3D-Reconstructed Retinal Pigment Epithelial Cells Provide Insights into the Anatomy of the Outer Retina

The retinal pigment epithelium (RPE) is located between the neuroretina and the choroid, and plays a critical role in vision. RPE cells internalise outer segments (OS) from overlying photoreceptors in the daily photoreceptor renewal. Changes to RPE structure are linked with age and retinopathy, which has been described in the past by conventional 2D electron microscopy. We used serial block face scanning electron microscopy (SBF-SEM) to reconstruct RPE cells from the central mouse retina. Three-dimensional-reconstructed OS revealed the RPE to support large numbers of photoreceptors (90–216 per RPE cell). Larger bi-nucleate RPE maintained more photoreceptors, although their cytoplasmic volume was comparable to smaller mono-nucleate RPE supporting fewer photoreceptors. Scrutiny of RPE microvilli and interdigitating OS revealed the angle and surface area of contact between RPE and photoreceptors. Bi-nucleate RPE contained more mitochondria compared to mono-nucleate RPE. Furthermore, bi-nucleate cells contained larger sub-RPE spaces, supporting a likely association with disease. Use of perfusion-fixed tissues ensured the highest possible standard of preservation, providing novel insights into the 3D RPE architecture and changes linked with retinopathy. This study serves as a benchmark for comparing retinal tissues from donor eyes with age-related macular degeneration (AMD) and other retinopathies.

Download Full-text

Genetic LAMP2 deficiency accelerates the age-associated formation of basal laminar deposits in the retina

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1906643116 ◽

2019 ◽

Vol 116 (47) ◽

pp. 23724-23734 ◽

Cited By ~ 11

Author(s):

Shoji Notomi ◽

Kenji Ishihara ◽

Nikolaos E. Efstathiou ◽

Jong-Jer Lee ◽

Toshio Hisatomi ◽

...

Keyword(s):

Basal Lamina ◽

Pigment Epithelium ◽

Retinal Pigment ◽

Critical Role ◽

Age Related Macular Degeneration ◽

Bruch's Membrane ◽

Bruch’S Membrane ◽

Rpe Cells ◽

Age Related ◽

Deficient Mice

The early stages of age-related macular degeneration (AMD) are characterized by the accumulation of basal laminar deposits (BLamDs). The mechanism for BLamDs accumulating between the retinal pigment epithelium (RPE) and its basal lamina remains elusive. Here we examined the role in AMD of lysosome-associated membrane protein-2 (LAMP2), a glycoprotein that plays a critical role in lysosomal biogenesis and maturation of autophagosomes/phagosomes. LAMP2 was preferentially expressed by RPE cells, and its expression declined with age. Deletion of the Lamp2 gene in mice resulted in age-dependent autofluorescence abnormalities of the fundus, thickening of Bruch’s membrane, and the formation of BLamDs, resembling histopathological changes occurring in AMD. Moreover, LAMP2-deficient mice developed molecular signatures similar to those found in human AMD—namely, the accumulation of APOE, APOA1, clusterin, and vitronectin—adjacent to BLamDs. In contrast, collagen 4, laminin, and fibronectin, which are extracellular matrix proteins constituting RPE basal lamina and Bruch’s membrane were reduced in Lamp2 knockout (KO) mice. Mechanistically, retarded phagocytic degradation of photoreceptor outer segments compromised lysosomal degradation and increased exocytosis in LAMP2-deficient RPE cells. The accumulation of BLamDs observed in LAMP2-deficient mice was eventually followed by loss of the RPE and photoreceptors. Finally, we observed loss of LAMP2 expression along with ultramicroscopic features of abnormal phagocytosis and exocytosis in eyes from AMD patients but not from control individuals. Taken together, these results indicate an important role for LAMP2 in RPE function in health and disease, suggesting that LAMP2 reduction may contribute to the formation of BLamDs in AMD.

Download Full-text

Hypoxia-induced metabolic stress in retinal pigment epithelial cells is sufficient to induce photoreceptor degeneration

eLife ◽

10.7554/elife.14319 ◽

2016 ◽

Vol 5 ◽

Cited By ~ 80

Author(s):

Toshihide Kurihara ◽

Peter D Westenskow ◽

Marin L Gantner ◽

Yoshihiko Usui ◽

Andrew Schultz ◽

...

Keyword(s):

Pigment Epithelium ◽

Retinal Pigment ◽

Metabolic Stress ◽

Retinal Pigment Epithelial Cells ◽

Age Related Macular Degeneration ◽

Photoreceptor Degeneration ◽

Rpe Cells ◽

Glucose And Lipid Metabolism ◽

Age Related ◽

Cellular Stresses

Photoreceptors are the most numerous and metabolically demanding cells in the retina. Their primary nutrient source is the choriocapillaris, and both the choriocapillaris and photoreceptors require trophic and functional support from retinal pigment epithelium (RPE) cells. Defects in RPE, photoreceptors, and the choriocapillaris are characteristic of age-related macular degeneration (AMD), a common vision-threatening disease. RPE dysfunction or death is a primary event in AMD, but the combination(s) of cellular stresses that affect the function and survival of RPE are incompletely understood. Here, using mouse models in which hypoxia can be genetically triggered in RPE, we show that hypoxia-induced metabolic stress alone leads to photoreceptor atrophy. Glucose and lipid metabolism are radically altered in hypoxic RPE cells; these changes impact nutrient availability for the sensory retina and promote progressive photoreceptor degeneration. Understanding the molecular pathways that control these responses may provide important clues about AMD pathogenesis and inform future therapies.

Download Full-text

Mitophagy in the Retinal Pigment Epithelium of Dry Age-Related Macular Degeneration Investigated in the NFE2L2/PGC-1α-/- Mouse Model

International Journal of Molecular Sciences ◽

10.3390/ijms21061976 ◽

2020 ◽

Vol 21 (6) ◽

pp. 1976 ◽

Cited By ~ 6

Author(s):

Iswariyaraja Sridevi Gurubaran ◽

Johanna Viiri ◽

Ali Koskela ◽

Juha M.T. Hyttinen ◽

Jussi J. Paterno ◽

...

Keyword(s):

Oxidative Stress ◽

Mouse Model ◽

Macular Degeneration ◽

Pigment Epithelium ◽

Retinal Pigment ◽

Retinal Pigment Epithelial Cells ◽

Age Related Macular Degeneration ◽

Double Knockout ◽

Rpe Cells ◽

Age Related

Increased oxidative stress and mitochondrial damage are observed in protein aggregation diseases, such as age-related macular degeneration (AMD). We have recently reported elevated levels of oxidative stress markers, damaged mitochondria, accumulating lysosomal lipofuscin and extracellular drusen-like structures in the retinal pigment epithelial cells (RPE) of the dry AMD-resembling NFE2L2/PGC1α double knockout (dKO) mouse model. Here, we provide evidence of a disturbance in the autolysosomal machinery handling mitochondrial clearance in the RPE cells of one-year-old NFE2L2/PGC1α-deficient mice. Confocal immunohistochemical analysis revealed an upregulation of autophagosome marker microtubule-associated proteins 1A/1B light chain 3B (LC3B) as well as numerous mitophagy markers, such as PTE-induced putative kinase 1 (PINK1) and E3 ubiquitin ligase (PARKIN) together with damaged mitochondria. However, we detected no evidence of increased autolysosome formation in transmission electron micrographs or of colocalization of lysosomal marker LAMP2 (lysosome-associated membrane protein 2) and the mitochondrial marker ATP synthase β in confocal micrographs. Interestingly, we observed an upregulation of late autolysosomal fusion Ras-related protein (Rab7) in the perinuclear space of RPE cells together with autofluorescence aggregates. Our results reveal that there is at least a relative decrease of mitophagy in the RPE cells of NFE2L2/PGC1α dKO mice. This further supports the hypothesis that mitophagy is a putative therapy target in AMD-like pathology.

Download Full-text

Protective Effect of Melatonin against Oxidative Stress-Induced Apoptosis and Enhanced Autophagy in Human Retinal Pigment Epithelium Cells

Oxidative Medicine and Cellular Longevity ◽

10.1155/2018/9015765 ◽

2018 ◽

Vol 2018 ◽

pp. 1-12 ◽

Cited By ~ 20

Author(s):

Chih-Chao Chang ◽

Tien-Yi Huang ◽

Hsin-Yuan Chen ◽

Tsui-Chin Huang ◽

Li-Chun Lin ◽

...

Keyword(s):

Oxidative Stress ◽

Oxidative Damage ◽

Pigment Epithelium ◽

Retinal Pigment ◽

Critical Role ◽

Age Related Macular Degeneration ◽

Cell Degeneration ◽

Rpe Cells ◽

Age Related ◽

Human Rpe Cells

Age-related macular degeneration (AMD) affects the retinal macula and results in loss of vision, and AMD is the primary cause of blindness and severe visual impairment among elderly people worldwide. AMD is characterized by the accumulation of drusen in the Bruch’s membrane and dysfunction of retinal pigment epithelial (RPE) cells and photoreceptors. The pathogenesis of AMD remains unclear, and no effective treatment exists. Accumulating evidence indicates that oxidative stress plays a critical role in RPE cell degeneration and AMD. Melatonin is an antioxidant that scavenges free radicals, and it has anti-inflammatory, antitumor, and antiangiogenic effects. This study investigated the antioxidative, antiapoptotic, and autophagic effects of melatonin on oxidative damage to RPE cells. We used hydrogen peroxide (H2O2) to stimulate reactive oxygen species production to cause cell apoptosis in ARPE-19 cell lines. Our findings revealed that treatment with melatonin significantly inhibited H2O2-induced RPE cell damage, decreased the apoptotic rate, increased the mitochondrial membrane potential, and increased the autophagy effect. Furthermore, melatonin reduced the Bax/Bcl-2 ratio and the expression levels of the apoptosis-associated proteins cytochrome c and caspase 7. Additionally, melatonin upregulated the expression of the autophagy-related proteins LC3-II and Beclin-1 and downregulated the expression of p62. Thus, melatonin’s effects on autophagy and apoptosis can protect against H2O2-induced oxidative damage in human RPE cells. Melatonin may have multiple protective effects on human RPE cells against H2O2-induced oxidative damage.

Download Full-text

The CD36 Ligand-Promoted Autophagy Protects Retinal Pigment Epithelial Cells from Oxidative Stress

Oxidative Medicine and Cellular Longevity ◽

10.1155/2021/6691402 ◽

2021 ◽

Vol 2021 ◽

pp. 1-14

Author(s):

Marie-France Dorion ◽

Mukandila Mulumba ◽

Shuya Kasai ◽

Ken Itoh ◽

William D. Lubell ◽

...

Keyword(s):

Oxidative Stress ◽

Pigment Epithelium ◽

Retinal Pigment ◽

Retinal Pigment Epithelial Cells ◽

Age Related Macular Degeneration ◽

Autophagic Flux ◽

Cytoprotective Effect ◽

Rpe Cells ◽

Age Related ◽

Cluster Of Differentiation

The retinal pigment epithelium (RPE) performs many functions that maintain photoreceptor health. Oxidative damage to the RPE is a critical component in the pathogenesis of eye diseases such as age-related macular degeneration (AMD). Ligands of the cluster of differentiation 36 (CD36) have previously preserved photoreceptor integrity in mouse models of AMD. The cytoprotective effect of the CD36 ligand MPE-001 on RPE cells has now been elucidated employing a model of oxidative stress. Sodium iodate (NaIO3) induced formation of reactive oxygen species and apoptosis in human RPE cells, which were decreased by MPE-001 without affecting antioxidant enzyme transcription. Immunoblotting and immunostaining assays showed a restorative effect of MPE-001 on the autophagic flux disrupted by NaIO3, which was associated with an increase in syntaxin 17-positive mature autophagosomes. The cytoprotective effect of MPE-001 was completely abolished by the autophagy inhibitors wortmannin and bafilomycin A1. In conclusion, we report for the first time an autophagy-dependent protection of RPE cells from oxidative stress by a CD36 ligand.

Download Full-text

Madecassoside protects retinal pigment epithelial cells against hydrogen peroxide-induced oxidative stress and apoptosis through the activation of Nrf2/HO-1 pathway

Bioscience Reports ◽

10.1042/bsr20194347 ◽

2020 ◽

Vol 40 (10) ◽

Author(s):

Jinzi Zhou ◽

Fenghua Chen ◽

Aimin Yan ◽

Xiaobo Xia

Keyword(s):

Oxidative Stress ◽

Hydrogen Peroxide ◽

Pigment Epithelium ◽

Retinal Pigment ◽

Retinal Pigment Epithelial Cells ◽

Age Related Macular Degeneration ◽

Triterpenoid Saponin ◽

Sod Activity ◽

Rpe Cells ◽

Age Related

Abstract Age-related macular degeneration (AMD) is a progressive and degenerative ocular disease associated with oxidative stress. Madecassoside (MADE) is a major bioactive triterpenoid saponin that possesses antioxidative activity. However, the role of MADE in AMD has never been investigated. In the current study, we aimed to evaluate the protective effect of MADE on retinal pigment epithelium (RPE) cells under oxidative stress condition. We used hydrogen peroxide (H2O2) to induce oxidative damage in human RPE cells (ARPE-19 cells). Our results showed that H2O2-caused significant decrease in cell viability and increase in lactate dehydrogenase (LDH) release were dose-dependently attenuated by MADE. MADE treatment also attenuated H2O2-induced reactive oxygen species (ROS) and malondialdehyde (MDA) production in RPE cells. The reduced glutathione (GSH) level and superoxide dismutase (SOD) activity in H2O2-induced ARPE-19 cells were elevated after MADE treatment. MADE also suppressed caspase-3 activity and bax expression, as well as increased bcl-2 expression. Furthermore, H2O2-induced increase in expression levels of HO-1 and nuclear Nrf2 were enhanced by MADE treatment. Finally, knockdown of Nrf2 reversed the protective effects of MADE on H2O2-induced ARPE-19 cells. In conclusion, these findings demonstrated that MADE protected ARPE-19 cells from H2O2-induced oxidative stress and apoptosis by inducing the activation of Nrf2/HO-1 signaling pathway.

Download Full-text

Scaffold-Free Retinal Pigment Epithelium Microtissues Exhibit Increased Release of PEDF

International Journal of Molecular Sciences ◽

10.3390/ijms222111317 ◽

2021 ◽

Vol 22 (21) ◽

pp. 11317

Author(s):

Abdullah Al-Ani ◽

Derek Toms ◽

Saud Sunba ◽

Kayla Giles ◽

Yacine Touahri ◽

...

Keyword(s):

Vision Loss ◽

Pigment Epithelium ◽

Retinal Pigment ◽

Critical Role ◽

Age Related Macular Degeneration ◽

Rpe Cells ◽

Age Related ◽

Wide Range ◽

And Function

The retinal pigmented epithelium (RPE) plays a critical role in photoreceptor survival and function. RPE deficits are implicated in a wide range of diseases that result in vision loss, including age-related macular degeneration (AMD) and Stargardt disease, affecting millions worldwide. Subretinal delivery of RPE cells is considered a promising avenue for treatment, and encouraging results from animal trials have supported recent progression into the clinic. However, the limited survival and engraftment of transplanted RPE cells delivered as a suspension continues to be a major challenge. While RPE delivery as epithelial sheets exhibits improved outcomes, this comes at the price of increased complexity at both the production and transplant stages. In order to combine the benefits of both approaches, we have developed size-controlled, scaffold-free RPE microtissues (RPE-µTs) that are suitable for scalable production and delivery via injection. RPE-µTs retain key RPE molecular markers, and interestingly, in comparison to conventional monolayer cultures, they show significant increases in the transcription and secretion of pigment-epithelium-derived factor (PEDF), which is a key trophic factor known to enhance the survival and function of photoreceptors. Furthermore, these microtissues readily spread in vitro on a substrate analogous to Bruch’s membrane, suggesting that RPE-µTs may collapse into a sheet upon transplantation. We anticipate that this approach may provide an alternative cell delivery system to improve the survival and integration of RPE transplants, while also retaining the benefits of low complexity in production and delivery.

Download Full-text

Histological Characterization of the Dicer1 Mutant Zebrafish Retina

Journal of Ophthalmology ◽

10.1155/2015/309510 ◽

2015 ◽

Vol 2015 ◽

pp. 1-8 ◽

Cited By ~ 5

Author(s):

Saeed Akhtar ◽

Sarita Rani Patnaik ◽

Rakesh Kotapati Raghupathy ◽

Turki M. Al-Mubrad ◽

John A. Craft ◽

...

Keyword(s):

Retinal Pigment Epithelium ◽

Developmental Stages ◽

Pigment Epithelium ◽

Retinal Pigment ◽

Critical Role ◽

Age Related Macular Degeneration ◽

Control Fish ◽

Rnase Iii ◽

Rpe Cells ◽

Age Related

DICER1, a multidomain RNase III endoribonuclease, plays a critical role in microRNA (miRNA) and RNA-interference (RNAi) functional pathways. Loss ofDicer1affects different developmental processes. Dicer1 is essential for retinal development and maintenance. DICER1 was recently shown to have another function of silencing the toxicity ofAluRNAs in retinal pigment epithelium (RPE) cells, which are involved in the pathogenesis of age related macular degeneration. In this study, we characterized aDicer1mutant fish line, which carries a nonsense mutation (W1457Ter) induced by N-ethyl-N-nitrosourea mutagenesis. Zebrafish DICER1 protein is highly conserved in the evolution. Zebrafish Dicer1 is expressed at the earliest stages of zebrafish development and persists into late developmental stages; it is widely expressed in adult tissues. HomozygousDicer1mutant fish (DICER1W1457Ter/W1457Ter) have an arrest in early growth with significantly smaller eyes and are dead at 14–18 dpf. HeterozygousDicer1mutant fish have similar retinal structure to that of control fish; the retinal pigment epithelium (RPE) cells are normal with no sign of degeneration at the age of 20 months.

Download Full-text

A Splice Variant in SLC16A8 Gene Leads to Lactate Transport Deficit in Human iPS Cell-Derived Retinal Pigment Epithelial Cells

Cells ◽

10.3390/cells10010179 ◽

2021 ◽

Vol 10 (1) ◽

pp. 179

Author(s):

Laurence Klipfel ◽

Marie Cordonnier ◽

Léa Thiébault ◽

Emmanuelle Clérin ◽

Frédéric Blond ◽

...

Keyword(s):

Genome Wide Association Study ◽

Pigment Epithelium ◽

Retinal Pigment ◽

Retinal Pigment Epithelial Cells ◽

Age Related Macular Degeneration ◽

Lactate Transport ◽

Ips Cell ◽

Risk Alleles ◽

Age Related ◽

A Genome

Age-related macular degeneration (AMD) is a blinding disease for which most of the patients remain untreatable. Since the disease affects the macula at the center of the retina, a structure specific to the primate lineage, rodent models to study the pathophysiology of AMD and to develop therapies are very limited. Consequently, our understanding relies mostly on genetic studies highlighting risk alleles at many loci. We are studying the possible implication of a metabolic imbalance associated with risk alleles within the SLC16A8 gene that encodes for a retinal pigment epithelium (RPE)-specific lactate transporter MCT3 and its consequences for vision. As a first approach, we report here the deficit in transepithelial lactate transport of a rare SLC16A8 allele identified during a genome-wide association study. We produced induced pluripotent stem cells (iPSCs) from the unique patient in our cohort that carries two copies of this allele. After in vitro differentiation of the iPSCs into RPE cells and their characterization, we demonstrate that the rare allele results in the retention of intron 2 of the SLC16A8 gene leading to the absence of MCT3 protein. We show using a biochemical assay that these cells have a deficit in transepithelial lactate transport.

Download Full-text

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.

Download Full-text