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GABRA3突变通过抑制TGF-β1/SMAD2信号通路调控神经元的凋亡、突触形成和递质释放
作者:付伦姣  罗成宏  杨媚  陆伟恒  廖成钜 
单位:东莞市松山湖中心医院 神经内科, 广东 东莞 523326
关键词:GABRA3突变 甲亢性低钾周期性麻痹 TGF-β1/SMAD2信号通路 人脑皮层神经元 突触形成 递质释放 
分类号:R338
出版年·卷·期(页码):2022·41·第一期(1-10)
摘要:

目的:探讨钙通道α3亚基基因(GABRA3)突变对人脑皮层神经元细胞凋亡、突触形成和递质释放的影响及其机制。方法:构建GABRA3基因野生型(wt-GABRA3组)和突变型(mut-GABRA3组)过表达的重组载体并将其转染到人脑皮层神经元中构建相应稳转细胞株。在机制研究中,使用转化生长因子(TGF)-β1/信号转导蛋白Sma-Mad族2(SMAD2)信号通路激活剂SRI-011381处理mut-GABRA3组细胞24 h (mut-GABRA3+SRI-011381组)。使用四唑化合物(MTS)测定法检测细胞恶性增殖能力。通过TUNEL和流式细胞术检测细胞凋亡能力。采用免疫荧光法,通过检测突触蛋白I的表达统计突触密度变化。蛋白质印迹法检测半胱氨酸天冬氨酸蛋白酶3(caspase3)、Bax、TGF-β1、SMAD2、磷酸化的SMAD2(P-SMAD2)的表达。ELISA法检测神经递质乙酰胆碱的水平。结果:与wt-GABRA3组比,mut-GABRA3组细胞增殖率降低,细胞凋亡率上调,差异均有统计学意义(均P<0.05);与wt-GABRA3组比,mut-GABRA3组caspase3和Bax的表达上调,TGF-β1、P-SMAD2的表达以及乙酰胆碱的分泌水平都下调,细胞突触密度也降低,差异均有统计学意义(均P<0.05)。与mut-GABRA3组比,mut-GABRA3+SRI-011381组细胞凋亡率、caspase3和Bax的表达均下调,而且乙酰胆碱的分泌水平和细胞突触密度都上调,差异均有统计学意义(均P<0.05)。结论:GABRA3突变通过抑制TGF-β1/SMAD2信号通路促进人脑皮层神经元的凋亡,并抑制突触形成和递质释放。

Objective: To investigate the effect and mechanism of the mutation of calcium channel α3 subunit gene(GABRA3)on apoptosis, synaptic formation, and transmitter release of human cortical neurons. Methods: The overexpressed recombinant vectors of GABRA3 in wild type(wt-GABRA3 group) and overexpressed mutant type(mut-GABRA3 group) were constructed and further stably transfected into human cortical neurons. In the mechanism study, the mut-GABRA3 cells were treated for 24 h with SRI-011381, an activator of transforming growth factor-β1(TGF)-β1/single transduction protein Sma-Mad family 2(SMAD2) signaling pathway(MUT-GABRA3+SRI-011381 group). The malignant proliferation of the cells was detected by MTS method. Apoptosis was detected by TUNEL and flow cytometry. Changes in synaptic density were measured by immunofluorescence by detecting Synapsin I expression. Western blot was used to detect the levels of caspase3, Bax, TGF-β1, SMAD2, and phosphorylated SMAD2(P-smad2). The levels of the neurotransmitter acetylcholine were measured by ELISA. Results: Compared with wt-GABRA3 group, the proliferation rate was decreased and the apoptosis rate was significantly up-regulated in mut-GABRA3 group(P<0.05). Compared with wt-GABRA3 group, the levels of caspase3 and Bax were up-regulated, the levels of TGF-β1, P-SMAD2, and acetylcholine were down-regulated, and the cell synaptic density was significantly decreased in mut-GABRA3 group(P<0.05). Compared with mut-GABRA3 group, the cell apoptosis rate, the levels of caspase3 and Bax were down-regulated in mut-GABRA3+SRI-011381 group, and the levels of acetylcholine and synaptic density were significantly up-regulated(P<0.05). Conclusion: GABRA3 mutation promotes the apoptosis of human cortical neurons and inhibits synaptic formation and transmitter release by inhibiting TGF-β1/SMAD2 signaling pathway.

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