Gene augmentation and read-through rescue channelopathy in an iPSC-RPE model of congenital blindness

PurposeMutations in the KCNJ13 gene are known to cause Leber’s Congenital Amaurosis (LCA16), an inherited pediatric blindness. KCNJ13 gene encodes the Kir7.1 subunit protein which acts as a tetrameric inwardly rectifying potassium ion channel in the retinal pigment epithelium to maintain ionic homeostasis thereby allowing photoreceptors to encode visual information. We sought to determine if genetic approaches might be effective in treating blindness due to mutations in KCNJ13.MethodsWe developed patient-derived hiPSC-RPE carrying an autosomal recessive nonsense mutation in the KCNJ13 gene (c.158G>A, p.Trp53*). We performed biochemical and electrophysiology assays of Kir7.1 function. Both small molecule read-through drug and gene-therapy approaches were tested using this disease-in-a-dish approach.ResultsWe found that the LCA16 hiPSC-RPE had normal morphology but did not express a functional Kir7.1 channel and was unable to demonstrate normal physiology. Following read-through drug treatment, the LCA16 hiPSC cells were hyperpolarized by 30 mV and Kir7.1 current was restored. Similarly, loss-of-function of Kir7.1 channel was circumvented by lentiviral gene delivery to the hiPSC-RPE cells. In either approach, Kir7.1 protein was expressing normally with restoration of membrane potential and Kir7.1 current.ConclusionLoss-of-function mutation in Kir7.1 is a cause of LCA. Using either read-through therapy or gene augmentation, we rescued Kir7.1 channel function in patient-derived iPSC-RPE cells via a precision medicine approach.

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

AJP Cell Physiology ◽

10.1152/ajpcell.00185.2011 ◽

2011 ◽

Vol 301 (5) ◽

pp. C1017-C1026 ◽

Cited By ~ 19

Author(s):

Xiaoming Zhang ◽

Dongli Yang ◽

Bret A. Hughes

Keyword(s):

Visual Information ◽

Pigment Epithelium ◽

Retinal Pigment ◽

Basal Membrane ◽

Cell Types ◽

Neural Retina ◽

Pcr Analysis ◽

Rod Photoreceptor ◽

Rpe Cells ◽

Channel Expression

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

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Distinct expression requirements and rescue strategies for BEST1 loss- and gain-of-function mutations

eLife ◽

10.7554/elife.67622 ◽

2021 ◽

Vol 10 ◽

Author(s):

Qingqing Zhao ◽

Yang Kong ◽

Alec Kittredge ◽

Yao Li ◽

Yin Shen ◽

...

Keyword(s):

Pigment Epithelium ◽

Retinal Pigment ◽

Genetic Mutation ◽

Dominant Negative ◽

Loss Of Function ◽

Gain Of Function ◽

Rpe Cells ◽

Degenerative Disorders ◽

Universal Treatment ◽

Human Rpe Cells

Genetic mutation of the human BEST1 gene, which encodes a Ca2+-activated Cl- channel (BEST1) predominantly expressed in retinal pigment epithelium (RPE), causes a spectrum of retinal degenerative disorders commonly known as bestrophinopathies. Previously, we showed that BEST1 plays an indispensable role in generating Ca2+-dependent Cl- currents in human RPE cells, and the deficiency of BEST1 function in patient-derived RPE is rescuable by gene augmentation (Li et al., 2017). Here, we report that BEST1 patient-derived loss-of-function and gain-of-function mutations require different mutant to wild-type (WT) molecule ratios for phenotypic manifestation, underlying their distinct epigenetic requirements in bestrophinopathy development, and suggesting that some of the previously classified autosomal dominant mutations actually behave in a dominant-negative manner. Importantly, the strong dominant effect of BEST1 gain-of-function mutations prohibits the restoration of BEST1-dependent Cl- currents in RPE cells by gene augmentation, in contrast to the efficient rescue of loss-of-function mutations via the same approach. Moreover, we demonstrate that gain-of-function mutations are rescuable by a combination of gene augmentation with CRISPR/Cas9-mediated knockdown of endogenous BEST1 expression, providing a universal treatment strategy for all bestrophinopathy patients regardless of their mutation types.

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Iron-mediated retinal degeneration in haemojuvelin-knockout mice

Biochemical Journal ◽

10.1042/bj20111148 ◽

2011 ◽

Vol 441 (2) ◽

pp. 599-608 ◽

Cited By ~ 14

Author(s):

Jaya P. Gnana-Prakasam ◽

Amany Tawfik ◽

Michelle Romej ◽

Sudha Ananth ◽

Pamela M. Martin ◽

...

Keyword(s):

Pigment Epithelium ◽

Genetic Disorder ◽

Retinal Pigment ◽

Growth Patterns ◽

Loss Of Function ◽

Iron Regulatory Proteins ◽

Rpe Cells ◽

Hepcidin Expression ◽

Morphogenetic Protein ◽

Bone Morphogenetic Protein 6

Haemochromatosis is a genetic disorder of iron overload resulting from loss-of-function mutations in genes coding for the iron-regulatory proteins HFE (human leucocyte antigen-like protein involved in iron homoeostasis), transferrin receptor 2, ferroportin, hepcidin and HJV (haemojuvelin). Recent studies have established the expression of all of the five genes in the retina, indicating their importance in retinal iron homoeostasis. Previously, we demonstrated that HJV is expressed in RPE (retinal pigment epithelium), the outer and inner nuclear layers and the ganglion cell layer. In the present paper, we report on the consequences of Hjv deletion on the retina in mice. Hjv−/− mice at ≥18 months of age had increased iron accumulation in the retina with marked morphological damage compared with age-matched controls; these changes were not found in younger mice. The retinal phenotype in Hjv−/− mice included hyperplasia of RPE. We isolated RPE cells from wild-type and Hjv−/− mice and examined their growth patterns. Hjv−/− RPE cells were less senescent and exhibited a hyperproliferative phenotype. Hjv−/− RPE cells also showed up-regulation of Slc7a11 (solute carrier family 7 member 11 gene), which encodes the ‘transporter proper’ subunit xCT in the heterodimeric amino acid transporter xCT/4F2hc (cystine/glutamate exchanger). BMP6 (bone morphogenetic protein 6) could not induce hepcidin expression in Hjv−/− RPE cells, confirming that retinal cells require HJV for induction of hepcidin via BMP6 signalling. HJV is a glycosylphosphatidylinositol-anchored protein, and the membrane-associated HJV is necessary for BMP6-mediated activation of hepcidin promoter in RPE cells. Taken together, these results confirm the biological importance of HJV in the regulation of iron homoeostasis in the retina and in RPE.

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A human model of Batten disease shows role of CLN3 in phagocytosis at the photoreceptor–RPE interface

Communications Biology ◽

10.1038/s42003-021-01682-5 ◽

2021 ◽

Vol 4 (1) ◽

Author(s):

Cynthia Tang ◽

Jimin Han ◽

Sonal Dalvi ◽

Kannan Manian ◽

Lauren Winschel ◽

...

Keyword(s):

Photoreceptor Cell ◽

Vision Loss ◽

Pigment Epithelium ◽

Retinal Pigment ◽

Cell Loss ◽

Human Model ◽

Lysosomal Storage ◽

Rpe Cells ◽

Cln3 Disease

AbstractMutations in CLN3 lead to photoreceptor cell loss in CLN3 disease, a lysosomal storage disorder characterized by childhood-onset vision loss, neurological impairment, and premature death. However, how CLN3 mutations cause photoreceptor cell death is not known. Here, we show that CLN3 is required for phagocytosis of photoreceptor outer segment (POS) by retinal pigment epithelium (RPE) cells, a cellular process essential for photoreceptor survival. Specifically, a proportion of CLN3 in human, mouse, and iPSC-RPE cells localized to RPE microvilli, the site of POS phagocytosis. Furthermore, patient-derived CLN3 disease iPSC-RPE cells showed decreased RPE microvilli density and reduced POS binding and ingestion. Notably, POS phagocytosis defect in CLN3 disease iPSC-RPE cells could be rescued by wild-type CLN3 gene supplementation. Altogether, these results illustrate a novel role of CLN3 in regulating POS phagocytosis and suggest a contribution of primary RPE dysfunction for photoreceptor cell loss in CLN3 disease that can be targeted by gene therapy.

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The cytoskeletal elements of human retinal pigment epithelium: in vitro and in vivo

Journal of Cell Science ◽

10.1242/jcs.91.2.303 ◽

1988 ◽

Vol 91 (2) ◽

pp. 303-312

Author(s):

N.M. McKechnie ◽

M. Boulton ◽

H.L. Robey ◽

F.J. Savage ◽

I. Grierson

Keyword(s):

Retinal Pigment Epithelium ◽

Pigment Epithelium ◽

Retinal Pigment ◽

Cytokeratin 18 ◽

Rpe Cells ◽

Human Retinal Pigment Epithelium ◽

Cytoskeletal Elements

The cytoskeletal elements of normal (in situ) and cultured human retinal pigment epithelium (RPE) were studied by a variety of immunocytochemical techniques. Primary antibodies to vimentin and cytokeratins were used. Positive immunoreactivity for vimentin was obtained with in situ and cultured material. The pattern of reactivity obtained with antisera and monoclonals to cytokeratins was more complex. Cytokeratin immunoreactivity could be demonstrated in situ and in cultured cells. The pattern of cytokeratin expression was similar to that of simple or glandular epithelia. A monoclonal antibody that specifically recognizes cytokeratin 18 identified a population of cultured RPE cells that had particularly well-defined filamentous networks within their cytoplasm. Freshly isolated RPE was cytokeratin 18 negative by immunofluorescence, but upon culture cytokeratin 18 positive cells were identifiable. Cytokeratin 18 positive cells were identified in all RPE cultures (other than early primaries), regardless of passage number, age or sex of the donor. In post-confluent cultures cytokeratin 18 cells were identified growing over cytokeratin 18 negative cells, suggesting an association of cytokeratin 18 immunoreactivity with cell proliferation. Immunofluorescence studies of retinal scar tissue from two individuals revealed the presence of numerous cytokeratin 18 positive cells. These findings indicate that RPE cells can be identified by their cytokeratin immunoreactivity and that the overt expression of cytokeratin 18 may be associated with proliferation of human RPE both in vitro and in vivo.

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Acadesine suppresses TNF-α induced complement component 3 (C3), in retinal pigment epithelial (RPE) cells

PLoS ONE ◽

10.1371/journal.pone.0244307 ◽

2020 ◽

Vol 15 (12) ◽

pp. e0244307

Author(s):

Nikolaos E. Efstathiou ◽

Giannis A. Moustafa ◽

Daniel E. Maidana ◽

Eleni K. Konstantinou ◽

Shoji Notomi ◽

...

Keyword(s):

Pigment Epithelium ◽

Retinal Pigment ◽

Complement Component ◽

Adenosine Kinase ◽

Age Related Macular Degeneration ◽

Dependent Manner ◽

Rpe Cells ◽

Age Related ◽

Tnf Α ◽

Complement Component 3

Rationale Age-related macular degeneration (AMD) is the most prevalent form of irreversible blindness in the developed world. Aging, inflammation and complement dysregulation affecting the retinal pigment epithelium (RPE), are considered significant contributors in its pathogenesis and several evidences have linked tumor necrosis factor alpha (TNF-α) and complement component 3 (C3) with AMD. Acadesine, an analog of AMP and an AMP-activated protein kinase (AMPK) activator, has been shown to have cytoprotective effects in human clinical trials as well as having anti-inflammatory and anti-vascular exudative effects in animals. The purpose of this study was to evaluate if acadesine is able to suppress TNF-α induced C3 in RPE cells. Methods ARPE-19 and human primary RPE cells were cultured and allowed to grow to confluence. TNF-α was used for C3 induction in the presence or absence of acadesine. Small molecule inhibitors and siRNA were used to determine if acadesine exerts its effect via the extracellular or intracellular pathway and to evaluate the importance of AMPK for these effects. The expression level of C3 was determined by immunoblot analysis. Results Acadesine suppresses TNF-α induced C3 in a dose dependent manner. When we utilized the adenosine receptor inhibitor dipyridamole (DPY) along with acadesine, acadesine’s effects were abolished, indicating the necessity of acadesine to enter the cell in order to exert it’s action. However, pretreatment with 5-iodotubericidin (5-Iodo), an adenosine kinase (AK) inhibitor, didn’t prevent acadesine from decreasing TNF-α induced C3 expression suggesting that acadesine does not exert its effect through AMP conversion and subsequent activation of AMPK. Consistent with this, knockdown of AMPK α catalytic subunit did not affect the inhibitory effect of acadesine on TNF-α upregulation of C3. Conclusions Our results suggest that acadesine suppresses TNF-α induced C3, likely through an AMPK-independent pathway, and could have potential use in complement over activation diseases.

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Pathogenic Effects of Mineralocorticoid Pathway Activation in Retinal Pigment Epithelium

International Journal of Molecular Sciences ◽

10.3390/ijms22179618 ◽

2021 ◽

Vol 22 (17) ◽

pp. 9618

Author(s):

Jérémie Canonica ◽

Min Zhao ◽

Tatiana Favez ◽

Emmanuelle Gelizé ◽

Laurent Jonet ◽

...

Keyword(s):

Extracellular Matrix ◽

Retinal Pigment Epithelium ◽

Barrier Function ◽

Pigment Epithelium ◽

Retinal Pigment ◽

Retinal Diseases ◽

Mesenchymal Transition ◽

Rpe Cells ◽

Pathway Activation ◽

And Migration

Glucocorticoids are amongst the most used drugs to treat retinal diseases of various origins. Yet, the transcriptional regulations induced by glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) activation in retinal pigment epithelium cells (RPE) that form the outer blood–retina barrier are unknown. Levels of endogenous corticoids, ligands for MR and GR, were measured in human ocular media. Human RPE cells derived from induced pluripotent stem cells (iRPE) were used to analyze the pan-transcriptional regulations induced by aldosterone—an MR-specific agonist, or cortisol or cortisol + RU486—a GR antagonist. The retinal phenotype of transgenic mice that overexpress the human MR (P1.hMR) was analyzed. In the human eye, the main ligand for GR and MR is cortisol. The iRPE cells express functional GR and MR. The subset of genes regulated by aldosterone and by cortisol + RU-486, and not by cortisol alone, mimics an imbalance toward MR activation. They are involved in extracellular matrix remodeling (CNN1, MGP, AMTN), epithelial–mesenchymal transition, RPE cell proliferation and migration (ITGB3, PLAUR and FOSL1) and immune balance (TNFSF18 and PTX3). The P1.hMR mice showed choroidal vasodilation, focal alteration of the RPE/choroid interface and migration of RPE cells together with RPE barrier function alteration, similar to human retinal diseases within the pachychoroid spectrum. RPE is a corticosteroid-sensitive epithelium. MR pathway activation in the RPE regulates genes involved in barrier function, extracellular matrix, neural regulation and epithelial differentiation, which could contribute to retinal pathology.

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Microfilament organization and wound repair in retinal pigment epithelium

Biochemistry and Cell Biology ◽

10.1139/o95-079 ◽

1995 ◽

Vol 73 (9-10) ◽

pp. 709-722 ◽

Cited By ~ 14

Author(s):

Vitauts. I. Kalnins ◽

Martin Sandig ◽

Greg J. Hergott ◽

Haruhiko Nagai

Keyword(s):

Cell Proliferation ◽

Cell Migration ◽

Retinal Pigment Epithelium ◽

Wound Repair ◽

Pigment Epithelium ◽

Retinal Pigment ◽

Wound Edge ◽

Rpe Cells ◽

Oriented Parallel ◽

Experimentally Induced

Several systems of microfilaments (MF) associated with adherens-type junctions between adjacent retinal pigment epithelial (RPE) cells and between these cells and the substratum play an important role in maintaining the integrity and organization of the RPE. They include prominent, contractile circumferential MF bundles that are associated with the zonula adherens (ZA) junctions. In chick RPE, these junctions are assembled from smaller subunits thus giving greater structural flexibility to the junctional region. Because the separation of the junctions requires trypsin and low calcium, both calcium-dependent and -independent mechanisms are involved in keeping adjacent RPE cells attached to one another. Another system of MF bundles that crosses the cell at the level of ZA junctions can be induced to form by stretching the epithelium. The MF bundles forming this system are oriented in the direction in which the RPE is stretched, thereby preventing the overextension of the cell in any one direction. The system may be useful as an indicator of the direction in which tension is experienced by RPE during development of the eye, in animal models of disease and during repair of experimentally induced wounds. Numerous single-cell wounds resulting from death of RPE cells by apoptosis at various stages of repair are normally present in developing chick and adult mammalian RPE. These wounds are repaired by the spreading of adjacent RPE cells and by the contraction of MF bundles oriented parallel to the wound edge, which develop during this time. As a result of the spreading in the absence of cell proliferation, the RPE cells increase in diameter with age. Experimentally induced wounds made by removing 5–10 RPE cells are repaired by a similar mechanism within 24 h. In repair of larger wounds, over 125 μm in width, the MF bundles oriented parallel to the wound edge characteristic of spreading cells are later replaced by stress fibers (SFs) that run perpendicularly to the wound edge and interact with the substratum at focal contacts (FCs) as RPE cells start to migrate. Cell proliferation is induced in cells along the wound edge only when the wounds are wide enough to require cell migration. In the presence of antibodies to beta-1-integrins, a component of FCs, cell spreading is not prevented but both cell migration and cell proliferation are inhibited. Thus, only the organization of the cytoskeleton characteristic of migrating RPE cells that have SFs that interact with the substratum at FCs, is associated with the induction of cell proliferation.Key words: retinal pigment epithelium, microfilaments, wound repair.

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Degradation of extracellular ATP by the retinal pigment epithelium

AJP Cell Physiology ◽

10.1152/ajpcell.00542.2004 ◽

2005 ◽

Vol 289 (3) ◽

pp. C617-C624 ◽

Cited By ~ 32

Author(s):

David Reigada ◽

Wennan Lu ◽

Xiulan Zhang ◽

Constantin Friedman ◽

Klara Pendrak ◽

...

Keyword(s):

Pigment Epithelium ◽

Retinal Pigment ◽

Adenosine Diphosphate ◽

Cell Types ◽

P2y Receptors ◽

Extracellular Atp ◽

Pcr Analysis ◽

Subretinal Space ◽

Rpe Cells ◽

Mrs 2179

Stimulation of ATP or adenosine receptors causes important physiological changes in retinal pigment epithelial (RPE) cells that may influence their relationship to the adjacent photoreceptors. While RPE cells have been shown to release ATP, the regulation of extracellular ATP levels and the production of dephosphorylated purines is not clear. This study examined the degradation of ATP by RPE cells and the physiological effects of the adenosine diphosphate (ADP) that result. ATP was readily broken down by both cultured human ARPE-19 cells and the apical membrane of fresh bovine RPE cells. The compounds ARL67156 and βγ-mATP inhibited this degradation in both cell types. RT-PCR analysis of ARPE-19 cells found mRNA message for multiple extracellular degradative enzymes; ectonucleotide pyrophosphatase/phosphodiesterase eNPP1, eNPP2, and eNPP3; the ectoATPase ectonucleoside triphosphate diphosphohydrolase NTPDase2, NTPDase3, and some message for NTPDase1. Considerable levels of ADP bathed RPE cells, consistent with a role for NTPDase2. ADP and ATP increased levels of intracellular Ca2+. Both responses were inhibited by thapsigargin and P2Y1 receptor inhibitor MRS 2179. Message for both P2Y1 and P2Y12 receptors was detected in ARPE-19 cells. These results suggest that extracellular degradation of ATP in subretinal space can result in the production of ADP. This ADP can stimulate P2Y receptors and augment Ca2+ signaling in the RPE.

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JAM-C maintains VEGR2 expression to promote retinal pigment epithelium cell survival under oxidative stress

Thrombosis and Haemostasis ◽

10.1160/th16-11-0885 ◽

2017 ◽

Vol 117 (04) ◽

pp. 750-757

Author(s):

Xin Jia ◽

Chen Zhao ◽

Qishan Chen ◽

Yuxiang Du ◽

Lijuan Huang ◽

...

Keyword(s):

Oxidative Stress ◽

Retinal Pigment Epithelium ◽

Cell Survival ◽

Pigment Epithelium ◽

Retinal Pigment ◽

Retinal Pigment Epithelium Cell ◽

Photoreceptor Cells ◽

Rpe Cells

SummaryJunctional adhesion molecule-C (JAM-C) has been shown to play critical roles during development and in immune responses. However, its role in adult eyes under oxidative stress remains poorly understood. Here, we report that JAM-C is abundantly expressed in adult mouse retinae and choroids in vivo and in cultured retinal pigment epithelium (RPE) and photoreceptor cells in vitro. Importantly, both JAM-C expression and its membrane localisation are downregulated by H2O2-induced oxidative stress. Under H2O2-induced oxidative stress, JAM-C is critically required for the survival of human RPE cells. Indeed, loss of JAM-C by siRNA knockdown decreased RPE cell survival. Mechanistically, we show that JAM-C is required to maintain VEGFR2 expression in RPE cells, and VEGFR2 plays an important role in keeping the RPE cells viable since overexpression of VEGFR2 partially restored impaired RPE survival caused by JAM-C knockdown and increased RPE survival. We further show that JAM-C regulates VEGFR2 expression and, in turn, modulates p38 phosphorylation. Together, our data demonstrate that JAM-C plays an important role in maintaining VEGR2 expression to promote RPE cell survival under oxidative stress. Given the vital importance of RPE in the eye, approaches that can modulate JAM-C expression may have therapeutic values in treating diseases with impaired RPE survival.

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