中国神经再生研究(英文版)

中国神经再生研究(英文版) ›› 2023, Vol. 18 ›› Issue (7): 1548-1552.doi: 10.4103/1673-5374.360246

• 原著:脑损伤修复保护与再生 • 上一篇    下一篇

抑制自噬可减轻爱拉斯汀诱导的HT22海马神经元铁死亡

  

  • 出版日期:2023-07-15 发布日期:2023-01-12
  • 基金资助:
    梅塞尔基金会研究基金

Inhibition of autophagy rescues HT22 hippocampal neurons from erastin-induced ferroptosis

Nora Hanke, Abdelhaq Rami*   

  1. Institut für Experimentelle Neurobiologie (Anatomie II), Klinikum der Johann Wolfgang von Goethe-Universität, Theodor-Stern-Kai 7, 60590 Frankfurt/Main, Germany
  • Online:2023-07-15 Published:2023-01-12
  • Contact: Abdelhaq Rami, PhD, Rami@em.uni-frankfurt.de.
  • Supported by:
    This work was supported in part by a research grant from the Messer Stiftung, No. 8571013 (to AR).

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摘要：

铁自噬参与了铁死亡，核受体辅助因子4的基因缺失抑制了铁自噬，并通过降低细胞内生物可利用铁的水平阻止了脂质过氧化和铁死亡。虽然新证据表明，谷胱甘肽过氧化酶4的抑制是许多癌细胞系中铁死亡的一个标志，但这一生化途径与神经元死亡关系仍不清楚。在此，实验旨在（1）了解铁死亡的关键因素是否参与了爱拉斯汀诱导的神经元细胞死亡的过程；（2）分析铁蛋白酶与自噬之间是否存在交叉作用；（3）观察神经元对铁死亡诱导剂爱拉斯汀的反应，特别关注铁蛋白和核受体辅助因子4介导的铁自噬。实验以爱拉斯汀（0.5-8 µM）处理海马HT22神经元16h。此外，在爱拉斯汀处理前，用自噬抑制剂3-甲基腺苷（10mM）和/或铁死亡抑制剂ferrostatin 1（10-20μM）或去铁胺（100-200μM）培养细胞。免疫荧光和Western blot检测结果显示，爱拉斯汀明显降低了谷胱甘肽过氧化酶4、钠依赖性胱氨酸-谷氨酸抗器（x-CT）和核受体辅助因子4的表达。在爱拉斯汀处理后，HT22海马神经元中的铁蛋白和线粒体铁蛋白（mtFT）的蛋白水平没有明显变化。此外，结果还显示，不仅铁死亡抑制剂（ferrostatin1/deferoxamine）能抑制海马HT22神经元中由爱拉斯汀诱导的铁死亡，而且还能消除有效的自噬抑制剂（3-MA）的作用。结论：（1）尽管核受体辅助因子4水平降低，但爱拉斯汀诱导了海马HT22神经元的铁死亡；（2）核受体辅助因子4介导的铁自噬没有发生；（3）铁死亡似乎与自噬性细胞死亡过程的某些特征相同。

https://orcid.org/0000-0002-4989-2690 (Abdelhaq Rami)

Abstract: Ferroptosis is a regulated form of cell death which is considered an oxidative iron-dependent process. The lipid hydroperoxidase glutathione peroxidase 4 prevents the iron (Fe2+)-dependent formation of toxic lipid reactive oxygen species. While emerging evidence indicates that inhibition of glutathione peroxidase 4 as a hallmark of ferroptosis in many cancer cell lines, the involvement of this biochemical pathway in neuronal death remains largely unclear. Here, we investigate, first whether the ferroptosis key players are involved in the neuronal cell death induced by erastin. The second objective was to examine whether there is a cross talk between ferroptosis and autophagy. The third main was to address neuron response to erastin, with a special focus on ferritin and nuclear receptor coactivator 4-mediated ferritinophagy. To test this in neurons, erastin (0.5–8 µM) was applied to hippocampal HT22 neurons for 16 hours. In addition, cells were cultured with the autophagy inhibitor, 3-methyladenin (10 mM) and/or ferroptosis inhibitors, ferrostatin 1 (10–20 µM) or deferoxamine (10–200 µM) before exposure to erastin. In this study, we demonstrated by immunofluorescence and western blot analysis, that erastin downregulates dramatically the expression of glutathione peroxidase 4, the sodium-independent cystine-glutamate antiporter and nuclear receptor coactivator 4. The protein levels of ferritin and mitochondrial ferritin in HT22 hippocampal neurons did not remarkably change following erastin treatment. In addition, we demonstrated that not only the ferroptosis inhibitor, ferrostatin1/deferoxamine abrogated the ferroptotic cell death induced by erastin in hippocampal HT22 neurons, but also the potent autophagy inhibitor, 3-methyladenin. We conclude that (1) erastin-induced ferroptosis in hippocampal HT22 neurons, despite reduced nuclear receptor coactivator 4 levels, (2) that either nuclear receptor coactivator 4-mediated ferritinophagy does not occur or is of secondary importance in this model, (3) that ferroptosis seems to share some features of the autophagic cell death process.

Key words: erastin, ferritin, ferritinophagy, ferroptosis, glutathione peroxidase 4, HT22 neurons, nuclear receptor coactivator 4