Gennadiy Moiseyev

Isorhodopsin rather than rhodopsin mediates rod function in RPE65 knock-out mice

The chromophore of visual pigments is 11-cis-retinal and, thus, in its absence, opsin is not photosensitive and no visual function exists. However, in the RPE65 knockout (Rpe65-/-) mouse, where synthesis of 11-cis-retinal does not occur, a minimal visual response from rod photoreceptors is obtained. We have examined if an alternative pathway exists for cis-retinoid generation in the absence of RPE65. Cyclic-light-reared, 2-month-old Rpe65-/- mice were placed in complete darkness. No exogenous retinoids were administered. After 4 weeks, enhanced a- and b-wave amplitudes were obtained, increasing >10-fold for the a-wave and >3-fold for the b-wave as compared with cyclic-light-reared Rpe65-/- mice. Visual-pigment levels increased to ≈10 pmol per retina, compared with no measurable pigment for cyclic-light-reared Rpe65-/- mice. The λmax of the isolated pigment was 487 nm, characteristic for isorhodopsin. Retinoid extractions confirmed the presence of 9-cis-retinal and the absence of 11-cis-retinal. Once the Rpe65-/- mice were returned to cyclic light, within 48 h the electroretinogram function returned to levels found in Rpe65-/- mice maintained in cyclic light. This dark-mediated pathway is also operational in older animals, because 13-month-old Rpe65-/- mice kept in prolonged darkness (12 weeks) had increased isorhodopsin levels and electroretinogram a- and b-wave amplitudes. These studies demonstrate that a pathway exists in the eye for the generation of 9-cis-retinal that is independent of RPE65 and light.

RPE65 is an iron(II)-dependent isomerohydrolase in the retinoid visual cycle

The isomerization of all-trans-retinyl ester to 11-cis-retinol in the retinal pigment epithelium (RPE) is a critical step in the visual cycle and is essential for normal vision. Recently, we have established that protein RPE65 is the isomerohydrolase catalyzing this reaction. The present study investigated if metal ions are required for the isomerohydrolase activity of RPE65. The conversion of all-trans-[3H]retinol to 11-cis-[3H]retinol was used as the measure for isomerohydrolase activity. Metal chelators 2,2′-bipyridine and 1,10-phenanthroline both showed dose-dependent inhibitions of the isomerohydrolase activity in bovine RPE microsomes, with IC50 values of 0.5 and 0.2 mm, respectively. In the same reaction systems, however, lecithin-retinol acyltransferase (LRAT) activity was not affected by these metal chelators. The isomerohydrolase activity inhibited by the metal chelators was restored by FeSO4 but not by CuSO4, ZnCl2, or MgCl2. Moreover, addition of Fe(III) citrate or FeCl3 did not restore the activity, indicating that Fe2+ is the metal ion essential for the isomerohydrolase activity. To confirm this result in recombinant RPE65, we expressed RPE65 in a 293A cell line stably expressing LRAT. In vitro activity assay showed that both metal chelators inhibited isomerohydrolase activity of recombinant RPE65. The addition of FeSO4 restored the enzymatic activity of the recombinant RPE65. Further, two specific iron-staining methods showed that purified RPE65 contains endogenous iron. Inductively coupled plasma mass spectrometry measurements showed that bovine RPE65 binds iron ion with a stoichiometry of 0.8 ± 0.1. These results indicate that RPE65 is an iron-dependent isomerohydrolase in the visual cycle

Retinol-Binding Protein 4 Induces Inflammation in Human Endothelial Cells by an NADPH Oxidase- and Nuclear Factor Kappa B-Dependent and Retinol-Independent Mechanism

ABSTRACT Serum retinol-binding protein 4 (RBP4) is the sole specific vitamin A (retinol) transporter in blood. Elevation of serum RBP4 in patients has been linked to cardiovascular disease and diabetic retinopathy. However, the significance of RBP4 elevation in the pathogenesis of these vascular diseases is unknown. Here we show that RBP4 induces inflammation in primary human retinal capillary endothelial cells (HRCEC) and human umbilical vein endothelial cells (HUVEC) by stimulating expression of proinflammatory molecules involved in leukocyte recruitment and adherence to endothelium, including vascular cell adhesion molecule 1 (VCAM-1), intercellular adhesion molecule 1 (ICAM-1), E-selectin, monocyte chemoattractant protein 1 (MCP-1), and interleukin-6 (IL-6). We demonstrate that these novel effects of RBP4 are independent of retinol and the RBP4 membrane receptor STRA6 and occur in part via activation of NADPH oxidase and NF-κB. Importantly, retinol-free RBP4 (apo-RBP4) was as potent as retinol-bound RBP4 (holo-RBP4) in inducing proinflammatory molecules in both HRCEC and HUVEC. These studies reveal that RBP4 elevation can directly contribute to endothelial inflammation and therefore may play a causative role in the development or progression of vascular inflammation during cardiovascular disease and microvascular complications of diabetes.

RDH10 is the primary enzyme responsible for the first step of embryonic Vitamin A metabolism and retinoic acid synthesis.

Retinoic acid (atRA) signaling is essential for regulating embryonic development, and atRA levels must be tightly controlled in order to prevent congenital abnormalities and fetal death which can result from both excessive and insufficient atRA signaling. Cellular enzymes synthesize atRA from Vitamin A, which is obtained from dietary sources. Embryos express multiple enzymes that are biochemically capable of catalyzing the initial step of Vitamin A oxidation, but the precise contribution of these enzymes to embryonic atRA synthesis remains unknown. Using Rdh10(trex)-mutant embryos, dietary supplementation of retinaldehyde, and retinol dehydrogenase (RDH) activity assays, we demonstrate that RDH10 is the primary RDH responsible for the first step of embryonic Vitamin A oxidation. Moreover, we show that this initial step of atRA synthesis occurs predominantly in a membrane-bound cellular compartment, which prevents inhibition by the cytosolic cellular retinol-binding protein (RBP1). These studies reveal that widely expressed cytosolic enzymes with RDH activity play a very limited role in embryonic atRA synthesis under normal dietary conditions. This provides a breakthrough in understanding the precise cellular mechanisms that regulate Vitamin A metabolism and the synthesis of the essential embryonic regulatory molecule atRA.

Identification of conserved histidines and glutamic acid as key residues for isomerohydrolase activity of RPE65, an enzyme of the visual cycle in the retinal pigment epithelium

We have recently reported that RPE65 from the retinal pigment epithelium is the isomerohydrolase, a critical enzyme in the visual cycle for regeneration of 11‐cis retinal, the chromophore for visual pigments. Here, we demonstrated that mutation of any one of the absolutely conserved four histidine and one glutamic acid residues to alanine in RPE65 abolished its isomerohydrolase activity. Substitution of the conserved glutamic acid with glutamine also resulted in loss of the activity. Moreover, these mutations significantly reduced protein stability of RPE65. These results indicate that these conserved residues are essential for the isomerohydrolase activity of RPE65 and its stability.

The 11-cis-Retinol Dehydrogenase Activity of RDH10 and Its Interaction with Visual Cycle Proteins

PURPOSE The final step in the retinoid visual cycle is catalyzed by 11-cis-retinol dehydrogenases (11-cis-RDHs) that oxidize 11-cis-retinol (11cROL) to 11-cis-retinaldehyde (11cRAL). Genetic studies in mice indicate that the full repertoire of 11-cis-RDH enzymes remains to be identified. This study was conducted to characterize the 11-cis-RDH activity of RDH10 in vitro and specifically to determine whether RDH10 can functionally and physically interact with visual cycle proteins. METHODS Human RDH10 was expressed in COS1 cells to measure its 11-cis-RDH activity in the presence or absence of purified recombinant cellular retinaldehyde-binding protein (CRALBP). The RPE visual cycle was reconstituted in HEK-293A cells by co-expressing RDH10, CRALBP, RPE-specific 65-kDa protein (RPE65) and lecithin retinol acyltransferase (LRAT). The cells were subsequently treated with all-trans-retinol (atROL), and retinoid profiles were quantified by HPLC. Immunocytochemical and co-immunoprecipitation analyses were performed to determine whether RDH10 physically interacts with other visual cycle proteins. RESULTS RDH10 oxidized 11cROL to generate 11cRAL in vitro in the presence of CRALBP. RDH10 can use both NAD(+) and NADP(+) as cofactors for 11-cis-RDH activity, although NAD(+) cofactor confers more robust activity. In a cell culture model co-expressing RDH10 with RPE65, LRAT and CRALBP, the visual chromophore 11cRAL was generated from atROL. Immunohistochemistry showed that RDH10 co-localizes with RPE65 and CRALBP in vivo in primary bovine RPE cells. Immunoprecipitation analysis demonstrated that RDH10 physically interacts with CRALBP and RPE65. CONCLUSIONS RDH10 may function in the RPE retinoid visual cycle as an 11-cis-RDH, and thereby partially compensate for the loss of RDH5 function in patients with fundus albipunctatus.

A1120, a Nonretinoid RBP4 Antagonist, Inhibits Formation of Cytotoxic Bisretinoids in the Animal Model of Enhanced Retinal Lipofuscinogenesis

PURPOSE Excessive accumulation of lipofuscin is associated with pathogenesis of atrophic age-related macular degeneration (AMD) and Stargardt disease. Pharmacologic inhibition of the retinol-induced interaction of retinol-binding protein 4 (RBP4) with transthyretin (TTR) in the serum may decrease the uptake of serum retinol to the retina and reduce formation of lipofuscin bisretinoids. We evaluated in vitro and in vivo properties of the new nonretinoid RBP4 antagonist, A1120. METHODS RBP4 binding potency, ability to antagonize RBP4-TTR interaction, and compound specificity were analyzed for A1120 and for the prototypic RBP4 antagonist fenretinide. A1120 ability to inhibit RPE65-mediated isomerohydrolase activity was assessed in the RPE microsomes. The in vivo effect of A1120 administration on serum RBP4, visual cycle retinoids, lipofuscin bisretinoids, and retinal visual function was evaluated using a combination of biochemical and electrophysiologic techniques. RESULTS In comparison to fenretinide, A1120 did not act as a RARα agonist, while exhibiting superior in vitro potency in RBP4 binding and RBP4-TTR interaction assays. A1120 did not inhibit isomerohydrolase activity in the RPE microsomes. A1120 dosing in mice induced 75% reduction in serum RBP4, which correlated with reduction in visual cycle retinoids and ocular levels of lipofuscin fluorophores. A1120 dosing did not induce changes in kinetics of dark adaptation. CONCLUSIONS A1120 significantly reduces accumulation of lipofuscin bisretinoids in the Abca4(-/-) animal model. This activity correlates with reduction in serum RBP4 and visual cycle retinoids confirming the mechanism of action for A1120. In contrast to fenretinide, A1120 does not act as a RARα agonist indicating a more favorable safety profile for this nonretinoid compound.

Inhibition of the visual cycle by A2E through direct interaction with RPE65 and implications in Stargardt disease

Stargardt disease (STGD) is the major form of inherited juvenile macular degeneration. Pyridinium bis-retinoid A2E is a major component of lipofuscin which accumulates in retinal pigment epithelium (RPE) cells in STGD and contributes to the disease pathogenesis. However, the precise role of A2E in vision loss is unclear. Here we report that A2E efficiently inhibits RPE65 isomerohydrolase, a key enzyme in the visual cycle. Subretinal injection of A2E significantly inhibited retinoid isomerohydrolase activity in mice. Likewise, A2E also inhibited isomerohydrolase activity in cells coexpressing RPE65, lecithin retinol acyltransferase (LRAT), and cellular retinaldehyde-binding protein. In vitro isomerohydrolase activity assays confirmed that A2E inhibited enzymatic activity of recombinant RPE65 in a concentration-dependent manner, but did not inhibit LRAT activity. The inhibition type for isomerohydrolase was found to be reversible and competitive with Ki = 13.6 μM. To determine the direct interaction of A2E with RPE65 protein, fluorescence binding assays were performed. As shown by fluorimetric titration, binding of purified RPE65 with A2E enhanced the bis-retinoid fluorescence. Consistently, the fluorescence of RPE65 decreased upon incubation with A2E. Both of these experiments suggest a direct, specific binding of A2E to RPE65. The binding constant for A2E and purified RPE65 was calculated to be 250 nM. These results demonstrate that A2E inhibits the regeneration of 11-cis retinal, the chromophore of visual pigments, which represents a unique mechanism by which A2E may impair vision in STGD.

Photoreceptor degeneration and retinal inflammation induced by very low-density lipoprotein receptor deficiency

Our previous studies have shown that very low-density lipoprotein receptor (VLDLR) is a negative regulator of the Wnt pathway. The present study showed that VLDLR gene knockout (Vldlr(-/-)) mice displayed impaired cone ERG responses at early ages. Immunostaining of mid-wavelength cones showed significantly decreased cone densities in the retina and shortened cone outer segments in Vldlr(-/-) mice. At older ages, Vldlr(-/-) mice displayed declined rod ERG responses, decreased layers of photoreceptor nuclei, reduced rhodopsin levels and decreased levels of 11-cis retinal, the chromophore of visual pigments. As shown by fluorescein angiography and permeability assay, Vldlr(-/-) mice had severe retinal vascular leakage. ZO-1, a tight junction protein, was down-regulated in Vldlr(-/-) mouse retinae, further supporting the impaired blood-retinal barrier. Double staining of pericytes and endothelial cells in retinal sections revealed that neovasculature in Vldlr(-/-) mice lacks pericyte coverage, suggesting impaired maturation of retinal vasculature in Vldlr(-/-) mice. Staining of adherent leukocytes in the retinal vasculature revealed significant leukostasis in Vldlr(-/-) mice. Moreover, Vldlr(-/-) mice displayed up-regulated expression of multiple pro-inflammatory factors and activated NF-kappaB and HIF-1 alpha, key regulators of inflammation. These findings suggest that deficiency of VLDLR leads to retinal degeneration and inflammation.

α-Phenyl-N-tert-butylnitrone (PBN) Prevents Light-induced Degeneration of the Retina by Inhibiting RPE65 Protein Isomerohydrolase Activity

α-Phenyl-N-tert-butylnitrone (PBN), a free radical spin trap, has been shown previously to protect retinas against light-induced neurodegeneration, but the mechanism of protection is not known. Here we report that PBN-mediated retinal protection probably occurs by slowing down the rate of rhodopsin regeneration by inhibiting RPE65 activity. PBN (50 mg/kg) protected albino Sprague-Dawley rat retinas when injected 0.5–12 h before exposure to damaging light at 2,700 lux intensity for 6 h but had no effect when administered after the exposure. PBN injection significantly inhibited in vivo recovery of rod photoresponses and the rate of recovery of functional rhodopsin photopigment. Assays for visual cycle enzyme activities indicated that PBN inhibited one of the key enzymes of the visual cycle, RPE65, with an IC50 = 0.1 mm. The inhibition type for RPE65 was found to be uncompetitive with Ki = 53 μm. PBN had no effect on the activity of other visual cycle enzymes, lecithin retinol acyltransferase and retinol dehydrogenases. Interestingly, a more soluble form of PBN, N-tert-butyl-α-(2-sulfophenyl) nitrone, which has similar free radical trapping activity, did not protect the retina or inhibit RPE65 activity, providing some insight into the mechanism of PBN specificity and action. Slowing down the visual cycle is considered a treatment strategy for retinal diseases, such as Stargardt disease and dry age-related macular degeneration, in which toxic byproducts of the visual cycle accumulate in retinal cells. Thus, PBN inhibition of RPE65 catalytic action may provide therapeutic benefit for such retinal diseases.