BACKGROUND: Retinitis pigmentosa, which affects one in 3000 people, causes blindness and has no treatment. Mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene cause 20% of all cases. Recent work suggests that RPGR, localised to the photoreceptor connecting cilium, regulates rhodopsin transport to the outer segment through its effect on the turnover of actin. We set out to establish a novel model for RPGR disease to test the hypothesis that RPGR mutations lead to retinal degeneration due to a dysregulation of the actin cytoskeleton.
METHODS: Patients with RPGR mutations and their unaffected relatives were recruited and skin biopsy samples taken. Fibroblast lines were established and reprogrammed to generate induced pluripotent stem cell (iPSC) lines. A three-dimensional organogenesis protocol was optimised whereby embryoid bodies were formed and patterned towards an eye field fate in a 100-day retinal differentiation protocol, allowing three-dimensional optic cups to form. RPGR-mutated cultures were compared with their healthy controls.
FINDINGS: Mutant and wild-type iPSC lines were generated and characterised. Differentiation of all lines resulted in the generation of optic cups in a self-organising manner after 100 days in culture. These cultures contained mature photoreceptors, as evidenced by morphology and both RNA and protein expression. Photoreceptor cultures from RGPR-mutated iPSCs had increased actin polymerisation compared with controls (mean confocal pixel intensity count 59·02 [SD 16·24] vs 23·70 [8·20], p=0·0081). This finding was confirmed by assessment of F-actin with western blot. Pathways regulating actin turnover were explored; western blot analysis showed a reduction in both Src and ERK phosphorylation in RGPR-mutated photoreceptor cultures. An unbiased protein array confirmed this reduction in ERK and Src activation. Several other pathways were also shown to be dysregulated in the RGPR-mutated photoreceptor cultures.
INTERPRETATION: This study supports the hypothesis that RPGR mutations lead to actin dysregulation. We have identified several pathways that are interrupted in RPGR-mutant photoreceptor cultures and could be contributing to disease. This study is the first use, to our knowledge, of human iPSCs with retinitis pigmentosa-causing mutations to look at pathophysiology of disease.
FUNDING: Wellcome Trust.