Abstract
Purpose :
Mitochondria are the cellular powerhouses that produce energy necessary for cellular function. Recent research has revealed that these organelles also play a crucial role in the expression of genes related to ferroptosis, a form of iron-dependent programmed cell death. It is characterized by the accumulation of lipid peroxides and iron-dependent reactive oxygen species (ROS), leading to oxidative damage and ultimately cell death. Ferroptosis is implicated in various eye diseases such as keratopathy, cataract, glaucoma, retinal ischemia-reperfusion injury, age-related macular degeneration, retinitis pigmentosa, diabetic retinopathy, and retinoblastoma. Our goal is to investigate the role of mitochondria in ferroptosis using Wildtype (WT) and Rho0 (lacking mtDNA) ARPE-19 cell lines.
Methods :
Cells were cultured in DMEM mixture 1:1 Ham's F-12 media. Rho0 cells were created by serial passage in 50 microM Ethidium Bromide. DNA was isolated and mtDNA copy number measured using qPCR using 18S as the nuclear reference gene and MT-ND2 as the mitochondrial target to verify Rho0 status. WT and Rho0 cells were pelleted, media removed and RNA isolated using the PureGene RNA Extraction Kit (ThermoFisher Scientific, Rockford, IL) following the manufacturer’s protocol. cDNA was synthesized with the SuperScript™ IV VILO™ Master Mix (ThermoFisher) according to the manufacturer’s protocol. All qRT-PCR was performed with PowerUp™ SYBR™ Green Master Mix on a QuantStudio™ 5 Real-Time PCR System. Pre-designed, bioinformatically verified SYBR™ Green primers were used (KiCqStart® SYBR Green Primers (Millipore Sigma) and QuantiTect Primer Assays (Qiagen)) with HPRT1 utilized as the reference gene.
Results :
Gene expression levels were analyzed for 3 ferroptosis-related pathways: Lipid (LPCAT3, ACSL4); xCT System (SLC7A11, GPX4, SLC3A2); iron transport (CISD1, TFRC, FTMT, SLC40A1). The Rho0 cells showed an increase of 7.5-fold (p<0.001) in SLC7A11 and decrease of 0.13-fold (p<0.001) in SLC40A1 compared to the WT ARPE-19 cells.
Conclusions :
Mitochondria play a crucial role in the expression of SLC7A11 (xCT system) and SLC40A1 (iron transport) genes related to ferroptosis. Understanding the mechanisms by which mitochondria contribute to ferroptosis could provide valuable insights into potential therapeutic targets for diseases associated with dysregulated cell death processes
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.