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 Mueller 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.

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

eLife ◽

10.7554/elife.71473 ◽

2021 ◽

Vol 10 ◽

Author(s):

Domino K Schlegel ◽

Srinivasagan Ramkumar ◽

Johannes von Lintig ◽

Stephan CF Neuhauss

Keyword(s):

Pigment Epithelium ◽

Retinal Pigment ◽

Cone Dystrophy ◽

Rod Photoreceptor ◽

Cone Photoreceptor ◽

Specific Expression ◽

Retinoid Metabolism ◽

Light Stimuli ◽

Cell Type Specific ◽

Retinal Thinning

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.

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Retinol dehydrogenases: Membrane-bound enzymes for the visual function

Biochemistry and Cell Biology ◽

10.1139/bcb-2014-0082 ◽

2014 ◽

Vol 92 (6) ◽

pp. 510-523 ◽

Cited By ~ 9

Author(s):

Mustapha Lhor ◽

Christian Salesse

Keyword(s):

Pigment Epithelium ◽

Retinal Pigment ◽

Membrane Binding ◽

Large Family ◽

Structural Features ◽

Visual Cycle ◽

Retinoid Metabolism ◽

Current State ◽

Membrane Bound ◽

Membrane Bound Enzymes

Retinoid metabolism is important for many physiological functions, such as differenciation, growth, and vision. In the visual context, after the absorption of light in rod photoreceptors by the visual pigment rhodopsin, 11-cis retinal is isomerized to all-trans retinal. This retinoid subsequently undergoes a series of modifications during the visual cycle through a cascade of reactions occurring in photoreceptors and in the retinal pigment epithelium. Retinol dehydrogenases (RDHs) are enzymes responsible for crucial steps of this visual cycle. They belong to a large family of proteins designated as short-chain dehydrogenases/reductases. The structure of these RDHs has been predicted using modern bioinformatics tools, which allowed to propose models with similar structures including a common Rossman fold. These enzymes undergo oxidoreduction reactions, whose direction is dictated by the preference and concentration of their individual cofactor (NAD(H)/NADP(H)). This review presents the current state of knowledge on functional and structural features of RDHs involved in the visual cycle as well as knockout models. RDHs are described as integral or peripheral enzymes. A topology model of the membrane binding of these RDHs via their N- and (or) C-terminal domain has been proposed on the basis of their individual properties. Membrane binding is a crucial issue for these enzymes because of the high hydrophobicity of their retinoid substrates.

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Voltage-Dependent Calcium Channel CaV1.3 Subunits Regulate the Light Peak of the Electroretinogram

Journal of Neurophysiology ◽

10.1152/jn.00146.2007 ◽

2007 ◽

Vol 97 (5) ◽

pp. 3731-3735 ◽

Cited By ~ 38

Author(s):

Jiang Wu ◽

Alan D. Marmorstein ◽

Jörg Striessnig ◽

Neal S. Peachey

Keyword(s):

Calcium Channel ◽

Pigment Epithelium ◽

Retinal Pigment ◽

Rod Photoreceptor ◽

Wild Type ◽

Fast Oscillation ◽

Light Peak ◽

Voltage Dependent Calcium Channel ◽

Voltage Dependent ◽

Voltage Dependent Calcium Channels

In response to light, the mouse retinal pigment epithelium (RPE) generates a series of slow changes in potential that are referred to as the c-wave, fast oscillation (FO), and light peak (LP) of the electroretinogram (ERG). The LP is generated by a depolarization of the basolateral RPE plasma membrane by the activation of a calcium-sensitive chloride conductance. We have previously shown that the LP is reduced in both mice and rats by nimodipine, which blocks voltage-dependent calcium channels (VDCCs) and is abnormal in lethargic mice, carrying a null mutation in the calcium channel β4 subunit. To define the α1 subunit involved in this process, we examined mice lacking CaV1.3. In comparison with wild-type (WT) control littermates, LPs were reduced in CaV1.3−/− mice. This pattern matched closely with that previously noted in lethargic mice, confirming a role for VDCCs in regulating the signaling pathway that culminates in LP generation. These abnormalities do not reflect a defect in rod photoreceptor activity, which provides the input to the RPE to generate the c-wave, FO, and LP, because ERG a-waves were comparable in WT and CaV1.3−/− littermates. Our results identify CaV1.3 as the principal pore-forming subunit of VDCCs involved in stimulating the ERG LP.

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KCNQ5/Kv7.5 potassium channel expression and subcellular localization in primate retinal pigment epithelium and neural retina

AJP Cell Physiology ◽

10.1152/ajpcell.00185.2011 ◽

2011 ◽

Vol 301 (5) ◽

pp. C1017-C1026 ◽

Cited By ~ 19

Author(s):

Xiaoming Zhang ◽

Dongli Yang ◽

Bret A. Hughes

Keyword(s):

Visual Information ◽

Pigment Epithelium ◽

Retinal Pigment ◽

Basal Membrane ◽

Cell Types ◽

Neural Retina ◽

Pcr Analysis ◽

Rod Photoreceptor ◽

Rpe Cells ◽

Channel Expression

Previous studies identified in retinal pigment epithelial (RPE) cells an M-type K+ current, which in many other cell types is mediated by channels encoded by KCNQ genes. The aim of this study was to assess the expression of KCNQ genes in the monkey RPE and neural retina. Application of the specific KCNQ channel blocker XE991 eliminated the M-type current in freshly isolated monkey RPE cells, indicating that KCNQ subunits contribute to the underlying channels. RT-PCR analysis revealed the expression of KCNQ1, KCNQ4, and KCNQ5 transcripts in the RPE and all five KCNQ transcripts in the neural retina. At the protein level, KCNQ5 was detected in the RPE, whereas both KCNQ4 and KCNQ5 were found in neural retina. In situ hybridization in frozen monkey retinal sections revealed KCNQ5 gene expression in the ganglion cell layer and the inner and outer nuclear layers of the neural retina, but results in the RPE were inconclusive due to the presence of melanin. Immunohistochemistry revealed KCNQ5 in the inner and outer plexiform layers, in cone and rod photoreceptor inner segments, and near the basal membrane of the RPE. The data suggest that KCNQ5 channels contribute to the RPE basal membrane K+ conductance and, thus, likely play an important role in active K+ absorption. The distribution of KCNQ5 in neural retina suggests that these channels may function in the shaping of the photoresponses of cone and rod photoreceptors and the processing of visual information by retinal neurons.

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A PRPH2 gene variant detected in retinitis punctata albescens with congenital hypertrophy of the retinal pigment epithelium

European Journal of Ophthalmology ◽

10.1177/1120672120962027 ◽

2020 ◽

pp. 112067212096202

Author(s):

Aowang Qiu ◽

Yan Yu ◽

Junlong Huang ◽

Qinghuai Liu ◽

Yannis M Paulus ◽

...

Keyword(s):

Retinal Pigment Epithelium ◽

Pigment Epithelium ◽

Retinal Pigment ◽

Cone Photoreceptor ◽

Blurred Vision ◽

Full Field ◽

Photoreceptor Outer Segments ◽

Pigmented Lesions ◽

Retinitis Punctata Albescens ◽

Congenital Hypertrophy

Retinitis punctata albescens (RPA) is generally diagnosed by the presence of numerous clusters of white punctate lesions in the retina that progress over time and are related to several gene variants. The multifocal variant of congenital hypertrophy of the retinal pigment epithelium (CHRPE) is characterized by multiple, grouped, sharply circumscribed, pigmented spots. The PRPH2 gene encodes a photoreceptor-specific glycoprotein, which is essential for the morphogenesis of rod and cone photoreceptor outer segments. A 39-year-old Chinese female with nyctalopia, complained about blurred vision, presented a unique co-existing feature of RPA and CHRPE. Dilated fundus exam demonstrated numerous porcelain white discrete dots in both eyes and multiple, small, flat clusters of round brown to black pigmented lesions in the left eye. The full field electroretinography (ERG) showed decreased responses after standard dark adaptation and normal b-wave amplitudes after a long (4-h) dark-adapted period. A heterozygous PRPH2 splicing variant was detected in the proband. In addition, the same variant was found in her mother, her son, and her daughter. We describe a PRPH2 variant in a rare case of RPA associated with multifocal CHRPE of the same individual.

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Light-Evoked Responses of the Retinal Pigment Epithelium: Changes Accompanying Photoreceptor Loss in the Mouse

Journal of Neurophysiology ◽

10.1152/jn.00088.2010 ◽

2010 ◽

Vol 104 (1) ◽

pp. 391-402 ◽

Cited By ~ 24

Author(s):

Ivy S. Samuels ◽

Gwen M. Sturgill ◽

Gregory H. Grossman ◽

Mary E. Rayborn ◽

Joe G. Hollyfield ◽

...

Keyword(s):

Retinal Pigment Epithelium ◽

Structural Changes ◽

Pigment Epithelium ◽

Retinal Pigment ◽

Retinal Disease ◽

Rod Photoreceptor ◽

Evoked Responses ◽

Photoreceptor Degeneration ◽

Rod Photoreceptors ◽

Slow Piii

Mutations in genes expressed in the retinal pigment epithelium (RPE) underlie a number of human inherited retinal disorders that manifest with photoreceptor degeneration. Because light-evoked responses of the RPE are generated secondary to rod photoreceptor activity, RPE response reductions observed in human patients or animal models may simply reflect decreased photoreceptor input. The purpose of this study was to define how the electrophysiological characteristics of the RPE change when the complement of rod photoreceptors is decreased. To measure RPE function, we used an electroretinogram (dc-ERG)-based technique. We studied a slowly progressive mouse model of photoreceptor degeneration ( Prph Rd2/+), which was crossed onto a Nyxnob background to eliminate the b-wave and most other postreceptoral ERG components. On this background, Prph Rd2/+ mice display characteristic reductions in a-wave amplitude, which parallel those in slow PIII amplitude and the loss of rod photoreceptors. At 2 and 4 mo of age, the amplitude of each dc-ERG component (c-wave, fast oscillation, light peak, and off response) was larger in Prph Rd2/+ mice than predicted by rod photoreceptor activity (RmP3) or anatomical analysis. At 4 mo of age, the RPE in Prph Rd2/+ mice showed several structural abnormalities including vacuoles and swollen, hypertrophic cells. These data demonstrate that insights into RPE function can be gained despite a loss of photoreceptors and structural changes in RPE cells and, moreover, that RPE function can be evaluated in a broader range of mouse models of human retinal disease.

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Human iPSC-derived retinal pigment epithelium: a model system for identifying and functionally characterizing causal variants at AMD risk loci

10.1101/440230 ◽

2018 ◽

Cited By ~ 1

Author(s):

Erin N. Smith ◽

Agnieszka D’Antonio-Chronowska ◽

William W. Greenwald ◽

Victor Borja ◽

Lana R. Aguiar ◽

...

Keyword(s):

Gene Expression ◽

Retinal Pigment Epithelium ◽

Pigment Epithelium ◽

Retinal Pigment ◽

Induced Pluripotent Stem Cell ◽

Age Related Macular Degeneration ◽

Molecular Characteristics ◽

Specific Expression ◽

Age Related ◽

Causal Variants

SummaryWe evaluate whether human induced pluripotent stem cell-derived retinal pigment epithelium (iPSC-RPE) cells can be used to prioritize and functionally characterize causal variants at age-related macular degeneration (AMD) risk loci. We generated iPSC-RPE from six subjects and show that they have morphological and molecular characteristics similar to native RPE. We generated RNA-seq, ATAC-seq, and H3K27ac ChIP-seq data and observe high similarity in gene expression and enriched transcription factor motif profiles between iPSC-RPE and human fetal-RPE. We performed fine-mapping of AMD risk loci by integrating molecular data from the iPSC-RPE, adult retina, and adult RPE, which identified rs943080 as the probable causal variant at VEGFA. We show that rs943080 is associated with altered chromatin accessibility of a distal ATAC-seq peak, decreased overall gene expression of VEGFA, and allele specific expression of a non-coding transcript. These results provide insight into the mechanism underlying the association of the VEGFA locus with AMD.

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CRB1-associated retinal dystrophies in a Belgian cohort: genetic characteristics and long-term clinical follow-up

British Journal of Ophthalmology ◽

10.1136/bjophthalmol-2020-316781 ◽

2021 ◽

pp. bjophthalmol-2020-316781

Author(s):

Mays Talib ◽

Caroline Van Cauwenbergh ◽

Julie De Zaeytijd ◽

David Van Wynsberghe ◽

Elfride De Baere ◽

...

Keyword(s):

Visual Function ◽

Pigment Epithelium ◽

Retinal Pigment ◽

Posterior Uveitis ◽

Cone Dystrophy ◽

Macular Dystrophy ◽

Genetic Characteristics ◽

Retinal Dystrophies ◽

Retinal Structure

AimTo investigate the natural history in a Belgian cohort of CRB1-associated retinal dystrophies.MethodsAn in-depth retrospective study focusing on visual function and retinal structure.ResultsForty patients from 35 families were included (ages: 2.5–80.1 years). In patients with a follow-up of >1 year (63%), the mean follow-up time was 12.0 years (range: 2.3–29.2 years). Based on the patient history, symptoms and/or electroretinography, 22 patients (55%) were diagnosed with retinitis pigmentosa (RP), 15 (38%) with Leber congenital amaurosis (LCA) and 3 (8%) with macular dystrophy (MD), the latter being associated with the p.(Ile167_Gly169del) mutation (in compound heterozygosity). MD later developed into a rod-cone dystrophy in one patient. Blindness at initial presentation was seen in the first decade of life in LCA, and in the fifth decade of life in RP. Eventually, 28 patients (70%) reached visual acuity-based blindness (<0.05). Visual field-based blindness (<10°) was documented in 17/25 patients (68%). Five patients (13%) developed Coats-like exudative vasculopathy. Intermediate/posterior uveitis was found in three patients (8%). Cystoid maculopathy was common in RP (9/21; 43%) and MD (3/3; 100%). Macular involvement, varying from retinal pigment epithelium alterations to complete outer retinal atrophy, was observed in all patients.ConclusionBi-allelic CRB1 mutations result in a range of progressive retinal disorders, most of which are generalised, with characteristically early macular involvement. Visual function and retinal structure analysis indicates a window for potential intervention with gene therapy before the fourth decade of life in RP and the first decade in LCA.

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Retinoid metabolism in cultured human retinal pigment epithelium

Biochemical Journal ◽

10.1042/bj2500459 ◽

1988 ◽

Vol 250 (2) ◽

pp. 459-465 ◽

Cited By ~ 25

Author(s):

S R Das ◽

P Gouras

Keyword(s):

Fatty Acid ◽

Culture Medium ◽

Cultured Cells ◽

Pigment Epithelium ◽

Retinal Pigment ◽

Primary Cultures ◽

Retinyl Palmitate ◽

Lipid Binding ◽

Retinoid Metabolism ◽

Hydrolysis Of

Uptake, esterification and release of all-trans-retinol in primary cultures of human retinal epithelium were studied. Cultured cells were supplemented with 3H-labelled 11,12-all-trans-retinol, using fatty-acid-free albumin as the carrier. This led to incorporation of retinal and the formation of all-trans- and 11-cis-retinyl palmitate. The metabolism of the all-trans ester was monitored in a medium containing various concentrations of foetal-bovine serum (FBS). In 20% (v/v) FBS, the ester was hydrolysed, and all-trans-retinol was released into the culture medium. In the absence of FBS, little ester was hydrolysed and no retinol was found in the medium. Dialysed or heat-inactivated FBS or fatty-acid-free albumin was as effective as FBS in provoking ester hydrolysis and retinol release. The concentration-dependency of this effect on FBS was matched by the corresponding concentrations of albumin alone. A linear relationship was also found between interphotoreceptor retinoid-binding protein and retinoid release. Haemoglobin, which does not bind retinoids, is ineffective in this capacity. It is concluded that lipid-binding substances, mainly albumin, in FBS act as acceptors for retinol and drain the cultured cells of this molecule. The release of the retinol is coupled to the hydrolysis of retinyl esters in the cell, so that there is little or no net hydrolysis of ester if there is no acceptor for retinol in the culture medium. This effect explains why cultured human retinal epithelial cells are depleted of their stores of retinoids when maintained in medium supplemented with FBS.

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