Department of Neurology (Xia Liu, Z.H., X. Li, L.Z., Xuan Liu, L.R.), The First Affiliated Hospital of Shenzhen University, Shenzhen Second People's Hospital, China.
Department of Neurology, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, China (Xia Liu, Y.-C.W.).
Stroke. 2023 Aug;54(8):2114-2125. doi: 10.1161/STROKEAHA.123.041783. Epub 2023 Jun 28.
The ubiquitin-proteasome system (UPS) and autophagy are 2 major protein degradation pathways in eukaryotic cells. We previously identified a switch from UPS to autophagy with changes in BAG3 (B-cell lymphoma 2-associated-athanogene 3) expression after cerebral ischemia in mice. BAG3 is an antiapoptotic-cochaperone that is directly involved in cellular protein quality control as a mediator for selective macroautophagy. Here, we aimed to investigate the role of BAG3 in ischemic stroke.
Middle cerebral artery occlusion/reperfusion (MCAO/R) and oxygen-glucose deprivation/reoxygenation were used to mimic cerebral ischemia in vivo and in vitro. The UPS inhibitor MG132 and autophagy inhibitor 3-MA (3-methyladenine) were administered to mice to identify how BAG3 was involved after MCAO/R. Adeno-associated virus and lentiviral vector were used to regulate BAG3 expression in vivo and in vitro, respectively. Behavioral tests, 2,3,5-triphenyltetrazolium chloride staining, and Hematoxylin & Eosin staining were performed to evaluate cerebral injury following MCAO/R, and a Cell Counting kit-8 assay was conducted to assess oxygen-glucose deprivation/reoxygenation-induced injury in cells. Brain tissues and cell lysates were collected and analyzed for UPS activation, autophagy, and apoptosis.
The UPS inhibitor alleviated MCAO injury in mice and increased autophagy and BAG3 expression, whereas the autophagy inhibitor exacerbated MCAO/R-induced injury. In addition, BAG3 overexpression significantly improved neurological outcomes, reduced infarct volume in vivo, and enhanced cell survival by activating autophagy and suppressing apoptosis in vitro.
Our findings indicate that BAG3 overexpression activates autophagy and inhibits apoptosis to prevent cerebral ischemia/reperfusion and hypoxia/reoxygenation injury, suggesting a potential therapeutic benefit of BAG3 expression in cerebral ischemia.
泛素-蛋白酶体系统（UPS）和自噬是真核细胞中两种主要的蛋白质降解途径。我们之前在小鼠脑缺血后发现 BAG3（B 细胞淋巴瘤 2 相关抗凋亡基因 3）表达的变化会导致 UPS 向自噬的转变。BAG3 是一种抗凋亡共伴侣，作为选择性巨自噬的介导物，直接参与细胞蛋白质质量控制。在这里，我们旨在研究 BAG3 在缺血性中风中的作用。
使用大脑中动脉闭塞/再灌注（MCAO/R）和氧葡萄糖剥夺/再氧合来模拟体内和体外的脑缺血。给予 UPS 抑制剂 MG132 和自噬抑制剂 3-MA（3-甲基腺嘌呤）以确定 MCAO/R 后 BAG3 是如何参与的。腺相关病毒和慢病毒载体分别用于体内和体外调节 BAG3 的表达。进行行为测试、2,3,5-三苯基四氮唑氯化物染色和苏木精和伊红染色，以评估 MCAO/R 后的脑损伤，并进行细胞计数试剂盒-8 测定以评估氧葡萄糖剥夺/再氧合诱导的细胞损伤。收集和分析脑组织和细胞裂解物，以评估 UPS 激活、自噬和细胞凋亡。
UPS 抑制剂减轻了小鼠的 MCAO 损伤并增加了自噬和 BAG3 的表达，而自噬抑制剂则加剧了 MCAO/R 诱导的损伤。此外，BAG3 的过表达显著改善了神经功能结局，减少了体内梗死体积，并通过在体外激活自噬和抑制细胞凋亡来增强细胞存活。
我们的研究结果表明，BAG3 的过表达激活自噬并抑制细胞凋亡，以防止脑缺血/再灌注和缺氧/再氧合损伤，这表明 BAG3 表达在脑缺血中具有潜在的治疗益处。
