Introduction Retinitis Pigmentosa affects 1 in 3000 people in the UK, causing photoreceptor degeneration and premature blindness. Mutations in the X-linked RPGR gene cause 20% of all disease and result in a particularly severe form of disease. The function of RPGR is unknown and it has no treatment. Methods Conflicting evidence from animal studies led me to develop a novel model for human disease with which to test the hypothesis that RPGR acts to regulate actin turnover in the photoreceptor connecting cilium. Skin biopsies were performed on patients with RPGR mutations and unaffected relatives. Subsequent fibroblast cultures were reprogrammed to generate induced pluripotent stem cell (iPSC) lines. A retinal differentiation protocol was optimised, resulting in healthy and RPGR-mutant in vitro photoreceptor cultures. Results Cultures were compared. RPGR-mutant iPSC-derived photoreceptors had increased actin polymerisation compared to wild-type control. Unbiased and hypothesis-driven experiments highlighted dysregulation of several key phospho-proteins involved in regulating actin turnover. Notably the RAC-PAK-LIMK-COFILIN pathway was dysregulated in RPGR-mutant cultures. A regulator of this pathway is the actin binding and severing protein, GELSOLIN. GELSOLIN activity was found to be perturbed in RPGR-mutant cultures. Examination of bovine retinal lysate showed an interaction between Rpgr and Gelsolin. Subsequent examination of iPSC-derived human photoreceptor cultures showed compromised interaction in RPGR-mutant cultures compared to controls. An Rpgr knock out mouse was obtained and characterised. Increased actin polymerisation in the connecting cilium and rhodopsin mislocalisation to the inner segment was seen prior to retinal degeneration. Gelsolin activity was perturbed. A Gelsolin knock out mouse was obtained and characterized. It, too, showed rhodopsin mislocalisation and retinal degeneration. Conclusion Results in this thesis confirm the hypothesis that RPGR acts to regulate actin turnover in the photoreceptor. Further, it suggests a mechanism through which this occurs. Further work is required to assess the extent of RPGR’s role in actin regulation in vivo.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:700060 |
| Date | January 2015 |
| Creators | Megaw, Roland David |
| Contributors | Ffrench-Constant, Charles ; Lako, Linda |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/17884 |
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