Ferroptosis: whERe is the critical site of lipid peroxidation?

Ferroptosis is a regulated form of cell death that is driven by a lethal accumulation of lipid peroxides in cell membranes (Dixon et al., 2012). Recent studies have revealed the involvement of ferroptosis in various physiological and pathological conditions (Stockwell, 2022). Understanding the precise mechanism of ferroptosis is necessary to explain this phenomenon in various contexts. Following the initial observation that lipid peroxides in cell membranes “execute” ferroptotic cell death, researchers have reported a more specific biochemical nature of these peroxides. For example, a genome-wide CRISPR screening identified acyl-CoA synthetase long-chain family member 4 (ACSL4) as an essential gene for executing ferroptosis, revealing that the ACSL4-mediated loading of the PUFAs to the membrane phospholipids is a critical event for executing ferroptosis (Doll et al., 2017). Conversely, unloaded, free PUFAs within cells were found to be neutral in modulating ferroptosis sensitivity. In addition, LC-MS/MS-based lipidomics analysis showed that phosphatidylethanolamines (PEs) containing polyunsaturated fatty acids (PUFAs) were more susceptible to oxidative damage during ferroptosis compared to other phospholipids in cell membranes (Kagan et al., 2017). Later, another genome-wide CRISPR screening discovered ether-phospholipid with PUFAs as a distinct functional lipid class mediating ferroptosis (Zou et al., 2020). Despite significant progress in identifying and refining lipid classes relevant to ferroptosis, there remined the question of whether specific subcellular locations of lipids that are more susceptible to ferroptosis initiation or play more important roles in ferroptosis execution. A recent study by von Krusenstiern and others (von Krusenstiern et al., 2023) addressed this question using fluorescence and stimulated Raman scattering (SRS) imaging techniques (Figure 1). Compared to fluorescent tags, Raman active tags offer an advantage in monitoring the subcellular location of small molecules, such as drugs or metabolites, due to their relatively tiny size (Shen et al., 2019). The authors first examined the cellular localization of deuterated-PUFA (D-PUFA), a specific ferroptosis inhibitor (vonKrusenstiern et al., 2023; Yang et al., 2016). Since deuterium is a Raman active tag, there was no need to modify D-PUFA further to visualize it within cells. SRS imaging revealed that D-PUFAs were primarily located in perinuclear regions and abundant puncta structures in the cells, and subsequent fluorescence imaging with Nile Red confirmed the puncta structures as lipid droplets. Treatment of cells with diglyceride acyltransferase (DGAT) inhibitors removed these lipid droplets. However, the absence of lipid droplets did not affect D-PUFA’s anti-ferroptotic activity, suggesting that lipid droplets are not functionally relevant to ferroptosis. Further analysis using fluorescence microscopy with ER-tracker Green dye revealed that the perinuclear region was the endoplasmic reticulum (ER), indicating that the ER is another cellular location where D-PUFAs mainly reside. Unlike lipid droplets, however, there was no straightforward way to alter the surface area of ER using pharmacological or genetic reagents. For example, induction of ER-phagy (Mochida and Nakatogawa, 2022) was unsuccessful in the cell lines used in this study, despite extensive attempts. Instead, the OPEN ACCESS