Flavins are essential cofactors for wide-ranging metabolic processes; hence they are particularly critical in highly metabolically active tissues. The retina, in which levels of flavins are several folds higher than in blood, is an important example. The physiological significance of modulating levels of retinal flavins is underscored by the observation that riboflavin deficiency results in photosensitivity and degeneration, a process thought to result from lipid peroxidation. Unbound flavins are toxic so, in practice, flavins are virtually always bound to flavin binding proteins. Although tight regulation of flavin levels is clearly critical for maintaining retinal homeostasis, nothing is known about the mechanisms governing their uptake, regulation, or binding proteins. We have recently begun studying a novel, and highly relevant candidate retinal flavin binding protein called Retbindin (Retb). Retb has homology to chicken riboflavin binding protein (RBP), and we have shown in vitro and in retinal explant that Retb binds flavins. This, coupled with the importance of flavins in the retina, led us to hypothesize that Retb's function is tied to flavin regulation. We recently reported that Retb is exclusively expressed by rods, secreted into the interphotoreceptor matrix (IPM) and maintained via electrostatic forces at the interface between photoreceptors and retinal pigment epithelium (RPE) microvilli, a region critical for retinal function and homeostasis. This localization combined with its ability to bind flavins, implicates Retb as a potential carrier of flavins between the retina and the RPE. To further assess Retb's function in the retina, we generated a knockout mouse (Retb-/-), in which Retb sequence was replaced with eGFP. Electroretinography revealed an age- and dose-dependent decline in rod and cone responses at postnatal days (P) 120 and 240 and a concurrent degeneration of rods and cones. We also show flavin levels significantly reduced in P45 Retb-/- retina, prior to the onset of degeneration. In light of the potential pathological consequences of elimination of Retb, coupled with a complete lack of knowledge of Retb function, we propose to explore the role of flavins in the retina and determine how Retb could regulate their levels. Specifically, we propose three aims. First, we will determine the functional role of Retb in the photoreceptor cells by evaluating Retb-/- and Retb+/- animals at different ages and under various lighting/dietary treatments as well as identify Retb binding partners. Second, we will take a metabolomic approach to identify the metabolic pathways that are affected by the absence of Retb. Third, because we observed levels of Retb are significantly elevated in the retinas of animal models of retinal diseases, we will test the role Retb plays in the development and progression of retinal degenerations such as retinitis pigmentosa. We have also initiated the generation of a Retb over-expresser mouse model to be used to assess the capability of Retb in ameliorating disease progression. These experiments are highly significant, not just to further our knowledge of a poorly understood protein critical for retinal function, but also to explore the role of metabolic dysregulation in retinal homeostasis and diseases.
Although flavins are essential cofactors for a vast number of metabolic processes and highly concentrated in the retina, little if any is known about their retinal uptake, regulation and binding partners. Unbound flavins in the extracellular space are toxic and dysregulation of retinal flavins leads to defects in vision, so flavin-proteins are critical for maintaining cellular homeostasis. In this application we plan to understand the role of a newly identified retinal flavin binding protein called retbindin, in the tight flavin regulation in the retina, in its protective role in myriad of retinal disorders in which metabolic dysregulation plays a part and how its absence leads to degeneration.
| Kelley, Ryan A; Al-Ubaidi, Muayyad R; Naash, Muna I (2018) Retbindin Is Capable of Protecting Photoreceptors from Flavin-Sensitized Light-Mediated Cell Death In Vitro. Adv Exp Med Biol 1074:485-490 |
| LaVail, Matthew M; Nishikawa, Shimpei; Steinberg, Roy H et al. (2018) Phenotypic characterization of P23H and S334ter rhodopsin transgenic rat models of inherited retinal degeneration. Exp Eye Res 167:56-90 |
| Kelley, Ryan A; Conley, Shannon M; Makkia, Rasha et al. (2018) DNA nanoparticles are safe and nontoxic in non-human primate eyes. Int J Nanomedicine 13:1361-1379 |
| Sinha, Tirthankar; Makia, Mustafa; Du, Jianhai et al. (2018) Flavin homeostasis in the mouse retina during aging and degeneration. J Nutr Biochem 62:123-133 |
| Kelley, Ryan A; Al-Ubaidi, Muayyad R; Sinha, Tirthankar et al. (2017) Ablation of the riboflavin-binding protein retbindin reduces flavin levels and leads to progressive and dose-dependent degeneration of rods and cones. J Biol Chem 292:21023-21034 |
