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Roly Megaw, Carla Mellough, Baljean Dhillon, Alan Wright, Majlinda Lako, Charles ffrench-Constant; Use of induced pluripotent stem cell technology to understand photoreceptor cytoskeletal dynamics in retinitis pigmentosa. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):3585.
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© ARVO (1962-2015); The Authors (2016-present)
Mutations in RPGR account for 20% of all Retinitis Pigmentosa (RP). RPGR’s function is unknown. It has no cure. Animal rpgr models provide conflicting evidence as to the nature of disease. We set out to establish a novel, human, cell-based model for RPGR disease to test the hypothesis that RPGR mutations lead to retinal degeneration due to a dysregulation of the actin cytoskeleton.
iPSCs were generated from skin biopsies of patients with RPGR mutations (g.ORF15+689_692del4) and unaffected relatives. Our published protocol was modified to produce self-organising 3 dimensional eye cups. RGPR-mutated and control cultures were compared.
Mutant and wild-type iPSC lines were generated and characterised. Differentiation of wild type and mutant lines resulted in the generation of optic cups in a self-organising manner. After 100 days in culture, these cups contained organised, mature photoreceptors (PRs), as evidenced by morphology and both RNA and protein expression (recoverin and rhodopsin). RPGR is localised to the PR connecting cilium. Western blot analysis shows expression of a truncated RPGRORF15 protein splice variant in mutated PR cultures compared to controls. PR cultures from RGPR-mutated iPS cells had increased actin polymerisation compared with controls (confocal pixel intensity counts : 59.02 (SD 16.24) vs 23.70 (SD 8.128) p<0·0081).This finding was confirmed by assessment of F-actin with western blot.<br /> <br /> Pathways regulating actin turnover were explored. Western blot analysis showed reduction in both Src and ERK phosphorylation in RGPR-mutated PR cultures. An unbiased protein array confirmed this reduction. Several other pathways were also shown to be dysregulated in the RGPR-mutated PR cultures; confirmed by western blots of repeat cultures from varying patient and control cell lines.
This study supports the hypothesis that RPGR mutations lead to actin dysregulation. We have identified several pathways which are interupted 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 RPGR mutations to look at pathophysiology of disease. It suggests a role for RPGR in facilitating Rhodopsin transport to PR outer segments by regulating actin turnover.
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