The Amphipathic Helix in Visual Cycle Proteins: A Review.

The visual cycle is a complex biological process that involves the sequential action of proteins in the retinal pigment epithelial (RPE) cells and photoreceptors to modify and shuttle visual retinoids. A majority of the visual cycle proteins are membrane proteins, either integral or peripheral membrane proteins. Despite significant progress in understanding their physiological function, very limited structural information is available for the visual cycle proteins. Moreover, the mechanism of membrane interaction is not yet clear in all cases. Here, we demonstrate the presence of an amphipathic helix in selected RPE visual cycle proteins, using in silico tools, and highlight their role in membrane association and function.

The downregulation of HSP90-controlled CRALBP expression is associated with age-related vision attenuation.

The dysfunction of CRALBP, a key regulator of the visual cycle, is associated with retinitis punctata albescens characterized by night vision loss and retinal degeneration. In this paper, we find that the expression of CRALBP is regulated by heat shock protein 90 (HSP90). Inhibition of HSP90α or HSP90ß expression by using the CRISPR-Cas9 technology downregulates CRALBP's mRNA and protein expression in ARPE-19 cells by triggering the degradation of transcription factor SP1 in the ubiquitin-proteasome pathway. SP1 can bind to CRALBP's promoter, and inhibition of SP1 by its inhibitor plicamycin or siRNA downregulates CRALBP's mRNA expression. In the zebrafish, inhibition of HSP90 by the intraperitoneal injection of IPI504 reduces the thickness of the retinal outer nuclear layer and Rlbp1b mRNA expression. Interestingly, the expression of HSP90, SP1, and CRALBP is correlatedly downregulated in the senescent ARPE-19 and Pig primary RPE cells in vitro and in the aged zebrafish and mouse retinal tissues in vivo. The aged mice exhibit the low night adaption activity. Taken together, these data indicate that the HSP90-SP1 is a novel regulatory axis of CRALBP transcriptional expression in RPE cells. The age-mediated downregulation of the HSP90-SP1-CRALBP axis is a potential etiology for the night vision reduction in senior people.

Disturbed retinoid metabolism upon loss of rlbp1a impairs cone function and leads to subretinal lipid deposits and photoreceptor degeneration in the zebrafish retina.

The RLBP1 gene encodes the 36 kDa cellular retinaldehyde-binding protein, CRALBP, a soluble retinoid carrier, in the visual cycle of the eyes. Mutations in RLBP1 are associated with recessively inherited clinical phenotypes, including Bothnia dystrophy, retinitis pigmentosa, retinitis punctata albescens, fundus albipunctatus, and Newfoundland rod-cone dystrophy. However, the etiology of these retinal disorders is not well understood. Here, we generated homologous zebrafish models to bridge this knowledge gap. Duplication of the rlbp1 gene in zebrafish and cell-specific expression of the paralogs rlbp1a in the retinal pigment epithelium and rlbp1b in Müller glial cells allowed us to create intrinsically cell type-specific knockout fish lines. Using rlbp1a and rlbp1b single and double mutants, we investigated the pathological effects on visual function. Our analyses revealed that rlbp1a was essential for cone photoreceptor function and chromophore metabolism in the fish eyes. rlbp1a-mutant fish displayed reduced chromophore levels and attenuated cone photoreceptor responses to light stimuli. They accumulated 11-cis and all-trans-retinyl esters which displayed as enlarged lipid droplets in the RPE reminiscent of the subretinal yellow-white lesions in patients with RLBP1 mutations. During aging, these fish developed retinal thinning and cone and rod photoreceptor dystrophy. In contrast, rlbp1b mutants did not display impaired vision. The double mutant essentially replicated the phenotype of the rlbp1a single mutant. Together, our study showed that the rlbp1a zebrafish mutant recapitulated many features of human blinding diseases caused by RLBP1 mutations and provided novel insights into the pathways for chromophore regeneration of cone photoreceptors.

YPIBP: A repository for phosphoinositide-binding proteins in yeast.

Phosphoinositides (PIs) are a family of eight lipids consisting of phosphatidylinositol (PtdIns) and its seven phosphorylated forms. PIs have important regulatory functions in the cell including lipid signaling, protein transport, and membrane trafficking. Yeast has been recognized as a eukaryotic model system to study lipid-protein interactions. Hundreds of yeast PI-binding proteins have been identified, but this research knowledge remains scattered. Besides, the complete PI-binding spectrum and potential PI-binding domains have not been interlinked. No comprehensive databases are available to support the lipid-protein interaction research on phosphoinositides. Here we constructed the first knowledgebase of Yeast Phosphoinositide-Binding Proteins (YPIBP), a repository consisting of 679 PI-binding proteins collected from high-throughput proteome-array and lipid-array studies, QuickGO, and a rigorous literature mining. The YPIBP also contains protein domain information in categories of lipid-binding domains, lipid-related domains and other domains. The YPIBP provides search and browse modes along with two enrichment analyses (PI-binding enrichment analysis and domain enrichment analysis). An interactive visualization is given to summarize the PI-domain-protein interactome. Finally, three case studies were given to demonstrate the utility of YPIBP. The YPIBP knowledgebase consolidates the present knowledge and provides new insights of the PI-binding proteins by bringing comprehensive and in-depth interaction network of the PI-binding proteins. YPIBP is available at http://cosbi7.ee.ncku.edu.tw/YPIBP/.

Retinitis Punctata Albescens and RLBP1-Allied Phenotypes: Phenotype-Genotype Correlation and Natural History in the Aim of Gene Therapy.

Purpose: To identify relevant criteria for gene therapy based on clinical and genetic characteristics of rod-cone dystrophy associated with RLBP1 pathogenic variants in a large cohort comprising children and adults. Design: Retrospective cohort study. Participants: Patients with pathogenic variants in RLBP1 registered in a single French reference center specialized in inherited retinal dystrophies. Methods: Clinical, multimodal imaging, and genetic findings were reviewed. Main Outcome Measures: Age of onset; visual acuity; ellipsoid line length; nasal, temporal, and foveal retinal thickness; and pathogenic variants and related phenotypes, including Newfoundland rod-cone and Bothnia dystrophies (NFRCDs), were reappraised. Results: Twenty-one patients (15 families) were included. The most frequent form was NFRCD with 12 patients (8 families) homozygous for the recurrent deletion of exons 7 through 9 in RLBP1 and 5 patients (4 families) with biallelic protein-truncating variants (2 novel: p.Gln16∗ and p.Tyr251∗). A novel combination of the p.Arg234Trp Bothnia variant with a nonsense variant in trans led to Bothnia dystrophy in 2 sisters. One proband carrying the p.Met266Lys Bothnia variant and in trans p.Arg121Trp and a second, with the p.Arg9Cys and p.Tyr111∗ combination, both demonstrated mild retinitis punctata albescens. Independently of genotype, all patients showed a visual acuity of worse than 20/200, an ellipsoid line width of less than 1000 µm, and a mean foveal thickness of less than 130 to 150 µm, with loss of both the interdigitation and ellipsoid lines. Conclusions: The eligibility for RLBP1 gene therapy first should be determined according to the biallelic variant combination using a robust classification as proposed herein. An ellipsoid line width of more than 1200 µm and a central thickness of more than 130 to 150 µm with detectable ellipsoid and interdigitation lines should be 2 prerequisite imaging indicators for gene therapy.

Effects of deficiency in the RLBP1-encoded visual cycle protein CRALBP on visual dysfunction in humans and mice.

Mutations in retinaldehyde-binding protein 1 (RLBP1), encoding the visual cycle protein cellular retinaldehyde-binding protein (CRALBP), cause an autosomal recessive form of retinal degeneration. By binding to 11-cis-retinoid, CRALBP augments the isomerase activity of retinoid isomerohydrolase RPE65 (RPE65) and facilitates 11-cis-retinol oxidation to 11-cis-retinal. CRALBP also maintains the 11-cis configuration and protects against unwanted retinaldehyde activity. Studying a sibling pair that is compound heterozygous for mutations in RLBP1/CRALBP, here we expand the phenotype of affected individuals, elucidate a previously unreported phenotype in RLBP1/CRALBP carriers, and demonstrate consistencies between the affected individuals and Rlbp1/Cralbp-/- mice. In the RLBP1/CRALBP-affected individuals, nonrecordable rod-specific electroretinogram traces were recovered after prolonged dark adaptation. In ultrawide-field fundus images, we observed radially arranged puncta typical of RLBP1/CRALBP-associated disease. Spectral domain-optical coherence tomography (SD-OCT) revealed hyperreflective aberrations within photoreceptor-associated bands. In short-wavelength fundus autofluorescence (SW-AF) images, speckled hyperautofluorescence and mottling indicated macular involvement. In both the affected individuals and their asymptomatic carrier parents, reduced SW-AF intensities, measured as quantitative fundus autofluorescence (qAF), indicated chronic impairment in 11-cis-retinal availability and provided information on mutation severity. Hypertransmission of the SD-OCT signal into the choroid together with decreased near-infrared autofluorescence (NIR-AF) provided evidence for retinal pigment epithelial cell (RPE) involvement. In Rlbp1/Cralbp-/- mice, reduced 11-cis-retinal levels, qAF and NIR-AF intensities, and photoreceptor loss were consistent with the clinical presentation of the affected siblings. These findings indicate that RLBP1 mutations are associated with progressive disease involving RPE atrophy and photoreceptor cell degeneration. In asymptomatic carriers, qAF disclosed previously undetected visual cycle deficiency.

A retinal-binding protein mediates olfactory attraction in the migratory locusts.

Cellular retinaldehyde-binding protein (CRALBP) is abundantly expressed in retina and its mutations are associated with visual impairments. The functions of CRALBP are less known in extra retinal tissues. Herein, we study the function of CRALBP in modulating olfactory behaviors in gregarious and solitary locusts. The expressions of cralbp mRNA and protein were enriched in locust brains and antennae. RNAi knockdown of cralbp in gregarious locusts decreased their attractive response to gregarious volatiles. RNA-seq and quantitative PCR confirmed that cralbp mRNA and protein expression levels were upregulated and downregulated after octopamine receptor α1 (OctαR1) activation and inhibition, respectively. Gene network analysis revealed that cralbp is the core hub gene in the interactive network among differentially expressed transcripts (DETs) resulting from activating and inhibiting OctαR1. Moreover, cralbp RNAi knockdown inhibited the induction of olfactory attraction by octopamine (OA)-OctαR1 signaling. CRALBP helped to transmit OA signals to mediate olfactory attraction response to guaiacol and veratrole, which are two odorant components in gregarious volatiles. This study suggested that CRALBP may act as a novel effector protein in OctαR1 signaling to mediate olfactory attraction. This study indicated that CRALBP modulates olfactory attraction in extra retina tissues and retinaldehyde metabolism may be crucial for olfactory attraction modulation.

Enhancing Understanding of the Visual Cycle by Applying CRISPR/Cas9 Gene Editing in Zebrafish.

During the vertebrate visual cycle, all-trans-retinal is exported from photoreceptors to the adjacent RPE or Müller glia wherein 11-cis-retinal is regenerated. The 11-cis chromophore is returned to photoreceptors, forming light-sensitive visual pigments with opsin GPCRs. Dysfunction of this process perturbs phototransduction because functional visual pigment cannot be generated. Mutations in visual cycle genes can result in monogenic inherited forms of blindness. Though key enzymatic processes are well characterized, questions remain as to the physiological role of visual cycle proteins in different retinal cell types, functional domains of these proteins in retinoid biochemistry and in vivo pathogenesis of disease mutations. Significant progress is needed to develop effective and accessible treatments for inherited blindness arising from mutations in visual cycle genes. Here, we review opportunities to apply gene editing technology to two crucial visual cycle components, RPE65 and CRALBP. Expressed exclusively in the human RPE, RPE65 enzymatically converts retinyl esters into 11-cis retinal. CRALBP is an 11-cis-retinal binding protein expressed in human RPE and Muller glia. Loss-of-function mutations in either protein results in autosomal recessive forms of blindness. Modeling these human conditions using RPE65 or CRALBP murine knockout models have enhanced our understanding of their biochemical function, associated disease pathogenesis and development of therapeutics. However, rod-dominated murine retinae provide a challenge to assess cone function. The cone-rich zebrafish model is amenable to cost-effective maintenance of a variety of strains. Interestingly, gene duplication in zebrafish resulted in three Rpe65 and two Cralbp isoforms with differential temporal and spatial expression patterns. Functional investigations of zebrafish Rpe65 and Cralbp were restricted to gene knockdown with morpholino oligonucleotides. However, transient silencing, off-target effects and discrepancies between knockdown and knockout models, highlight a need for more comprehensive alternatives for functional genomics. CRISPR/Cas9 in zebrafish has emerged as a formidable technology enabling targeted gene knockout, knock-in, activation, or silencing to single base-pair resolution. Effective, targeted gene editing by CRISPR/Cas9 in zebrafish enables unprecedented opportunities to create genetic research models. This review will discuss existing knowledge gaps regarding RPE65 and CRALBP. We explore the benefits of CRISPR/Cas9 to establish innovative zebrafish models to enhance knowledge of the visual cycle.

Nonclinical Safety Evaluation of scAAV8-RLBP1 for Treatment of RLBP1 Retinitis Pigmentosa.

Retinitis pigmentosa is a form of retinal degeneration usually caused by genetic mutations affecting key functional proteins. We have previously demonstrated efficacy in a mouse model of RLBP1 deficiency with a self-complementary AAV8 vector carrying the gene for human RLBP1 under control of a short RLBP1 promoter (CPK850).1 In this article, we describe the nonclinical safety profile of this construct as well as updated efficacy data in the intended clinical formulation. In Rlbp1-/- mice dosed at a range of CPK850 levels, a minimum efficacious dose of 3 × 107 vg in a volume of 1 µL was observed. For safety assessment in these and Rlbp1+/+ mice, optical coherence tomography (OCT) and histopathological analysis indicated retinal thinning that appeared to be dose-dependent for both Rlbp1 genotypes, with no qualitative difference noted between Rlbp1+/+ and Rlbp1-/- mice. In a non-human primate study, RLBP1 mRNA expression was detected and dose dependent intraocular inflammation and retinal thinning were observed. Inflammation resolved slowly over time and did not appear to be exacerbated in the presence of anti-AAV8 antibodies. Biodistribution was evaluated in rats and satellite animals in the non-human primate study. The vector was largely detected in ocular tissues and low levels in the optic nerve, superior colliculus, and lateral geniculate nucleus, with limited distribution outside of these tissues. These data suggest that an initial subretinal dose of ∼3 × 107 vg/µL CPK850 can safely be used in clinical trials.

Vitamin A and Vision.

Visual systems detect light by monitoring the effect of photoisomerization of a chromophore on the release of a neurotransmitter from sensory neurons, known as rod and cone photoreceptor cells in vertebrate retina. In all known visual systems, the chromophore is 11-cis-retinal complexed with a protein, called opsin, and photoisomerization produces all-trans-retinal. In mammals, regeneration of 11-cis-retinal following photoisomerization occurs by a thermally driven isomerization reaction. Additional reactions are required during regeneration to protect cells from the toxicity of aldehyde forms of vitamin A that are essential to the visual process. Photochemical and phototransduction reactions in rods and cones are identical; however, reactions of the rod and cone visual pigment regeneration cycles differ, and perplexingly, rod and cone regeneration cycles appear to use different mechanisms to overcome the energy barrier involved in converting all-trans- to 11-cis-retinoid. Abnormal processing of all-trans-retinal in the rod regeneration cycle leads to retinal degeneration, suggesting that excessive amounts of the retinoid itself or its derivatives are toxic. This line of reasoning led to the development of various approaches to modifying the activity of the rod visual cycle as a possible therapeutic approach to delay or prevent retinal degeneration in inherited retinal diseases and perhaps in the dry form of macular degeneration (geographic atrophy). In spite of great progress in understanding the functioning of rod and cone regeneration cycles at a molecular level, resolution of a number of remaining puzzling issues will offer insight into the amelioration of several blinding retinal diseases.

cis Retinol oxidation regulates photoreceptor access to the retina visual cycle and cone pigment regeneration.

KEY POINTS: This study explores the nature of the cis retinol that Müller cells in the retina provide to cones for the regeneration of their visual pigment. We report that the retina visual cycle provides cones exclusively with 11-cis chromophore in both salamander and mouse and show that this selectivity is dependent on the 11-cis-specific cellular retinaldehyde binding protein (CRALBP) present in Müller cells. Even though salamander blue cones and green rods share the same visual pigment, only blue cones but not green rods are able to dark-adapt in the retina following a bleach and to use exogenous 9-cis retinol for pigment regeneration, suggesting that access to the retina visual cycle is cone-specific and pigment-independent. Our results show that the retina produces 11-cis retinol that can be oxidized and used for pigment regeneration and dark adaptation selectively in cones and not in rods. ABSTRACT: Chromophore supply by the retinal Müller cells (retina visual cycle) supports the efficient pigment regeneration required for cone photoreceptor function in bright light. Surprisingly, a large fraction of the chromophore produced by dihydroceramide desaturase-1, the putative all-trans retinol isomerase in Müller cells, appears to be 9-cis retinol. In contrast, the canonical retinal pigment epithelium (RPE) visual cycle produces exclusively 11-cis retinal. Here, we used the different absorption spectra of 9-cis and 11-cis pigments to identify the isoform of the chromophore produced by the visual cycle of the intact retina. We found that the spectral sensitivity of salamander and mouse cones dark-adapted in the isolated retina (with only the retina visual cycle) was similar to that of cones dark-adapted in the intact eye (with both the RPE and retina visual cycles) and consistent with pure 11-cis pigment composition. However, in mice lacking the cellular retinaldehyde binding protein (CRALBP), cone spectral sensitivity contained a substantial 9-cis component. Thus, the retina visual cycle provides cones exclusively with 11-cis chromophore and this process is mediated by the 11-cis selective CRALBP in Müller cells. Finally, despite sharing the same pigment, salamander blue cones, but not green rods, recovered their sensitivity in the isolated retina. Exogenous 9-cis retinol produced robust sensitivity recovery in bleached red and blue cones but not in red and green rods, suggesting that cis retinol oxidation restricts access to the retina visual cycle to cones.

Intracellular transport of fat-soluble vitamins A and E.

Vitamins are compounds that are essential for the normal growth, reproduction and functioning of the human body. Of the 13 known vitamins, vitamins A, D, E and K are lipophilic compounds and are therefore called fat-soluble vitamins. Because of their lipophilicity, fat-soluble vitamins are solubilized and transported by intracellular carrier proteins to exert their actions and to be metabolized properly. Vitamin A and its derivatives, collectively called retinoids, are solubilized by intracellular retinoid-binding proteins such as cellular retinol-binding protein (CRBP), cellular retinoic acid-binding protein (CRABP) and cellular retinal-binding protein (CRALBP). These proteins act as chaperones that regulate the metabolism, signaling and transport of retinoids. CRALBP-mediated intracellular retinoid transport is essential for vision in human. α-Tocopherol, the main form of vitamin E found in the body, is transported by α-tocopherol transfer protein (α-TTP) in hepatic cells. Defects of α-TTP cause vitamin E deficiency and neurological disorders in humans. Recently, it has been shown that the interaction of α-TTP with phosphoinositides plays a critical role in the intracellular transport of α-tocopherol and is associated with familial vitamin E deficiency. In this review, we summarize the mechanisms and biological significance of the intracellular transport of vitamins A and E.

Stem cells as source for retinal pigment epithelium transplantation.

Inherited maculopathies, age related macular degeneration and some forms of retinitis pigmentosa are associated with impaired function or loss of the retinal pigment epithelium (RPE). Among potential treatments, transplantation approaches are particularly promising. The arrangement of RPE cells in a well-defined tissue layer makes the RPE amenable to cell or tissue sheet transplantation. Different cell sources have been suggested for RPE transplantation but the development of a clinical protocol faces several obstacles. The source should provide a sufficient number of cells to at least recover the macula area. Secondly, cells should be plastic enough to be able to integrate in the host tissue. Tissue sheets should be considered as well, but the substrate on which RPE cells are cultured needs to be carefully evaluated. Immunogenicity can also be an obstacle for effective transplantation as well as tumorigenicity of not fully differentiated cells. Finally, ethical concerns may represent drawbacks when embryo-derived cells are proposed for RPE transplantation. Here we discuss different cell sources that became available in recent years and their different properties. We also present data on a new source of human RPE. We provide a protocol for RPE differentiation of retinal stem cells derived from adult ciliary bodies of post-mortem donors. We show molecular characterization of the in vitro differentiated RPE tissue and demonstrate its functionality based on a phagocytosis assay. This new source may provide tissue for allogenic transplantation based on best matches through histocompatibility testing.

Photodynamic therapy for polypoidal choroidal vasculopathy.

The first effective therapy for exudative macular degeneration (AMD) was Photodynamic Therapy (PDT). Diagnosis of the disease was to a large extent by fluorescein angiography (FA). Distinguishing between the leaky choroidal neovessels (CNV) associated with exudative AMD, and the polypoidal structures associated with Polypoidal Choroidal Vasculopathy (PCV) is not always easy using FA alone. The switch to Indocyanine Green angiography helped to pinpoint PCV, and thus to study the efficacy of photodynamic therapy of this particular form of retinal disease, which is more frequently encountered among pigmented individuals. The results appear to be quite promising, and in the year following treatment only a small fraction of the patients had to be retreated. Alternatively, treating PCV with repeated intravitreal VEGF blocking agents was not as successful as it was in the treatment of wet AMD. However, combining PDT-induced angio-occlusion of the polypoidal lesions with anti-vascular endothelial growth factor therapy was shown to be quite effective, and the combination of PDT with an anti-angiogenic agent as well as a steroid, in a triple therapy, was recently also shown to be a quite promising option. In the present article we review the data on PDT of PCV, including combination therapies and alternative treatments. We also report on similarities and differences between AMD and PCV.