Deciphering the role of Rab28 in cone vision and outer segment phagocytosis

Abstract

Purpose : Rab28 is a small farnesylated G-protein associated with human autosomal recessive cone-rod dystrophy (CRD). Rab28 localises to cone photoreceptor outer segments (OS) which are modified cilia. Knockout of murine or zebrafish Rab28 diminishes OS phagocytosis (OSP) by the retinal pigment epithelium (RPE). As Rab28 function and regulation of cone OSP are poorly understood, this study further investigates the molecular mechanism by which Rab28 functions in zebrafish cone photoreceptors and its role in OSP.

Methods : Genetic, biochemical, behavioural and imaging approaches interrogated the function of Rab28 in zebrafish cone photoreceptors using a rab28 knockout (KO) model and transgenic strains overexpressing gnat2:egfp-rab28. Visual behaviour was analysed using optokinetic response, contrast sensitivity and visual acuity assays. For TEM analysis of shed OS, phagosomes were manually counted, and the density calculated as phagosomes per μm of RPE. Proteomic profiling of rab28 knockout and sibling eyes collected at ZT 4, ZT 9 and ZT 17 was performed by mass spectrometry (MS). Retinoid profiling was carried out by HPLC.

Results : At 15 days post fertilisation (dpf), rab28 KO zebrafish display a 55% reduction (N=3, p<0.001) of shed OS material at morning (ZT 4) and evening (ZT 17) OSP peaks. Overexpression of Rab28 specifically in cones rescues the OS shedding defect in rab28 KO fish (N=3, p<0.001). Real-time PCR identified cerkl, mertka and abcf1 genes to maximise expression prior to one or both OSP peaks. Proteomic analysis demonstrates loss of zebrafish Rab28 leads to reduced levels of phototransduction cascade components and core ciliary BBSome proteins (N=3, p<0.05). Visual cycle retinoid levels are also reduced (N=3, p<0.03) in larval and adult rab28 KO retinae inferring a direct or indirect role of Rab28 in the visual cycle. We observed no visual impairment in juvenile rab28 KO fish.

Conclusions : Our findings indicate that Rab28 regulates phagocytic processes at the ciliary/OS membrane, thereby providing insight into the mechanisms of RAB28-associated cone-rod dystrophy and cone OSP regulation.

This is a 2021 ARVO Annual Meeting abstract.