Both vitamin A (retinol, ROL) deficiency and excess are known to cause severe visual defects including blindness and retinal degenerative diseases. ROL serves as the precursor for 11-cis-retinaldehyde (11-cis RAL), which functions as the visual chromophore. Therefore a sustained supply of ROL is required for systemic and ocular retinoid production in supporting visual function. Mammals, are unable to synthesize vitamin A de novo, and must obtain vitamin A precursors via the diet. Vitamin A precursors are predicted to follow the fate of cholesterol/lipids in the intestine; i.e. facilitated uptake. It was long assmed intestinal uptake of vitamin A precursors occurs by passive diffusion; however a receptor mediated or facilitated uptake of dietary ROL is now proposed as this would not only provide physiological doses of ROL but would also prevent systemic and eye malformations known to be associated with excessive vitamin A accumulation. Even given the importance of a continued supply of dietary vitamin A for visual function, information related to the uptake and storage of dietary vitamin A still constitutes a knowledge gap; therefore study of receptors facilitating ROL entry into intestinal cells for ocular retinoid homeostasis could remedy this. In this study, we ai to use the larva of the zebrafish, which is well established for studies of genes involved in ocula retinoid homeostasis. Knockdown of such genes have recapitulated visual phenotypes similar to those observed in humans with corresponding loss-of- function mutations. The zebrafish larval eye shows the typical vertebrate morphology and larvae have visual performance already at 5.5 days post fertilization (dpf). The larval zebrafish also possess many of the same gastrointestinal organs present in humans as well as specialized cell types involved in absorption and processing (intestinal enterocytes, ENTs) of lipids and vitamin A precursors. Therefore, functional studies of receptors, proposed in the dietary uptake and supply of vitamin A for ocular retinoid homeostasis in a vertebrate model such as the zebrafish are feasible. Our preliminary results, using in vitro cell based assays showed plasma membrane localization patterns for the Rbpr2 receptor, consistent with a localization pattern for a receptor involved in facilitated uptak of ROL. To determine tissue distribution patterns for rbpr2, we performed whole mount in-situ hybridization (WISH) in zebrafish. We found that Rbpr2 is expressed in intestinal enterocytes and liver hepatocytes, tissues and cells proposed to mediate dietary ROL uptake and storage respectively. Using a morpholino gene knockdown approach, we observed that at 5.5 days post fertilization rbpr2-morphants display reduced saccade frequency and gain in optokinetic response (OKR) tests for visual function. This indicated that reduced Rbpr2 expression in late stage zebrafish larvae, which are dependent on exogenous dietary sources for vitamin A, have suboptimal chromophore concentrations, manifesting in decreased visual function. Building on these preliminary data, we have now generated F0 rbpr2- mutants using the TALEN technology. Rbpr2-mutants are viable and show no severe developmental or eye defects, thus making functional analysis feasible. Based on these findings, we will test our hypothesis that rbpr2 facilitates dietary ROL uptake in the intestine and loss of rbpr2 will impact ocular retinoid level manifesting in loss of visual function. This hypothesis will be addressed in both in vitro and an i vivo model in the experiments of the following Specific Aims:
Specific Aim 1 : Determine the biochemical capabilities of Rbpr2 for retinol uptake.
Specific Aim 2 : Determine if rbpr2 facilitate intestinal ROL uptake for ocular chromophore production in the support of photoreception. The proposed studies will reveal how loss of rbpr2 in zebrafish decreases ROL supply for the production of retinoids in sustaining visual function. Should this study reveal a role of the rbpr2 receptor for retinol homeostasis in the support of vision and should an absence of such a receptor affects chromophore production and visual function, strategies aimed at addressing visual defects due to vitamin A deficiency or excess in humans could be better explored by modulating receptors involved in dietary retinol uptake and supply.
Vitamin A (retinol, ROL) deficiency causes impaired vision and blindness. This project will characterize the physiological role of the retinol receptor rbpr2 n zebrafish, and provide new information on the mechanisms influencing retinol uptake and storage for ocular retinoid homeostasis in the support of vision.
|Lobo, Glenn P; Pauer, Gayle; Lipschutz, Joshua H et al. (2018) The Retinol-Binding Protein Receptor 2 (Rbpr2) Is Required for Photoreceptor Survival and Visual Function in the Zebrafish. Adv Exp Med Biol 1074:569-576|
|Shi, Yi; Obert, Elisabeth; Rahman, Bushra et al. (2017) The Retinol Binding Protein Receptor 2 (Rbpr2) is required for Photoreceptor Outer Segment Morphogenesis and Visual Function in Zebrafish. Sci Rep 7:16207|
|Lobo, Glenn P; Fulmer, Diana; Guo, Lilong et al. (2017) The exocyst is required for photoreceptor ciliogenesis and retinal development. J Biol Chem 292:14814-14826|