Ferroptosis: whERe is the critical site of lipid peroxidation?
铁死亡：脂质过氧化的关键部位在哪里？

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.
铁死亡是一种受调节的细胞死亡形式，由细胞膜中脂质过氧化物的致命积累驱动。 Dixon et al., 2012 ）。最近的研究揭示了铁死亡与多种生理和病理条件有关（ Stockwell, 2022 ）。了解铁死亡的精确机制对于在各种情况下解释这种现象是必要的。

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).
继初步观察到细胞膜中的脂质过氧化物“执行”铁死亡细胞死亡后，研究人员报告了这些过氧化物更具体的生化性质。例如，全基因组 CRISPR 筛选发现酰基辅酶 A 合成酶长链家族成员 4 (ACSL4) 是执行铁死亡的必需基因，揭示了 ACSL4 介导的 PUFA 加载到膜磷脂上是执行铁死亡的关键事件。执行铁死亡（ Doll et al., 2017 ）。相反，细胞内未加载的游离 PUFA 在调节铁死亡敏感性方面呈中性。此外，基于 LC-MS/MS 的脂质组学分析表明，与细胞膜中的其他磷脂相比，含有多不饱和脂肪酸 (PUFA) 的磷脂酰乙醇胺 (PE) 在铁死亡过程中更容易受到氧化损伤。 Kagan et al., 2017 ）。后来，另一项全基因组 CRISPR 筛选发现含有 PUFA 的醚磷脂是介导铁死亡的独特功能脂质类。 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).
尽管在识别和精炼与铁死亡相关的脂质类别方面取得了重大进展，但仍然有人提出一个问题：脂质的特定亚细胞位置是否更容易受到铁死亡的启动或在铁死亡的执行中发挥更重要的作用。 von Krusenstiern 等人最近的一项研究（ von Krusenstiern et al., 2023 ）使用荧光和受激拉曼散射（SRS）成像技术解决了这个问题（ Figure 1 ）。与荧光标签相比，拉曼活性标签由于其相对较小的尺寸，在监测药物或代谢物等小分子的亚细胞位置方面具有优势。 Shen et al., 2019 ）。

The authors first examined the cellular localization of deuterated-PUFA (D-PUFA), a specific ferroptosis inhibitor (von Krusenstiern 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 authors treated cells with pro-ferroptotic PUFAs or anti-ferroptotic MUFAs and determined their cellular locations using SRS imaging. Both fatty acids were mainly located in the ER, with small percentages located in plasma membrane. The authors then overexpressed ACSL4 in the cells to increase the loading of pro-ferroptotic PUFAs or anti-ferroptotic MUFAs to the ER phospholipids. This approach demonstrated a solid correlation between the ER membrane composition and ferroptosis sensitivity, highlighting the ER as a site of ferroptosis modulation.
作者首先检查了氘化多不饱和脂肪酸 (D-PUFA) 的细胞定位，这是一种特定的铁死亡抑制剂。 von Krusenstiern et al., 2023 ; Yang et al., 2016 ）。由于氘是拉曼活性标签，因此无需进一步修改 D-PUFA 即可在细胞内可视化它。 SRS 成像显示 D-PUFA 主要位于核周区域和细胞中丰富的点状结构，随后尼罗红荧光成像证实这些点状结构为脂滴。用甘油二酯酰基转移酶（DGAT）抑制剂处理细胞可以去除这些脂滴。然而，脂滴的缺失并不影响 D-PUFA 的抗铁死亡活性，表明脂滴与铁死亡没有功能相关性。使用带有 ER-tracker Green 染料的荧光显微镜进一步分析表明，核周区域是内质网 (ER)，表明 ER 是 D-PUFA 主要驻留的另一个细胞位置。然而，与脂滴不同的是，没有直接的方法可以使用药理学或遗传试剂来改变内质网的表面积。例如，诱导内质网自噬（ Mochida and Nakatogawa, 2022 尽管进行了广泛的尝试，但在本研究中使用的细胞系中仍不成功。相反，作者用促铁死亡的 PUFA 或抗铁死亡的 MUFA 处理细胞，并使用 SRS 成像确定它们的细胞位置。两种脂肪酸主要位于内质网，少量位于质膜。然后，作者在细胞中过表达 ACSL4，以增加内质网磷脂中促铁死亡的 PUFA 或抗铁死亡的 MUFA 的负载。 这种方法证明了内质网膜组成与铁死亡敏感性之间的紧密相关性，突出了内质网作为铁死亡调节的位点。

Next, the authors explored the site of action for FINO₂, a canonical ferroptosis inducer, which directly oxidizes cellular iron and indirectly inhibits GPx4 to induce ferroptosis (Gaschler et al., 2018a). Structurally, FINO₂ is a lipophilic compound that contains an endoperoxide moiety, allowing it to accumulate in cell membranes and induce lipid peroxidation directly at those locations. Taking advantage of this feature, the authors synthesized a series of FINO₂ analogs with fluorescent tags that were distributed to different cellular locations to investigate whether ferroptosis can be initiated by oxidative damage in a particular cell membrane. The original FINO₂ with a fluorescent tag displayed ER distribution and induced ferroptosis. Interestingly, FINO₂ analogs distributed to mitochondria and lysosomes also induced ferroptosis, indicating that ferroptosis can be initiated from various organelles. More importantly, cells treated with mitochondrial and lysosomal FINO₂ analogs were protected by ferrostatin-1 (Fer-1). A previous report demonstrated that Fer-1 primarily acted at the ER site, rather than in mitochondria and lysosomes which were dispensable to Fer-1’s anti-ferroptotic activity (Gaschler et al., 2018b). These data suggest that while ferroptosis can be initiated at multiple sites, the ER is the critical site for ferroptosis protection.
接下来，作者探索了 FINO ₂的作用位点，FINO 2 是一种典型的铁死亡诱导剂，它直接氧化细胞铁并间接抑制 GPx4 以诱导铁死亡。 Gaschler et al., 2018a ）。从结构上看，FINO ₂是一种亲脂性化合物，含有内过氧化物部分，使其能够在细胞膜中积聚并直接在这些位置诱导脂质过氧化。利用这一特性，作者合成了一系列带有荧光标签的 FINO ₂类似物，将其分布到不同的细胞位置，以研究特定细胞膜的氧化损伤是否可以引发铁死亡。带有荧光标签的原始 FINO ₂显示 ER 分布并诱导铁死亡。有趣的是，分布到线粒体和溶酶体的FINO ₂类似物也诱导铁死亡，表明铁死亡可以从多种细胞器启动。更重要的是，用线粒体和溶酶体 FINO ₂类似物处理的细胞受到 Ferrostatin-1 (Fer-1) 的保护。之前的一份报告表明，Fer-1 主要作用于 ER 位点，而不是作用于线粒体和溶酶体，而线粒体和溶酶体对于 Fer-1 的抗铁死亡活性来说是可有可无的。 Gaschler et al., 2018b ）。这些数据表明，虽然铁死亡可以在多个部位启动，但内质网是铁死亡保护的关键部位。

Lastly, the authors examined time-dependent changes in the location of lipid peroxidation using BODIPY™-C11 dye and fluorescence microscopy. Upon treatment with ferroptosis inducers, cells underwent initial lipid peroxidation events in the ER, followed by lipid peroxidation in the plasma membrane. This time-dependent change was observed in all four classes of ferroptosis inducers (RSL3, FIN56, FINO₂, or IKE). These findings complement a previous study, which reported that both erastin2 and RSL3 caused intracellular lipid peroxidation before the onset of lipid peroxidation in the plasma membrane (Magtanong et al., 2022). Altogether, these data strongly point towards the ER membrane as the most critical site for inducing ferroptosis.
最后，作者使用 BODIPY™-C11 染料和荧光显微镜检查了脂质过氧化位置随时间的变化。在用铁死亡诱导剂处理后，细胞在内质网中经历最初的脂质过氧化事件，随后在质膜中发生脂质过氧化。在所有四类铁死亡诱导剂（RSL3、FIN56、FINO ₂或 IKE）中均观察到这种时间依赖性变化。这些发现补充了之前的一项研究，该研究报告在质膜中脂质过氧化发生之前，erastin2 和 RSL3 都会引起细胞内脂质过氧化。 Magtanong et al., 2022 ）。总而言之，这些数据强烈表明内质网膜是诱导铁死亡的最关键部位。

In summary, the findings outlined above emphasized the essential role of the ER in ferroptotic cell death. The conclusion from the current research raises several intriguing questions. First, what makes the ER so critical in executing ferroptosis? Does the ER membrane contain a higher level of ferroptosis-relevant lipids compared to other organelles? Are there any microenvironmental factors in the ER that can affect ferroptosis? A refined lipidomics analysis with subcellular fractionation samples may provide answers to these questions. Second, how is the ferroptosis signal initiated from other organelles transduced to the ER? For example, it is unclear how the lipid peroxides in mitochondria or lysosomes spread to the ER when FINO₂ analogs are used. This question also applies to the later stage of ferroptosis. How are the lipid peroxides in the ER transduced or transported to the plasma membrane before the onset of cell death? Could it be the conventional vesicle-mediated transport or some other mechanism? Third, can we find more specific regions within the ER, such as rough ER, smooth ER, ER lumen, or microdomains in the ER membrane, that are responsible for the initiation of ferroptosis? Lastly, what are the ER-specific genes that regulate ferroptosis? Xiaodong Wang’s group recently discovered that the ER-resident oxidoreductases POR and CYB5R1 produced initial hydrogen peroxides, which subsequently induced lipid peroxidation and ferroptosis (Yan et al., 2021). Future research focused on the ER should broaden our knowledge about the natural triggers of ferroptosis and help identify better target proteins for ferroptosis targeted therapy.
总之，上述研究结果强调了 ER 在铁死亡细胞死亡中的重要作用。当前研究的结论提出了几个有趣的问题。首先，是什么使得 ER 在执行铁死亡过程中如此重要？与其他细胞器相比，内质网膜是否含有更高水平的铁死亡相关脂质？内质网中是否有任何微环境因素可以影响铁死亡？使用亚细胞分级样品进行精细的脂质组学分析可以为这些问题提供答案。其次，其他细胞器引发的铁死亡信号是如何转导到内质网的？例如，当使用 FINO ₂类似物时，尚不清楚线粒体或溶酶体中的脂质过氧化物如何扩散到 ER。这个问题也适用于铁死亡的后期。在细胞死亡开始之前，内质网中的脂质过氧化物如何转导或转运到质膜？它可能是传统的囊泡介导的运输还是其他机制？第三，我们能否找到内质网内更具体的区域，例如粗糙内质网、光滑内质网、内质网管腔或内质网膜上的微结构域，这些区域负责铁死亡的启动？最后，调节铁死亡的 ER 特异性基因是什么？王晓东团队最近发现，内质网驻留的氧化还原酶 POR 和 CYB5R1 最初产生过氧化氢，随后诱导脂质过氧化和铁死亡。 Yan et al., 2021 ）。未来针对内质网的研究应该拓宽我们对铁死亡自然触发因素的了解，并帮助识别铁死亡靶向治疗的更好靶蛋白。