Characterisation of the small ciliary GTPase Rab28 using Danio rerio and C. elegans models

RAB28 is a farnesylated, ciliary G-protein. Patient variants in RAB28 are causative of autosomal recessive cone-rod dystrophy (CRD), an inherited human blindness. Primary cilia are microtubule-based, hair-like, structures which emanate from the surface of eukaryotic cells. The conserved organelles are considered the ‘antennae of the cell’, capable of both receiving and transmitting signals. Cilia are also remarkably well conserved, enabling the use of diverse animal models, such as C. elegans and zebrafish, to dissect the molecular mechanisms of ciliary dysfunction. Using these two leading animal models (C. elegans, zebrafish) of cilia, ciliopathy and vision research, together with genetic, cell biology and biochemical tools in this thesis, I endeavoured to further elucidate the molecular mechanisms of RAB28-associated CRD.
In Chapter III, the zebrafish model was used to demonstrate that Rab28 is required for dawn and dusk peaks of outer segment phagocytosis (OSP), a waste removal process in the retina, which aims to prevent the accumulation of photo-oxidative compounds and maintains photoreceptor health and viability. This study further elucidated the molecular mechanisms by which Rab28 controls OSP and inherited blindness. Proteomic profiling identifies factors whose expression in the eye or whose expression at dawn and dusk peaks of OSP is dysregulated by loss of Rab28. Notably, transgenic overexpression of Rab28, solely in zebrafish cones, rescues the OSP defect in rab28 KO fish, suggesting rab28 gene replacement in cone photoreceptors is sufficient to regulate Rab28-OSP. Rab28 loss also perturbs function of the visual cycle as retinoid levels of 11-cRAL, 11cRP, and atRP are significantly reduced in larval and adult rab28 KO retinae. This data gives further understanding on the molecular mechanisms of RAB28-associated CRD, highlighting roles of Rab28 in both peaks of OSP, in vitamin A metabolism and in retinoid recycling. The mechanisms instigating OSP in photoreceptors, particularly in cones, remains poorly characterised, leaving a knowledge gap in retinal biology and disease. As such, this aspect of the study investigates extrinsic environmental and intrinsic molecular regulators of OSP in zebrafish. Proteomic profiling of wildtype eyes was performed by mass spectrometry which revealed potential novel regulators of OSP. Exposure to light:dark transition was proven necessary to fully initiate the burst of OSP at the expected dawn and dusk peak time in the zebrafish retina. Pharmacological modulation with cigarette smoke extract was also shown to induce significantly higher levels of RPE phagosomes in wildtype zebrafish larvae. This data provides models for insights into the regulators of OSP in a cone-rich retina, and may hold biomedical significance as defective OSP could be part of the disease pathway for many retinal and macular degenerations. Previous work on C. elegans uncovered that RAB-28 negatively regulates EV levels in the sensory organs of Caenorhabditis elegans in a cilia specific manner. In Chapter IV the sequential targeting of lipidated RAB-28 to periciliary and ciliary membranes in EV releasing neurons was investigated and shown to be highly dependent on the BBSome and the prenyl-binding protein phosphodiesterase 6 subunit delta (PDE6D), respectively. Building on the work in Akella et al., 2020, where RAB-28 and BBSome loss was revealed to cause excessive and ectopic EV production, the role of BBSome genes in EV regulation was further investigated. Lastly, using a filtration approach, a method for EV isolation and purification was established.