bims-mideyd 2023-11-12 papers

J Biomed Sci. 2023 Nov 07. 30(1): 91

Spatiotemporal roles of AMPK in PARP-1- and autophagy-dependent retinal pigment epithelial cell death caused by UVA.

Anthony Yan-Tang Wu, Ponarulselvam Sekar, Duen-Yi Huang, Shu-Hao Hsu, Chi-Ming Chan, Wan-Wan Lin.

BACKGROUND: Although stimulating autophagy caused by UV has been widely demonstrated in skin cells to exert cell protection, it remains unknown the cellular events in UVA-treated retinal pigment epithelial (RPE) cells.
METHODS: Human ARPE-19 cells were used to measure cell viability, mitochondrial reactive oxygen species (ROS), mitochondrial membrane potential (MMP), mitochondrial mass and lysosomal mass by flow cytometry. Mitochondrial oxygen consumption rate (OCR) was recorded using Seahorse XF flux analyzer. Confocal microscopic images were performed to indicate the mitochondrial dynamics, LC3 level, and AMPK translocation after UVA irradiation.
RESULTS: We confirmed mitochondrial ROS production and DNA damage are two major features caused by UVA. We found the cell death is prevented by autophagy inhibitor 3-methyladenine and gene silencing of ATG5, and UVA induces ROS-dependent LC3II expression, LC3 punctate and TFEB expression, suggesting the autophagic death in the UVA-stressed RPE cells. Although PARP-1 inhibitor olaparib increases DNA damage, ROS production, and cell death, it also blocks AMPK activation caused by UVA. Interestingly we found a dramatic nuclear export of AMPK upon UVA irradiation which is blocked by N-acetylcysteine and olaparib. In addition, UVA exposure gradually decreases lysosomal mass and inhibits cathepsin B activity at late phase due to lysosomal dysfunction. Nevertheless, cathepsin B inhibitor, CA-074Me, reverses the death extent, suggesting the contribution of cathepsin B in the death pathway. When examining the role of EGFR in cellular events caused by UVA, we found that UVA can rapidly transactivate EGFR, and treatment with EGFR TKIs (gefitinib and afatinib) enhances the cell death accompanied by the increased LC3II formation, ROS production, loss of MMP and mass of mitochondria and lysosomes. Although AMPK activation by ROS-PARP-1 mediates autophagic cell death, we surprisingly found that pretreatment of cells with AMPK activators (A769662 and metformin) reverses cell death. Concomitantly, both agents block UVA-induced mitochondrial ROS production, autophagic flux, and mitochondrial fission without changing the inhibition of cathepsin B.
CONCLUSION: UVA exposure rapidly induces ROS-PARP-1-AMPK-autophagic flux and late lysosomal dysfunction. Pre-inducing AMPK activation can prevent cellular events caused by UVA and provide a new protective strategy in photo-oxidative stress and photo-retinopathy.

Keywords: AMPK; Autophagic cell death; EGFR; Lysosome dysfunction; PARP; ROS; RPE; UVA

DOI: https://doi.org/10.1186/s12929-023-00978-4

Exp Eye Res. 2023 Nov 06. pii: S0014-4835(23)00311-1. [Epub ahead of print] 109690

Circ-CARD6 inhibits oxidative stress-induced apoptosis and autophagy in ARPE-19 cells via the miR-29b-3p/PRDX6/PI3K/Akt axis.

Xinyu Fan, Yanni Yang, Guojiu Wu, Yanbo Kong, Yuanping Zhang, Xu Zha.

BACKGROUND: Oxidative stress-induced damage to and dysfunction of retinal pigment epithelium (RPE) cells are important pathogenetic factors of age-related macular degeneration (AMD) and hereditary retinopathy diseases (HRDs). This study aimed to elucidate the roles and mechanisms of circ-CARD6 and miR-29b-3p in oxidative stress-induced RPE and provide new ideas for the diagnosis and treatment of retinopathy disease (RD).
METHODS: A model of oxidative stress-induced RPE (ARPE-19) was established, and the level and concentration of reactive oxygen species (ROS) were detected by a DCFH-DA fluorescent probe, malondialdehyde (MDA) and ELISA. The cell viability was measured by a CCK-8 assay. The expression of PRDX6/PI3K/Akt axis genes and proteins related to apoptosis and autophagy were determined by RT‒qPCR and Western blot analyses. The dual-luciferase reporter system confirmed the targeting relationship between miR-29b-3p and circ-CARD6 and between circ-CARD6 and PRDX6.
RESULTS: In H2O2-treated ARPE-19 cells, the expression of circ-CARD6 and PRDX6 was decreased, while the expression of miR-29b-3p was increased. The overexpression of circ-CARD6 inhibits oxidative stress-induced increases in ROS, apoptosis and autophagy in ARPE-19 cells. circ-CARD6 targets miR-29b-3p, miR-29b-3p targets PRDX6, and circ-CARD6 regulates PRDX6 via miR-29b-3p. Further studies showed that circ-CARD6 acts as a competitive endogenous RNA of miR-29b-3p to affect the expression of PRDX6, thereby inhibiting autophagy and apoptosis in ARPE-19 cells.
CONCLUSION: circ-CARD6 can inhibit oxidative stress and apoptosis by regulating the miR-29b-3p/PRDX6/PI3K/Akt axis.

Keywords: Circ-CARD6; Oxidative stress; PRDX6/PI3K/Akt; Retinopathy; miR-29b-3p

DOI: https://doi.org/10.1016/j.exer.2023.109690

Front Mol Neurosci. 2023 ;16 1279457

A novel quantification method for retinal pigment epithelium phagocytosis using a very-long-chain polyunsaturated fatty acids-based strategy.

Fangyuan Gao, Emily Tom, Stephanie A Lieffrig, Silvia C Finnemann, Dorota Skowronska-Krawczyk.

Introduction: The vertebrate retinal pigment epithelium (RPE) lies adjacent to the photoreceptors and is responsible for the engulfment and degradation of shed photoreceptor outer segment fragments (POS) through receptor-mediated phagocytosis. Phagocytosis of POS is critical for maintaining photoreceptor function and is a key indicator of RPE functionality. Popular established methods to assess RPE phagocytosis rely mainly on quantifying POS proteins, especially their most abundant protein rhodopsin, or on fluorescent dye conjugation of bulk, unspecified POS components. While these approaches are practical and quantitative, they fail to assess the fate of POS lipids, which make up about 50% of POS by dry weight and whose processing is essential for life-long functionality of RPE and retina.
Methods: We have developed a novel very-long-chain polyunsaturated fatty acids (VLC-PUFA)-based approach for evaluating RPE phagocytic activity by primary bovine and rat RPE and the human ARPE-19 cell line and validated its results using traditional methods.
Results and discussion: This new approach can be used to detect in vitro the dynamic process of phagocytosis at varying POS concentrations and incubation times and offers a robust, unbiased, and reproducible assay that will have utility in studies of POS lipid processing.

Keywords: liquid chromatography-mass spectrometer; phagocytosis; quantification; retinal pigment epithelium; very-long-chain polyunsaturated fatty acids

DOI: https://doi.org/10.3389/fnmol.2023.1279457

Sci Rep. 2023 11 07. 13(1): 19345

Co-cultivation of primary porcine RPE cells and neuroretina induces inflammation: a potential inflammatory AMD-model.

Agnes Fietz, Sven Schnichels, José Hurst.

One common aspect in the pathology of many retinal diseases like age-related macular degeneration (AMD) is the death of retinal pigment epithelium (RPE) cells. RPE cells are essential for photoreceptor survival as they recycle and remove compounds of the visual cycle and secrete protective cytokines. Studying RPE cells is crucial to improve our understanding of retinal pathologies, yet only a few retinal ex vivo models include them or do so only indirectly. Besides the positive effects in indirect co-cultivation models, also a slight inflammation was observed. In this study we developed an ex vivo model consisting of a primary porcine RPE monolayer directly co-cultured with porcine retinal organ cultures, to investigate and simulate inflammatory retinal diseases, such as (dry) AMD. The direct co-cultivation resulted in immune reactivity (enhanced expression of pro-inflammatory cytokines e.g., IL-1β, IL-6, IL-8) and cell death. These effects were evaluated for the retinal explant as well as for the RPE-monolayer to further understand the complex interactions between these two compartments. Taken together, this ex vivo model can be used to study inflammatory retinal diseases like AMD as well as the rejection observed after RPE-transplantation.

DOI: https://doi.org/10.1038/s41598-023-46029-8

Biochim Biophys Acta Mol Cell Res. 2023 Sep 15. pii: S0167-4889(23)00146-5. [Epub ahead of print] 119573

Reactive oxygen species and cell signaling. Mini review.

Diana Averill-Bates.

Reactive oxygen species (ROS) is a term encompassing a group of highly reactive oxygen-derived molecules. In physiological systems, ROS production exists in concert with antioxidant defenses, which safeguard cells against higher, toxic levels of ROS. Oxidative stress, coined as "oxidative distress", is "a serious imbalance between the generation of ROS and antioxidant defenses in favor of ROS, causing excessive oxidative damage to biomolecules". At physiological levels, ROS are essential for many cellular processes, which is known as "oxidative eustress". Oxidants like hydrogen peroxide (H2O2) activate signaling pathways like mitogen-activated protein kinases (MAPK)s and phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt). ROS activate transcription factors like nuclear factor erythroid 2-related factor 2 (Nrf2), hypoxia-inducible factor 1α (HIF-1α), activator protein 1 (AP-1), and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). Redox signaling through H2O2 mainly occurs through reversible oxidation of protein cysteine thiolate residues (RS-) to form sulfenic acids (RSOH). An unresolved question is that the reaction rate of H2O2 with protein thiols is very low. In cells, the reaction of H2O2 with protein thiols is likely to be outcompeted by faster reactions of H2O2 with peroxiredoxins and glutathione peroxidases. A novel mechanism being explored is that H2O2 could react with peroxiredoxins that act as reactive redox sensor proteins, leading to peroxiredoxin-mediated relays. Very few redox signaling pathways have been well characterized. Improved understanding of precise mechanisms by which ROS regulate signaling pathways and the role of cellular sensors, is essential for deciphering their roles in physiological and pathological conditions.

Keywords: Cell signaling; Nrf2; Oxidative stress; ROS; Thiol oxidation; Transcription factor

DOI: https://doi.org/10.1016/j.bbamcr.2023.119573