Determining the role of peroxidation of subcellular lipid membranes in ferroptosis
确定亚细胞脂质膜过氧化在铁死亡中的作用

Ferroptosis is a non-apoptotic form of cell death that is driven by iron-dependent lipid peroxidation. Ferroptosis has been implicated in diseases including neurodegeneration and ischemia-reperfusion injury, and initiation of ferroptosis is a promising avenue for cancer treatment¹. There remain questions about the different players and stages of ferroptotic death. Although it is known that lipid peroxidation in membranes drives ferroptosis¹, whether certain membranes are essential and at what point they are oxidized has been unclear. A deeper understanding of the mechanisms that drive ferroptosis will facilitate the development of new medicines and biomarkers and strengthen our insight into how cells die in diverse diseases.
铁死亡是一种非凋亡形式的细胞死亡，由铁依赖性脂质过氧化驱动。铁死亡与神经变性和缺血再灌注损伤等疾病有关，铁死亡的启动是癌症治疗的一个有前途的途径¹ 。关于铁死亡的不同参与者和阶段仍然存在疑问。尽管已知膜中的脂质过氧化会导致铁死亡¹ ，但某些膜是否是必需的以及它们在什么时候被氧化尚不清楚。更深入地了解铁死亡的驱动机制将有助于新药物和生物标志物的开发，并加强我们对细胞在不同疾病中如何死亡的了解。

In this project, we sought to elucidate the subcellular dynamics of lipid peroxidation in ferroptosis. We approached this question by exploring the subcellular distribution of different ferroptosis-modulating compounds: fatty acids that make cells susceptible to or protect against ferroptosis, and FINO₂, a 1,2-dioxolane that contains an endoperoxide moiety that induces ferroptosis². Polyunsaturated fatty acids (PUFAs), such as arachidonic acid and docosahexaenoic acid (DHA), are excellent substrates for peroxidation and, therefore, drive ferroptosis, whereas monounsaturated fatty acids (MUFAs), such as oleic acid, block ferroptosis through an unknown mechanism¹. We used both Raman microscopy and confocal fluorescence microscopy to explore the subcellular distributions of these compounds (Fig. 1). The distribution of ferroptosis-modulating fatty acids helped to identify which membranes are likely to be key targets of lipid peroxidation. By targeting FINO₂ to different subcellular membranes, we evaluated whether peroxidation of certain membranes is a prerequisite for initiating ferroptosis. Finally, we used a fluorescent fatty acid analog that detects lipid peroxides (BODIPY 581/591 C11) to capture the evolution of lipid peroxidation in different cellular membranes over time.
在这个项目中，我们试图阐明铁死亡中脂质过氧化的亚细胞动力学。我们通过探索不同铁死亡调节化合物的亚细胞分布来解决这个问题：使细胞容易受到铁死亡的影响或防止铁死亡的脂肪酸，以及 FINO ₂ ，​​一种 1,2-二氧戊环，含有诱导铁死亡的内过氧化物部分² 。多不饱和脂肪酸 (PUFA)，例如花生四烯酸和二十二碳六烯酸 (DHA)，是过氧化的极好底物，因此会导致铁死亡，而单不饱和脂肪酸 (MUFA)，例如油酸，则通过未知机制阻止铁死亡¹ 。我们使用拉曼显微镜和共焦荧光显微镜来探索这些化合物的亚细胞分布（图1 ）。铁死亡调节脂肪酸的分布有助于确定哪些膜可能是脂质过氧化的关键目标。通过将 FINO ₂靶向不同的亚细胞膜，我们评估了某些膜的过氧化是否是启动铁死亡的先决条件。最后，我们使用检测脂质过氧化物的荧光脂肪酸类似物 (BODIPY 581/591 C11) 来捕获不同细胞膜中脂质过氧化随时间的演变。

Fluorescence microscopy imaging (left) and stimulated Raman scattering (SRS) imaging (right) of cells treated with deuterated (in which hydrogen has been replaced by deuterium) DHA (DHA-d₁₀) show a DHA-d₁₀ distribution that is consistent with ER localization. Treatment with diglyceride acyltransferase (DGAT) inhibitors results in elimination of lipid droplets. DMSO, dimethyl sulfoxide. © 2023, von Krusenstiern, A. N. et al.
用氘化（其中氢已被氘取代）DHA (DHA- d ₁₀ ) 处理的细胞的荧光显微镜成像（左）和受激拉曼散射 (SRS) 成像（右）显示 DHA- d ₁₀分布与急诊室本地化。用甘油二酯酰基转移酶 (DGAT) 抑制剂治疗可消除脂滴。 DMSO，二甲基亚砜。 © 2023，von Krusenstiern, AN 等人。

Full size image  全尺寸图像

Our findings converged on common key subcellular sites. We observed both PUFAs and MUFAs primarily accumulating in the ER, with some incorporation in the plasma membrane and a small amount in the mitochondria. We found that modulating the fatty acid content of these sites by addition of MUFAs or PUFAs to the medium influenced cell sensitivity to ferroptosis. These results pointed to the ER and plasma membrane as essential sites of peroxidation. We then explored the distribution of direct peroxide-forming ferroptosis inducer FINO₂ and found that it was targeted to the ER. However, re-localizing FINO₂-like compounds to mitochondria or lysosomes also induced ferroptosis, and we were surprised to discover that ferroptosis could be induced by FINO₂ in any of these membranes. Finally, we observed that for all four canonical classes of ferroptosis inducers (that is, inhibitors of the cystine/glutamate transporter, which deplete glutathione, GPX4 (phospholipid hydroperoxide glutathione peroxidase) inhibitors and degraders, which block repair of lipid peroxides, and endoperoxides, which directly trigger lipid peroxidation), ER peroxidation occurred before plasma membrane or mitochondrial peroxidation. As mitochondria are not required for all types of ferroptosis^3,4, these findings again point to the ER followed by the plasma membrane as key sites of lipid peroxidation that drive ferroptotic cell death.
我们的研究结果集中在共同的关键亚细胞位点上。我们观察到 PUFA 和 MUFA 主要积累在 ER 中，部分掺入质膜中，少量掺入线粒体中。我们发现通过向培养基中添加 MUFA 或 PUFA 来调节这些位点的脂肪酸含量会影响细胞对铁死亡的敏感性。这些结果表明内质网和质膜是过氧化的重要位点。然后，我们探索了直接过氧化物形成铁死亡诱导剂 FINO ₂的分布，发现它针对 ER。然而，将 FINO ₂样化合物重新定位到线粒体或溶酶体也会诱导铁死亡，我们惊讶地发现 FINO ₂在任何这些膜中都可以诱导铁死亡。最后，我们观察到，对于所有四种经典类别的铁死亡诱导剂（即消耗谷胱甘肽的胱氨酸/谷氨酸转运蛋白抑制剂、GPX4（磷脂氢过氧化物谷胱甘肽过氧化物酶）抑制剂和降解剂（阻止脂质过氧化物和内过氧化物的修复），直接引发脂质过氧化），内质网过氧化发生在质膜或线粒体过氧化之前。由于并非所有类型的铁死亡都需要线粒体^{3 , 4} ，这些发现再次指出内质网和质膜是驱动铁死亡细胞死亡的脂质过氧化的关键位点。

The two important findings in this work are that lipid peroxidation can be initiated in several different organelles and results in ferroptosis, and that the ER is an essential early site of lipid peroxidation for ferroptosis-inducing compounds. These findings indicate that the ER should be considered a crucial target for compounds that block ferroptosis, and that dysregulation of lipid content and peroxidation in the ER is likely to be an important determinant of pathological cell death in disease⁵.
这项工作的两个重要发现是，脂质过氧化可以在几种不同的细胞器中启动并导致铁死亡，并且内质网是诱导铁死亡化合物的脂质过氧化的重要早期位点。这些发现表明，内质网应被视为阻止铁死亡的化合物的重要靶点，并且内质网中脂质含量的失调和过氧化可能是疾病中病理性细胞死亡的重要决定因素⁵ 。

Although we were able to visualize compounds at a subcellular level, we were limited by the tools and technology that were available. Selectively modulating the size and lipid content of these organelles individually as well as more sophisticated tools for measuring live lipid peroxidation would enable a deeper understanding of these mechanisms. Ultimately, some membranes, such as those of the ER and plasma membrane, are difficult or impossible to eliminate, and require alternative techniques to evaluate their involvement.
尽管我们能够在亚细胞水平上可视化化合物，但我们受到可用工具和技术的限制。选择性地单独调节这些细胞器的大小和脂质含量以及用于测量活脂质过氧化的更复杂的工具将使人们能够更深入地了解这些机制。最终，一些膜，例如内质网和质膜的膜，很难或不可能消除，需要替代技术来评估它们的参与情况。

One question that emerges from this work is how lipid peroxidation spreads from membrane to membrane within cells, or whether it even spreads at all. It will be interesting to determine whether each membrane is oxidized independently or whether there is a role for lipid trafficking in the spread of oxidation.
这项工作提出的一个问题是脂质过氧化如何在细胞内从一个膜传播到另一个膜，或者它是否真的扩散。确定每个膜是否独立氧化，或者脂质运输是否在氧化扩散中发挥作用，将会很有趣。

A. Nikolai von Krusenstiern & Brent R. Stockwell
A. 尼古拉·冯·克鲁森斯蒂恩 & 布伦特·斯托克韦尔

Columbia University, New York, NY, USA
哥伦比亚大学，纽约，纽约州，美国

“von Krusenstiern and colleagues use conventional fluorescence and Raman scattering imaging to understand which cellular membranes are peroxidized during ferroptosis. They conclude that the ER is the primary site of lipid peroxidation and the plasma membrane is a secondary site; mitochondrial membranes can also be sites of ferroptosis, but protecting the ER from lipid peroxidation is sufficient to block mitochondria-initiated ferroptosis. This study is of high quality and will be important to the field.” An anonymous reviewer.
“von Krusenstiern 及其同事使用传统的荧光和拉曼散射成像来了解哪些细胞膜在铁死亡过程中被过氧化。他们的结论是，内质网是脂质过氧化的主要部位，质膜是次要部位；线粒体膜也可能是铁死亡的部位，但保护内质网免受脂质过氧化作用足以阻止线粒体引发的铁死亡。这项研究质量很高，对该领域很重要。”一位匿名审稿人。

The questions of where lipid peroxidation occurs in ferroptosis and which membranes are required have been longstanding in the field. This work arose out of two different collaborations that led all our groups in the same direction. With Dr. Wei Min’s group, we had been exploring the distribution of fatty acids throughout different cell membranes. With Dr. Keith Woerpel’s group, we redistributed FINO₂ to different cellular locales to determine whether ferroptosis could still be induced. These projects came together in a meeting in which we realized that all our work pointed to the ER as a central site of lipid peroxidation. Once combined, these projects morphed into a two-pronged approach that enabled us to build a more convincing case. We were also excited to show the utility of evaluating the structure–activity–distribution relationship of multiple chemical probes using two different imaging techniques. B.R.S.
铁死亡中脂质过氧化发生在何处以及需要哪种膜的问题在该领域长期以来一直存在。这项工作源于两次不同的合作，这两次合作引导我们所有的团队朝着同一个方向发展。我们与魏敏博士的团队一起，一直在探索脂肪酸在不同细胞膜上的分布。我们与 Keith Woerpel 博士的团队一起，将 FINO ₂重新分配到不同的细胞区域，以确定是否仍然可以诱导铁死亡。这些项目在一次会议上汇集在一起​​，我们意识到我们所有的工作都指向内质网作为脂质过氧化的中心部位。一旦合并，这些项目就变成了双管齐下的方法，使我们能够建立一个更有说服力的案例。我们还很高兴展示使用两种不同成像技术评估多种化学探针的结构-活性-分布关系的实用性。 BRS

“This work by von Krusenstiern et al. stands out because the smart use of imaging methods to visualize the lipid and ferroptosis modulators enables the monitoring of the lipid flow and the identification of subcellular sites essential for ferroptosis induction. These findings promote the mechanistic understanding of ferroptosis and provide clues for future drug development targeting this biological process.” Editorial Team, Nature Chemical Biology.
“von Krusenstiern 等人的这项工作。之所以脱颖而出，是因为巧妙地使用成像方法来可视化脂质和铁死亡调节剂，从而能够监测脂质流并识别铁死亡诱导所必需的亚细胞位点。这些发现促进了对铁死亡机制的理解，并为未来针对这一生物过程的药物开发提供了线索。”编辑团队， 《自然化学生物学》。