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.

Retinitis Pigmentosa (RP) refers to a group of inherited retinal dystrophies resulting from progressive photoreceptor degeneration and accumulation of intra-retinal pigment-like deposits. X-linked forms of RP are frequently caused by mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene. The RPGR transcript undergoes complex alternative splicing to express both constitutive (RPGR^ex1-19) and RPGR^ORF15 variants. Although RPGR is thought to play a role in ciliary function, little is known about the physiological significance of expressing two distinct groups of variants. This study compares Rpgr^ex1-19 and Rpgr^ORF15 expression in developing photoreceptors using immunoblot analysis and immunohistochemistry, assesses ciliary affinity in adult photoreceptors by protein fractionation, examines Rpgr function in transgenic mouse models and identifies a novel Rpgr^ORF15 binding partner using a yeast two-hybrid screen. Our data reveal that Rpgr expression undergoes dynamic temporal regulation during retinal development and indicates variability in ciliary localization of Rpgr variants in adult photoreceptors. Utilization of distinct Rpgr variants during stages of photoreceptor development suggests independent roles. Further examination of Rpgr function using transgenic mouse models over-expressing either the Rpgr^ex1-19 or Rpgr^ORF15 variant reveals that despite normal ciliary localization, an excess of RPGR^ex1-19 results in atypical accumulation of Rpgr in photoreceptor outer segments, abnormal photoreceptor morphology and severe retinal degeneration. The data indicate that the constitutive variant cannot substitute for Rpgr function in photoreceptors and suggest that proper maintenance of the Rpgr isoform ratio is critical to photoreceptor viability. Using mouse retinal cDNA in a yeast two-hybrid screen with the C-terminus of the Rpgr^ORF15 variant, we identified a novel variant of whirlin as an interacting partner. Mutations in whirlin result in Usher syndrome, a disorder characterized by hearing loss and RP. RT-PCR and immunoblot analysis were used to confirm the presence of selected candidate partners in the retina and interaction was confirmed by pull-down assays and co-immunoprecipitation from retinal homogenate. Immunohistochemistry showed co-localization of RPGR and whirlin within photoreceptors and identified isoform specific localization of whirlin. These findings indicate that whirlin binds Rpgr^ORF15 and that this novel isoform may be required for photoreceptor function, thus providing a potential mechanism for the RP phenotype observed in Usher syndrome.