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Photoreceptor cells and their related gene expression were maintained in Mitf−/− mice treated with 9-cis-retinal. (A) RHODOPSIN and OPSIN expression in superior retina of WT and Mitf− /− treated with solvent or 9-cis-retinal. Rod RHODOPSIN was localized to the rod outer segment (ROS), and cone OPSIN was localized to the ONL and the rod outer segment in WT neural retina. In Mitf− /− mice treated with solvent, the RHODOPSIN and OPSIN localization in ROS were decreased and strikingly mislocalized to the cone cell bodies in the ONL. By contrast, RHODOPSIN and OPSIN localization in ROS, especially Rhodopsin, were maintained in Mitf− /− mice treated with 9-cis-retinal. (B) Quantification of OPSIN-labeled cells in retinal ROS of WT, Mitf− /− treated with solvent or treated with 9-cis-retinal. In contrast to WT, the OPSIN-labeled cells in retinal ROS were almost completely lost in Mitf− /− mice treated with solvent, whereas OPSIN-positive cells were partially preserved in Mitf− /− mice treated with 9-cis-retinal. (C) The expression of photoreceptor marker genes in WT, Mitf− /− treated with solvent, and Mitf− /− mice treated with 9-cis-retinal at P22 by RT-PCR. For photoreceptor marker genes, 9-cis retinal administration partially restored S-opsin, M-opsin and Gnat2 expression. Moreover, 9-cis retinal administration also restored expression of the rod-specific genes Rho and Gnat1, as well as genes expressed by both rod and cone cells, including Crx, Nrl, and Nr2e3 expression. Scale bar: 50 μ m.
Source publication
Regeneration of the visual pigment by cells of the retinal pigment epithelium (RPE) is fundamental to vision. Here we show that the microphthalmia-associated transcription factor, MITF, which plays a central role in the development and function of RPE cells, regulates the expression of two visual cycle genes, Rlbp1 which encodes retinaldehyde bindi...
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Purpose:
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.
Methods:
We cultivated porcine primary RPE cells isolated from cadaver eyes from the slaughterhouse...
Citations
... In one study, western blot analysis of HEK-293 cells transfected with RLBP1-expressing plasmids clearly resulted in two CRALBP bands, but these were not commented on by the authors (42). Similarly, western blot analyses of human ARPE19 cells overexpressing MITF (43) showed the presence of two CRALBP bands that were not highlighted. Particularly interesting was the study on human donor RPE that showed the presence of two CRALBP bands following western blot analysis of fresh RPE culture (44). ...
Inherited retinal diseases (IRDs) are clinically and genetically heterogeneous disorders characterised by progressive vision loss. Over 270 causative genes have been identified and variants within the same gene can give rise to clinically distinct disorders. Human induced pluripotent stem cells (iPSCs) have revolutionised disease modelling, by allowing pathophysiological and therapeutic studies in the patient and tissue context. The IRD gene RLBP1 encodes CRALBP, an actor of the rod and cone visual cycles in the retinal pigment epithelium (RPE) and Müller cells, respectively. Variants in RLBP1 lead to three clinical subtypes: Bothnia dystrophy, Retinitis punctata albescens and Newfoundland rod-cone dystrophy. We modelled RLBP1 -IRD subtypes by patient-specific iPSC-derived RPE and identified pertinent therapeutic read-outs. We developed an AAV2/5-mediated gene replacement strategy and provided a proof-of-concept in the ex vivo human models that was validated in an in vivo Rlbp1 −/− murine model. Most importantly, we identified a previously unsuspected smaller CRALBP isoform that is naturally and differentially expressed in both human and murine retina. The new isoform arises from an alternative methionine initiation site and plays a role in the visual cycle. This work provides novel insights into CRALBP expression and RLBP1 -associated pathophysiology and raises important considerations for successful gene supplementation therapy.
... However, this issue had to be balanced with the potential for non-cell autonomous effects on retinal development from disruptions in RPE development caused by the mi allele in a homozygous state. [76][77][78] Genetic redundancy is also possible since Tfeb, a related bHLH-zip gene, was up-regulated in the orJ retina. These issues are somewhat mitigated with the mi allele since the mutant protein functions in a dominant negative manner by heterodimerizing with wild-type Mitf and Tfeb, 43 and because a large cohort of Mitf-dependent genes were identified. ...
Background
A goal of developmental genetics is to identify functional interactions that underlie phenotypes caused by mutations. We sought to identify functional interactors of Vsx2, which when mutated, disrupts early retinal development. We utilized the Vsx2 loss‐of‐function mouse, ocular retardation J (orJ), to assess interactions based on principles of positive and negative epistasis as applied to bulk transcriptome data. This was first tested in vivo with Mitf, a target of Vsx2 repression, and then to cultures of orJ retina treated with inhibitors of Retinoid‐X Receptors (RXR) to target Rxrg, an up‐regulated gene in the orJ retina, and gamma‐Secretase, an enzyme required for Notch signaling, a key mediator of retinal proliferation and neurogenesis.
Results
Whereas Mitf exhibited robust positive epistasis with Vsx2, it only partially accounts for the orJ phenotype, suggesting other functional interactors. RXR inhibition yielded minimal evidence for epistasis between Vsx2 and Rxrg. In contrast, gamma‐Secretase inhibition caused hundreds of Vsx2‐dependent genes associated with proliferation to deviate further from wild‐type, providing evidence for convergent negative epistasis with Vsx2 in regulating tissue growth.
Conclusions
Combining in vivo and ex vivo testing with transcriptome analysis revealed quantitative and qualitative characteristics of functional interaction between Vsx2, Mitf, RXR, and gamma‐Secretase activities.
... We have recently shown that MITF regulates a number of factors in the RPE that are of importance in retinal physiology. They include PEDF (pigment epithelium derived factor), RDH5 (retinol dehydrogenase-5) and RLBP1 (retinaldehyde binding protein-1) [29][30][31], and NRF2 and PGC1α, both of which involved in regulating the antioxidant defense system of the RPE [32,33]. Interestingly, deliberate overexpression of MITF in the RPE can protect the retina against oxidative damage-induced retinal degeneration [33]. ...
... In fact, MITF is considered a master regulator of pigment cells and a major factor in their development and function. In the RPE, it regulates pigmentation genes but also Pedf and visual cycle genes such as Rlbp1 and Rdh5 [29][30][31]. Interestingly, in AMD patients, the expression of PEDF is lower in RPE cells, the RPE basal lamina, Bruch's membrane, and choroidal stroma, thereby potentially promoting choroidal neovascularization [54]. In mice, Pedf deficiency leads to an increase in susceptibility of retinal degeneration associated with the rd10 mutation [55]. ...
The decreased antioxidant capacity in the retinal pigment epithelium (RPE) is the hallmark of retinal degenerative diseases including age-related macular degeneration (AMD). Nevertheless, the exact regulatory mechanisms underlying the pathogenesis of retinal degenerations remain largely unknown. Here we show in mice that deficiencies in Dapl1, a susceptibility gene for human AMD, impair the antioxidant capacity of the RPE and lead to age-related retinal degeneration in the 18-month-old mice homozygous for a partial deletion of Dapl1. Dapl1-deficiency is associated with a reduction of the RPE's antioxidant capacity, and experimental re-expression of Dapl1 reverses this reduction and protects the retina from oxidative damage. Mechanistically, DAPL1 directly binds the transcription factor E2F4 and inhibits the expression of MYC, leading to upregulation of the transcription factor MITF and its targets NRF2 and PGC1α, both of which regulate the RPE's antioxidant function. When MITF is experimentally overexpressed in the RPE of DAPL1 deficient mice, antioxidation is restored and retinas are protected from degeneration. These findings suggest that the DAPL1-MITF axis functions as a novel regulator of the antioxidant defense system of the RPE and may play a critical role in the pathogenesis of age-related retinal degenerative diseases.
... 17,18 How CRALBP is regulated in RPE cells and whether the downregulation of CRALBP in the senescent RPE is associated with the attenuation of agerelated scotopic vision have not been determined yet. The transcriptional factors such as MITF, 19 Sox9, 20 and Pax6 21 are involved in regulating CRALBP's mRNA expression. Mice lacking MITF cause the degeneration of photoreceptor and RPE cells with the downregulation of CRALBP and RDH5 transcriptional expression in RPE cells. ...
... Mice lacking MITF cause the degeneration of photoreceptor and RPE cells with the downregulation of CRALBP and RDH5 transcriptional expression in RPE cells. 19 The promoter analysis defines an SP1 binding motif in the Rlbp1's promoter, which can drive GFP expression in retinal Muller cells in transgenic mice. 9,22 However, the regulatory effect of SP1 on CRALBP expression has not been completely analyzed yet. ...
... 22 In addition to SP1, CRALBP expression is regulated by other transcriptional factors such as MITF, SOX9, Pax6, and TR2F2. [19][20][21] We did not analyze the regulatory association between HSP90 and transcription factors of MITF or SOX9 on the CRALBP expression because we did not observe the association of MITF or SOX9 to CRALBP's promoter in the DNA pull-down combining MS assay (data not shown). However, due to the limitation of CRALBP's promoter (450 bp) used in the pull-down assay, the present data could not exclude the potential regulation of MITF or SOX9 on CRALBP expression. ...
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 HSP90β 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.
... MITF is a basic helix-loop-helix leucine zipper (bHLH-Zip), a dimeric transcription factor involved in the generation and function of mast cells [7], melanocytes [8], osteoclasts [9,10], and retinal pigmented epithelium [11]. Mutations in MITF lead to defects in melanocytes, the retinal pigmented epithelium, mast cells, and osteoclasts [12]. ...
Gastrointestinal stromal tumors (GISTs) are the most common neoplasms of mesenchymal origin, and most of them emerge due to the oncogenic activation of KIT or PDGFRA receptors. Despite their relevance in GIST oncogenesis, critical intermediates mediating the KIT/PDGFRA transforming program remain mostly unknown. Previously, we found that the adaptor molecule SH3BP2 was involved in GIST cell survival, likely due to the co-regulation of the expression of KIT and Microphthalmia-associated transcription factor (MITF). Remarkably, MITF reconstitution restored KIT expression levels in SH3BP2 silenced cells and restored cell viability. This study aimed to analyze MITF as a novel driver of KIT transforming program in GIST. Firstly, MITF isoforms were characterized in GIST cell lines and GIST patients’ samples. MITF silencing decreases cell viability and increases apoptosis in GIST cell lines irrespective of the type of KIT primary or secondary mutation. Additionally, MITF silencing leads to cell cycle arrest and impaired tumor growth in vivo. Interestingly, MITF silencing also affects ETV1 expression, a linage survival factor in GIST that promotes tumorigenesis and is directly regulated by KIT signaling. Altogether, these results point to MITF as a key target of KIT/PDGFRA oncogenic signaling for GIST survival and tumor growth.
... 17 HSD family has a total of 14 members, most of which are involved in the metabolism of sex hormones, such as HSD17B5. Some members are also involved in the oxidation process of fatty acid in peroxisome, and HSD17B9 is involved in retinol metabolism [3]. ...
... COL I is involved in ECM formation and is one of the important components of RPE tissue [57]. MIFT is involved in the production of melanocytes and is required for normal RPE development [58]. The GG9/HA1 group exhibited the highest expression of RPE-related genes. ...
Cell therapies for age-related macular degeneration (AMD) treatment have been developed by integrating hydrogel-based biomaterials. Until now, cell activity has been observed only in terms of the modulus of the hydrogel. In addition, cell behavior has only been observed in the 2D environment of the hydrogel and the 3D matrix. As time-dependent stress relaxation is considered a significant mechanical cue for the control of cellular activities, it is important to optimize hydrogels for retinal tissue engineering (TE) by applying this viewpoint. Herein, a gellan Gum (GG)/Hyaluronic acid (HA) hydrogel was fabricated using a facile physical crosslinking method. The physicochemical and mechanical properties were controlled by forming a different composition of GG and HA. The characterization was performed by conducting a mass swelling study, a sol fraction study, a weight loss test, a viscosity test, an injection force study, a compression test, and a stress relaxation analysis. The biological activity of the cells encapsulated in 3D constructs was evaluated by conducting a morphological study, a proliferation test, a live/dead analysis, histology, immunofluorescence staining, and a gene expression study to determine the most appropriate material for retinal TE biomaterial. Hydrogels with moderate amounts of HA showed improved physicochemical and mechanical properties suitable for injection into the retina. Moreover, the time-dependent stress relaxation property of the GG/HA hydrogel was enhanced when the appropriate amount of HA was loaded. In addition, the cellular compatibility of the GG/HA hydrogel in in vitro experiments was significantly improved in the fast-relaxing hydrogel. Overall, these results demonstrate the remarkable potential of GG/HA hydrogel as an injectable hydrogel for retinal TE and the importance of the stress relaxation property when designing retinal TE hydrogels. Therefore, we believe that GG/HA hydrogel is a prospective candidate for retinal TE biomaterial.
... Accumulating evidence suggests that macular clusters with high expression of EGFR may have more important functions, such as autophagy, phagocytosis and cell proliferation, in the RPE. Interestingly, TYRP1 has been reported to be involved in melanin synthesis (Rai et al., 2020), and RLBP1 is related to the visual cycle (Wen et al., 2016). Our study showed that both of these genes were highly expressed in the peripheral P2-1 cluster ( Figure 3G). ...
Human retinal pigment epithelium cells are arranged in a monolayer that plays an important supporting role in the retina. Although the heterogeneity of specific retinal cells has been well studied, the diversity of hRPE cells has not been reported. Here, we performed a single-cell RNA sequencing on 9,302 hRPE cells from three donors and profiled a transcriptome atlas. Our results identified two subpopulations that exhibit substantial differences in gene expression patterns and functions. One of the clusters specifically expressed ID3 , a macular retinal pigment epithelium marker. The other cluster highly expressed CRYAB , a peripheral RPE marker. Our results also showed that the genes associated with oxidative stress and endoplasmic reticulum stress were more enriched in the macular RPE. The genes related to light perception, oxidative stress and lipid metabolism were more enriched in the peripheral RPE. Additionally, we provided a map of disease-related genes in the hRPE and highlighted the importance of the macular RPE and peripheral RPE clusters P4 and P6 as potential therapeutic targets for retinal diseases. Our study provides a transcriptional landscape for the human retinal pigment epithelium that is critical to understanding retinal biology and disease.
... MITF plays multiple roles in regulating RPE cell development and differentiation, including its effects on antioxidant systems, growth factor expression, visual cycle activities, proliferation, and melanogenesis. 29,[41][42][43][44][45] In RPE cells, transcriptional control of MITF expression has been shown to be regulated by signaling by bone morphogenetic protein (BMP), Wnt/β-catenin, and fibroblast growth factor (FGF), as well as transcription factors VSX2, PAX6, PAX2, OTX2, and ZEB1, 17 but the posttranscriptional regulation of MITF splicing in RPE cells is still poorly understood. Alternative splicing plays a critical role in providing protein diversity and functional activity. ...
Purpose:
Retinal pigment epithelium (RPE) cell proliferation is precisely regulated to maintain retinal homoeostasis. Microphthalmia-associated transcription factor (MITF), a critical transcription factor in RPE cells, has two alternatively spliced isoforms: (+)MITF and (-)MITF. Previous work has shown that (-)MITF but not (+)MITF inhibits RPE cell proliferation. This study aims to investigate the role of long non-coding RNA (lncRNA) nuclear-enriched abundant transcript 1 (NEAT1) in regulating MITF splicing and hence proliferation of RPE cells.
Methods:
Mouse RPE, primary cultured mouse RPE cells, and different proliferative human embryonic stem cell (hESC)-RPE cells were used to evaluate the expression of (+)MITF, (-)MITF, and NEAT1 by reverse-transcription PCR (RT-PCR) or quantitative RT-PCR. NEAT1 was knocked down using specific small interfering RNAs (siRNAs). Splicing factor proline- and glutamine-rich (SFPQ) was overexpressed with the use of lentivirus infection. Cell proliferation was analyzed by cell number counting and Ki67 immunostaining. RNA immunoprecipitation (RIP) was used to analyze the co-binding between the SFPQ and MITF or NEAT1.
Results:
NEAT1 was highly expressed in proliferative RPE cells, which had low expression of (-)MITF. Knockdown of NEAT1 in RPE cells switched the MITF splicing pattern to produce higher levels of (-)MITF and inhibited cell proliferation. Mechanistically, NEAT1 recruited SFPQ to bind directly with MITF mRNA to regulate its alternative splicing. Overexpression of SFPQ in ARPE-19 cells enhanced the binding enrichment of SFPQ to MITF and increased the splicing efficiency of (+)MITF. The binding affinity between SFPQ and MITF was decreased after NEAT1 knockdown.
Conclusions:
NEAT1 acts as a scaffold to recruit SFPQ to MITF mRNA and promote its binding affinity, which plays an important role in regulating the alternative splicing of MITF and RPE cell proliferation.
... Morphological characterization and histological studies showed that the Ge/GG/CS gel provided an optimal microenvironment for encapsulated cells for cell-cell and cell-matrix interactions. Gene expression analysis of encapsulated RPE cells also showed greater expression of RPE pertinent genes (RPE65, CRALBP, MITF, NPR-A, RHODO, COL I) [68][69][70][71][72] in GE/GG/CS hydrogels, which are indicative of higher amounts of proliferative cells. Despite the promising results, in vivo studies must be conducted to fully determine its biocompatibility and efficacy. ...
With the prevalence of eye diseases, such as cataracts, retinal degenerative diseases, and glaucoma, different treatments including lens replacement, vitrectomy, and stem cell transplantation have been developed; however, they are not without their respective shortcomings. For example, current methods to seal corneal incisions induced by cataract surgery, such as suturing and stromal hydration, are less than ideal due to the potential for surgically induced astigmatism or wound leakage. Vitrectomy performed on patients with diabetic retinopathy requires an artificial vitreous substitute, with current offerings having many shortcomings such as retinal toxicity. The use of stem cells has also been investigated in retinal degenerative diseases; however, an optimal delivery system is required for successful transplantation. The incorporation of hydrogels into ocular therapy has been a critical focus in overcoming the limitations of current treatments. Previous reviews have extensively documented the use of hydrogels in drug delivery; thus, the goal of this review is to discuss recent advances in hydrogel technology in surgical applications, including dendrimer and gelatin-based hydrogels for ocular adhesives and a variety of different polymers for vitreous substitutes, as well as recent advances in hydrogel-based retinal pigment epithelium (RPE) and retinal progenitor cell (RPC) delivery to the retina.
