Retinaldehyde-binding Protein Research Articles

Dominant role for pigment epithelial CRALBP in supplying visual chromophore to photoreceptors

Cellular retinaldehyde-binding protein (CRALBP) supports production of 11-cis-retinaldehyde and its delivery to photoreceptors. It is found in the retinal pigment epithelium (RPE) and Müller glia (MG), but the relative functional importance of these two cellular pools is debated. Here, we report RPE- and MG-specific CRALBP knockout (KO) mice and examine their photoreceptor and visual cycle function. Bulk visual chromophore regeneration in RPE-KO mice is 15-fold slower than in controls, accounting for their delayed rod dark adaptation and protection against retinal phototoxicity, whereas MG-KO mice have normal bulk visual chromophore regeneration and retinal light damage susceptibility. Cone pigment regeneration is significantly impaired in RPE-KO mice but mildly affected in MG-KO mice, disclosing an unexpectedly strong reliance of cone photoreceptors on the RPE-based visual cycle. These data reveal a dominant role for RPE-CRALBP in supporting rod and cone function and highlight the importance of RPE cell targeting for CRALBP gene therapies.

A novel RLBP1 gene mutation associated to a flecked retina syndrome.

To present an unusual fleck retina condition associated to a novel RLBP1 gene mutation.Methods/Results: A 25-year old male presented flecks on fundoscopic examination. Clinical presentation, multimodal imaging and ERG were compatible with the diagnosis of benign familial fleck retina. Genetic analysis detected an RLBP1 gene, a gene commonly associated with more severe retinal disease. Flecked retina syndromes and other genetic retinal diseases have a complex genotype-phenotype relation and need further research for their pathophysiology to be fully understood.

Retinal Dystrophy Associated With RLBP1 Retinitis Pigmentosa: A Five-Year Prospective Natural History Study.

To assess the progression in functional and structural measures over a five-year period in patients with retinal dystrophy caused by RLBP1 gene mutation. This prospective, noninterventional study included patients with biallelic RLBP1 mutations from two clinical sites in Sweden and Canada. Key assessments included ocular examinations, visual functional measures (best-corrected visual acuity [BCVA], contrast sensitivity [CS], dark-adaptation [DA] kinetics up to six hours for two wavelengths [450 and 632 nm], Humphrey visual fields [HVF], full-field flicker electroretinograms), and structural ocular assessments. Of the 45 patients enrolled, 38 completed the full five years of follow-up. At baseline, patients had BCVA ranging from -0.2 to 1.3 logMAR, poor CS, HVF defects, and prominent thinning in central foveal thickness. All patients had extremely prolonged DA rod recovery of approximately six hours at both wavelengths. The test-retest repeatability was high across all anatomic and functional endpoints. Cross-sectionally, poorer VA was associated with older age (right eye, correlation coefficient [CC]: 0.606; left eye, CC: -0.578; P < 0.001) and HVF MD values decreased with age (right eye, CC: -0.672, left eye, CC: -0.654; P < 0.001). However, no major changes in functional or structural measures were noted longitudinally over the five-year period. This natural history study, which is the first study to monitor patients with RLBP1 RD for five years, showed that severely delayed DA sensitivity recovery, a characteristic feature of this disease, was observed in all patients across all age groups (17-69 years), making it a potentially suitable efficacy assessment for gene therapy treatment in this patient population.

CD40 Upregulation in the Retina of Patients With Diabetic Retinopathy: Association With TRAF2/TRAF6 Upregulation and Inflammatory Molecule Expression.

CD40 is upregulated in the retinas of diabetic mice, drives pro-inflammatory molecule expression, and promotes diabetic retinopathy. The role of CD40 in diabetic retinopathy in humans is unknown. Upregulation of CD40 and its downstream signaling molecules TNF receptor associated factors (TRAFs) is a key feature of CD40-driven inflammatory disorders. We examined the expression of CD40, TRAF2, and TRAF6 as well as pro-inflammatory molecules in retinas from patients with diabetic retinopathy. Posterior poles from patients with diabetic retinopathy and non-diabetic controls were stained with antibodies against von Willebrand factor (labels endothelial cells), cellular retinaldehyde-binding protein (CRALBP), or vimentin (both label Müller cells) plus antibodies against CD40, TRAF2, TRAF6, ICAM-1, CCL2, TNF-α, and/or phospho-Tyr783 phospholipase Cγ1 (PLCγ1). Sections were analyzed by confocal microscopy. CD40 expression was increased in endothelial and Müller cells from patients with diabetic retinopathy. CD40 was co-expressed with ICAM-1 in endothelial cells and with CCL2 in Müller cells. TNF-α was detected in retinal cells from these patients, but these cells lacked endothelial/Müller cell markers. CD40 in Müller cells from patients with diabetic retinopathy co-expressed activated phospholipase Cγ1, a molecule that induces TNF-α expression in myeloid cells in mice. CD40 upregulation in endothelial cells and Müller cells from patients with diabetic retinopathy was accompanied by TRAF2 and TRAF6 upregulation. CD40, TRAF2, and TRAF6 are upregulated in patients with diabetic retinopathy. CD40 associates with expression of pro-inflammatory molecules. These findings suggest that CD40-TRAF signaling may promote pro-inflammatory responses in the retinas of patients with diabetic retinopathy.

Microglia activation and neuronal alterations in retinas from COVID-19 patients: correlation with clinical parameters

BackgroundDifferent ocular alterations have been described in patients with coronavirus disease 2019 (COVID-19). Our aim was to determine whether COVID-19 affected retinal cells and establish correlations with clinical parameters.MethodsRetinal sections and flat-mount retinas from human donors with COVID-19 (n = 16) and controls (n = 15) were immunostained. The location of angiotensin-converting enzyme 2 (ACE2) and the morphology of microglial cells, Müller cells, astrocytes, and photoreceptors were analyzed by confocal microscopy. Microglial quantification and the area occupied by them were measured. Correlations among retinal and clinical parameters were calculated.ResultsACE2 was mainly located in the Müller cells, outer segment of cones and retinal pigment epithelium. Cell bodies of Müller cells in COVID-19 group showed greater staining of ACE2 and cellular retinaldehyde-binding protein (CRALBP). The 81.3% of COVID-19 patients presented disorganization of honeycomb-like pattern formed by Müller cells. Gliosis was detected in 56.3% of COVID-19 patients compared to controls (40%) as well as epiretinal membranes (ERMs) or astrocytes protruding (50%). Activated or ameboid-shape microglia was the main sign in the COVID-19 group (93.8%). Microglial migration towards the vessels was greater in the COVID-19 retinas (P < 0.05) and the area occupied by microglia was also reduced (P < 0.01) compared to control group. Cone degeneration was more severe in the COVID-19 group. Duration of the disease, age and respiratory failure were the most relevant clinical data in relation with retinal degeneration.ConclusionsThe retinas of patients with COVID-19 exhibit glial activation and neuronal alterations, mostly related to the inflammation, hypoxic conditions, and age.

Clinical parameters related to the glial activation and neuronal alterations present in the retinas of COVID‐19 patients

AbstractPurpose: Ocular and eye fundus alterations have been described in COVID‐19 patients. In this study, the aim was to establish several stages of response to damage in the retinal cells from human donors with COVID‐19 and analyse their relationship with clinical parameters.Methods: Retinal sections and flat‐mount retinas from human donors with COVID‐19 (n = 16) and control (n = 12) were analysed with immunohistochemistry. The location of angiotensin‐converting enzyme 2 (ACE2) and the morphology of microglial cells, Müller cells, astrocytes, and photoreceptors were evaluated by confocal microscopy. Microglial analysis and cell death were measured. Clinical data were correlated with different retinal parameters.Results: Müller cells, outer segment of cones and retinal pigment epithelium presented the ACE2 protein. Larger staining of ACE2 and cellular retinaldehyde–binding protein (CRALBP) was found in the cell bodies of Müller cells in COVID‐19 group. Disorganization of honeycomb‐like pattern formed by Müller cells and disruption of external limiting membrane were observed in the 81.3% of COVID‐19 patients. The 53.3% of COVID‐19 patients showed reactive gliosis as well as astrocytes protruding and epiretinal membranes. Cone degeneration was aggravated in the COVID‐19 group. Activated or ameboid‐shape microglia was found in the 93.8% of COVID‐19 patients. Most retinas showed a reduction of the area occupied by microglia and presented microglial nodules around vessels. Retinal degeneration in COVID‐19 group correlated with the duration of the disease (ro: 0.655; p &lt; 0.01) and age (ro: −0.5; p &lt; 0.05). Moreover, microglia activation was correlated with blood oxygen levels (ro: −0.554; p &lt; 0.05).Conclusions: Different stages of glial activation and neuronal alterations are present in COVID‐19 retinas and are significantly correlated with the duration of the disease, age and respiratory failure.

Expression of SH3 and Multiple Ankyrin Repeat Domains Protein 3 in Mouse Retina.

Synapse-associated gene mutations of SH3 and multiple ankyrin repeat domains protein 3 (SHANK3) may lead to autism spectrum disorder (ASD). In some ASD cases, patients may also have vision disorders. However, the effects of SHANK3 in the retina are barely mentioned in the literature. In this study, we used wild-type mice to systematically map the distribution of SHANK3 expression in entire mouse retinas. Using Western blot analysis and immunofluorescence double labeling, we identified a large number of prominent cells expressing high levels of SHANK3 in the inner retina, in particular, the ganglion cell layer (GCL) and inner nucleus layer. The inner plexiform layer and outer nucleus layer were also exhibited positive SHANK3 signals. In the inner layer, GABAergic amacrine cells (ACs) labeled by glutamate decarboxylase were colocalized with SHANK3-positive cells. Dopaminergic ACs (labeled by tyrosine hydroxylase) and cholinergic ACs (labeled by choline acetyltransferase) were also found to contain SHANK3-positive signals. Additionally, most GCs (labeled by Brn3a) were also found to be SHANK3 positive. In the outer retina, bipolar cells (labeled by homeobox protein ChX10) and horizontal cells (labeled by calbindin) were SHANK3 positive. In the outer nucleus layers, the somata of blue cones (labeled by S-opsin) were weekly co-labeled with SHANK3. The inner segments of blue cones and the outer segments of red/green cones (labeled by L/M-opsin) were partially colocalized with SHANK3 and the outer segments of rods (labeled by Rho4D2) were partially SHANK3 positive too. Moreover, SHANK3-positive labeling was also observed in Müller cells (labeled by cellular retinaldehyde-binding protein). These wide expression patterns indicate that SHANK3 may play an important role in the visual signaling pathway.

Advantages of nanofibrous membranes for culturing of primary RPE cells compared to commercial scaffolds.

Dysfunction of the retinal pigment epithelium (RPE) causes numerous forms of retinal degeneration. RPE replacement is a modern option to save vision. We aimed to test the results of transplanting cultured RPEs on biocompatible membranes. We cultivated porcine primary RPE cells isolated from cadaver eyes from the slaughterhouse on two types of membranes: commercial polyester scaffolds Transwell (Corning Inc., Kenneburg, ME, USA) with 0.4 µm pore size and prepared Poly (L-lactide-co-DL-lactide) (PDLLA) nanofibrous membranes with an average pore size of 0.4 µm. Five types of assays were used for the analysis: immunocytochemistry (ICC), phagocytosis assay, Western blotting, real-time qPCR (RT-qPCR) and electron microscopy. RT-qPCR demonstrated that RPEs cultured on nanofibrous membranes have higher expressions of BEST1 (bestrophin 1), RLBP1 (retinaldehyde-binding protein 1), RPE65 (retinal pigment epithelium-specific 65 kDa protein), PAX6 (transcription factor PAX6), SOX9 (transcription factor SOX9), DCT (dopachrome tautomerase) and MITF (microphthalmia-associated transcription factor). ICC of the RPEs cultured on nanofibrous membranes showed more intensive staining of markers such as BEST1, MCT1 (monocarboxylate transporter 1), Na+ /K+ ATPase, RPE65 and acetylated tubulin in comparison with commercial ones. Additionally, the absence of α-SMA proved the stability of the RPE polarization state and the absence of epithelial-to-mesenchymal transition. RPE possessed high phagocytic activity. Electron microscopy of both membranes confirmed a confluent layer of RPE cells and their genuine morphological structure, which was comparable to native RPEs. Retinal pigment epitheliums cultured on polylactide nanofibrous membranes improved the final quality of the cell product by having better maturation and long-term survival of the RPE monolayer compared to those cultured on commercial polyester scaffolds. PDLLA-cultured RPEs are a plausible source for the replacement of non-functioning RPEs during cell therapy.

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.

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

PurposeTo 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.DesignRetrospective cohort study.ParticipantsPatients with pathogenic variants in RLBP1 registered in a single French reference center specialized in inherited retinal dystrophies.MethodsClinical, multimodal imaging, and genetic findings were reviewed.Main Outcome MeasuresAge 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.ResultsTwenty-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.ConclusionsThe 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.

Shared Features in Retinal Disorders With Involvement of Retinal Pigment Epithelium.

When using spectral domain optical coherence tomography (SD-OCT) to inform the status of outer retina, we have noted discrete hyperreflective lesions extending through photoreceptor-attributable bands that have a similar presentation in multiple retinal diseases. These lesions present as either corrugated thickenings of interdigitation zone and ellipsoid zone bands or in later stages as rectangular or pyramidal shaped foci that extend radially through photoreceptor cell-attributable bands. In ABCA4-related and peripherin-2/RDS-disease (PRPH2/RDS), monogenic forms of retinopathy caused by mutations in proteins expressed in photoreceptor cells, these punctate lesions colocalize with fundus flecks in en face images. In fundus albipunctatus and retinitis punctata albescens, diseases caused by mutations in genes (retinol dehydrogenase 5, RDH5; and retinaldehyde-binding protein 1, RLBP1) encoding proteins of the visual cycle, these lesions manifest as white dot-like puncta. Similar aberrations in photoreceptor cell-attributable SD-OCT reflectivity layers manifest as reticular pseudodrusen (RPD) in short-wavelength fundus autofluorescence and near-infrared fundus autofluorescence fundus images and are linked to age-related macular degeneration a complex disease. Despite differences in the etiologies of retinal diseases presenting as fundus flecks, dots and RPD, underlying degenerative processes in photoreceptor cells are signified in SD-OCT scans by the loss of structural features that would otherwise define healthy photoreceptor cells at these foci.

PSS14 Qualitative Exploration of Patient Experiences of Visual Function Impairments and Health-Related Quality of Life Impacts Associated with RLBP1 Retinitis Pigmentosa in a Sample of Canadian Patients

RLBP1 retinitis pigmentosa (RP) is a rare inherited retinal degeneration caused by biallelic mutations in the RLBP1 gene. Visual impairments associated with RP result in significant impacts on patients’ vision-dependent activities of daily living (ADL) and broader health-related quality of life (HRQoL). However, there is a paucity of research exploring the patient experience of RP in general and RLBP1 RP. The objective of this qualitative study was to explore the experience of RLBP1 RP from the patient perspective in Canada. Semi-structured qualitative concept elicitation telephone interviews were conducted with eight Canadian RLBP1 RP adult patients at a site in Newfoundland. All patients had a genetic diagnosis of RLBP1 RP. Qualitative thematic analysis of verbatim interview transcripts was performed. Findings contributed to refinement of a conceptual model of RP detailing relevant visual function symptom and impact measurement concepts. All eight RLBP1 RP patients (33-70 years) reported experiencing night blindness, reduced peripheral vision, difficulty seeing in very bright lighting and difficulty adapting to new lighting conditions. Visual function symptoms led to proximal vision-dependent impacts on mobility and ADLs. Visual aids commonly used included sunglasses, white cane and assistive devices. Impacts on broader HRQoL domains included social functioning, emotional well-being, work/school functioning and financial. Visual functioning and impacts on vision-dependent activities were moderated by lighting conditions and familiarity of environment. Findings from this study provide in-depth insights into the visual function symptoms and impacts on vision-dependent ADLs and broader HRQoL experienced by RLBP1 RP patients. Results are consistent with evidence generated in other RP genotypes and suggest that the experience of RP is similar across different mutations, such as RPE65 and RPGR. Interview findings supported the development of a RP specific patient-reported outcome instrument for use in clinical trials and to track disease severity in future clinical practice.

Molecular genetics of inherited retinal degenerations in Icelandic patients.

The study objective was to delineate the genetics of inherited retinal degenerations (IRDs) in Iceland, a small nation of 364.000 and a genetic isolate. Benefits include delineating novel pathogenic genetic variants and defining genetically homogenous patients as potential investigative molecular therapy candidates. The study sample comprised patients with IRD in Iceland ascertained through national centralized genetic and ophthalmological services at Landspitali, a national social support institute, and the Icelandic patient association. Information on patients' disease, syndrome, and genetic testing was collected in a clinical registry. Variants were reevaluated according to ACMG/AMP guidelines. Overall, 140 IRD patients were identified (point prevalence of 1/2.600), of which 70 patients had a genetic evaluation where two‐thirds had an identified genetic cause. Thirteen disease genes were found in patients with retinitis pigmentosa, with the RLBP1 gene most common (n = 4). The c.1073 + 5G > A variant in the PRPF31 gene was homozygous in two RP patients. All tested patients with X‐linked retinoschisis (XLRS) had the same possibly unique RS1 pathogenic variant, c.441G > A (p.Trp147X). Pathologic variants and genes for IRDs in Iceland did not resemble those described in ancestral North‐Western European nations. Four variants were reclassified as likely pathogenic. One novel pathogenic variant defined a genetically homogenous XLRS patient group.

Pathogenesis Study Based on High-Throughput Single-Cell Sequencing Analysis Reveals Novel Transcriptional Landscape and Heterogeneity of Retinal Cells in Type 2 Diabetic Mice.

Diabetic retinopathy (DR) is the leading cause of acquired blindness in middle-aged people. The complex pathology of DR is difficult to dissect, given the convoluted cytoarchitecture of the retina. Here, we performed single-cell RNA sequencing (scRNA-seq) of retina from a model of type 2 diabetes, induced in leptin receptor-deficient (db/db) and control db/m mice, with the aim of elucidating the factors mediating the pathogenesis of DR. We identified 11 cell types and determined cell-type-specific expression of DR-associated loci via genome-wide association study (GWAS)-based enrichment analysis. DR also impacted cell-type-specific genes and altered cell-cell communication. Based on the scRNA-seq results, retinaldehyde-binding protein 1 (RLBP1) was investigated as a promising therapeutic target for DR. Retinal RLBP1 expression was decreased in diabetes, and its overexpression in Müller glia mitigated DR-associated neurovascular degeneration. These data provide a detailed analysis of the retina under diabetic and normal conditions, revealing new insights into pathogenic factors that may be targeted to treat DR and related dysfunctions.

Induction of Differentiation of Mesenchymal Stem Cells into Retinal Pigment Epithelial Cells for Retinal Regeneration by Using Ciliary Neurotrophic Factor in Diabetic Rats.

Diabetic retinopathy (DR) is a common cause of blindness all over the world. Bone marrow mesenchymal stem cells (BMSCs) have been considered as a promising strategy for retinal regeneration in the treatment of DR. However, the poor viability and low levels of BMSCs engraftment limit the therapeutic potential of BMSCs. The present study aimed to examine the direct induction of BMSCs differentiation into the cell types related to retinal regeneration by using soluble cytokine ciliary neurotrophic factor (CNTF). We observed remarkably increased expression of cellular retinaldehyde-binding protein (CRALBP) and retinoid isomerohydrolase (RPE65) in BMSCs treated with CNTF in vitro, indicating the directional differentiation of BMSCs into the retinal pigment epithelium (RPE) cells, which are crucial for retinal healing. In vivo, the diabetic rat model was established by use of streptozotocin (STZ), and animals treated with BMSCs+CNTF exhibited better viability and higher delivery efficiency of the transplanted cells than those treated with BMSCs injection alone. Similar to the in-vitro result, treatment with BMSCs and CNTF combined led to the differentiation of BMSCs into beneficial cells (RPE cells), and accelerated retinal healing characterized by the activation of rod photoreceptor cells and phagocytosis function of RPE cells. In conclusion, CNTF contributes to the differentiation of BMSCs into RPE cells, which may help overcome the current stem cell therapy limitations in the field of retinal regeneration.

Neurosphere-Free Transdifferentiation of Rat Bone Marrow Stromal Stem Cells Into Retinal Cells and Retinal Pigment Epithelium.

Introduction:Neurosphere-free transdifferentiation of bone marrow stem cells into Retinal Pigment Epithelium (RPE) and Retinal Cells (RCs) in vitro could offer an exceptional opportunity to study cell replacement in degenerative eye diseases. Thus, a simple and efficient protocol for retinal cells production from transdifferentiation of rat BMSCs in the neurosphere-free state is reported.Methods:Extracted BMSCs from hooded pigment rats were exposed to a single-step protocol, including neurosphere-free, containing a cocktail medium that induced transdifferentiation into Retinal Pigment Epithelium (RPE) and retinal cells.Results:The results showed morphological differentiation changes in vitro. Also, the expressed retinal pigment epithelium and retinal cell markers, such as retinal orthodenticle homeobox 2 (23.45%), retinal pigment epithelium protein 65 (91.54%), cellular retinaldehyde-binding protein (91.21%), vascular endothelial growth factor (94.79%), rhodopsin (57.19%), glial fibrillary acidic protein (28.33%), and neurofilament 200 (24.55%).Conclusion:Overall, these findings showed that a protocol without using basic fibroblast growth factor, epidermal growth factor, and B-27 supplements could generate RPE and retinal cells in vitro.

Absence of ephrin-A2/A3 increases retinal regenerative potential for Müller cells in Rhodopsin knockout mice.

Müller cells (MC) are considered dormant retinal progenitor cells in mammals. Previous studies demonstrated ephrin-As act as negative regulators of neural progenitor cells in the retina and brain. It remains unclear whether the lack of ephrin-A2/A3 is sufficient to promote the neurogenic potential of MC. Here we investigated whether the MC is the primary retinal cell type expressing ephrin-A2/A3 and their role on the neurogenic potential of Müller cells. In this study, we showed that ephrin-A2/A3 and their receptor EphA4 were expressed in retina and especially enriched in MC. The level of ephrinAs/EphA4 expression increased as the retina matured that is correlated with the reduced proliferative and progenitor cell potential of MC. Next, we investigated the proliferation in primary MC cultures isolated from wild-type and A2-/- A3-/- mice by 5-ethynyl-2'-deoxyuridine (EdU) incorporation. We detected a significant increase of EdU+ cells in MC derived from A2-/- A3-/- mice. Next, we investigated the role of ephrin-A2/A3 in mice undergoing photoreceptor degeneration such as Rhodopsin knockout (Rho-/-) mice. To further evaluate the role of ephrin-A2/A3 in MC proliferation in vivo, EdU was injected intraperitoneally to adult wild-type, A2-/- A3-/- , Rho-/- and Rho-/- A2-/- A3-/- mice and the numbers of EdU+ cells distributed among different layers of the retina. EphrinAs/EphA4 expression was upregulated in the retina of Rho-/- mice compared to the wild-type mice. In addition, cultured MC derived from ephrin-A2-/- A3-/- mice also expressed higher levels of progenitor cell markers and exhibited higher proliferation potential than those from wild-type mice. Interestingly, we detected a significant increase of EdU+ cells in the retinas of adult ephrin-A2-/- A3-/- mice mainly in the inner nuclear layer; and these EdU+ cells were co-localized with MC marker, cellular retinaldehyde-binding protein, suggesting some proliferating cells are from MC. In Rhodopsin knockout mice (Rho-/- A2-/- A3-/- mice), a significantly greater amount of EdU+ cells were located in the ciliary body, retina and RPE than that of Rho-/- mice. Comparing between 6 and 12 weeks old Rho-/- A2-/- A3-/- mice, we recorded more EdU+ cells in the outer nuclear layer in the 12-week-old mice undergoing severe retinal degeneration. Taken together, Ephrin-A2/A3 are negative regulators of the proliferative and neurogenic potentials of MC. Absence of ephrin-A2/A3 promotes the migration of proliferating cells into the outer nuclear layer and may lead to retinal cell regeneration. All experimental procedures were approved by the Animal Care and Use Committee at Schepens Eye Research Institute, USA (approval No. S-353-0715) on October 24, 2012.

Function of mammalian M-cones depends on the level of CRALBP in Müller cells.

Cone photoreceptors mediate daytime vision in vertebrates. The rapid and efficient regeneration of their visual pigments following photoactivation is critical for the cones to remain photoresponsive in bright and rapidly changing light conditions. Cone pigment regeneration depends on the recycling of visual chromophore, which takes place via the canonical visual cycle in the retinal pigment epithelium (RPE) and the Müller cell–driven intraretinal visual cycle. The molecular mechanisms that enable the neural retina to regenerate visual chromophore for cones have not been fully elucidated. However, one known component of the two visual cycles is the cellular retinaldehyde-binding protein (CRALBP), which is expressed both in the RPE and in Müller cells. To understand the significance of CRALBP in cone pigment regeneration, we examined the function of cones in mice heterozygous for Rlbp1, the gene encoding CRALBP. We found that CRALBP expression was reduced by ∼50% in both the RPE and retina of Rlbp1+/− mice. Electroretinography (ERG) showed that the dark adaptation of rods and cones is unaltered in Rlbp1+/− mice, indicating a normal RPE visual cycle. However, pharmacologic blockade of the RPE visual cycle revealed suppressed cone dark adaptation in Rlbp1+/− mice in comparison with controls. We conclude that the expression level of CRALPB specifically in the Müller cells modulates the efficiency of the retina visual cycle. Finally, blocking the RPE visual cycle also suppressed further cone dark adaptation in Rlbp1−/− mice, revealing a shunt in the classical RPE visual cycle that bypasses CRALBP and allows partial but unexpectedly rapid cone dark adaptation.

Vitamin A aldehyde-taurine adduct and the visual cycle

Visual pigment consists of opsin covalently linked to the vitamin A-derived chromophore, 11-cis-retinaldehyde. Photon absorption causes the chromophore to isomerize from the 11-cis- to all-trans-retinal configuration. Continued light sensitivity necessitates the regeneration of 11-cis-retinal via a series of enzyme-catalyzed steps within the visual cycle. During this process, vitamin A aldehyde is shepherded within photoreceptors and retinal pigment epithelial cells to facilitate retinoid trafficking, to prevent nonspecific reactivity, and to conserve the 11-cis configuration. Here we show that redundancy in this system is provided by a protonated Schiff base adduct of retinaldehyde and taurine (A1-taurine, A1T) that forms reversibly by nonenzymatic reaction. A1T was present as 9-cis, 11-cis, 13-cis, and all-trans isomers, and the total levels were higher in neural retina than in retinal pigment epithelium (RPE). A1T was also more abundant under conditions in which 11-cis-retinaldehyde was higher; this included black versus albino mice, dark-adapted versus light-adapted mice, and mice carrying the Rpe65-Leu450 versus Rpe65-450Met variant. Taurine levels paralleled these differences in A1T. Moreover, A1T was substantially reduced in mice deficient in the Rpe65 isomerase and in mice deficient in cellular retinaldehyde-binding protein; in these models the production of 11-cis-retinal is compromised. A1T is an amphiphilic small molecule that may represent a mechanism for escorting retinaldehyde. The transient Schiff base conjugate that the primary amine of taurine forms with retinaldehyde would readily hydrolyze to release the retinoid and thus may embody a pool of 11-cis-retinal that can be marshalled in photoreceptor cells.

Prolonged Melanopsin-based Photoresponses Depend in Part on RPE65 and Cellular Retinaldehyde-binding Protein (CRALBP)

ABSTRACT Purpose Intrinsically photosensitive retinal ganglion cells (ipRGCs) contain the photopigment melanopsin and can signal light continuously for many hours. Melanopsin is excited when its chromophore 11-cis-retinal absorbs a photon and becomes all-trans-retinal, which must be reisomerized to 11-cis-retinal to regenerate photoexcitable melanopsin. Due to the great distance separating ipRGCs from the retinal pigment epithelium (RPE) whose retinoid cycle produces 11-cis-retinal, ipRGCs had been assumed to regenerate all melanopsin molecules autonomously. Surprisingly, we previously found that pharmacologically inhibiting the retinoid cycle rendered melanopsin-based responses to prolonged illumination less sustained, suggesting that the RPE may supply retinoids to help ipRGCs regenerate melanopsin during extended photostimulation. However, the specificity of those drugs is unclear. Here, we reexamined the role of the retinoid cycle, and tested whether the RPE-to-ipRGC transport of retinoids utilizes cellular retinaldehyde-binding protein (CRALBP), present throughout the RPE and Müller glia. Methods To measure melanopsin-mediated photoresponses in isolation, all animals were 8- to 12-month-old rod/cone-degenerate mice. We genetically knocked out RPE-specific 65 kDa protein (RPE65), a critical enzyme in the retinoid cycle. We also knocked out the CRALBP gene rlbp1 mainly in Foxg1-expressing Müller cells. We obtained multielectrode-array recordings from ipRGCs in a novel RPE-attached mouse retina preparation, and imaged pupillary light reflexes in vivo. Results Melanopsin-based ipRGC responses to prolonged light became less tonic in both knockout lines, and pupillary light reflexes were also less sustained in RPE65-knockout than control mice. Conclusions These results confirm that ipRGCs rely partly on the retinoid cycle to continuously regenerate melanopsin during prolonged photostimulation, and suggest that CRALBP in Müller glia likely transports 11-cis-retinal from the RPE to ipRGCs – this is the first proposed functional role for CRALBP in the inner retina.