Retinal Responses to Visual Stimuli in Interphotoreceptor Retinoid Binding-Protein Knock-Out Mice.

Interphotoreceptor retinoid-binding protein (IRBP) is an abundant glycoprotein in the subretinal space bound by the photoreceptor (PR) outer segments and the processes of the retinal pigmented epithelium (RPE). IRBP binds retinoids, including 11-cis-retinal and all-trans-retinol. In this study, visual function for demanding visual tasks was assessed in IRBP knock-out (KO) mice. Surprisingly, IRBP KO mice showed no differences in scotopic critical flicker frequency (CFF) compared to wildtype (WT). However, they did have lower photopic CFF than WT. IRBP KO mice had reduced scotopic and photopic acuity and contrast sensitivity compared to WT. IRBP KO mice had a significant reduction in outer nuclear layer (ONL) thickness, PR outer and inner segment, and full retinal thickness (FRT) compared to WT. There were fewer cones in IRBP KO mice. Overall, these results confirm substantial loss of rods and significant loss of cones within 30 days. Absence of IRBP resulted in cone circuit damage, reducing photopic flicker, contrast sensitivity, and spatial frequency sensitivity. The c-wave was reduced and accelerated in response to bright steps of light. This result also suggests altered retinal pigment epithelium activity. There appears to be a compensatory mechanism such as higher synaptic gain between PRs and bipolar cells since the loss of the b-wave did not linearly follow the loss of rods, or the a-wave. Scotopic CFF is normal despite thinning of ONL and reduced scotopic electroretinogram (ERG) in IRBP KO mice, suggesting either a redundancy or plasticity in circuits detecting (encoding) scotopic flicker at threshold even with substantial rod loss.

Novel Functions of the Fatty Acid and Retinol Binding Protein (FAR) Gene Family Revealed by Fungus-Mediated RNAi in the Parasitic Nematode, Aphelenchoides besseyi.

RNA interference (RNAi) is a powerful tool for the analysis of gene function in nematodes. Fatty acid and retinol binding protein (FAR) is a protein that only exists in nematodes and plays an important role in their life activities. The rice white-tip nematode (RWTN), Aphelenchoides besseyi, is a migratory endoparasitic plant nematode that causes serious damage in agricultural production. In this study, the expression levels of eight RWTN genes were effectively decreased when RWTN was fed Ab-far-n (n: 1-8) hairpin RNA transgenic Botrytis cinerea (ARTBn). These functions of the far gene family were identified to be consistent and diverse through phenotypic changes after any gene was silenced. Such consistency indicates that the body lengths of the females were significantly shortened after silencing any of the eight Ab-far genes. The diversities were mainly manifested as follows: (1) Reproduction of nematodes was clearly inhibited after Ab-far-1 to Ab-far-4 were silenced. In addition, silencing Ab-far-2 could inhibit the pathogenicity of nematodes to Arabidopsis; (2) gonad length of female nematodes was significantly shortened after Ab-far-2 and Ab-far-4 were silenced; (3) proportion of male nematodes significantly increased in the adult population after Ab-far-1, Ab-far-3, and Ab-far-5 were silenced, whereas the proportion of adult nematodes significantly decreased in the nematode population after Ab-far-4 were silenced. (4) Fat storage of nematodes significantly decreased after Ab-far-3, Ab-far-4, and Ab-far-7 were silenced. To our knowledge, this is the first study to demonstrate that Ab-far genes affect sex formation and lipid metabolism in nematodes, which provides valuable data for further study and control of RWTNs.

Interphotoreceptor retinoid-binding protein removes all-trans-retinol and retinal from rod outer segments, preventing lipofuscin precursor formation.

Interphotoreceptor retinoid-binding protein (IRBP) is a specialized lipophilic carrier that binds the all-trans and 11-cis isomers of retinal and retinol, and this facilitates their transport between photoreceptors and cells in the retina. One of these retinoids, all-trans-retinal, is released in the rod outer segment by photoactivated rhodopsin after light excitation. Following its release, all-trans-retinal is reduced by the retinol dehydrogenase RDH8 to all-trans-retinol in an NADPH-dependent reaction. However, all-trans-retinal can also react with outer segment components, sometimes forming lipofuscin precursors, which after conversion to lipofuscin accumulate in the lysosomes of the retinal pigment epithelium and display cytotoxic effects. Here, we have imaged the fluorescence of all-trans-retinol, all-trans-retinal, and lipofuscin precursors in real time in single isolated mouse rod photoreceptors. We found that IRBP removes all-trans-retinol from individual rod photoreceptors in a concentration-dependent manner. The rate constant for retinol removal increased linearly with IRBP concentration with a slope of 0.012 min-1 µm-1 IRBP also removed all-trans-retinal, but with much less efficacy, indicating that the reduction of retinal to retinol promotes faster clearance of the photoisomerized rhodopsin chromophore. The presence of physiological IRBP concentrations in the extracellular medium resulted in lower levels of all-trans-retinal and retinol in rod outer segments following light exposure. It also prevented light-induced lipofuscin precursor formation, but it did not remove precursors that were already present. These findings reveal an important and previously unappreciated role of IRBP in protecting the photoreceptor cells against the cytotoxic effects of accumulated all-trans-retinal.

Adipokines and cardiovascular disease: A comprehensive review.

Adipokines are peptides that signal the functional status of adipose tissue to the brain and other target organs. In adipose tissue dysfunction, adipokine secretion is altered, and this can contribute to a spectrum of obesity-associated conditions including cardiovascular disease. Some adipokines have anti-inflammatory and cardioprotective effects (omentin, apelin, adiponectin). Others are pro-inflammatory with negative impact on cardiovascular function (leptin, visfatin, resistin, adipocyte fatty-acid-binding protein). In the first part, this article reviews the endocrine functions of adipose tissue in general, effects of the distribution and composition of fat tissue, and the roles of cortisol and the renin-angiotensin-aldosterone system in the development of the inflammatory state of addipose tissue. In the second part, the known cardiovascular effects of different adipokines and their clinical potential are discussed in detail.

Fold conservation and proteolysis in zebrafish IRBP structure: Clues to possible enzymatic function?

Multiple functions for Interphotoreceptor Retinoid-Binding Protein (IRBP) may explain its localization in the retina, vitreous and pineal gland and association with retinitis pigmentosa and myopia. We have been engaged in uncovering the structure-function relationships of this interesting protein long thought to bind visual-cycle retinoids and fatty acids in the subretinal space. Although hydrophobic domains capable of binding such ligands have now been found, we ask what other structural domains might be present that could predict new functions? Interestingly, IRBP possesses a fold similar to C-terminal processing proteases (CTPases) but is missing the PDZ domain. Here we present structural evidence that this fold may have a role in a recently observed autoproteolytic activity of the two-module zebrafish (z) IRBP (Ghosh et al. Exp. Eye Res., 2015). When the structure of Scenedesmus obliquus D1 CTPase (D1P) is superimposed with the first module of zIRBP (z1), the PDZ domain of D1P occupies roughly the same position in the amino acid sequence as the inter-domain tether in z1, between residues P71 and P85. The catalytic triad K397, S372 and E375 of D1P is located at the inter-domain interfacial cleft, similarly as the tetrad K241, S243, D177 and T179 of z1 residues, presumed to have proteolytic function. Packing of two adjacent symmetry-related molecules within the z1 crystal show that the helix α8 penetrates the interfacial cleft underneath the inter-domain tether, forming a simple intermolecular "knot". The full-length zIRBP is cleaved at or immediately after T309, which is located at the end of α8 and is the ninth residue of the second module z2. We propose that the helix α8 within intact zIRBP bends at P301, away from the improbable knotted fold, and positions the cleavage site T309 near the putative catalytic tetrad of the neighboring zIRBP to be proteolytically cleaved. The conservation of this functional catalytic domain suggests that possible physiological roles of IRBP as a hydrolase needs to be considered.

The peptidic middle molecules: is molecular weight doing the trick?

Chronic kidney disease (CKD) is characterized by a gradual endogenous intoxication caused by the progressive accumulation of bioactive compounds that in normal conditions would be excreted and/or metabolized by the kidney. Uremic toxicity now is understood as one of the potential causes for the excess of cardiovascular disease and mortality observed in CKD. An important family of uremic toxins is that of the peptidic middle molecules, with a molecular weight ranging between 500 and 60,000 Da, which makes them, as a consequence, difficult to remove in the process of dialysis unless the dialyzer pore size is large enough. This review provides an overview of the main and best-characterized peptidic middle molecules and their role as potential culprits of the cardiometabolic complications inherent to CKD patients.

The use of cultured human fetal retinal pigment epithelium in studies of the classical retinoid visual cycle and retinoid-based disease processes.

Human fetal retinal pigment epithelium (hfRPE), when harvested by mechanical dissection and cultured initially under low calcium conditions, will proliferate and tolerate cryopreservation for future use. Cryopreserved cells can be subsequently thawed and cultured in standard calcium and in the presence of appropriate nutrients to a high state of differentiation, allowing recapitulation of multiple in vivo functions. In this review we briefly discuss some of our previous studies of the classical retinoid visual cycle and introduce current studies in our laboratory that involve two new areas of investigation; the dynamic response of the receptor for retinol binding protein, STRA6 to the addition of holo-retinol binding protein to the culture medium and the protective complement-based response of hfRPE to the ingestion of toxic byproducts of the visual cycle. This response is studied in the context of genotyped hfRPE expressing either predisposing or protective variants of complement factor H.

Early involvement of nitric oxide in mechanisms of pathogenesis of experimental autoimmune uveitis induced by interphotoreceptor retinoid-binding protein (IRBP).

INTRODUCTION: Autoimmune uveitis is a group of HLA-associated inflammatory diseases of the eye, prevalent worldwide, that may cause blindness. It can be limited to the eye, or associated with a systemic syndrome. Furthermore, patients suffering from uveitis exhibit high serum and local nitric oxide (NO) levels as a consequence of cellular responses to immunologically privileged antigens within the eye such as interphotoreceptor retinoid binding protein (IRBP). To investigate NO production kinetics in autoimmune uveitis and its implication in mechanisms of ocular pathogenesis, we first attempted to develop an experimental model of autoimmune uveitis (EAU) on the Wistar rat, using the whole bovine retinal interphotoreceptor matrix extract (IPMe) and isolated IRBP. MATERIAL AND METHODS: Female Wistar rats (n=24) were divided into three experimental groups: "control rats" (n=3) consisting of non-immunized animals, "IRBP-immunized rats" (n=12) and "IPMe-immunized rats" (n=9), which received a subcutaneous injection, respectively, of 13 µg IRBP and 100 µg IPMe emulsified in complete Freund's adjuvant. On days 7, 14 and 21 post immunization, the rats were sacrificed. Nitrites were assessed in plasma and in homogenate of eyes using the Griess reaction. Meanwhile, eyes were collected for histological studies. RESULTS: Our results show the sensitivity of the Wistar strain to both IPMe and IRBP-induced EAU. In fact, we observed histological disorders affecting the retinal tissue in both models of EAU. On the other hand, a significantly increased production of NO in plasma and homogenate of eyes was also observed in comparison to the control group. Moreover, we noted with interest that maximal production of NO occurs prior to the alteration of retinal tissue. CONCLUSION: In summary, our results suggest the early involvement of NO in the mechanisms of pathogenesis of EAU. NO can be considered as a key bio-marker of poor prognosis in ocular autoimmune inflammation.

Normal cone function requires the interphotoreceptor retinoid binding protein.

11-cis-retinal is the light-sensitive component in rod and cone photoreceptors, and its isomerization to all-trans retinal in the presence of light initiates the visual response. For photoreceptors to function normally, all-trans retinal must be converted back into 11-cis-retinal through a series of enzymatic steps known as the visual cycle. The interphotoreceptor retinoid-binding protein (IRBP) is a proposed retinoid transporter in the visual cycle, but rods in Irbp(-/-) mice have a normal visual cycle. While rods are primarily responsible for dim light vision, the ability of cones to function in constant light is essential to human vision and may be facilitated by cone-specific visual cycle pathways. We analyzed the cones in Irbp(-/-) mice to determine whether IRBP has a cone-specific visual cycle function. Cone electroretinogram (ERG) responses were reduced in Irbp(-/-) mice, but similar responses from Irbp(-/-) mice at all ages suggest that degeneration does not underlie cone dysfunction. Furthermore, cone densities and opsin levels in Irbp(-/-) mice were similar to C57BL/6 (wild-type) mice, and both cone opsins were properly localized to the cone outer segments. To test for retinoid deficiency in Irbp(-/-) mice, ERGs were analyzed before and after intraperitoneal injections of 9-cis-retinal. Treatment with 9-cis-retinal produced a significant recovery of the cone response in Irbp(-/-) mice and shows that retinoid deficiency underlies cone dysfunction. These data indicate that IRBP is essential to normal cone function and demonstrate that differences exist in the visual cycle of rods and cones.

Retinoid induction of alveolar regeneration: from mice to man?

The use of retinoids to induce human lung regeneration is under investigation in a number of studies in patients with chronic obstructive pulmonary disease (COPD). Retinoic acid (RA) has complex pleiotropic functions during vertebrate patterning and development and can induce regeneration in a number of different organ systems. Studies of retinoid signalling during lung development might provide a molecular basis to explain pharmacological induction of alveolar regeneration in adult models of lung disease. In this review the role of endogenous RA signalling during alveologenesis is explored and data suggesting that a number of exogenous retinoids can induce regeneration in the adult lung are discussed. Current controversies in this area are highlighted and a hypothesis of lung regeneration is put forward. Understanding the cellular and molecular mechanisms of induction of regeneration will be central for effective translation into patients with lung disease and may reveal novel insights into the pathogenesis of alveolar disease and senescence.

The role of interphotoreceptor retinoid-binding protein on the translocation of visual retinoids and function of cone photoreceptors.

The first event in light perception is absorption of a photon by the retinaldehyde chromophore of an opsin pigment in a rod or cone photoreceptor cell. This induces isomerization of the chromophore, rendering the bleached pigment insensitive to light. Restoration of light sensitivity requires chemical reisomerization of retinaldehyde via a multistep enzyme pathway, called the visual cycle, in cells of the retinal pigment epithelium (RPE). Interphotoreceptor retinoid-binding protein (IRBP) is present in the extracellular space between photoreceptors and the RPE. IRBP is known to bind visual retinoids. Previous studies on irbp(-/-) mice suggested that IRBP plays an insignificant role in opsin-pigment regeneration. However, the mice in these studies were uncontrolled for a severe mutation in the rpe65 gene. Rpe65 catalyzes the rate-limiting step in the visual cycle. Here, we examined the phenotype in irbp(-/-) mice homozygous for the wild-type (Leu450) rpe65 gene. We show that lack of IRBP causes delayed transfer of newly synthesized chromophore from RPE to photoreceptors. Removal of bleached chromophore from photoreceptors is also delayed in irbp(-/-) retinas after light exposure. It was previously shown that rods degenerate in irbp(-/-) mice. Here, we show that cones and rods degenerate at similar rates. However, cones are more affected functionally and show greater reductions in outer segment length than rods in irbp(-/-) mice. The disproportionate reductions in cone function and outer-segment length appear to result from mistrafficking of cone opsins due to impaired delivery of retinaldehyde chromophore, which functions as a chaperone for cone opsins but not rhodopsin.

Subfunctionalization of a retinoid-binding protein provides evidence for two parallel visual cycles in the cone-dominant zebrafish retina.

In vertebrates, the absorption of a photon results in an 11-cis to all-trans isomerization of the retinylidene chromophore of cone and rod visual pigments. To sustain vision, metabolic pathways (visual cycles) have evolved that recycle all-trans-retinal back to 11-cis-retinal. The canonical visual cycle takes place in photoreceptor cells and the adjacent retinal pigment epithelium (RPE). Biochemical analyses provided evidence for the existence of an additional cone-specific visual cycle involving Müller glia cells, but none of its molecular components has yet been identified. Here we took advantage of the zebrafish retina to investigate the role of the cellular retinaldehyde-binding protein CRALBP in this process. We found that the zebrafish genome encodes two cralbp paralogs: cralbp a and cralbp b. These paralogs are differentially expressed in the retina. Cralbp a is exclusively expressed in the RPE, and Cralbp b is localized to Müller cells. We used an antisense morpholino approach to knock down each cralbp paralog. Analysis of 11-cis-retinal levels revealed that visual chromophore regeneration is diminished under both conditions. Visual performance, as assessed by electroretinography, revealed reduced light sensitivity in both Cralbp a- and Cralbp b-deficient larvae, but it was more pronounced in Cralbp b-deficient larvae. Cralbp b-deficient larvae also exhibited significant deficits in their visual behavior. Together, these data demonstrate that Cralbp expression in Müller cells is essential for cone vision, thereby providing evidence that both the canonical and the alternative visual cycle depend on the same type of retinoid-binding protein.

Module structure of interphotoreceptor retinoid-binding protein (IRBP) may provide bases for its complex role in the visual cycle - structure/function study of Xenopus IRBP.

BACKGROUND: Interphotoreceptor retinoid-binding protein's (IRBP) remarkable module structure may be critical to its role in mediating the transport of all-trans and 11-cis retinol, and 11-cis retinal between rods, cones, RPE and Müller cells during the visual cycle. We isolated cDNAs for Xenopus IRBP, and expressed and purified its individual modules, module combinations, and the full-length polypeptide. Binding of all-trans retinol, 11-cis retinal and 9-(9-anthroyloxy) stearic acid were characterized by fluorescence spectroscopy monitoring ligand-fluorescence enhancement, quenching of endogenous protein fluorescence, and energy transfer. Finally, the X-ray crystal structure of module-2 was used to predict the location of the ligand-binding sites, and compare their structures among modules using homology modeling. RESULTS: The full-length Xenopus IRBP cDNA codes for a polypeptide of 1,197 amino acid residues beginning with a signal peptide followed by four homologous modules each approximately 300 amino acid residues in length. Modules 1 and 3 are more closely related to each other than either is to modules 2 and 4. Modules 1 and 4 are most similar to the N- and C-terminal modules of the two module IRBP of teleosts. Our data are consistent with the model that vertebrate IRBPs arose through two genetic duplication events, but that the middle two modules were lost during the evolution of the ray finned fish. The sequence of the expressed full-length IRBP was confirmed by liquid chromatography-tandem mass spectrometry. The recombinant full-length Xenopus IRBP bound all-trans retinol and 11-cis retinaldehyde at 3 to 4 sites with Kd's of 0.2 to 0.3 microM, and was active in protecting all-trans retinol from degradation. Module 2 showed selectivity for all-trans retinol over 11-cis retinaldehyde. The binding data are correlated to the results of docking of all-trans-retinol to the crystal structure of Xenopus module 2 suggesting two ligand-binding sites. However, homology modeling of modules 1, 3 and 4 indicate that both sites may not be available for binding of ligands in all four modules. CONCLUSION: Although its four modules are homologous and each capable of supporting ligand-binding activity, structural differences between their ligand-binding domains, and interactions between the modules themselves will be critical to understanding IRBP's complex role in the visual cycle.

Visceral and subcutaneous adiposity: are both potential therapeutic targets for tackling the metabolic syndrome?

The metabolic syndrome represents a constellation of co-morbidities that include central adiposity, insulin resistance, dyslipidemia and hypertension, which results from an elevated prevalence of obesity. An increased abdominal adiposity is observed in upper-body obesity with preferential accumulation of fat in the visceral depot, which renders these individuals more prone to metabolic and cardiovascular problems. The pathophysiology of the metabolic syndrome seems to be closely associated to an elevated efflux of free fatty acids from the visceral fat compartment and a dysregulation of the expression of adipose tissue-derived factors (also termed "adipokines"). Weight reduction and increased physical activity represent the main approach to tackle the "diabesity" epidemic. Nonetheless, taking advantage of the different biochemical and molecular characteristics of visceral and subcutaneous adipose tissue may open up novel pharmacological strategies to combat the metabolic and cardiovascular derangements accompanying the metabolic syndrome.

RBP2 is an MRG15 complex component and down-regulates intragenic histone H3 lysine 4 methylation.

MRG15 is a conserved chromodomain protein that associates with histone deacetylases (HDACs) and Tip60-containing histone acetyltransferase (HAT) complexes. Here we further characterize MRG15-containing complexes and show a functional link between MRG15 and histone H3K4 demethylase activity in mammalian cells. MRG15 was predominantly localized to discrete nuclear subdomains enriched for Ser(2)-phosphorylated RNA polymerase II, suggesting it is involved specifically with active transcription. Protein analysis of the MRG15-containing complexes led to the identification of RBP2, a JmjC domain-containing protein. Remarkably, over-expression of RBP2 greatly reduced the H3K4 methylation in culture human cells in vivo, and recombinant RBP2 efficiently removed H3K4 methylation of histone tails in vitro. Knockdown of RBP2 resulted in increased H3K4 methylation levels within transcribed regions of active genes. Our findings demonstrate that RBP2 associated with MRG15 complex to maintain reduced H3K4 methylation at transcribed regions, which may ensure the transcriptional elongation state.