Abstract
Purpose :
The small G-protein Rab28 is the first Rab GTPase associated with human autosomal recessive cone-rod dystrophy (CRD). We previously reported C. elegans RAB-28 functions in sensory neuronal cilia. In functional studies, rab-28 loss in C. elegans causes abnormal accumulation of extracellular vesicles in ciliated sensory pores. Consistent with these findings, zebrafish Rab28 localises to the outer segments (OS; modified cilia) of cone photoreceptors and regulates OS shedding. Here, we further interrogate the cellular and molecular mechanism by which RAB28 functions in cone photoreceptors.
Methods :
Genetic, biochemical, behavioural and imaging approaches interrogated the function of RAB28 in zebrafish cone photoreceptors. rab28 knock out zebrafish were generated using CRISPR-Cas9 gene editing. Transgenic zebrafish expressing eGFP-Rab28 in cone photoreceptors were generated by microinjection of plasmids containing a Tol2-gnat2:eGFP-rab28(cDNA)-Tol2 construct, together with Tol2 transposase mRNA. GDP and GTP-preferring mutants of Rab28 were generated by site-directed mutagenesis of the cDNA. Visual behaviour was analysed using optokinetic response and visual motor response assays. For TEM analysis of shed OS, phagosomes were manually counted,and the density calculated as phagosomes per μm of RPE.
Results :
CRISPR-Cas9 generated rab28 mutant zebrafish display a 60% reduction (N=3, p<0.001) of shed OS material in the retinal pigment epithelium (RPE) at 15 days post fertilisation (dpf). Cone photoreceptor-specific transgenic reporter lines show eGFP variants of Rab28 localise almost exclusively to OS. Analysis of Rab-28 transgenic variants in the knockout background show normal visual function and retinal structure up until 15 dpf (N=3, n=29-36). Overexpression of Rab28 specifically in cone photoreceptors rescues the OS shedding defect in rab28 knockouts(N=3, p<0.001), suggesting Rab28 function in cone photoreceptors alone is sufficient to regulate Rab28-OS shedding. There was no evidence of retinal degeneration or visual impairment in rab28 knockout zebrafish.
Conclusions :
In conclusion, this work furthers our understanding of how RAB28 regulates ciliary membrane homeostasis. The data provides a model for RAB28-associated cone-rod dystrophy in addition to insights into molecular regulators of cone OS phagocytosis. We propose that dysregulation of phagocytic processes at the ciliary/OS membrane underpins RAB28-CRD.
This is a 2020 ARVO Annual Meeting abstract.