Guo, Y., Wang, Y., Ni, Y., Bo, B., He, J., Zhu, Y., Qin, A., Zhou, X., Du, H., Liu, Y. and Wang, T., 2025. Iron Overload Mediates the Differential Cell Fate of Astrocytes from Neurons and Its Regulatory Mechanisms in Ischemic Stroke. Advanced Science, p.e07384.

Iron Overload Mediates the Differential Cell Fate of Astrocytes from Neurons and Its Regulatory Mechanisms in Ischemic Stroke

Abstract
Iron accumulation and ferroptosis occur in the brain following ischemic stroke. However, the relationship between iron overload and cell type-specific fates remains largely unclear. Here, iron deposition and neuronal loss are reported within the perilesional cortex of three patients with ischemic stroke at both acute and subacute stages. It is identified that ischemia/reperfusion-induced iron overload triggers ferroptosis predominantly in neurons and to a lesser extent in astrocytes, whereas most astrocytes undergo reactive proliferation. Mechanistically, the reduced or elevated Nrf2/GPX4 and SLC7A11 levels in neurons or astrocytes, respectively, account for these distinct iron overload-induced cellular fates. Moreover, iron overload promotes astrogliosis by enhancing the transcriptional activities of several proliferation-related genes. Using mice with partial knockout of the transferrin receptor 1 (TfR1) gene Tfrc, astrocyte-specific Tfrc knockdown, and conditional astrocytic Cpt1a partial knockout (to induce fatty acid metabolism disorders), it is revealed that increased TfR1 palmitoylation and clathrin-mediated endocytosis drive astrocytic iron overload. Notably, ischemia/reperfusion-induced elevation of palmitic acid is associated with enhanced TfR1 palmitoylation. Treatment with antioxidants or iron chelators mitigates ischemic brain injury. Together, these findings provide a comprehensive framework linking ischemia/reperfusion-induced iron overload to cell type-specific fates. TfR1 palmitoylation emerges as a potential target for ischemic stroke therapy.

铁过载介导缺血性脑卒中星形胶质细胞与神经元的差异性细胞命运及其调控机制

摘要
缺血性脑卒中后，大脑会出现铁蓄积与 ferroptosis（ ferroptosis，即铁死亡，是一种铁依赖性的、区别于凋亡、坏死等传统细胞死亡方式的新型调控性细胞死亡形式）。然而，铁过载与细胞类型特异性命运之间的关系在很大程度上仍不明确。本研究发现，在 3 例处于急性期和亚急性期的缺血性脑卒中患者的病灶周围皮层内，均存在铁沉积与神经元丢失现象。研究证实，缺血 / 再灌注诱导的铁过载主要引发神经元发生铁死亡，对星形胶质细胞的铁死亡诱导作用较弱，而大多数星形胶质细胞则会发生反应性增殖。
从机制上看，神经元中 Nrf2/GPX4（Nrf2，即核因子 E2 相关因子 2，是重要的抗氧化应激转录因子；GPX4，即谷胱甘肽过氧化物酶 4，是铁死亡关键调控酶）和 SLC7A11（即溶质载体家族 7 成员 11，是负责转运胱氨酸的关键蛋白，对维持细胞内谷胱甘肽水平至关重要）水平降低，而星形胶质细胞中上述因子水平升高，这正是铁过载导致两类细胞出现不同命运的原因。此外，铁过载通过增强多个增殖相关基因的转录活性，促进星形胶质细胞增生（astrogliosis）。
研究人员利用转铁蛋白受体 1（TfR1，负责介导细胞对转铁蛋白结合铁的摄取）基因 Tfrc 部分敲除小鼠、星形胶质细胞特异性 Tfrc 敲低模型以及星形胶质细胞条件性肉碱棕榈酰转移酶 1A（Cpt1a，是脂肪酸 β- 氧化的关键限速酶）部分敲除模型（用于诱导脂肪酸代谢紊乱），发现 TfR1 棕榈酰化水平升高以及网格蛋白介导的内吞作用（clathrin-mediated endocytosis，一种重要的细胞内吞方式，负责特定分子的摄取）共同驱动了星形胶质细胞的铁过载。值得注意的是，缺血 / 再灌注诱导的棕榈酸水平升高与 TfR1 棕榈酰化增强相关。
此外，抗氧化剂或铁螯合剂治疗可减轻缺血性脑损伤。综上，这些研究结果构建了一个完整的机制框架，揭示了缺血 / 再灌注诱导的铁过载与细胞类型特异性命运之间的关联，同时表明 TfR1 棕榈酰化有望成为缺血性脑卒中治疗的潜在靶点。

实验方法部分：

Palmitoylation of TfR1 Assays—Click Chemistry Reaction (CCR) – Based Method
Initially, cells were metabolically labeled with a chemical palmitic acid probe, alkylene palmitic acid (Alkyl-C16; HY-W040304, MedChemExpress, USA) for 24 h.
For immunofluorescence analysis, the cells were fixed in 4% paraformaldehyde in PBS for 10 min, and then subjected to click chemistry using Click-iT Cell Reaction Buffer Kit (C10276, Invitrogen, USA) to conjugate Alexa Fluor 594-azide (A10270, Invitrogen, USA) to Alkyl-C16 for visualizing the palmitoylated proteins. Subsequently, cells were subjected to immunostaining to label TfR1, Na+-K+-ATPase, and Hoechst. Eventually, the images were captured by a confocal microscope (LSM 710, Carl Zeiss Co. Ltd., Germany, 63 × oil immersion objective, pixel sizes: 2048 × 2048).
For Western blots, cells were lysed and then divided into two groups. One group was subjected to click chemistry using Click-iT Protein Reaction Buffer Kit (Thermo Fisher Scientific, C10276) to conjugate Alkyl-C16 to Azide magnetic bead (PuriMag G Series, PuriMag Biotech, China) to pull down all palmitoylated protein, and then was probed with an anti-TfR1 antibody to identify the palmitoylated TfR1. Another one was directly detected for total TfR1 protein level by Western blots without CCR. Finally, the data were expressed as palmitoylated TfR1/total TfR1 to assess the level of palmitoylated TfR1.

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