TMEM230-A110T突变体对多巴胺能神经细胞自噬、增殖及凋亡的影响  

Effects of TMEM230-A110T mutant on autophagy,proliferation and apoptosis of dopaminergic neurons

作　　者：杨百元[1] 李柯麓 刘彬[2] 朱永云 殷康福 尹蔚芳 任惠[2] 杨兴隆 YANG Baiyuan;LI Kelu;LIU Bin;ZHU Yongyun;YIN Kangfu;YIN Weifang;REN Hui;YANG Xinglong(不详;Department of Geriatric Neurology,The First Affiliated Hospital of Kunming Medical University,Kunming Yunnan 650032,China)

机构地区：[1]成都市第七人民医院神经内科,610041 [2]昆明医科大学第一附属医院老年神经内科,650032

出　　处：《中国神经免疫学和神经病学杂志》2022年第1期45-51,共7页Chinese Journal of Neuroimmunology and Neurology

基　　金：国家自然科学基金地区基金(81960242);昆明医科大学-云南省科技厅联合专项(2019FE001-048);四川省应用基础项目(2020YJ0484);云南省应用基础项目(202001AT070001)。

摘　　要：目的探讨跨膜蛋白230(TMEM230)的突变体TMEM230-A110T对多巴胺能(DA)神经细胞增殖、凋亡和自噬的影响。方法采用全反式维甲酸(RA)/脑源性神经营养因子(BDNF)序贯分化法构建DA能神经细胞模型,并随机分为WT组(野生型对照组)、MUT组(转染TMEM230-A110T突变体载体)、SP600125组(给予JNK通路抑制剂SP600125处理)、SB203580组(给予p38 MAPK通路抑制剂SB203580处理)、si-Beclin-1组(转染si-Becline-1)和SP600125+pcDNA-Beclin-1组(给予SP600125干预+转染pcDNA-Beclin-1)组(n=3)。采用Western blotting检测各组细胞TMEM230、JNK/p38MAPK/Beclin-1途径、凋亡和自噬相关蛋白的表达。采用CCK-8和Annexin V-FITC/PI试剂盒分别检测细胞模型的增殖和凋亡。采用透射电镜观察细胞模型中的自噬小体。GFP-LC3试剂盒监测细胞模型中的LC3斑点。结果成功构建DA能神经细胞模型。转染TMEM230-A110T突变体显著降低DA能神经细胞增殖,并增加其凋亡和自噬水平(P<0.05)。TMEM230-A110T突变体能够激活JNK/p38 MAPK通路并上调Beclin-1的表达(P<0.05)。阻断JNK/p38 MAPK/Beclin-1途径显著增加DA能神经细胞增殖,并下调凋亡和自噬水平,而过表达Beclin-1能够逆转这个过程(P<0.05)。结论TMEM230-A110T突变体可通过激活JNK/p38 MAPK/Beclin-1途径促进DA能神经细胞自噬,从而抑制其增殖并诱导其凋亡。Objective To explore the effect of the mutant of transmembrane protein 230(TMEM230),TMEM230-A110T on the proliferation,apoptosis and autophagy of dopamine(DA)nerve cells.Methods We constructed a DA nerve cell model by all trans retinoic acid(RA)/brain-derived neurotrophic factor(BDNF),and the cells were randomly divided into a WT(wild-type control)group,a MUT(transfection of TMEM230-A110T mutant vector)group,an SP600125(treatment with JNK pathway inhibitor SP600125)group,an SB203580(treatment with p38 MAPK pathway inhibitor SB203580)group,an si-Beclin-1(transfection with si-Becline-1)group,and an SP600125+pcDNA-Beclin-1(transfection with pcDNA-Beclin-1)group(n=3).Western blotting was performed to detect TMEM230,JNK/p38MAPK/Beclin-1 pathway-related proteins,apoptosis-related proteins and autophagy-related proteins expression of the cells in each group.The proliferation and apoptosis of the model cells were detected by CCK-8 and Annexin V-FITC/PI Kit,respectively.Transmission electron microscope was used to observe autophagosomes in the cells.The GFP-LC3 kit monitored the LC3 spots in the cells.Results DA nerve cell model was successfully constructed.Transfected TMEM230-A110T mutant significantly reduced the proliferation of DA nerve cells,and increased their apoptosis and autophagy(P<0.05).The TMEM230-A110T mutant activated JNK/p38 MAPK pathway and up-regulated Beclin-1 expression(P<0.05).Blocking the JNK/p38 MAPK/Beclin-1 pathway significantly increased the proliferation,and down-regulate the apoptosis and autophagy of the DA nerve cells.However,overexpression of Beclin-1 could reversed this process(P<0.05).Conclusions TMEM230-A110T mutant can promote the autophagy of DA nerve cells by activating the JNK/p38 MAPK/Beclin-1 pathway,thereby inhibiting their proliferation and inducing their apoptosis.

关 键 词：帕金森病 多巴胺 神经元 TMEM230-A110T突变体 MAP激酶信号系统 JNK丝裂原活化蛋白激酶类 细胞凋亡 自噬 

分 类 号：R742.5[医药卫生—神经病学与精神病学]