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Title: Functional analysis of RP2 and ARL3 in X-linked retinitis pigmentosa
Author: Little, Abigail May
ISNI:       0000 0004 7969 1632
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2019
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Retinitis Pigmentosa (RP) is a disease of the retina, which causes progressive retinal degeneration. X-linked RP is one of the most severe subtypes with an estimated 15% of cases caused by mutations in RP2. RP2 functions as a GTPase Activating Protein (GAP) for the small G protein ARL3, which is proposed to regulate the traffic of lipid-modified proteins within photoreceptors. It is hypothesised that mutations in RP2 result in dysregulation of ARL3 and therefore protein mis-trafficking. In order to elucidate the contribution of ARL3 dysregulation to the pathogenesis of RP, I have established new mouse models by CRISPR-mediated genome editing. These include an Rp2h knockout line and a line, which harbours a human pathogenic missense mutation, E135G, which abolishes interaction with ARL3. Furthermore, I have generated mice carrying a Q71L missense mutation in Arl3. This mutation locks ARL3 in the active GTP-bound state, and hence is predicted to phenocopy Rp2h knockout. Histological examination has revealed that Rp2h knockout, Rp2h E135G and Arl3 Q71L/+ mutant animals display progressive retinal degeneration evident from age 6 months. Arl3 Q71L/Q71L animals display retinal degeneration at age 3 months demonstrating that elevated levels of ARL3-GTP is a driver of retinal degeneration in mice. Immunofluorescence analysis has shown ARL3 Q71L mice, Rp2h knockout mice and Rp2h E135G/Y mice show mislocalisation of lipid modified proteins likely driving retinal degeneration, however further analysis has shown that these mice do not completely phenocopy each other suggesting that levels of ARL3-GTP may not be the only mechanism contributing to retinal degeneration in Rp2h mutant mice. The mechanisms of RP2 regulation are not well understood; therefore to identify potential interactors of RP2 a BIO-ID proximity labelling assay in RPE-1 cells was performed. A top hit from this assay was palmitoyltransferase ZDHHC5. I confirmed this interaction in cells and using a click chemistry based approach demonstrated that it is unlikely that this enzyme functions to palmitoylate RP2. Using immunofluorescence in HeLa cells I have shown that overexpression of ZDHHC5 can rescue the localisation of human pathogenic RP2 mutants C3S and G2A, which are normally mislocalised in vivo, independent of its catalytic activity. SiRNA knockdown of ZDHHC5 in cells leads to mislocalisation of RP2 demonstrating ZDHHC5 has a role in trafficking RP2. Results from these studies have provided new knowledge regarding the mechanisms that cause retinal degeneration and new insights into the potential mechanisms that regulate the trafficking of lipid-modified proteins in photoreceptors.
Supervisor: Hurd, Toby ; Davey, Megan Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Inherited Retinal Degenerations ; Retinitis Pigmentosa ; RP2 ; ARL3 ; photoreceptor death ; CRISPR editing