Regulation of the rhythmic diversity of daily photoreceptor outer segment phagocytosis in vivo.

Outer segment phagocytosis (OSP) is a highly-regulated, biological process wherein photoreceptor outer segment (OS) tips are cyclically phagocytosed by the adjacent retinal pigment epithelium (RPE) cells. Often an overlooked retinal process, rhythmic OSP ensures the maintenance of healthy photoreceptors and vision. Daily, the photoreceptors renew OS at their base and the most distal, and likely oldest, OS tips, are phagocytosed by the RPE, preventing the accumulation of photo-oxidative compounds by breaking down phagocytosed OS tips and recycling useful components to the photoreceptors. Light changes often coincide with an escalation of OSP and within hours the phagosomes formed in each RPE cell are resolved. In the last two decades, individual molecular regulators were elucidated. Some of the molecular machinery used by RPE cells for OSP is highly similar to mechanisms used by other phagocytic cells for the clearance of apoptotic cells. Consequently, in the RPE, many molecular regulators of retinal phagocytosis have been elucidated. However, there is still a knowledge gap regarding the key regulators of physiological OSP in vivo between endogenous photoreceptors and the RPE. Understanding the regulation of OSP is of significant clinical interest as age-related macular degeneration (AMD) and inherited retinal diseases (IRD) are linked with altered OSP. Here, we review the in vivo timing of OSP peaks in selected species and focus on the reported in vivo environmental and molecular regulators of OSP.

Dawn and dusk peaks of outer segment phagocytosis, and visual cycle function require Rab28.

RAB28 is a farnesylated, ciliary G-protein. Patient variants in RAB28 are causative of autosomal recessive cone-rod dystrophy (CRD), an inherited human blindness. In rodent and zebrafish models, the absence of Rab28 results in diminished dawn, photoreceptor, outer segment phagocytosis (OSP). Here, we demonstrate that Rab28 is also required for dusk peaks of OSP, but not for basal OSP levels. This study further elucidated the molecular mechanisms by which Rab28 controls OSP and inherited blindness. Proteomic profiling identified 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 (p < .05). These data give 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.

Transport and barrier mechanisms that regulate ciliary compartmentalization and ciliopathies.

Primary cilia act as cell surface antennae, coordinating cellular responses to sensory inputs and signalling molecules that regulate developmental and homeostatic pathways. Cilia are therefore critical to physiological processes, and defects in ciliary components are associated with a large group of inherited pleiotropic disorders - known collectively as ciliopathies - that have a broad spectrum of phenotypes and affect many or most tissues, including the kidney. A central feature of the cilium is its compartmentalized structure, which imparts its unique molecular composition and signalling environment despite its membrane and cytosol being contiguous with those of the cell. Such compartmentalization is achieved via active transport pathways that bring protein cargoes to and from the cilium, as well as gating pathways at the ciliary base that establish diffusion barriers to protein exchange into and out of the organelle. Many ciliopathy-linked proteins, including those involved in kidney development and homeostasis, are components of the compartmentalizing machinery. New insights into the major compartmentalizing pathways at the cilium, namely, ciliary gating, intraflagellar transport, lipidated protein flagellar transport and ciliary extracellular vesicle release pathways, have improved our understanding of the mechanisms that underpin ciliary disease and associated renal disorders.

Gene therapy for RAB28: What can we learn from zebrafish?

The eye is particularly suited to gene therapy due to its accessibility, immunoprivileged state and compartmentalised structure. Indeed, many clinical trials are underway for therapeutic gene strategies for inherited retinal degenerations (IRDs). However, as there are currently 281 genes associated with IRD, there is still a large unmet need for effective therapies for the majority of IRD-causing genes. In humans, RAB28 null and hypomorphic alleles cause autosomal recessive cone-rod dystrophy (arCORD). Previous work demonstrated that restoring wild type zebrafish Rab28 via germline transgenesis, specifically in cone photoreceptors, is sufficient to rescue the defects in outer segment phagocytosis (OSP) observed in zebrafish rab28-/- knockouts (KO). This rescue suggests that gene therapy for RAB28-associated CORD may be successful by RAB28 gene restoration to cones. It also inspired us to critically consider the scenarios in which zebrafish can provide informative preclinical data for development of gene therapies. Thus, this review focuses on RAB28 biology and disease, and delves into both the opportunities and limitations of using zebrafish as a model for both gene therapy development and as a diagnostic tool for patient variants of unknown significance (VUS).

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

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. Co-immunoprecipitation 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.

Ciliary Rab28 and the BBSome negatively regulate extracellular vesicle shedding.

Cilia both receive and send information, the latter in the form of extracellular vesicles (EVs). EVs are nano-communication devices that influence cell, tissue, and organism behavior. Mechanisms driving ciliary EV biogenesis are almost entirely unknown. Here, we show that the ciliary G-protein Rab28, associated with human autosomal recessive cone-rod dystrophy, negatively regulates EV levels in the sensory organs of Caenorhabditis elegans in a cilia specific manner. Sequential targeting of lipidated Rab28 to periciliary and ciliary membranes is highly dependent on the BBSome and the prenyl-binding protein phosphodiesterase 6 subunit delta (PDE6D), respectively, and BBSome loss causes excessive and ectopic EV production. We also find that EV defective mutants display abnormalities in sensory compartment morphogenesis. Together, these findings reveal that Rab28 and the BBSome are key in vivo regulators of EV production at the periciliary membrane and suggest that EVs may mediate signaling between cilia and glia to shape sensory organ compartments. Our data also suggest that defects in the biogenesis of cilia-related EVs may contribute to human ciliopathies.