As the demand and application of engineered nanomaterials have increased, their potential toxicity to the central nervous system has drawn increasing attention. Tunneling nanotubes (TNTs) are novel cell–cell communication that plays a crucial role in pathology and physiology. However, the relationship between TNTs and nanomaterials neurotoxicity remains unclear. Here, three types of commonly used engineered nanomaterials, namely cobalt nanoparticles (CoNPs), titanium dioxide nanoparticles (TiO2NPs), and multi-walled carbon nanotubes (MWCNTs), were selected to address this limitation. After the complete characterization of the nanomaterials, the induction of TNTs formation with all of the nanomaterials was observed using high-content screening system and confocal microscopy in both primary astrocytes and U251 cells. It was further revealed that TNT formation protected against nanomaterial-induced neurotoxicity due to cell apoptosis and disrupted ATP production. We then determined the mechanism underlying the protective role of TNTs. Since oxidative stress is a common mechanism in nanotoxicity, we first observed a significant increase in total and mitochondrial reactive oxygen species (namely ROS, mtROS), causing mitochondrial damage. Moreover, pretreatment of U251 cells with either the ROS scavenger N-acetylcysteine or the mtROS scavenger mitoquinone attenuated nanomaterial-induced neurotoxicity and TNTs generation, suggesting a central role of ROS in nanomaterials-induced TNTs formation. Furthermore, a vigorous downstream pathway of ROS, the PI3K/AKT/mTOR pathway, was found to be actively involved in nanomaterials-promoted TNTs development, which was abolished by LY294002, Perifosine and Rapamycin, inhibitors of PI3K, AKT, and mTOR, respectively. Finally, western blot analysis demonstrated that ROS and mtROS scavengers suppressed the PI3K/AKT/mTOR pathway, which abrogated TNTs formation. Despite their biophysical properties, various types of nanomaterials promote TNTs formation and mitochondrial transfer, preventing cell apoptosis and disrupting ATP production induced by nanomaterials. ROS/mtROS and the activation of the downstream PI3K/AKT/mTOR pathway are common mechanisms to regulate TNTs formation and mitochondrial transfer. Our study reveals that engineered nanomaterials share the same molecular mechanism of TNTs formation and intercellular mitochondrial transfer, and the proposed adverse outcome pathway contributes to a better understanding of the intercellular protection mechanism against nanomaterials-induced neurotoxicity.

中文翻译：

---

ROS/mtROS 通过 PI3K/AKT/mTOR 途径促进 TNT 形成，以防止工程纳米材料诱导的神经胶质细胞线粒体损伤

随着工程纳米材料的需求和应用的增加，它们对中枢神经系统的潜在毒性引起了越来越多的关注。隧道纳米管（TNT）是一种新型的细胞间通讯方式，在病理学和生理学中发挥着至关重要的作用。然而，TNT 与纳米材料神经毒性之间的关系仍不清楚。这里，选择了三种常用的工程纳米材料，即钴纳米颗粒（CoNP）、二氧化钛纳米颗粒（TiO2NP）和多壁碳纳米管（MWCNT）来解决这一限制。在对纳米材料进行完整表征后，使用高内涵筛选系统和共聚焦显微镜在原代星形胶质细胞和 U251 细胞中观察到所有纳米材料诱导 TNT 形成的情况。进一步表明，TNT 的形成可以防止纳米材料因细胞凋亡和破坏 ATP 产生而引起的神经毒性。然后我们确定了 TNT 保护作用的机制。由于氧化应激是纳米毒性的常见机制，我们首先观察到总活性氧和线粒体活性氧（即ROS、mtROS）显着增加，导致线粒体损伤。此外，用ROS清除剂N-乙酰半胱氨酸或mtROS清除剂米托醌预处理U251细胞可减弱纳米材料诱导的神经毒性和TNT的生成，表明ROS在纳米材料诱导的TNT形成中发挥核心作用。此外，还发现 ROS 的一条活跃的下游途径，即 PI3K/AKT/mTOR 途径，积极参与纳米材料促进的 TNT 发育，该途径分别被 PI3K、AKT 和 mTOR 抑制剂 LY294002、Perifosine 和 Rapamycin 消除。最后，蛋白质印迹分析表明 ROS 和 mtROS 清除剂抑制 PI3K/AKT/mTOR 通路，从而消除 TNT 的形成。尽管具有生物物理特性，各种类型的纳米材料仍可促进 TNT 形成和线粒体转移，防止细胞凋亡并破坏纳米材料诱导的 ATP 产生。ROS/mtROS 和下游 PI3K/AKT/mTOR 通路的激活是调节 TNT 形成和线粒体转移的常见机制。我们的研究表明，工程纳米材料具有相同的 TNT 形成和细胞间线粒体转移的分子机制，并且所提出的不良结果途径有助于更好地理解针对纳米材料诱导的神经毒性的细胞间保护机制。