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Oxidative stress induces mitochondrial dysfunction and a protective unfolded protein response in RPE cells

ABSTRACT: How retinal pigmented epithelial (RPE) cells degenerate from oxidative stress in age-related macular degeneration (AMD) is incompletely understood. The study's intent was to identify key cytoprotective pathways activated by oxidative stress, and to determine the extent of their protection. Immunohistochemistry was used to identify the unfolded protein response (UPR) and mitochondria in the RPE of AMD samples. Maculas with early AMD had prominent IRE1α, but minimal mitochondrial TOM20 immunolabeling in mildly degenerated RPE. RPE cells treated with cigarette smoke extract (CSE), by microarray analysis, had over-represented genes involved in the antioxidant and unfolded protein response, and mitochondrial location. CSE induced the UPR sensors IRE1α, p-PERK, and ATP6, which activated CHOP. CHOP knockdown compromised cell viability after CSE exposure. At the same CSE doses, mitochondria generated superoxide anion and produced less ATP. In mice given intravitreal CSE, the RPE had increased IRE1α and decreased ATP, which elicited RPE epithelial-mesenchymal transition, as suggested by altered ZO1 immunolabeling of RPE flatmounts. Our experiments indicate that RPE cells exposed to oxidative stress respond with a cytoprotective antioxidant and unfolded protein response, but develop mitochondrial impairment that contributed to epithelial mesenchymal transition. With similar responses in the RPE of early AMD samples, these results suggest that mitochondria are vulnerable to oxidative stress while the ER elicits a protective response during early AMD.

ORGANISM(S): Homo sapiens

PROVIDER: GSE49107 | GEO | 2014/01/30

SECONDARY ACCESSION(S): PRJNA213007

REPOSITORIES: GEO

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Oxidative stress induces mitochondrial dysfunction and a protective unfolded protein response in RPE cells

Project description:How retinal pigmented epithelial (RPE) cells degenerate from oxidative stress in age-related macular degeneration (AMD) is incompletely understood. The study's intent was to identify key cytoprotective pathways activated by oxidative stress, and to determine the extent of their protection. Immunohistochemistry was used to identify the unfolded protein response (UPR) and mitochondria in the RPE of AMD samples. Maculas with early AMD had prominent IRE1α, but minimal mitochondrial TOM20 immunolabeling in mildly degenerated RPE. RPE cells treated with cigarette smoke extract (CSE), by microarray analysis, had over-represented genes involved in the antioxidant and unfolded protein response, and mitochondrial location. CSE induced the UPR sensors IRE1α, p-PERK, and ATP6, which activated CHOP. CHOP knockdown compromised cell viability after CSE exposure. At the same CSE doses, mitochondria generated superoxide anion and produced less ATP. In mice given intravitreal CSE, the RPE had increased IRE1α and decreased ATP, which elicited RPE epithelial-mesenchymal transition, as suggested by altered ZO1 immunolabeling of RPE flatmounts. Our experiments indicate that RPE cells exposed to oxidative stress respond with a cytoprotective antioxidant and unfolded protein response, but develop mitochondrial impairment that contributed to epithelial mesenchymal transition. With similar responses in the RPE of early AMD samples, these results suggest that mitochondria are vulnerable to oxidative stress while the ER elicits a protective response during early AMD. A total of 9 samples were analyzed: 3 control samples, 3 samples treated with 100ug/ml of Cigarette Smoke Condensate, and 3 samples treated with 250ug/ml of Cigarettes Smoke Condensate.

2014-01-30 | E-GEOD-49107 | biostudies-arrayexpress

Deficient Pink1 Retrograde Mitochondrial-Nrf2 Signaling Induces EMT That Is Mitigated By A Mitochondria-tropic ROS Reducing Dendrimer

Project description:Age-related macular degeneration (AMD), the leading cause of blindness among the elderly, is without effective treatment for early, dry disease. Retinal pigment epithelial (RPE) cell degeneration is a cardinal feature of dry AMD. Given the reliance of photoreceptors on the RPE for maintaining health and vision, rejuvenating dysfunctional RPE is a logical treatment strategy. Here, the interplay between two cytoprotective pathways, Pink1 mediated mitophagy and the Nrf2 stress-response, was studied in human AMD tissue samples, RPE cells, and relevant mouse models. Pink1 immunolabeling was decreased in the RPE of early AMD eyes. The RPE from Pink1-/- mice and Pink1 deficient cultured human RPE cells displayed impaired mitophagy, decreased aerobic respiration, and increased mitochondrial reactive oxygen species (ROS) generation, and coincidently, developed morphological, molecular, and functional features of epithelial-mesenchymal transition (EMT). This change was triggered by novel retrograde mitochondrial to nuclear signaling that was initiated by mitochondrial ROS, which activated Nrf2 to induce TXNRD1, PI3K/AKT, and canonical EMT transcription factors Zeb1 and Snail. Nrf2 signaling was pivotal since its deficiency prevented EMT and increased cell death. A dendrimer containing N-acetyl cysteine that is tropic for the RPE and contains the mitochondrial targeting ligand Triphenyl phosphinium abrogates EMT in human RPE cells in vitro and in Pink1-/- mice. This study describes a novel interdependency of mitophagy and the Nrf2 antioxidant response in determining cell fate and introduces an RPE and mitochondrial specific ROS reducing dendrimer that can rescue RPE cells in EMT, a change recognized in early AMD.

2022-01-01 | GSE186751 | GEO

Retinal pigment epithelium extracellular vesicles are potent inducers of age-related macular degeneration disease phenotype in the outer retina

Project description:Age-related macular degeneration (AMD) is a leading cause of blindness. Vision loss is caused by the loss of the retinal pigment epithelium (RPE) and photoreceptors and/or retinal and choroidal angiogenesis. Here we use AMD patient specific RPE cells with the Y402H high-risk polymorphism in the complement factor H to perform a comprehensive analysis of EVs, their cargo and role in disease pathology. We show that AMD RPE is characterised by enhanced and polarised EV secretion. Transcriptomic, proteomic and lipidomic analyses demonstrate that AMD RPE EVs carry RNA, proteins and lipids that reflect changes in the parental RPE and mediate key AMD pathological processes including oxidative stress, cytoskeletal dysfunction, angiogenesis and drusen accumulation. We demonstrate that exposure of control RPE to AMD RPE apical EVs leads to the formation of stress vacuoles, cytoskeletal destabilization, and abnormalities in the morphology of the nucleus in the recipient cells. Treatment of retinal organoids with apical AMD RPE EVs leads to disrupted neuroepithelium and appearance of cytoprotective alpha B crystallin immunopositive cells, with some co-expressing retinal progenitor cell markers Pax6 or Vsx2, suggesting activation of regenerative pathways upon injury. These findings indicate that AMD RPE EVs mediated signalling have an important role in disease progression.

2022-01-21 | MSV000088713 | MassIVE

Extracellular vesicles confer disease phenotype in Y402H-retinal pigment epithelium cell model of age-related macular degeneration.

Project description:Age related macular degeneration (AMD) is a leading cause of legal blindness. Vision loss is caused by the retinal pigment epithelium (RPE) and photoreceptors loss and/or retinal and choroidal angiogenesis. Here we report enhanced polarised secretion of extracellular vesicles (EVs) in complement factor H (CFH) Y402H AMD patient specific RPE. As indicated by multi ‘omics analyses, AMD RPE EVs carry a repertoire of RNA, proteins and lipids that reflect disease changes in the RPE cells of origin and mediate pathological processes such as oxidative stress, cytoskeletal dysfunction and angiogenesis. We demonstrate that exposure of control RPE to AMD RPE EVs leads to the formation of numerous cytoplasmic stress vacuoles, cytoskeletal destabilization, and abnormalities in the morphology of the nucleus in the recipient cells. Treatment of laminated retinal organoids containing photoreceptor cells with apical AMD RPE EVs leads to disrupted neuroepithelium and appearance of enlarged cells immunopositive for cytoprotective alpha B crystallin. The presence of cells expressing alpha B crystallin and progenitor cells markers Pax6 or Vsx2 are consistent with the activation of regenerative pathways upon injury. These findings suggest that AMD RPE EVs act as signalling messengers in the outer retina and may be involved in disease progression.

2022-12-28 | GSE190263 | GEO

Large-Scale Quantitative Proteomics of Retinal Mitochondrial Fractions from Age-Related Macular Degeneration (AMD) Patients

Project description:Age-related macular degeneration (AMD) is the leading cause of blindness in seniors, with no effective treatment options available for most patients. AMD is characterized by the death of retinal pigment epithelium (RPE) and photoreceptors, resulting in central vision loss causing tremendous difficulties in performing daily tasks requiring visualization of fine details visualization. Dysfunction of RPE mitochondria is a critical early event involved in AMD pathogenesis, and we previously reported that maintaining normal mitochondrial functions in RPE could be a viable option for AMD treatment. We hypothesize that dysregulation of RPE mitochondrial proteome is highly relevant to the loss of RPE mitochondrial function during the transition from healthy aging to early AMD. The hypothesis was examined by quantitative proteomics using UHR-IonStar.

2022-07-13 | PXD033413 | Pride

Dual role of the unfolded protein response in the therapeutic effect of AZD1775 on TP53 mutant high grade serous ovarian cancer

Project description:we demonstrate that the WEE1 inhibitor AZD1775 triggers endoplasmic reticulum (ER) stress and activates the PERK and IRE1α branches of the unfolded protein response (UPR) in TP53 mutant HGSOC cells. Upon AZD1775 treatment, PERK facilitates apoptotic signaling in these cells via activating CHOP, whereas IRE1α-induced spliced XBP1 (XBP1s) confers survival in response to WEE1 inhibition. Our data uncover an important dual role of UPR in TP53 mutant HGSOC cells in response to AZD1775, where additional inhibition of IRE1α-XBP1s signaling may offer synergistic efficacy.

2021-02-10 | GSE166417 | GEO

Deep RNA-sequencing of the RPE Reveals a Suppressed Innate Immune Response and Activation of Dedifferentiation Pathway in Response to Chronic Smoking

Project description:Cigarette smoking is the strongest environmental risk factor for developing age-related macular degeneration (AMD), the most common cause of blindness among the elderly in western societies. Despite intensive study, the full impact of smoking on the retinal pigment epithelium (RPE), a central cell type involved in AMD pathobiology, remains unknown. The relative contribution of the known dysfunctional pathways to AMD, at what stage they are most pathogenic, or whether other processes are relevant, is poorly understood, and furthermore, whether they are activated by smoking, is unknown. We performed global RNA-sequencing of the RPE from C57BL/6J mice exposed to chronic cigarette smoke or air for 6 months to identify potential pathogenic and cytoprotective pathways. The RPE transcriptome induced by chronic cigarette smoking exhibited a mixed response of marked suppression of the innate immune response that included the antiviral response with type I and II interferons concurrent with upregulation of cell differentiation and morphogenic gene clusters, suggesting an attempt by the RPE to maintain its differentiated state despite smoke-induced injury. Given that mice exposed to chronic smoke develop early features of AMD, these novel findings are potentially relevant to the transition from aging to AMD.

2020-08-26 | GSE149386 | GEO

Single cell RNA sequencing reveals transcriptional changes of human choroid and retinal pigment epithelium cells during development, in healthy adult and intermediate age-related macular degeneration

Project description:Age related macular degeneration (AMD) is the most prominent cause of blindness in the developed countries. Vision loss in the advanced stages of the disease is caused by atrophy of retinal photoreceptors, overlaying retinal pigment epithelium (RPE) and choroid endothelial cells. The molecular events that underline the development of these cell types from in utero to adult as well as the progression from the asymptomatic, to intermediate and advanced stages AMD are not yet fully understood. We performed single cell RNA-Sequencing (Seq) of human fetal and adult RPE and choroid, profiling in detail all the cell types and elucidating proliferation, differentiation, antigen pathway presentation and immunomodulation events that characterise the first half of human gestation. Our data demonstrate that progression from fetal to adult state is characterised by an increase in expression of genes involved in oxidative stress response and detoxification from heavy metals, suggesting a better defence against oxidative stress in the adult RPE and choroid tissues. Single cell comparative transcriptional analysis between an intermediate AMD patient and unaffected subject revealed specific loss of RPE and melanocytes in the macular region. While the CECs loss was not supported by our single cell RNA-Seq data, pathway enrichment analyses supported the onset of apoptosis in these cells, suggesting that during the course of AMD pathogenesis, endothelial cells are the most likely cell type to atrophy in the macula of AMD patients. Together these finds provide a rich resource for enhancing our understanding the development of RPE and choroid and the role they play in AMD progression and pathogenesis

2023-01-19 | GSE210543 | GEO

Expression data from GFP- or GFP Satellite alpha-transfected human retinal pigment epithelia cell line ARPE19

Project description:Oxidative stress (OS) and inflammation play pivotal roles in retinal pigment epithelium (RPE) degeneration and age-related macular degeneration (AMD) pathogenesis. Interleukin 17 (IL17) signaling is a founding pathway regulating inflammatory response, the IL17 ligand has been shown to induce RPE degeneration and is emerging as a potential target for AMD treatment. However, the regulation and function of IL17 receptors are largely unexplored in RPE cells under OS. Here, we identified that IL17RA was significantly upregulated in the presence of OS both in human and mouse RPE cells. We found that OS upregulates expression of reprogramming factor Kruppel-like factor 4 (KLF4), which directly binds to the IL17RA promoter and activates IL17RA transcription, therefore inducing RPE proinflammatory response. Furthermore, our results reveal that KLF4 isoform 2, but not the canonical KLF4 isoform1, is the major isoform in RPE cells that activates IL17RA transcription. Finally, we demonstrate that mRNA level of IL17RA was positively correlated with that of KLF4 isoform2 in AMD patients. Together, our data demonstrates a new regulatory mechanism of IL17RA by KLF4 upon OS exposure, which is likely to play a role in AMD pathogenesis.

2018-11-08 | GSE122270 | GEO

Polarized Desmosome and Hemidesmosome Shedding via Exosomes is an Early Indicator of Outer Blood-Retina Barrier Dysfunction

Project description:The retinal pigmented epithelium (RPE) makes up the outer blood-retinal barrier, supports photoreceptor function of the eye and is constantly damaged by oxidative stress. Dysfunction of the RPE underlies pathology leading to development of age-related macular degeneration (AMD), the leading cause of vision loss among the elderly in industrialized nations. A major function of the RPE is to process photoreceptor outer segments which relies on the proper functioning of the RPE endocytic pathways and endosomal trafficking. RPE-released exosomes and other extracellular vesicles are essential parts of these pathways and may be early indicators of cellular stress. To test this, we used a polarized primary RPE cell culture model under chronic subtoxic oxidative stress to study the role of exosomes in the extracellular matrix (ECM) changes that underlie the early and late stages of AMD. Resulting unbiased proteomic analyses of highly purified basolateral exosomes from oxidatively stressed RPE cultures revealed changes to proteins involved in epithelial barrier integrity, that were detectable prior to overt cellular dysfunction. There were also changes to proteins accumulating in the basal-side sub-RPE ECM during oxidative stress, that could be prevented in the presence of an inhibitor of exosome release. We show for the first time that chronic subtoxic oxidative stress in primary RPE cultures induces proteomic changes in exosomes including basal-side specific desmosome and hemidesmosome shedding via exosomes. We also show that release of these exosomes correlates with ECM changes that can be partially prevented by inhibition of exosome release. These findings open a completely novel avenue for therapeutic intervention and access to early biomarkers of cellular dysfunction in aging-related retinal diseases, in particular AMD, and broadly from blood-CNS barriers in other neurodegenerative diseases.

2023-07-07 | MSV000092372 | MassIVE

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