Genetic Deletion of Zebrafish Rab28 Causes Defective Outer Segment Shedding, but Not Retinal Degeneration

Abstract

The photoreceptor outer segment is the canonical example of a modified and highly specialized cilium, with an expanded membrane surface area in the form of disks or lamellae for efficient light detection. Many ciliary proteins are essential for normal photoreceptor function and cilium dysfunction often results in retinal degeneration leading to impaired vision. Herein, we investigate the function and localization of the ciliary G-protein RAB28 in zebrafish cone photoreceptors. CRISPR-Cas9 generated rab28 mutant zebrafish display significantly reduced shed outer segment material/phagosomes in the RPE at 1 month post fertilization (mpf), but otherwise normal visual function up to 21 dpf and retinal structure up to 12 mpf. Cone photoreceptor-specific transgenic reporter lines show Rab28 localizes almost exclusively to outer segments, independently of GTP/GDP nucleotide binding. Coimmunoprecipitation analysis demonstrates tagged Rab28 interacts with components of the phototransduction cascade, including opsins, phosphodiesterase 6C and guanylate cyclase 2D. Our data shed light on RAB28 function in cones and provide a model for RAB28-associated cone-rod dystrophy.

Expanding the Clinical and Genetic Spectrum of RAB28-Related Cone-Rod Dystrophy: Pathogenicity of Novel Variants in Italian Families

“…In summary, our results suggested that the concerted effect of the structural perturbation induced by the T26N variant on the proximal switch 1 region (Figure 8) and on the distant β5-β6 region would affect both GTP-GDP exchange and Mg 2+ -binding, thus classifying the substitution as a partial loss-of-function missense mutation. Overall, our findings are perfectly in line with several previous lines of evidence on Rab family proteins [17], as the T26N mutation affects the last residue of the broadly conserved G motif among GTPases, and it is widely used to mimic a "GDP-locked" state of Rab proteins [10,13,16,17] due to the disruption of Mg 2+ binding site and the resultant 100-fold decrease in affinity for GTP. Concerning the nonsense p.(Arg137*) variant, no nonsense-mediated decay of mRNA was previously reported [2] on CORD patients carrying such variant, suggesting potential functional defects as the pathological mechanism.…”

“…Recent studies on RAB28 knockout mice showed that Rab-28 plays an essential role in cone-specific disc shedding and phagocytosis and an impaired membrane shedding at the distal COS and/or failed phagocytosis by the RPE has been proposed as possible pathogenetic mechanism for CORD [9]. The role of Rab-28 in regulating membrane shedding from cone outer segment tips has been confirmed in a RAB28 knockout zebrafish model where, however, a longer visual function with no sign of retinal degeneration up to 12 months has been observed [10]. In humans, only six RAB28 variants have been reported to be associated with CORD.…”

“…Reports on animal models showed a different impact of RAB28 variants on retinal function among species. Studies on mouse and zebrafish confirmed the role of RAB28 in cone outer segment shedding mechanism, but in contrast to the mouse RAB28 knockout [9], RAB28 knockout zebrafish display decreased RPE phagosomes, but normal visual function up to 21 days post fertilization and no retinal degeneration up to 12 months post fertilization [10]. One explanation of this different impact of RAB28 variants on retinal function could be that the level of outer segment shedding/phagocytosis remaining in zebrafish RAB28 mutants is enough to support photoreceptor survival.…”

“…From a molecular standpoint, the structural analysis of the nonsense and splicing variants here identified suggested that the lack of several key structural elements would either lead to protein degradation or mRNA-mediated decay. As far as patient 5 is concerned, the missense variant p.(Thr26Gln) found in compound heterozygosis affects one of the most highly conserved residues of the P-loop, resulting in a partial loss-of function due to the impaired Mg 2+ -coordination, which locks Rab-28 in a GDP-bound inactive state [10,13,16,17], ultimately leading to the altered membrane trafficking. Interestingly, the P-loop seems to be a hotspot region for CORD-associated missense variants, as also p.(Gly19Arg) [5] and p.(Ser23Phe) [4] belong to the same conserved 7-residues stretch.…”

“…This led to the proposition of an impaired membrane shedding at the distal cone outer segment (COS) and/or failed phagocytosis by the RPE as the possible pathogenetic mechanism for CORD [9]. Such evidence has been confirmed in a recent paper reporting altered COS shedding in the zebrafish knockout model [10]. To date, only six variants have been reported worldwide, confirming the extreme rarity of RAB28-related CORD.…”