Résumé
Purpose: Retinitis pigmentosa (RP) is characterized by progressive visual loss due to degeneration of the light-sensing photoreceptors and/or their underlying support tissue, the retinal pigment epithelium (RPE). We recently detected mutations in a TBC1D32 gene, previously associated with Oral-Facial-Digital syndrome, in a patient presenting with an autosomal recessive RP. In order to decipher the nature of anomalies provoked by TBC1D32 gene defects, we generated induced pluripotent stem cells (iPSCs) from the patient and differentiated them into RPE and mature retinal organoids containing a layer of photoreceptors. Methods: We used immunofluorescence, qPCR and Western-blot assays to study the morphology and gene expression in patient derived RPE and retinal organoids. The transmission electron microscopy was used to analyze cell microstructure. VEGF and PEDF secretion of human iPSC-derived RPE was assayed by ELISA. Results We detected significantly lower levels of TBC1D32 gene expression in the patient than in control iPSC-derived RPE. We noted that the primarycilium in patient cells was significantly longer (5.79 μm ± 0.07 μm) in comparison with the control (4.55 μm ± 0.09 μm). The microstructural analysis shown an important accumulation of lipid droplets in patient RPE accompanied by increase of Perilipin expression. Furthermore, the RPE monolayer shows signs of losing its tight junctions which indicates the triggering of partial EMT which was confirmed by decrease of E-cadherin expression and increase of Vimentin and SMA expression. We also noticed the delocalization of beta-catenin protein from membrane to cytosol in patient RPE. Moreover we observed some significant modifications in polarized VEGF and PEDF secretion in patient RPE. Characterization of the retinal organoids show a markedly reduced expression of several photoreceptor specific proteins (CRX, RCVRN, ZO1 and CRALBP) even if CPH-RP and control retinal organoids had a similar morphology on bright-field imaging. On the other side, the expression profiles of RK, RHO, ABCA4 and PDE6B in patient retinal organoids suggest that the trafficking to the outer segments of photoreceptors was impaired. Conclusion: Taken together, we show that mutations in TBC1D32 gene can cause RP. We suggest that the disease development may be linked to abnormal cilium formation, which can affect RPE function and ciliary transport in the photoreceptors.