Li Ping, Luo Xue, Luo Zhen, He Gen-Lin, Shen Ting-Ting, Yu Xue-Ting, Wang Ze-Ze, Tan Yu-Long, Liu Xiao-Qian, Yang Xue-Sen
Department of Tropical Medicine, Army Medical University, Chongqing, China.
Key Laboratory of Extreme Environmental Medicine, Ministry of Education of China, Army Medical University, Chongqing, China.
Front Cell Neurosci. 2022 May 11;16:865568. doi: 10.3389/fncel.2022.865568. eCollection 2022.
Heat stroke is the outcome of excessive heat stress, which results in core temperatures exceeding 40°C accompanied by a series of complications. The brain is particularly vulnerable to damage from heat stress. In our previous studies, both activated microglia and increased neuronal autophagy were found in the cortices of mice with heat stroke. However, whether activated microglia can accelerate neuronal autophagy under heat stress conditions is still unknown. In this study, we aimed to investigate the underlying mechanism that caused neuronal autophagy upregulation in heat stroke from the perspective of exosome-mediated intercellular communication.
In this study, BV2 and N2a cells were used instead of microglia and neurons, respectively. Exosomes were extracted from BV2 culture supernatants by ultracentrifugation and then characterized transmission electron microscopy, nanoparticle tracking analysis and Western blotting. N2a cells pretreated with/without miR-155 inhibitor were cocultured with microglial exosomes that were treated with/without heat stress or miR-155 overexpression and subsequently subjected to heat stress treatment. Autophagy in N2a cells was assessed by detecting autophagosomes and autophagy-related proteins through transmission electron microscopy, immunofluorescence, and Western blotting. The expression of miR-155 in BV2 and BV2 exosomes and N2a cells was measured using real-time reverse transcription polymerase chain reaction. Target binding analysis was verified a dual-luciferase reporter assay.
N2a autophagy moderately increased in response to heat stress and accelerated by BV2 cells through transferring exosomes to neurons. Furthermore, we found that neuronal autophagy was positively correlated with the content of miR-155 in microglial exosomes. Inhibition of miR-155 partly abolished autophagy in N2a cells, which was increased by coculture with miR-155-upregulated exosomes. Mechanistic analysis confirmed that Rheb is a functional target of miR-155 and that microglial exosomal miR-155 accelerated heat stress-induced neuronal autophagy mainly by regulating the Rheb-mTOR signaling pathway.
Increased miR-155 in microglial exosomes after heat stroke can induce neuronal autophagy their transfer into neurons. miR-155 exerted these effects by targeting Rheb, thus inhibiting the activity of mTOR signaling. Therefore, miR-155 could be a promising target for interventions of neuronal autophagy after heat stroke.
中暑是过度热应激的结果，会导致核心体温超过40°C并伴有一系列并发症。大脑特别容易受到热应激的损害。在我们之前的研究中，在中暑小鼠的皮层中发现了活化的小胶质细胞和神经元自噬增加。然而，在热应激条件下活化的小胶质细胞是否能加速神经元自噬仍不清楚。在本研究中，我们旨在从外泌体介导的细胞间通讯角度探讨中暑时神经元自噬上调背后的机制。
在本研究中，分别使用BV2和N2a细胞代替小胶质细胞和神经元。通过超速离心从BV2培养上清液中提取外泌体，然后通过透射电子显微镜、纳米颗粒跟踪分析和蛋白质印迹对其进行表征。将用/不用miR-155抑制剂预处理的N2a细胞与经/未经热应激或miR-155过表达处理的小胶质细胞外泌体共培养，随后进行热应激处理。通过透射电子显微镜、免疫荧光和蛋白质印迹检测自噬体和自噬相关蛋白来评估N2a细胞中的自噬。使用实时逆转录聚合酶链反应测量BV2、BV2外泌体和N2a细胞中miR-155的表达。通过双荧光素酶报告基因测定验证靶标结合分析。
N2a自噬在热应激下适度增加，并通过BV2细胞将外泌体转移到神经元而加速。此外，我们发现神经元自噬与小胶质细胞外泌体中miR-155的含量呈正相关。抑制miR-155部分消除了N2a细胞中的自噬，与miR-155上调的外泌体共培养可增加自噬。机制分析证实Rheb是miR-155的功能靶标，小胶质细胞外泌体miR-155主要通过调节Rheb-mTOR信号通路加速热应激诱导的神经元自噬。
中暑后小胶质细胞外泌体中miR-155增加可通过将其转移到神经元中诱导神经元自噬。miR-155通过靶向Rheb发挥这些作用，从而抑制mTOR信号的活性。因此，miR-155可能是中暑后神经元自噬干预的一个有前景的靶点。
