LRRK2G2019S位点突变通过抑制自噬相关蛋白诱发驱铁处理后小胶质细胞的活化  

LRRK2G2019S mutation induced microglia activation after iron removal by inhibiting autophagy associated proteins

作　　者：刘政 郑子键 刘昕杰 薛成 吴潇 张欣然 李建蔚 卢立轩 卢国辉[1] Liu Zheng;Zheng Zijian;Liu Xinjie;Xue Cheng;Wu Xiao;Zhang Xinran;Li Jianwei;Lu Lixuan;Lu Guohui(Department of Neurosurgery,First Affiliated Hospital of Nanchang University,Nanchang 330006,China;First Clinical Medical School,Nanchang University,Nanchang 330006,China)

机构地区：[1]南昌大学第一附属医院神经外科,南昌330006 [2]南昌大学第一临床医学院,南昌330006

出　　处：《中华神经医学杂志》2023年第11期1098-1110,共13页Chinese Journal of Neuromedicine

基　　金：国家自然科学基金(82060249);江西省主要学科学术和技术带头人培养计划青年人才项目(20204BCJ23019)。

摘　　要：目的探讨LRRK2G2019S位点突变对驱铁处理后小胶质细胞活化的影响及其机制。方法(1)利用人类诱导多能干细胞(IPSC)经造血祖细胞(HPC)分化产生小胶质细胞,免疫荧光染色进行鉴定,利用蛋白纯化技术获取α-突触核蛋白(α-syn)A53T突变蛋白。(2)将小胶质细胞分为对照组、α-syn组、α-syn+甲磺酸去铁胺(DFO)组,分别加入磷酸盐缓冲液(PBS)、1μmol/L纯化的α-syn A53T突变蛋白、1μmol/L纯化的α-syn A53T突变蛋白+30 mmol/L DFO作用24 h。采用比色法检测细胞Fe2+浓度,Western blotting实验检测细胞Rab35蛋白的表达,流式细胞仪检测细胞内活性氧(ROS)水平,实时定量聚合酶链式反应(RT-PCR)检测细胞白细胞介素(IL)-6、肿瘤坏死因子α(TNF-α)、转化生长因子β(TGF-β)mRNA的表达。将3组小胶质细胞培养上清液(MCS)转移到SH-SY5Y细胞中,采用流式细胞仪检测SH-SY5Y细胞的凋亡情况。(3)双向DNA测序检测1μmol/L纯化的α-syn A53T突变蛋白处理后小胶质细胞富亮氨酸重复激酶2(LRRK2)基因的突变。将小胶质细胞分为对照组、α-syn组、α-syn+GSK3357679A组,分别加入对应的药物处理24 h(LRRK2抑制剂GSK3357679A浓度为10 nmol/L),采用Western blotting实验检测细胞LRRK2蛋白的表达。将小胶质细胞分为对照组、α-syn组、α-syn+GSK3357679A、α-syn+GSK3357679A+DFO组,分别加入对应的药物处理24 h后采用Western blotting实验检测细胞Rab35蛋白的表达,流式细胞仪检测细胞内ROS水平,RT-PCR检测细胞IL-6、TNF-a、TGF-βmRNA的表达。(4)将小胶质细胞分为对照组、α-syn组、α-syn+雷帕霉素(RAPA)组,分别加入对应的药物处理24 h(自噬诱导剂RAPA的浓度为50 nmol/L),采用Western blotting实验检测细胞Rab35、P62、微管相关蛋白轻链3Ⅱ(LC3Ⅱ)蛋白的表达,流式细胞仪检测细胞内ROS水平,RT-PCR检测细胞IL-6、TNF-α、TGF-βmRNA的表达。(5)将小胶质细胞分为对照组、α-syn组、α-syn+Rab35组,分别加入对�Objective To investigate the effect of LRRK2G2019S mutation on activation of microglia after iron deprivation and its mechanism.Methods(1)Microglia were differentiated from human induced pluripotent stem cells(IPSC)with the help of hematopoietic progenitor cells(HPC)and identified by immunofluorescent staining,andα-synuclein(α-syn)A53T mutant protein was obtained by protein purification technology.(2)Microglia were divided into control group,α-syn group,α-syn+deferoxamine(DFO)group;phosphate buffer solution(PBS),1μmol/L purifiedα-syn A53T mutant protein,1μmol/L purifiedα-syn A53T mutant protein+30 mmol/L DFO were given respectively for 24 h.Fe2+concentration was detected by colorimetry,Rab35 protein expression was detected by Western blotting,intracellular reactive oxygen species(ROS)level was detected by flow cytometry,and interleukin-6(IL-6),tumor necrosis factor-α(TNF-α)and transforming growth factor-β(TGF-β)mRNA expressions were detected by real time-PCR(RT-PCR);microglia culture supernatant(MCS)in the 3 groups were transfered to SH-SY5Y cells,and SH-SY5Y cell apoptosis was detected by flow cytometry.(3)Bidirectional DNA sequencing was used to detect leucine rich repeat kinase 2(LRRK2)gene mutations in microglia treated with 1μmol/L purifiedα-syn A53T mutant protein.Microglia were divided into control group,α-syn group andα-syn+GSK3357679A group,and treated with corresponding drugs for 24 h,respectively(LRRK2 inhibitor GSK3357679A concentration:10 nmol/L),and LRRK2 protein expression was detected by Western blotting;microglia were divided into control group,α-syn group,α-syn+GSK3357679A,andα-syn+GSK3357679A+DFO group,and treated with corresponding drugs for 24 h,Rab35 protein expression was detected by Western blotting,intracellular ROS level was detected by flow cytometry,and IL-6,TNF-αand TGF-βmRNA expressions were detected by RT-PCR.(4)Microglia were divided into control group,α-syn group,α-syn+rapamycin(RAPA)group,and treated with corresponding drugs for 24 h(concentration of autoph

关 键 词：小胶质细胞 神经炎症 驱铁处理 富亮氨酸重复激酶2 Rab35 

分 类 号：R741[医药卫生—神经病学与精神病学]