bims-mideyd 2024-10-06 papers

Issue of 2024‒10‒06
five papers selected by
Rajalekshmy “Raji” Shyam, Indiana University Bloomington

Phytomedicine. 2024 Sep 22. pii: S0944-7113(24)00745-1. [Epub ahead of print]135 156088

Melatonin protects RPE cells from necroptosis and NLRP3 activation via promoting SERCA2-related intracellular Ca2+ homeostasis.

Chengda Ren, Chengyu Hu, Ming Hu, Yan Wu, Yang Yang, Fang Lu.

BACKGROUND: Melatonin is an antioxidant that also has anti-inflammatory effects. It has been reported to delay the progression of age-related macular degeneration (AMD), however, the mechanism has not been fully recognized.PURPOSE: The aim of the present study was to investigate the effects of melatonin on sodium iodate (SI)-induced retinal degeneration and elucidate the specific mechanisms, then, provide novel targets in AMD treatment.
METHODS: Retinal degeneration mouse model and in vitro retinal pigment epithelium (RPE) death model were established by SI treatment. Melatonin was administrated intraperitoneally at a concentration of 20, 40 or 80 mg/kg for in vivo study or treated at 48 h before SI treatment. To confirm the therapeutic effects of melatonin on mouse, the retinal structure and visual function were evaluated. The specific cell death rates were determined by CCK-8 assay, PI staining and protein level of RIPK3. The cytosolic or mitochondrial calcium levels were determined by Fluo-4AM or Rhod-2AM staining. Mitochondrial functions including mitochondrial dynamics, mitochondrial membrane potential, or mitochondrial permeability pore opening were evaluated. The proteins involved in endoplasmic reticulum (ER) stress were measured by western blot assay while the genes expression in calcium signaling pathway were measured by RT-qPCR.
RESULTS: We show that melatonin protects RPE cells from necroptosis and NLRP3 inflammasome activation induced by SI. Mechanistically, melatonin suppresses ER stress and intracellular calcium overload triggered by SI through restoring the function of SERCA2. Silencing of SERCA2 or blocking of melatonin receptors inhibit the protective effects of melatonin. Melatonin reduces mitochondrial Ca2+ levels and restores mitochondrial membrane potential. Constant mitochondrial Ca2+ overload directly promote cell necroptosis through mitochondrial fission. Inhibition of mitochondrial fission by Mdivi-1 prevent necroptosis induced by SI without altering the level of mitochondrial Ca2+.
CONCLUSIONS: The results confirmed that melatonin protects RPE cells from SI-induced injury by regulates MT2/SERCA2/Ca2+ axis. This study highlighted the potential of melatonin in the treatment of AMD and elucidated the mechanism and signaling pathway that mediate the protective effects.

Keywords: Calcium signaling; ER-stress; Inflammation; Melatonin; Necroptosis; Retinal degeneration

DOI: https://doi.org/10.1016/j.phymed.2024.156088

Sci Rep. 2024 09 27. 14(1): 22391

Clustered ARPE-19 cells distinct in mitochondrial membrane potential may play a pivotal role in cell differentiation.

Takafumi Miyatani, Hiroshi Tanaka, Kosaku Numa, Asako Uehara, Yohei Otsuki, Junji Hamuro, Shigeru Kinoshita, Chie Sotozono.

Age-related macular degeneration (AMD) is associated with the dysfunction and degeneration of retinal pigment epithelium (RPE) cells. Here, we examined how the formation and expansions of cell clusters are regulated by the differentiation of the RPE　cells. In this study, ARPE-19 cells were cultivated in standard or differentiation media, i.e., without or with nicotinamide, to evaluate the spreading of cell clusters specified with differentiated cell phenotypes. Mitochondria membrane potential (MMP) and the distribution of the RPE cell clusters was also monitored with or without rotenone, a mitochondrial electron transport chain (ETC) complex I inhibitor. Cultured ARPE-19 cells generated scattered cell clusters composed mostly of smaller size cells expressing the differentiation markers mouse anti-cellular retinaldehyde-binding protein (CRALBP) and Bestrophin only in differentiation medium. After the increase of the number of clusters, the clusters appeared to paracellularly merge, resulting in expansion of the area occupied by the clusters. Of note, the cells within the clusters selectively had high MMP and were in accordance with the expression of RPE differentiation markers. Rotenone repressed the formation of the clusters and decreased intracellular MMP. The above results suggest that clustering of RPE cells with functional mitochondria plays a pivotal role in RPE cell differentiation process and the ETC complex I inhibition greatly influences the composition of RPE cells that are degenerated or differentiation disposed.

Keywords: Cell cluster formation; Electron transport chain (ETC) complex I inhibitor; Mitochondrial membrane potential (MMP); Retinal pigment epithelial (RPE) cell differentiation

DOI: https://doi.org/10.1038/s41598-024-73145-w

J Ocul Pharmacol Ther. 2024 Oct 02.

Detection of Residual iPSCs Following Differentiation of iPSC-Derived Retinal Pigment Epithelial Cells.

Matthew Hill, Cynthia Andrews-Pfannkoch, Evan Atherton, Travis Knudsen, Emma Trncic, Alan D Marmorstein.

Purpose: The goal of this study was to develop a lot release assay for iPSC residuals following directed differentiation of iPSCs to retinal pigment epithelial (RPE) cells. Methods: RNA Sequencing (RNA Seq) of iPSCs and RPE derived from them was used to identify pluripotency markers downregulated in RPE cells. Quantitative real time PCR (qPCR) was then applied to assess iPSC residuals in iPSC-derived RPE. The limit of detection (LOD) of the assay was determined by performing spike-in assays with known quantities of iPSCs serially diluted into an RPE suspension. Results: ZSCAN10 and LIN28A were among 8 pluripotency markers identified by RNA Seq as downregulated in RPE. Based on copy number and expression of pseudogenes and lncRNAs ZSCAN10 and LIN28A were chosen for use in qPCR assays for residual iPSCs. Reverse transcription PCR indicated generally uniform expression of ZSCAN10 and LIN28A in 21 clones derived from 8 iPSC donors with no expression of either in RPE cells derived from 5 donor lines. Based on qPCR, ZSCAN10, and LIN28A expression in iPSCs was generally uniform. The LOD for ZSCAN10 and LIN28A in qPCR assays was determined using spike in assays of RPE derived from 2 iPSC lines. Analysis of ΔΔCt found the limit of detection to be <0.01% of cells, equivalent to <1 iPSC/10,000 RPE cells in both iPSC lines. Conclusions: qPCR for ZSCAN10 and LIN28A detects <1 in 10,000 residual iPSCs in a population of iPSC-derived RPE providing an adequate LOD of iPSC residuals for lot release testing.

Keywords: cGMP; gene expression; induced pluripotent stem cell; macular degeneration; retinal pigment epithelium

DOI: https://doi.org/10.1089/jop.2024.0130

Biochim Biophys Acta Mol Basis Dis. 2024 Sep 27. pii: S0925-4439(24)00524-6. [Epub ahead of print]1871(1): 167530

TMCO1 promotes ferroptosis and ECM deposition in glaucomatous trabecular meshwork via ERK1/2 signaling.

Yang Zhang, Ruiqi Han, Shushu Xu, Bingqiao Shen, Huan Yu, Junjue Chen, Huiping Yao, Shouyue Huang, Yisheng Zhong.

Glaucoma, a leading cause of global blindness, is marked by irreversible retinal ganglion cells (RGCs) loss, elevated intraocular pressure (IOP), and extracellular matrix (ECM) deposition in the trabecular meshwork (TM). Transmembrane and coiled-coil domain protein 1 (TMCO1), implicated in calcium regulation, has potential links to primary open-angle glaucoma (POAG). Ferroptosis, an iron-dependent cell death mechanism driven by lipid peroxidation, is also observed in glaucoma. This study investigates the role of TMCO1 in POAG, focusing on its involvement in TM ECM deposition via ferroptosis induction and ERK1/2 phosphorylation inhibition. In both in vivo and in vitro models, we demonstrated that dexamethasone (DEX) stimulation upregulates TMCO1, leading to increased ECM deposition and ferroptosis in human trabecular meshwork cells (HTMCs). Furthermore, treatment with ferrostatin-1 (Fer-1), a ferroptosis inhibitor, significantly reduced ECM deposition and ferroptosis in HTMCs. These findings establish TMCO1 as a critical regulator of ferroptosis and ECM deposition through the ERK/MAPK pathway, positioning it as a promising therapeutic target for glaucoma.

Keywords: ECM deposition; ERK/MAPK; ferroptosis; glaucoam; trabecular meshwork

DOI: https://doi.org/10.1016/j.bbadis.2024.167530

Theranostics. 2024 ;14(15): 5762-5777

ROCK inhibitor enhances mitochondrial transfer via tunneling nanotubes in retinal pigment epithelium.

Jing Yuan, Fangxuan Chen, Dan Jiang, Zehua Xu, Hang Zhang, Zi-Bing Jin.

Rationale: Tunnel nanotube (TNT)-mediated mitochondrial transport is crucial for the development and maintenance of multicellular organisms. Despite numerous studies highlighting the significance of this process in both physiological and pathological contexts, knowledge of the underlying mechanisms is still limited. This research focused on the role of the ROCK inhibitor Y-27632 in modulating TNT formation and mitochondrial transport in retinal pigment epithelial (RPE) cells. Methods: Two types of ARPE19 cells (a retinal pigment epithelial cell line) with distinct mitochondrial fluorescently labeled, were co-cultured and treated with ROCK inhibitor Y-27632. The formation of nanotubes and transport of mitochondria were assessed through cytoskeletal staining and live cell imaging. Mitochondrial dysfunction was induced by light damage to establish a model, while mitochondrial function was evaluated through measurement of oxygen consumption rate. The effects of Y-27632 on cytoskeletal and mitochondrial dynamics were further elucidated through detailed analysis. Results: Y-27632 treatment led to an increase in nanotube formation and enhanced mitochondrial transfer among ARPE19 cells, even following exposure to light-induced damage. Our analysis of cytoskeletal and mitochondrial distribution changes suggests that Y-27632 promotes nanotube-mediated mitochondrial transport by influencing cytoskeletal remodeling and mitochondrial movement. Conclusions: These results suggest that Y-27632 has the ability to enhance mitochondrial transfer via tunneling nanotubes in retinal pigment epithelium, and similarly predict that ROCK inhibitor can fulfill its therapeutic potential through promoting mitochondrial transport in the retinal pigment epithelium in the future.

Keywords: ARPE19; Y-27632; cytoskeletal remodeling; light damage; mitochondrial transfer; nanotubes

DOI: https://doi.org/10.7150/thno.96508