. We also show key differences between two cellular models of RPE, describing advantages and disadvantages of each model for the use of different assays and technical approaches. In the future, we aim to achieve optimized protocols for hiPSc-RPE to overcome technical challenges and make hiPSc-RPE an even better model of human RPE to investigate the molecular and cellular mechanisms of choroideremia as well as other diseases of the RPE.