RPE65 mRNA Research Articles

Lentiviral mediated RPE65 gene transfer in healthy hiPSCs-derived retinal pigment epithelial cells markedly increased RPE65 mRNA, but modestly protein level

The retinal pigment epithelium (RPE) is a monolayer of cobblestone-like epithelial cells that accomplishes critical functions for the retina. Several protocols have been published to differentiate pluripotent stem cells into RPE cells suitable for disease modelling and therapy development. In our study, the RPE identity of human induced pluripotent stem cell (hiPSC)-derived RPE (iRPE) was extensively characterized, and then used to test a lentiviral-mediated RPE65 gene augmentation therapy. A dose study of the lentiviral vector revealed a dose-dependent effect of the vector on RPE65 mRNA levels. A marked increase of the RPE65 mRNA was also observed in the iRPE (100-fold) as well as in an experimental set with RPE derived from another hiPSC source and from foetal human RPE. Although iRPE displayed features close to bona fide RPE, no or a modest increase of the RPE65 protein level was observed depending on the protein detection method. Similar results were observed with the two other cell lines. The mechanism of RPE65 protein regulation remains to be elucidated, but the current work suggests that high vector expression will not produce an excess of the normal RPE65 protein level.

In vivo Genome Editing for Vision

Background & Aim Leber congenital amaurosis (LCA), one of the leading causes of childhood-onset blindness, is caused by autosomal recessive mutations in several genes including RPE65, resulting in retinal degeneration. For the recovery of retinal function in patients with LCA, it is required to repair the gene to replace intact protein and to restore physiological responses in the retinal tissues. In this study, we investigated the therapeutic potential of RPE65 replacement through CRISPR/Cas9-mediated therapeutic genome editing including homology-directed repair and in-frame deletion in rd12 mice, a model of human LCA, which have a homozygous nonsense mutation in the Rpe65 gene. Methods, Results & Conclusion Through subretinal delivery of the dual adeno-associated virus system containing transcripts for CRISPR/Cas9 and donor DNA, we achieved >1% homology-directed repair and ∼1.6% deletion of the pathogenic stop codon in Rpe65 gene in retinal pigment epithelial tissues of rd12 mice. This treatment resulted in ∼10% of Rpe65 mRNA expression and 20-30% of dark-adapted electroretinographic responses in rd12 mice at seven months after the injection. There was no definite evidence of histologic abnormalities or tumorigenesis related to potential off-target effects during seven months of follow-up periods. Taken together, we suggest the results of the therapeutic genome editing of a Rpe65 nonsense mutation by CRISPR/Cas9, providing new insight for developing therapeutics for LCA.

Stable Intronic Sequences and Exon Skipping Events in the Human RPE65 Gene: Analysis of Expression in Retinal Pigment Epithelium Cells and Cell Culture Models.

Currently, there is much interest in intronic sequence-containing long non-coding RNAs and the role of intronic transcription in regulation of cellular metabolism and fate. Several stable intronic sequence RNAs (sisRNAs) were recently implicated in regulation of parental genes. To investigate transcription from introns of the RPE65 gene, we analyzed RNA-seq and Nanopore sequencing data from different cell models of human retinal pigment epithelium (RPE) and native bovine RPE. We discovered putative stable poly-adenylated transcripts with sequences corresponding to intronic regions of the RPE65 gene in the cytoplasm of RPE cells. These stable intronic sequences could be important for RPE65 transcription, splicing or translation. We also analyzed alternative splicing events in RPE65. Frequent exon skipping events involving exons 2, 3, and 7 were detected. The rate of these events was much higher in human RPE cell cultures compared with native RPE , consistent with lack of translation of RPE65 mRNA in cell cultures.

Aberrant RNA splicing is the major pathogenic effect in a knock-in mouse model of the dominantly inherited c.1430A>G human RPE65 mutation.

Human RPE65 mutations cause a spectrum of retinal dystrophies that result in blindness. While RPE65 mutations have been almost invariably recessively inherited, a c.1430A>G (p.(D477G)) mutation has been reported to cause autosomal dominant retinitis pigmentosa (adRP). To study the pathogenesis of this human mutation, we have replicated the mutation in a knock-in (KI) mouse model using CRISPR/Cas9-mediated genome editing. Significantly, in contrast to human patients, heterozygous KI mice do not exhibit any phenotypes in visual function tests. When raised in regular vivarium conditions, homozygous KI mice display relatively undisturbed visual functions with minimal retinal structural changes. However, KI/KI mouse retinae are more sensitive to light exposure and exhibit signs of degenerative features when subjected to light stress. We find that instead of merely producing a missense mutant protein, the A>G nucleotide substitution greatly affects appropriate splicing of Rpe65 mRNA by generating an ectopic splice site in comparable context to the canonical one, thereby disrupting RPE65 protein expression. Similar splicing defects were also confirmed for the human RPE65 c.1430G mutant in an in vitro Exontrap assay. Our data demonstrate that a splicing defect is associated with c.1430G pathogenesis, and therefore provide insights in the therapeutic strategy for human patients.

Improving the Transduction of Bone Marrow-Derived Cells with an Integrase-Defective Lentiviral Vector.

In lentiviral vector (LV) applications where transient transgene expression is sufficient, integrase-defective lentiviral vectors (IDLVs) are beneficial for reducing the potential for off-target effects associated with insertional mutagenesis. It was previously demonstrated that human RPE65 mRNA expression from an integrating lentiviral vector (ILV) induces endogenous Rpe65 and Cralbp mRNA expression in murine bone marrow–derived cells (BMDCs), initiating programming of the cells to retinal pigment epithelium (RPE)-like cells. These cells regenerate RPE in retinal degeneration models when injected systemically. As transient expression of RPE65 is sufficient to activate endogenous RPE-associated genes for programming BMDCs, use of an ILV is an unnecessary risk. In this study, an IDLV expressing RPE65 (IDLV3-RPE65) was generated. Transduction with IDLV3-RPE65 is less efficient than the integrating vector (ILV3-RPE65). Therefore, IDLV3-RPE65 transduction was enhanced with a combination of preloading 20 × -concentrated viral supernatant on RetroNectin at a multiplicity of infection of 50 and transduction of BMDCs by low-speed centrifugation. RPE65 mRNA levels increased from ∼12-fold to ∼25-fold (p < 0.05) after modification of the IDLV3-RPE65 transduction protocol, achieving expression similar to the ∼27-fold (p < 0.05) increase observed with ILV3-RPE65. Additionally, the study shows that the same preparation of RetroNectin can be used to coat up to three wells with no reduction in transduction. Critically, IDLV3-RPE65 transduction initiates endogenous Rpe65 mRNA expression in murine BMDCs and Cralbp/CRALBP mRNA in both murine and human BMDCs, similar to expression observed in ILV3-RPE65-transduced cells. Systemic administration of ILV3-RPE65 or IDLV3-RPE65 programmed BMDCs in a mouse model of retinal degeneration is sufficient to retain visual function and reduce retinal degeneration compared to mice receiving no treatment or naïve BMDC. It is concluded that IDLV3-RPE65 is appropriate for programming BMDCs to RPE-like cells.

Rapid differentiation of human ESCs into RPE cells by defined xeno-free culture system

Background Subretinal transplantation of retinal pigment epithelium (RPE) cells for the treatment of age-related macular degeneration (AMD) have accelerated the drive to develop xeno-free cultivation system that support the rapid differentiation of human embryonic stem cells (hESCs) into ES-RPE cells. Objective This study was to report a modified xeno-free culture system and method for accelerating derivation of hESCs to differentiate into RPE cells. Methods This study was approved by Ethic Committee of Zhongshan Ophthalmic Center.HESC H1 line was cloned and cultured in Vitronectin XF™-coated 6-well dish with xenogenetic-free medium.Cells were cultured in 50 ng/ml noggin, 10 ng/ml DKK-1 and 10 ng/ml insulin like growth factor-1 (IGF-1) medium for 2 days, and then the concentration of noggin was decreased to 10 ng/ml and 5 ng/ml basic fibroblast growth factor (bFGF) and cultured for the following 2 days.Sequentially, noggin and bFGF were removed and cultured for 2 days.Finally, 1 μmol/L CHIR99021 was added in medium for 6 days.Morphological changes in the progress of ESCs differentiation into RPE were observed by Living Cell Imaging System.The expression of Mitf and RPE65, RPE cells-specific markers, in the cells were detected by immunofluorescence technique, and the relative expression levels of RPE cells-specific marker mRNA were assayed using real time fluorescent quantitation PCR. Results Polygonal-shape monolayer cells which contained pigments were initially observed at day 14 after cultured with the cobblestone- like arrangement.Mitf and RPE65 were strongly expressed in the hES-derived RPE cells 35 days after induced, showing red fluorescence, and the cells presented hexagonal shape at cultured day 60 with numerous pigment granules in cytoplasm.Compared with before differentiation, the expression levels of Mitf mRNA in hES-RPE cells increased by (3.43±2.77) folds and (8.91±2.83) folds, and the expression levels of RPE65 mRNA increased by (14.60±3.94) folds and (87.16±9.32) folds at day 7 and day 14 after differentiation, respectively (all at P<0.05). Conclusions A defined xeno-free culture system is successfully established by adding niacinamide, DKK-1, noggin, IGF-1 and CHIR99021 in xeno-free medium, and this system can accelerate the derivation and differentiation of hESCs into RPE-like cells. Key words: Embryonic stem cells/drug effect; Cell culture techniques/methods; Cell differentiation; Retinal pigment epithelium; Culture media/drug effects; Cell line; Humans; Xeno-free

The pilot study on rapamycin retarding the differentiation of RPE cells in vitro

Background Retinal pigment epithelial (RPE) cell transplantation is a novel approach to the treatment of hereditary retinal diseases, however, human-derived RPE cell line occurs de-differentiation during in vitro cell culture.Studies showed that early abnormal activation of Akt/mammalian target of rapamycin (mTOR) signal pathway is the primary cause of RPE cell line de-differentiation, therefore, the inhibition of mTOR pathway will be helpful for the retard of de-differentiation of RPE cells. Objective This study aimed to investigate whether rapamycin can suppress the activation of mTOR pathway and promote differentiation of ARPE19 cells. Methods ARPE-19 cells were incubated in 12-well plate and divided into control group and rapamycin-treated group.DMSO or rapamycin with the final concentration of 400 nmol/L was added in the medium of the control group and the rapamycin-treated group, respectively.The cells of each group were collected 24 hours and 48 hours after cultured.The expression of zonula occludens-1 (ZO-1) in the cells was examined by immunofluorescence.The relative expression levels of RPE cell specific genes and proteins were assayed by using real-time quantitative PCR and Western blot, respectively.The detected results were compared between the two groups. Results ZO-1 was expressed in both groups, but the fluorescence intensity was evidently enhanced in the rapamycin-treated group.The relative expression levels of RPE65, MERKT and LRAT mRNA in the cells increased by 25.97%, 29.71% and 13.00% in the rapamycin-treated group compared with the control group 24 hours after cultured (P=0.04, 0.04, 0.04), and the expression levels of RPE65, LRAT, rLBP1, BEST1, keratin18 and MERKT mRNA elevated by 174.00%, 88.00%, 56.18%, 193.81%, 10.83% and 35.02% in the rapamycin-treated group in comparison with the control group 48 hours after cultured (P=0.00, 0.04, 0.01, 0.04, 0.04, 0.03). In addition, the expressions of p-mTOR, p-P70S6 and p-S6 protein were weaker in the rapamycin-treated group than those in the control group both 24 hours and 48 hours after cultured.Twenty four hours after cultured, the expression level of ZO-1 protein raised by 40% in the rapamycin-treated group compared with the control group (P=0.01); while 48 hours after cultured, the expression levels of ZO-1, MERKT, catenin and LRAT proteins elevated by 36.00%, 57.37%, 13.68% and 41.07% in the rapamycin-treated group in comparison with the control group (P=0.01, 0.00, 0.04, 0.04). Conclusions Rapamycin can suppress the activation of mTOR signaling pathway and up-regulate the expressions of RPE specific genes in ARPE19 cells.Inhibition of mTOR pathway might be an effective way for culturing RPE cells in vitro. Key words: ARPE19 cells; Rapamycin; mTOR signaling pathway; Differentiation

In Vitro Differentiation of Human Bone Marrow Stem Cells into Retinal Pigment Epithelium

Introduction: Dysfunction, degeneration, and loss of retinal pigment epithelial (RPE) cells are prominent features of subtypes of retinitis pigmentosa (RP) which is a cause of visual disability. In this condition, there is progressive visual loss that often leads to blindness. A variety of therapeutic approaches to delay the degenerative process are under development, the most promising of which is the stem cell regenerative therapy. Stem cells are self-renewing cells capable of transdifferentiation into a number of cell lineages. Mesenchymal stem cells (MSCs) represent a type of stem cell with remarkable proliferative, regenerative and transdifferentiation ability. Aim of the Work: The aim of this work was to evaluate the ability of bone marrow derived MSCs to differentiate into retinal pigmented epithelial cells in vitro as a step towards regeneration of degenerated retinal cells as in cases of retinitis pigmentosa. Patients and Methods: The present study was carried out on 20 bone marrow samples obtained from donors undergoing bone marrow aspirations, at EL-Maadi Military Hospital. 3 ml bone marrow were aspirated, mesenchymal stem cells were separated and induced to RPE lineage through culture in the presence of nicotinamide NIC and factors from the TGF-b superfamily (Activin A). The BMSC- derived pigmented-like cells were evaluated for the morphology, function, and marker expression of authentic RPE using RT-PCR for RPE65 mRNA. Results: Bone marrow derived mesenchymal stem cells in all samples demonstrated the morphologic characteristics as well as molecular markers of RPE cells after 8 weeks of culture with activin A and nicotinamide. Conclusion: In the present study, compelling evidences support the possibility of differentiation of bone marrow stem cells into retinal pigmented epithelial cells (RPE) which have the morphology, function, and marker expression of authentic RPE.

The Rpe65 rd12 allele exerts a semidominant negative effect on vision in mice.

The rd12 mouse was reported as a recessively inherited Rpe65 mutation. We asked if the rd12 mutation resides in Rpe65 and how the mutation manifests itself. A complementation test was performed by mating Rpe65(KO) (KO/KO) and rd12 mice together to determine if the rd12 mutation is in the Rpe65 gene. Visual function of wild-type (+/+), KO/+, rd12/+, KO/KO, rd12/rd12, and KO/rd12 mice was measured by optokinetic tracking (OKT) and ERG. Morphology was assessed by retinal cross section. qRT-PCR quantified Rpe65 mRNA levels. Immunoblotting measured the size and level of RPE65 protein. Rpe65 mRNA localization was visualized with RNA fluorescence in situ hybridization (FISH). Fractions of Rpe65 mRNA-bound proteins were separated by linear sucrose gradient fractionation. The KO and rd12 alleles did not complement. The rd12 allele induced a negative semidominant effect on visual function; OKT responses became undetectable 120 days earlier in rd12/rd12 mice compared with KO/KO mice. rd12/+ mice lost approximately 21% visual acuity by P210. rd12/rd12 mice had fewer cone photoreceptor nuclei than KO/KO mice at P60. rd12/rd12 mice expressed 71% +/+ levels of Rpe65 mRNA, but protein was undetectable. Mutant mRNA was appropriately spliced, exported to the cytoplasm, trafficked, and contained no other coding mutation aside from the known nonsense mutation. Mutant mRNA was enriched on ribosome-free messenger ribonucleoproteins (mRNPs), whereas wild-type mRNA was enriched on actively translating polyribosomes. The rd12 lesion is in Rpe65. The rd12 mutant phenotype inherits in a semidominant manner. The effects of the mutant mRNA on visual function may result from inefficient binding to ribosomes for translation.

Complementation Test ofRpe65Knockout and Tvrm148

A mouse mutation, tvrm148, was previously reported as resulting in retinal degeneration. Tvrm148 and Rpe65 map between markers D3Mit147 and D3Mit19 on a genetic map, but the physical map places RPE65 outside the markers. We asked if Rpe65 or perhaps another nearby gene is mutated and if the mutant reduced 11-cis-retinal levels. We studied the impact of the tvrm148 mutation on visual function, morphology, and retinoid levels.Normal phase HPLC was used to measure retinoid levels. Rpe65(+/+), tvrm148/+ (T(+/-)), tvrm148/tvrm148 (T(-/-)), RPE65(KO/KO) (Rpe65(-/-)), and Rpe65(T/-) mice visual function was measured by optokinetic tracking (OKT) and electroretinography (ERG). Morphology was assessed by light microscopy and transmission electron microscopy (TEM). qRT-PCR was used to measure Rpe65 mRNA levels. Immunoblotting measured the size and amount of RPE65 protein.The knockout and tvrm148 alleles did not complement. No 11-cis-retinal was detected in T(-/-) or Rpe65(-/-) mice. Visual acuity in Rpe65(+/+) and T(+/-) mouse was -0.382 c/d, but 0.037 c/d in T(-/-) mice at postnatal day 210 (P210). ERG response in T(-/-) mice was undetectable except at bright flash intensities. Outer nuclear layer (ONL) thickness in T(-/-) mice was -70% of Rpe65(+/+) by P210. Rpe65 mRNA levels in T(-/-) mice were unchanged, yet 14.5% of Rpe65(+/+) protein levels was detected. Protein size was unchanged.A complementation test revealed the RPE65 knockout and tvrm148 alleles do not complement, proving that the tvrm148 mutation is in Rpe65. Behavioral, physiological, molecular, biochemical, and histological approaches indicate that tvrm148 is a null allele of Rpe65.

Cell-Deposited Matrix Improves Retinal Pigment Epithelium Survival on Aged Submacular Human Bruch's Membrane

To determine whether resurfacing submacular human Bruch's membrane with a cell-deposited extracellular matrix (ECM) improves retinal pigment epithelial (RPE) survival. Bovine corneal endothelial (BCE) cells were seeded onto the inner collagenous layer of submacular Bruch's membrane explants of human donor eyes to allow ECM deposition. Control explants from fellow eyes were cultured in medium only. The deposited ECM was exposed by removing BCE. Fetal RPE cells were then cultured on these explants for 1, 14, or 21 days. The explants were analyzed quantitatively by light microscopy and scanning electron microscopy. Surviving RPE cells from explants cultured for 21 days were harvested to compare bestrophin and RPE65 mRNA expression. Mass spectroscopy was performed on BCE-ECM to examine the protein composition. The BCE-treated explants showed significantly higher RPE nuclear density than did the control explants at all time points. RPE expressed more differentiated features on BCE-treated explants than on untreated explants, but expressed very little mRNA for bestrophin or RPE65. The untreated young (<50 years) and African American submacular Bruch's membrane explants supported significantly higher RPE nuclear densities (NDs) than did the Caucasian explants. These differences were reduced or nonexistent in the BCE-ECM-treated explants. Proteins identified in the BCE-ECM included ECM proteins, ECM-associated proteins, cell membrane proteins, and intracellular proteins. Increased RPE survival can be achieved on aged submacular human Bruch's membrane by resurfacing the latter with a cell-deposited ECM. Caucasian eyes seem to benefit the most, as cell survival is the worst on submacular Bruch's membrane in these eyes.

Towards a blood test for diabetic retinopathy

Abstract Purpose Diabetic retinopathy (DR) is the leading cause of blindness among adults of working age in the UK. Early detection and appropriate management can prevent severe visual loss in 95% of cases. However, current screening methods are costly and suffer from subjective grading. Nucleic acids have been shown to circulate in plasma, with levels raised in conditions of cell death such as cancer and trauma. This study aimed to quantify circulatory retinal‐specific nucleic acids, and to evaluate potential for their use in the assessment of DR. Methods Diabetic patients (n = 106) and healthy controls (n = 24) were recruited from the Diabetes and Ophthalmology departments at St Thomas’ Hospital. RNA was extracted from whole blood, with quantitative real‐time RT‐PCR used to quantify mRNA levels for rhodopsin and RPE65, two proteins expressed exclusively in the retina. Beta‐actin mRNA was used for normalisation. Results Rhodopsin, RPE65 and beta‐actin mRNA were detected in 100% of subjects. Circulating rhodopsin and RPE65 mRNA levels were higher in diabetic patients than healthy individuals (p &lt; 0.02). Circulating rhodopsin mRNA was raised in all DR groups compared to healthy individuals, irrespective of presence or severity of DR (p &lt; 0.02). With respect to healthy controls, circulating RPE65 mRNA levels were higher in diabetic patients with background and proliferative DR (p &lt; 0.02). Patients with active proliferative DR (neovascularisation, vitreous and pre‐retinal haemorrhage, or retinal detachment) possessed higher RPE65 mRNA and lower rhodopsin mRNA levels than those with quiescent disease (p &lt; 0.01). Conclusion There is significant potential for use of these markers to screen for the presence of DR in diabetic patients in a quantitative manner using a blood test.

Retina‐Specific mRNA in the Assessment of Diabetic Retinopathy

In a previous study we demonstrated the presence and diagnostic usefulness of circulating rhodopsin mRNA in the assessment of diabetic retinopathy (DR). In the present study we investigated three further retina-specific markers in blood to determine their suitability as markers of DR. The markers were RPE65, retinoschisin, and melanopsin. Whole blood was collected from diabetic patients and healthy controls into PAXgene Blood RNA tubes and RNA was extracted using the PAXgene Blood RNA System. Quantitative real-time PCR was used to quantify mRNA for RPE65, retinoschisin, and melanopsin. beta-actin mRNA was used for normalization. RPE65, retinoschisin, and beta-actin mRNA were detected in 100% of subjects; melanopsin was not detected in either controls or diabetic patients. Circulating RPE65 mRNA concentration was 63% higher in diabetic patients than in healthy individuals (P= 0.019), whereas retinoschisin showed no change between the two groups. Compared with healthy controls, circulating RPE65 mRNA concentration was higher in diabetics with no retinopathy (30%; P= NS), background DR (93%; P= 0.01), preproliferative DR (20%; P= NS), and proliferative DR (107%; P= 0.004). Compared with diabetics with no retinopathy, levels of RPE65 mRNA were also significantly higher (60%) in the presence of proliferative DR (P= 0.029). In contrast, levels of retinoschisin mRNA were lower in background DR (34%; P= 0.033), preproliferative DR (43%; P= 0.026), and proliferative DR (47%; P= 0.038) compared to that in diabetics without retinopathy. We conclude that not all retina-specific mRNA species are detectable in circulation (e.g., melanopsin). This may be related to differences in expression levels for the individual markers. Both RPE65 and retinoschisin were detectable and demonstrated contrasting trends in diabetics with and without retinopathy. In combination with rhodopsin, RPE65, and retinoschisin, mRNA may offer a useful tool in developing a blood test for DR.

Effect of Hypoxia on Circulating Levels of Retina‐Specific Messenger RNA in Type 2 Diabetes Mellitus

Previously it was shown that the circulating rhodopsin mRNA level was higher in diabetic retinopathy (DR). Recent evidence suggests that hypoxia may also be associated with DR. The aim of this study was to investigate the effect of oxygen desaturation on circulating retina-specific mRNA in type 2 diabetic patients. Thirty-five type 2 diabetic patients underwent overnight oximetry. Two parameters from oximetry were used to measure oxygen desaturation: the number of times per hour the oxygen saturation decreased by 4% or greater (number of dips/hr) and percentage of sleep time with oxygen saturation (SpO(2)) <90%. Blood samples were collected into PAXgene Blood RNA tubes. Total RNA was extracted from the samples and reverse-transcribed into cDNA, and retina-specific markers were measured by quantitative real time PCR. In patients with >/=5 dips/hr, mRNA values for rhodopsin (P= 0.05) and RPE65 (P= 0.044) were significantly higher than in patients with <5 dips/hr. No change was seen in retinoschisin mRNA expression. In patients with preproliferative or proliferative DR, median levels for rhodopsin mRNA and RPE65 mRNA were 30% and 80% higher and retinoschisin mRNA was lower in patients with >/=5 dips/hr when compared to patients with <5 dips/hr. These results indicate that hypoxia may modulate expression of genes in the retina.

The level of thymic expression of RPE65 inversely correlates with its capacity to induce experimental autoimmune uveitis (EAU) in different rodent strains

We have previously shown that immunization with RPE65 produces in rats of four strains a severe inflammatory eye disease, designated experimental autoimmune uveitis (EAU). Here, we examined the uveitogenicity of RPE65 in six strains of mice. Only one strain, C57Bl/6, was found to develop consistently moderate levels of EAU, whereas other strains (BALB/c, B10.A, B10.BR, B10.RIII, C57BL/10J) were found to be essentially resistant to disease induced by RPE65. Analysis of the expression of RPE65 mRNA in thymi of the six mouse strains revealed detectable levels of the transcript in all strains, but with remarkable quantitative differences, with the lowest levels seen in thymi of C57Bl/6 mice, the only strain susceptible to RPE65-induced EAU. Moreover, unlike the finding with the mice, no RPE65 mRNA was detected in thymi of any of the four rat strains (Lewis, BN, F344, SHR) all of which are susceptible to the disease. These data thus indicate that the susceptibility to RPE65-induced EAU is inversely related to the thymic expression of the molecule. The data also suggest that this disease can be induced only in mice in which thymic expression of RPE65 is sufficiently low to allow the escape from deletion of T-cells with the adequate capacity to initiate the pathogenic immune response.

Expression of RPE65, a putative receptor for plasma retinol-binding protein, in nonmelanocytic skin tumours

In a recent report we described RPE65, a protein originally characterized in retinal pigment epithelium, to be expressed in normal human epidermis. RPE65 is suspected to be involved in cellular uptake of retinol which is transported in the bloodstream complexed with plasma retinol-binding protein. To evaluate protein and mRNA expression of RPE65 in actinic keratosis (AK), squamous cell carcinoma (SCC) and basal cell carcinoma (BCC) compared with normal skin. RPE65 mRNA expression in skin tumours relative to normal skin of the respective donor was studied by real-time polymerase chain reaction in AK (n = 15), invasive SCC (n = 30) and BCC (n = 18). A peptide-specific anti-RPE65 antibody was used for immunohistochemical staining of formalin-fixed and paraffin-embedded tissue sections of the respective tumours. RPE65 mRNA expression was reduced in AK. A highly significant reduction of RPE65 mRNA was observed in invasive SCC relative to normal skin of the respective donors. Immunohistochemistry revealed a continuous staining of basal and suprabasal keratinocytes in normal human epidermis. RPE65 in AK shown by immunohistochemical staining was reduced and quite irregular, whereas invasive SCC revealed no staining of tumour cells with the anti-RPE65 antibody. RPE65 mRNA values were elevated, whereas immunohistochemical staining for RPE65 protein was heterogeneous in BCC. These results suggest progressive downregulation of RPE65 from AK to invasive SCC.

RPE65 of retinal pigment epithelium, a putative receptor molecule for plasma retinol-binding protein, is expressed in human keratinocytes.

Retinoids are important modulators for cell growth and differentiation of normal skin. In plasma, retinol is transported coupled to plasma retinol-binding protein. In this study, we investigated gene and protein expression of RPE65, a putative receptor for plasma retinol-binding protein in human epidermal keratinocytes. We performed real-time PCR analysis to evaluate expression of RPE65 mRNA in proliferating and differentiating keratinocytes. Immunoblotting with anti-RPE65 antibody shows distinct reactivity to a 61-kDa protein. Indirect immunofluorescence on normal human epidermis reveals cell surface labeling of keratinocytes. Laser scan microscopy exhibits colocalization of plasma retinol-binding protein and RPE65 on cultured keratinocytes. Internalization experiments with [3H]retinoic acid-retinol-binding protein complex in the presence and absence of excess of retinol-binding protein indicates receptor-dependent uptake of retinoids. We further show isolation of RPE65 protein by affinity chromatography from lysates of keratinocytes using a retinol-binding protein-matrix gel column. In summary, we demonstrate mRNA and protein expression of RPE65 in epidermal keratinocytes. Colocalization of plasma retinol-binding protein with RPE65 and affinity binding suggest a direct interaction of RPE65 with plasma retinol-binding protein in cultured human keratinocytes that might be involved in retinoid uptake of keratinocytes.

Light Damage Susceptibility and RPE65 in Rats

A sequence variation in the pigment epithelial protein RPE65 has been shown to correlate with RPE65 protein levels, rhodopsin regeneration kinetics and light damage susceptibility in different mouse strains. Here, we tested whether such a correlation can also be found in rats. We examined four rat strains for RPE65 protein levels and the Rpe65 gene sequence. In two strains, we additionally determined Rpe65 mRNA levels, rhodopsin regeneration and light damage susceptibility (LDS).RPE65 protein levels were higher in Lewis and Brown Norway rats compared to Wistar and Long Evans. The albino strains Wistar and Lewis were investigated further. Lewis had higher Rpe65 mRNA levels than Wistar. Sequence analysis of the coding region of the Rpe65 cDNA revealed no relevant sequence variations in the two strains. Content and regeneration of rhodopsin were comparable in both strains. However, Wistar rats were more susceptible to light damage than Lewis. We conclude that lower RPE65 protein levels in Wistar may have been caused by decreased gene expression and not by a sequence variation as suggested for mice. In rats, RPE65 may not be a limiting factor for rhodopsin regeneration. Since LDS in rats did not directly correlate with RPE65 protein levels and rhodopsin regeneration, other yet unidentified (genetic) factors may account for the susceptibility differences observed in rats.

Light Damage Susceptibility and RPE65 in Rats

A sequence variation in the pigment epithelial protein RPE65 has been shown to correlate with RPE65 protein levels, rhodopsin regeneration kinetics and light damage susceptibility in different mouse strains. Here, we tested whether such a correlation can also be found in rats. We examined four rat strains for RPE65 protein levels and the Rpe65 gene sequence. In two strains, we additionally determined Rpe65 mRNA levels, rhodopsin regeneration and light damage susceptibility (LDS). RPE65 protein levels were higher in Lewis and Brown Norway rats compared to Wistar and Long Evans. The albino strains Wistar and Lewis were investigated further. Lewis had higher Rpe65 mRNA levels than Wistar. Sequence analysis of the coding region of the Rpe65 cDNA revealed no relevant sequence variations in the two strains. Content and regeneration of rhodopsin were comparable in both strains. However, Wistar rats were more susceptible to light damage than Lewis. We conclude that lower RPE65 protein levels in Wistar may have been caused by decreased gene expression and not by a sequence variation as suggested for mice. In rats, RPE65 may not be a limiting factor for rhodopsin regeneration. Since LDS in rats did not directly correlate with RPE65 protein levels and rhodopsin regeneration, other yet unidentified (genetic) factors may account for the susceptibility differences observed in rats.

46] Genetic analysis of RPE65: From human disease to mouse model

RPE65 is a highly expressed, conserved retinal pigment epithelium (RPE)-specific protein preferentially associated with the RPE microsomal membrane fraction, although it is not an integral membrane protein. Comparison of bovine, human, rat, mouse, dog, and salamander RPE65s shows a rather high degree of conservation of this protein and Northern analysis detects RPE65 mRNA only in RPE. In neonatal rats, morphologically well-differentiated RPE cells do not express RPE65 at birth but it is detectable on postnatal day 4 (P4), that is, 1–2 days before the photoreceptors develop their outer segments (OS). This chapter describes the methods and reagents that have been used for the characterization of mutations in human RPE65 and for the genetic analysis of knockout mice and how such studies are helping in our understanding of the function of RPE65. RPE65 mutations resulting in amino acid substitutions can cause early- or late-onset disease depending on the residue changed, while null mutations invariably cause severe early-onset changes in the vision of both humans and mice.