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Journal of Cell Science Sep 2020Human retinal pigment epithelial-1 (RPE-1) cells are increasingly being used as a model to study mitosis because they represent a non-transformed alternative to cancer...
Human retinal pigment epithelial-1 (RPE-1) cells are increasingly being used as a model to study mitosis because they represent a non-transformed alternative to cancer cell lines, such as HeLa cervical adenocarcinoma cells. However, the lack of an efficient method to synchronize RPE-1 cells in mitosis precludes their application for large-scale biochemical and proteomics assays. Here, we report a protocol to synchronize RPE-1 cells based on sequential treatments with the Cdk4 and Cdk6 inhibitor PD 0332991 (palbociclib) and the microtubule-depolymerizing drug nocodazole. With this method, the vast majority (80-90%) of RPE-1 cells arrested at prometaphase and exited mitosis synchronously after release from nocodazole. Moreover, the cells fully recovered and re-entered the cell cycle after the palbociclib-nocodazole block. Finally, we show that this protocol could be successfully employed for the characterization of the protein-protein interaction network of the kinetochore protein Ndc80 by immunoprecipitation coupled with mass spectrometry. This synchronization method significantly expands the versatility and applicability of RPE-1 cells to the study of cell division and might be applied to other cell lines that do not respond to treatments with DNA synthesis inhibitors.
Topics: Humans; Kinetochores; Mitosis; Nocodazole; Prometaphase; Retinal Pigments
PubMed: 32878943
DOI: 10.1242/jcs.247940 -
The FEBS Journal Apr 2022Retinal pigment epithelium (RPE) cell damage is implicated in the pathogenesis of age-related macular degeneration (AMD). An increase of interferon-γ (IFN-γ) levels...
Retinal pigment epithelium (RPE) cell damage is implicated in the pathogenesis of age-related macular degeneration (AMD). An increase of interferon-γ (IFN-γ) levels was observed in patients with AMD, but whether inflammatory factors are causally related to AMD progression is unclear. Here, we demonstrate a direct causal relationship between IFN-γ and RPE cell death. IFN-γ induced human retinal pigment epithelial cell (ARPE-19) death accompanied by increases in Fe , reactive oxygen species, lipid peroxidation, and glutathione (GSH) depletion, which are main characteristics of ferroptosis. Mechanistically, IFN-γ upregulates the level of intracellular Fe through inhibiting Fe efflux protein SLC40A1 and induces GSH depletion by blocking cystine/glutamate antiporter, System xc-. At the same time, treatment with IFN-γ decreases the level of glutathione peroxidase 4 (GPx4), rendering the cells more sensitive to ferroptosis. JAK1/2 and STAT1 inhibitors could reverse the reduction of SLC7A11, GPx4 and GSH expression induced by IFN-γ, indicating IFN-γ induces ARPE-19 cell ferroptosis via activation of the JAK1-2/STAT1/SLC7A11 signaling pathway. The above results were largely confirmed in IFN-γ-treated mice in vivo. Finally, we used sodium iodate (NaIO )-induced retinal degeneration to further explore the role of ferroptosis in AMD in vivo. Consistent with the role of IFN-γ, treatment with NaIO decreased SLC7A11, GPx4 and SLC40A1 expressions. NaIO -induced RPE damage was accompanied by increased iron, lipid peroxidation products (4-hydroxynonenal, malondialdehyde), and GSH depletion, and ferroptosis inhibitors could reverse the above phenomenon. Taken together, our findings suggest that inhibiting ferroptosis or reducing IFN-γ may serve as a promising target for AMD.
Topics: Amino Acid Transport System y+; Animals; Epithelial Cells; Ferroptosis; Humans; Interferon-gamma; Janus Kinase 1; Macular Degeneration; Mice; Oxidative Stress; Retinal Pigment Epithelium; Retinal Pigments; STAT1 Transcription Factor; Signal Transduction
PubMed: 34741776
DOI: 10.1111/febs.16272 -
Autophagy Mar 2023Age-related macular degeneration (AMD), the leading cause of blindness among the elderly, is without treatment for early disease. Degenerative retinal pigment epithelial...
Age-related macular degeneration (AMD), the leading cause of blindness among the elderly, is without treatment for early disease. Degenerative retinal pigment epithelial (RPE) cell heterogeneity is a well-recognized but understudied pathogenic factor. Due to the daily phagocytosis of photoreceptor outer segments, unique photo-oxidative stress, and high metabolism for maintaining vision, the RPE has robust macroautophagy/autophagy, and mitochondrial and antioxidant networks. However, the autophagy subtype, mitophagy, in the RPE and AMD is understudied. Here, we found decreased PINK1 (PTEN induced kinase 1) in perifoveal RPE of early AMD eyes. PINK1-deficient RPE have impaired mitophagy and mitochondrial function that triggers death-resistant epithelial-mesenchymal transition (EMT). This reprogramming is mediated by novel retrograde mitochondrial-nuclear signaling (RMNS) through superoxide, NFE2L2 (NFE2 like bZIP transcription factor 2), TXNRD1 (thioredoxin reductase 1), and phosphoinositide 3-kinase (PI3K)-AKT (AKT serine/threonine kinase) that induced canonical transcription factors ZEB1 (zinc finger E-box binding homeobox 1) and SNAI1 (Snail family transcriptional repressor 1) and an EMT transcriptome. NFE2L2 deficiency disrupted RMNS that paradoxically normalized morphology but decreased function and viability. Thus, RPE heterogeneity is defined by the interaction of two cytoprotective pathways that is triggered by mitophagy function. By neutralizing the consequences of impaired mitophagy, an antioxidant dendrimer tropic for the RPE and mitochondria, EMT (a recognized AMD alteration) was abrogated to offer potential therapy for early AMD, a stage without treatment.: ACTB: actin beta; AKT: AKT serine/threonine kinase; AMD: age-related macular degeneration; CCCP: cyanide m-chlorophenyl hydrazone; CDH1: cadherin 1; DAVID: Database for Annotation, Visualization and Integrated Discovery; DHE: dihydroethidium; D-NAC: N-acetyl-l-cysteine conjugated to a poly(amido amine) dendrimer; ECAR: extracellular acidification rate; EMT: epithelial-mesenchymal transition; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GSEA: Gene Set Enrichment Analysis; HSPD1: heat shock protein family D (Hsp60) member 1; IVT: intravitreal; KD: knockdown; LMNA, lamin A/C; MAP1LC3B: microtubule associated protein 1 light chain 3 beta; MMP: mitochondrial membrane potential; NAC: N-acetyl-l-cysteine; NQO1: NAD(P)H quinone dehydrogenase 1; NFE2L2: NFE2 like bZIP transcription factor 2; O: superoxide anion; OCR: oxygen consumption rate; PI3K: phosphoinositide 3-kinase; PINK1: PTEN induced kinase 1; RMNS: retrograde mitochondrial-nuclear signaling; ROS: reactive oxygen species; RPE: retinal pigment epithelium; SNAI1: snail family transcriptional repressor 1; TJP1: tight junction protein 1; TPP-D-NAC: triphenyl phosphinium and N-acetyl-l-cysteine conjugated to a poly(amido amine) dendrimer; TIMM23: translocase of inner mitochondrial membrane 23; TOMM20: translocase of outer mitochondrial membrane 20; Trig: trigonelline; TXNRD1: thioredoxin reductase 1; VIM: vimentin; WT: wild-type; ZEB1: zinc finger E-box binding homeobox 1.
Topics: Humans; Aged; Mitophagy; Autophagy; Thioredoxin Reductase 1; Antioxidants; Acetylcysteine; Dendrimers; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Retinal Pigment Epithelium; Macular Degeneration; Phosphatidylinositol 3-Kinase; Basic-Leucine Zipper Transcription Factors; Amines; Retinal Pigments; Serine
PubMed: 35921555
DOI: 10.1080/15548627.2022.2109286 -
Autophagy Feb 2022Diabetic retinopathy (DR) is a serious complication of diabetes mellitus and currently one of the major causes of blindness. Several previous studies have demonstrated...
Diabetic retinopathy (DR) is a serious complication of diabetes mellitus and currently one of the major causes of blindness. Several previous studies have demonstrated that autophagy, which is regulated by HMGB1 (high mobility group box 1), is involved in DR development. However, the role of autophagy in DR is quite complicated in that it promotes pericyte survival in early DR, whereas excessive autophagy causes excess stress and leads to necrosis. Therefore, this study aimed to investigate the relationship between HMGB1, the macroautophagy/autophagy-lysosome pathway, and DR, as well as their underlying molecular mechanisms. In brief, the relationship between high glucose (HG) and the autophagy-lysosome pathway was examined in retinal pigment epithelial (RPE) cells. The relationship was studied by detecting classical autophagic features, and siRNAs targeting HMGB1 and pharmacological regulators were used to explore the role of the autophagy-lysosome pathway in DR development. The results demonstrated that HG inhibited autophagy and diminished the degradative capacity of autophagy due to lysosome membrane permeabilization (LMP). In addition, HMGB1 was found to be involved in LMP via the CTSB (cathepsin B)-dependent pathway, but not the CTSL (cathepsin L)-dependent pathway. Knockdown of HMGB1 expression rescued LMP, restored the degradative capacity of autophagy, decreased the expression of inflammatory factors and VEGF (vascular endothelial growth factor), and protected against apoptosis in RPE cells in the early stages of DR.
Topics: Autophagy; Diabetes Mellitus; Diabetic Retinopathy; Down-Regulation; Epithelial Cells; HMGB1 Protein; Humans; Lysosomes; Retinal Pigments; Vascular Endothelial Growth Factor A
PubMed: 34024230
DOI: 10.1080/15548627.2021.1926655 -
Bioengineered Apr 2022Astragaloside-IV (AS-IV) (CHO) is a high-purity natural product extracted from , which has demonstrated biological activities. However, the effect of AS-IV on retinal...
Astragaloside-IV (AS-IV) (CHO) is a high-purity natural product extracted from , which has demonstrated biological activities. However, the effect of AS-IV on retinal pigment epithelial (RPE) cells in diabetic retinopathy (DR) remains unclear. In this study, high glucose (HG) was shown to promote ARPE-19 RPE cell death, increase the contents of reactive oxygen species (ROS) and oxidized glutathione (GSSG), and enhance lipid peroxidation density of mitochondrial membrane. In contrast, AS-IV decreased glutathione (GSH) content, mitochondria size and ridge. Addition of iron death inhibitor Ferrostatin-1 (Fer-1) to RPE cells decreased cell dead rate, thus indicating that HG-induced mitochondrial damage occurred due to ferroptosis. AS-IV alleviated HG-induced RPE cell damage. Furthermore, HG decreased levels of silent information regulator 1 (Sirt1) and nuclear factor (erythroid-derived 2)-like 2 (Nrf2) in the nucleus of RPE cells; AS-IV could alleviate these effects and increased expression of glutathione peroxidase 4 (GPX4), glutamate cysteine ligase (GCLM) and glutamate cysteine ligase catalytic subunit (GCLC), which are Nrf2 downstream genes. Mechanistically, AS-IV was shown to alleviate the effects of HG by increasing mir-138-5p expression in RPE cells and promoting expression of Sirt1 and Nrf2 in the nucleus. Transfection of mir-138-5p agonist inhibited the regulatory effects of AS-IV on Sirt1 and Nrf2, accompanied by decreased GPX4, GCLM and GCLC levels, and restoration of ferroptosis-related changes. Collectively, HG increased ferroptosis rate in RPE cells. In addition, AS-IV inhibited miR-138-5p expression, subsequently increasing Sirt1/Nrf2 activity and cellular antioxidant capacity to alleviate ferroptosis, resulting decreased cell death, which potentially inhibits the DR pathological process.
Topics: Epithelial Cells; Ferroptosis; Glucose; Glutamate-Cysteine Ligase; MicroRNAs; NF-E2-Related Factor 2; Oxidative Stress; Reactive Oxygen Species; Retinal Pigment Epithelium; Retinal Pigments; Sirtuin 1
PubMed: 35302431
DOI: 10.1080/21655979.2022.2049471 -
Journal of Neuroinflammation Jul 2022We previously reported higher plasma levels of complement fragments C3a and C5a in neovascular Age-related Macular Degeneration (nAMD) patients with macular fibrosis....
BACKGROUND
We previously reported higher plasma levels of complement fragments C3a and C5a in neovascular Age-related Macular Degeneration (nAMD) patients with macular fibrosis. This study aimed to understand whether complement activation contributes to the development of macular fibrosis and the underlying mechanisms involved.
METHODS
Complement activation was blocked using a C5 neutralizing antibody (BB5.1) in C57BL/6J mice after induction of subretinal fibrosis using the two-stage laser protocol. Fibrotic lesions were examined 10 days after the 2nd laser through fundus examination and immunohistochemistry. The expression of C5aR in fibrotic lesions and retinal pigment epithelial (RPE) cultures were examined by confocal microscopy. Primary murine RPE cells were treated with C3a or C5a (10-100 ng/mL) or TGF-β2 (10 ng/mL). Epithelial-to-mesenchymal transition (EMT) was assessed through various readouts. The expression of E-cadherin, vimentin, fibronectin, α-SMA, Slug, ERK/AKT and pSMAD2/3 were determined by Western blot and immunocytochemistry. Collagen contraction and wound-healing assays were used as functional readouts of EMT. The production of IL-6, TGF-β1, TGF-β2 and VEGF by RPE cells were determined by ELISA. PMX53 was used to block C5aR in RPE cultures and in vivo in mice with subretinal fibrosis.
RESULTS
Extensive C5b-9 deposition was detected at the site of subretinal fibrosis. BB5.1 treatment completely abrogated complement activation and significantly reduced subretinal fibrosis. C5aR was detected in RPE and infiltrating MHC-II cells in subretinal fibrosis. In vitro, RPE cells constitutively express C5/C5a and C5aR, and their expression was increased by TGF-β2 treatment. C5a but not C3a increased fibronectin, α-SMA, vimentin and Slug expression, and decreased E-cadherin expression in RPE cells. C5a treatment also increased the contractility and migration of RPE cells and enhanced the production of VEGF and TGF-β1/2. C5a treatment induced pSmad2/3 and pERK1/2 expression in RPE cells and this was blocked by PMX53. PMX53 treatment significantly reduced sodium fluorescein leakage in the subretinal fibrosis model, while collagen-I lesions only mildly reduced.
CONCLUSIONS
Complement activation is critically involved in the development of subretinal fibrosis, partially through C5a-C5aR-mediated EMT in RPE cells. Targeting complement activation rather than C5a may be a novel approach for the management of macular fibrosis.
Topics: Animals; Cadherins; Collagen; Complement Activation; Epithelial Cells; Epithelial-Mesenchymal Transition; Fibronectins; Fibrosis; Mice; Mice, Inbred C57BL; Retinal Pigment Epithelium; Retinal Pigments; Transforming Growth Factor beta1; Transforming Growth Factor beta2; Vascular Endothelial Growth Factor A; Vimentin
PubMed: 35831910
DOI: 10.1186/s12974-022-02546-3 -
BMC Biology Sep 2019As a "holy grail" of neuroscience, optical imaging of membrane potential could enable high resolution measurements of spiking and synaptic activity in neuronal... (Review)
Review
As a "holy grail" of neuroscience, optical imaging of membrane potential could enable high resolution measurements of spiking and synaptic activity in neuronal populations. This has been partly achieved using organic voltage-sensitive dyes in vitro, or in invertebrate preparations yet unspecific staining has prevented single-cell resolution measurements from mammalian preparations in vivo. The development of genetically encoded voltage indicators (GEVIs) and chemogenetic sensors has enabled targeting voltage indicators to plasma membranes and selective neuronal populations. Here, we review recent advances in the design and use of genetic voltage indicators and discuss advantages and disadvantages of three classes of them. Although genetic voltage indicators could revolutionize neuroscience, there are still significant challenges, particularly two-photon performance. To overcome them may require cross-disciplinary collaborations, team effort, and sustained support by large-scale research initiatives.
Topics: Animals; Cell Membrane; Fluorescent Dyes; Luminescent Proteins; Neurons; Rhodopsin; Single-Cell Analysis; Voltage-Dependent Anion Channels
PubMed: 31514747
DOI: 10.1186/s12915-019-0682-0 -
Molecular Biology and Evolution Mar 2022Opsins, the protein moieties of animal visual photo-pigments, have emerged as moonlighting proteins with diverse, light-dependent and -independent physiological...
Opsins, the protein moieties of animal visual photo-pigments, have emerged as moonlighting proteins with diverse, light-dependent and -independent physiological functions. This raises the need to revise some basic assumptions concerning opsin expression, structure, classification, and evolution.
Topics: Animals; Evolution, Molecular; Opsins; Phylogeny; Retinal Pigments; Rod Opsins
PubMed: 35143663
DOI: 10.1093/molbev/msac033 -
Cell Proliferation Apr 2022'Human retinal pigment epithelial cells' is the first set of guidelines on human retinal pigment epithelial cells in China, jointly drafted and agreed upon by experts...
'Human retinal pigment epithelial cells' is the first set of guidelines on human retinal pigment epithelial cells in China, jointly drafted and agreed upon by experts from the Chinese Society for Stem Cell Research. This standard specifies technical requirements, test methods, inspection rules, instructions for usage, labelling requirements, packaging requirements, storage requirements and transportation requirements and waste disposal requirements for human retinal pigment epithelial cells, which is applicable to quality control during the process of manufacturing and testing of human retinal pigment epithelial cells. It was originally released by the Chinese Society for Cell Biology on 9 January 2021. We hope that publication of these guidelines will promote institutional establishment, acceptance and execution of proper protocols and accelerate the international standardization of human retinal pigment epithelial cells for applications.
Topics: China; Epithelial Cells; Humans; Neurons; Retinal Pigments
PubMed: 34773310
DOI: 10.1111/cpr.13153 -
The Journal of Experimental Biology Dec 2021Melanopsin is a visual pigment that is expressed in a small subset of intrinsically photosensitive retinal ganglion cells (ipRGCs). It is involved in regulating... (Review)
Review
Melanopsin is a visual pigment that is expressed in a small subset of intrinsically photosensitive retinal ganglion cells (ipRGCs). It is involved in regulating non-image forming visual behaviors, such as circadian photoentrainment and the pupillary light reflex, while also playing a role in many aspects of image-forming vision, such as contrast sensitivity. Melanopsin was initially discovered in the melanophores of the skin of the frog Xenopus, and subsequently found in a subset of ganglion cells in rat, mouse and primate retinas. ipRGCs were initially thought to be a single retinal ganglion cell population, and melanopsin was thought to activate a single, invertebrate-like Gq/transient receptor potential canonical (TRPC)-based phototransduction cascade within these cells. However, in the 20 years since the discovery of melanopsin, our knowledge of this visual pigment and ipRGCs has expanded dramatically. Six ipRGC subtypes have now been identified in the mouse, each with unique morphological, physiological and functional properties. Multiple subtypes have also been identified in other species, suggesting that this cell type diversity is a general feature of the ipRGC system. This diversity has led to a renewed interest in melanopsin phototransduction that may not follow the canonical Gq/TRPC cascade in the mouse or in the plethora of other organisms that express the melanopsin photopigment. In this Review, we discuss recent findings and discoveries that have challenged the prevailing view of melanopsin phototransduction as a single pathway that influences solely non-image forming functions.
Topics: Animals; Light Signal Transduction; Mice; Rats; Retina; Retinal Ganglion Cells; Rod Opsins
PubMed: 34842918
DOI: 10.1242/jeb.226522