Background

Alzheimer's disease (AD) is a complex, multifactorial neurodegenerative disease. However, the pathogenesis remains unclear. Recently, an increasing number of studies have demonstrated that ferroptosis is a new type of iron-dependent programmed cell death, contributes to the death of nerve cells in AD. By controlling iron homeostasis and mitochondrial function, the particular protein called frataxin (FXN), which is situated in the mitochondrial matrix, is a critical regulator of ferroptosis disease. It is encoded by the nuclear gene FXN. Here, we identified a novel underlying mechanism through which ferroptosis mediated by FXN contributes to AD.

Methods

Human neuroblastoma cells (SH-SY5Y) were injured by L-glutamate (L-Glu). Overexpression of FXN by lentiviral transfection. In each experimental group, we assessed the ultrastructure of the mitochondria, the presence of iron and intracellular Fe2 + , the levels of reactive oxygen species, the mitochondrial membrane potential (MMP), and lipid peroxidation. Quantification was done for malondialdehyde (MDA) and reduced glutathione (GSH), as well as reactive oxygen species (ROS). Western blot and cellular immunofluorescence assays were used to detect the expression of xCT and GPX4 proteins which in System Xc-/GPX4 pathway, and the protein expressions of ACSL4 and TfR1 were investigated by Western blot.

Results

The present work showed: (1) The expression of FXN was reduced in the L-Glu group; (2) Compared with the Control group, MMP was reduced in the L-Glu group, and mitochondria were observed to shrink and cristae were deformed, reduced or disappeared by transmission electron microscopy, and after FXN overexpression and ferrostatin-1 (Fer-1) (10 μmol/L) intervened, MMP was increased and mitochondrial morphology was significantly improved, suggesting that mitochondrial function was impaired in the L-Glu group, and overexpression of FXN could improve the manifestation of mitochondrial function impairment. (3) In the L-Glu group, ROS, MDA, iron ion concentration and Fe²⁺ levels were increased, GSH was decreased. Elevated expression of ACSL4 and TfR1, important regulatory proteins of ferroptosis, was detected by Western blot, and the expression of xCT and GPX4 in the System Xc-/GPX4 pathway was reduced by Western blot and cellular immunofluorescence. However, the above results were reversed when FXN overexpression and Fer-1 intervened.

Conclusion

To conclude, our research demonstrates that an elevated expression of FXN effectively demonstrates a robust neuroprotective effect against oxidative damage induced by L-Glu. Moreover, it mitigates mitochondrial dysfunction and lipid metabolic dysregulation associated with ferroptosis. FXN overexpression holds promise in potential therapeutic strategies for AD by inhibiting ferroptosis in nerve cells and fostering their protection.

中文翻译：

---

FXN过表达对L-Glu诱导的SH-SY5Y细胞铁死亡的保护作用

背景

阿尔茨海默病（AD）是一种复杂的、多因素的神经退行性疾病。然而，发病机制仍不清楚。近年来，越来越多的研究表明铁死亡是一种新型的铁依赖性程序性细胞死亡，导致AD神经细胞死亡。通过控制铁稳态和线粒体功能，位于线粒体基质中的称为 frataxin (FXN) 的特殊蛋白质是铁死亡疾病的关键调节因子。它由核基因 FXN 编码。在这里，我们发现了 FXN 介导的铁死亡促进 AD 的一种新的潜在机制。

方法

人神经母细胞瘤细胞 (SH-SY5Y) 被 L-谷氨酸 (L-Glu) 损伤。通过慢病毒转染过度表达 FXN。在每个实验组中，我们评估了线粒体的超微结构、铁和细胞内 Fe2+ 的存在、活性氧水平、线粒体膜电位 (MMP) 和脂质过氧化。对丙二醛(MDA) 和还原型谷胱甘肽(GSH) 以及活性氧 (ROS)进行了定量。采用Western blot和细胞免疫荧光法检测System Xc-/GPX4通路中xCT和GPX4蛋白的表达，并采用Western blot检测ACSL4和TfR1蛋白的表达。

结果

目前的工作表明：（1）L-Glu组FXN表达降低；(2)与对照组相比，L-Glu组MMP减少，透射电镜观察线粒体收缩，嵴变形、减少或消失，过表达FXN和ferrostatin-1(Fer-1)后，L-Glu组MMP减少，线粒体收缩，嵴变形、减少或消失。 )(10 μmol/L)干预后，MMP增加，线粒体形态明显改善，提示L-Glu组线粒体功能受损，过表达FXN可改善线粒体功能受损的表现。(3)L-Glu组ROS、MDA、铁离子浓度和Fe ²⁺水平升高，GSH降低。Western blot检测发现铁死亡的重要调节蛋白ACSL4和TfR1表达升高，Western blot和细胞免疫荧光检测System Xc-/GPX4途径中xCT和GPX4的表达降低。然而，当 FXN 过表达和 Fer-1 干预时，上述结果发生了逆转。

结论

总之，我们的研究表明，FXN 表达的升高有效地证明了对 L-Glu 诱导的氧化损伤具有强大的神经保护作用。此外，它还能减轻与铁死亡相关的线粒体功能障碍和脂质代谢失调。FXN 过度表达通过抑制神经细胞中的铁死亡并促进其保护，为 AD 的潜在治疗策略带来了希望。