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TFEB activator 1增强小胶质细胞中β淀粉样蛋白寡聚体的自噬降解

解雨琪¹, 朱俐¹, 王雪婷^1,*

¹南通大学特种医学研究院，南通 226019

摘要

本文旨在研究TFEB activator 1 (TA1)改善小胶质细胞自噬降解β淀粉样蛋白寡聚体(oligomeric amyloid-β, oAβ)的机制，探讨TA1对于阿尔茨海默病(Alzheimer’s disease, AD)体外小胶质细胞模型的治疗效果。在体外培养的原代小胶质细胞中，分别给予oAβ (1 μmol/L)处理0、3、12、24 h，或以oAβ (1 μmol/L)和TA1 (1 μmol/L)联合处理细胞12 h后，巴弗洛霉素A1 (100 nmol/L)继续处理细胞1 h。采用荧光示踪法检测小胶质细胞内吞或降解oAβ1-42的水平；采用mCherry-EGFP-LC3的慢病毒转染原代小胶质细胞，检测自噬通量的变化；利用免疫荧光法检测oAβ1-42与溶酶体相关膜蛋白1 (lysosome-associated membrane protein 1, LAMP1)或微管相关蛋白轻链3 (microtuble-associated protein light chain 3, LC3)的共定位、TFEB核表达及自噬小体数量的变化；采用qRT-PCR法检测自噬基因Lamp1、Atg5、Map1lc3b的表达变化。结果显示：长时程oAβ暴露后，小胶质细胞内吞与降解oAβ的能力明显下降，细胞内自噬小体数量及自噬通量降低，自噬调节因子TFEB的核表达降低，自噬基因表达降低，引发oAβ的自噬降解受损；给予上述细胞TA1治疗后，可观察到TFEB的核表达明显上调，细胞内自噬基因表达上调，自噬通量恢复，小胶质细胞内吞与降解oAβ的能力得到明显恢复。因此，TA1可以通过上调小胶质细胞TFEB介导的自噬，改善AD小胶质细胞清除oAβ的能力，提示TA1可作为治疗AD的潜在药物。

关键词： TFEB activator 1; β淀粉样蛋白寡聚体; 小胶质细胞; 自噬

TFEB activator 1 enhances autophagic degradation of oligomeric amyloid-β in microglia

XIE Yu-Qi¹, ZHU Li¹, WANG Xue-Ting^1,*

¹Institute of Special Environmental Medicine, Nantong University, Nantong 226019, China

Abstract

The purpose of the study was to investigate the mechanism of TFEB activator 1 (TA1) improving the autophagic degradation of oligomeric amyloid-β (oAβ) in microglia, and to explore the therapeutic effect of TA1 on an in vitro model of microglia in Alzheimer’s disease (AD). Primary microglia were exposed to 1 μmol/L oAβ for 0, 3, 12, and 24 h respectively to construct the in vitro model of microglia in AD. In order to explore the therapeutic effect of TA1, primary microglia were co-treated with 1 μmol/L oAβ and 1 μmol/L TA1 for 12 h. To determine the autophagy flux, the above cells were further treated with 100 nmol/L Bafilomycin A1 for 1 h before fixation. Fluorescent probes were used to detect the endocytosis or degradation of oAβ1-42 by microglia. The autophagic flux was determined by infection of lentivirus mCherry-EGFP-LC3. The nuclear TFEB intensity, the autophagosomes number, and the colocalization ratio of oAβ1-42 with lysosome-associated membrane protein 1 (LAMP1) or microtubule-associated protein light chain 3 (LC3), were detected by immunofluorescence assay. Expressions of autophagy-related-genes, including Lamp1, Atg5, and Map1lc3b, were detected by qRT-PCR. Results showed that prolonged oAβ exposure inhibited the endocytosis and degradation of oAβ by microglia. Meanwhile, the number of autophagosomes and autophagy flux in microglia decreased after 12 h of oAβ treatment. We further found that the nuclear expression of autophagy regulator TFEB decreased after 12 h of oAβ exposure, resulting in the decrease of autophagy genes, thus leading to the damage of autophagic degradation of oAβ. Therefore, long-term oAβ exposure was considered to construct the in vitro model of microglia in AD. After TA1 treatment, the nuclear expression of TFEB in cells was obviously upregulated. TA1 treatment upregulated the expressions of autophagy-related genes, leading to the recovery of autophagy flux. TA1 also recovered the endocytosis and degradation of oAβ by microglia. In conclusion, TA1 could improve oAβ clearance by microglia in AD by upregulating microglial TFEB-mediated autophagy, suggesting TA1 as a potential therapeutic drug for AD.

Key words: TFEB activator 1; oligomeric amyloid-β; microglia; autophagy

收稿日期：　　录用日期：

通讯作者：王雪婷　　E-mail:

DOI: 10.13294/j.aps.2024.0029

引用本文：

解雨琪, 朱俐, 王雪婷. TFEB activator 1增强小胶质细胞中β淀粉样蛋白寡聚体的自噬降解[J]. 生理学报 2024; 76 (3): 365-375.

XIE Yu-Qi, ZHU Li, WANG Xue-Ting. TFEB activator 1 enhances autophagic degradation of oligomeric amyloid-β in microglia. Acta Physiol Sin 2024; 76 (3): 365-375 (in Chinese with English abstract).