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

Abstract

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.

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

“…A similar profile is observed in zebrafish, albeit raised under a different environmental light–dark cycle of 14 h light: 10 h dark. 2 prominent OSP peaks occur, the first after light exposure, and a second shortly after darkness 16,28 . In contrast to the goldfish study, the zebrafish were shown by immunolabeling to display mixed rod and cone OSP peaks, with the morning peak slightly favoring the phagocytosis of distal rod cell OS tips and the evening peak favoring the phagocytosis of distal cone cell OS tips 16 …”

“…This dysregulation of OSP also occurs in genetic models. In Rab28 and Kif17 knockout zebrafish and mice, the peak levels of OSP become reduced 16,28 . OSP peaks can also be increased in amplitude.…”

“…Like frogs, goldfish OSP is inhibited by constant light and a massive shedding event happens after constant light treated animals are exposed to a dark period and then back to light 27 . In zebrafish, morning and night peaks of OSP, quantified by transmission electron microscopy (TEM), are inhibited in animals held in constant darkness suggesting a lack of circadian control 28 …”

“…To date, the majority of OSP regulators characterized, carry out their primary function in the RPE. However, in recent years several studies began to elucidate the role of photoreceptor signaling in the promotion and regulation of OSP 4,16,28,69 . A study focusing on ciliary protein Kif17 (kinesin family member 17) shows a potential role for Kif17 phosphorylation in both OS localization as well as promoting phagocytosis of zebrafish and mouse cone photoreceptors in a cell‐autonomous manner 16 .…”

Differences in Diurnal Rhythm of Rod Outer Segment Renewal between 129T2/SvEmsJ and C57BL/6J Mice

“…A robust morning peak shortly after light onset and low activity at other times is a rhythm that has been reported for over 50 years in laboratory mice and rats that are entrained to a 12 h light–12 h dark cycle, i.e., with light on from zeitgeber time 0 (ZT0) to ZT12 and light off from ZT12 to ZT0 [ 4 , 5 , 6 ]. Recently, a previously unseen evening peak of rod POS phagocytosis following onset of darkness has been reported in both mice and zebrafish [ 7 , 8 , 9 ]. In these studies, both electron microscopy and rhodopsin immunofluorescence microscopy were used to identify and quantify POS phagosomes in the RPE in situ.…”