自噬相关基因4a在超氧化物歧化酶1- G93A突变型肌萎缩侧索硬化症转基因鼠大脑皮层和纹状体的表达
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摘要
目的检测自噬相关基因(Atg)4a在不同时期超氧化物歧化酶(SOD)1-G93A突变型肌萎缩侧索硬化症(ALS)转基因鼠大脑皮层和纹状体中表达以探究Atg4a与ALS发病的关系。方法 SOD1-G93A突变型ALS转基因鼠和同窝野生型WT鼠于95 d(发病早期)、108 d(中期)和122 d(晚期)分别取材,运用定量实时聚合酶链反应(qRT-PCR)检测大脑皮层和纹状体中Atg4a mRNA水平,运用Western印迹技术检测蛋白水平,运用免疫荧光双标技术进行形态学检测。结果与WT鼠相比,ALS鼠大脑皮层中Atg4a mRNA和蛋白在发病95 d和108 d表达上调,122 d下调,而纹状体中Atg4a mRNA在95 d、108 d和122 d均上调,Atg4a蛋白则在95 d、108 d表达上调,122 d下调,差异均有统计学意义(P<0.05)。Atg4a在大脑皮层和纹状体中与神经元共表达。108 d,与WT鼠相比,ALS鼠大脑皮层和纹状体中Atg4a免疫反应性均显著增强(P<0.05)。结论 Atg4a在大脑皮层和纹状体中的异常表达与SOD1-G93A突变型ALS转基因鼠发病关系密切。
Objective To explore the relationship between Atg4 a and ALS by detecting the expression of Atg4 a in the cerebral cortex and striatum of SOD1-G93 A mutant amyotrophic lateral sclerosis(ALS) transgenic mice in different periods.Methods SOD1-G93 A mutant ALS transgenic mice and littermate wild-type mice were killed at 95 d(the onset of early stage), 108 d(middle stage) and 122 d(late stage), qRT-PCR technique was used to detect Atg4 a mRNA level of cerebral cortex and striatum, Western blot to detect the change of the protein level and double immunofluorescence labeled technique to detect the change of morphology. Results The levels of Atg4 a mRNA and protein increased in the early and middle stage but decreased in the late stage in the cerebral cortex of ALS mice compared with wide-type mice. The levels of Atg4 a mRNA increased in the striatum of ALS mice in the early, middle and late stage, however, the levels of Atg4 a protein increased in the early and middle stage but decreased in the late stage. It was found that Atg4 a was expressed with neurons in the cerebral cortex and striatum by double immunofluorescence staining in the middle stage. Compared with wild-type mice, the Atg4 a immunoreactivities in the cerebral cortex and striatum of ALS mice were enhanced.Conclusions The abnormal expression of Atg4 a is closely related to the pathogenesis of SOD1-G93 A mutant ALS transgenic mice.
Objective To explore the relationship between Atg4 a and ALS by detecting the expression of Atg4 a in the cerebral cortex and striatum of SOD1-G93 A mutant amyotrophic lateral sclerosis(ALS) transgenic mice in different periods.Methods SOD1-G93 A mutant ALS transgenic mice and littermate wild-type mice were killed at 95 d(the onset of early stage), 108 d(middle stage) and 122 d(late stage), qRT-PCR technique was used to detect Atg4 a mRNA level of cerebral cortex and striatum, Western blot to detect the change of the protein level and double immunofluorescence labeled technique to detect the change of morphology. Results The levels of Atg4 a mRNA and protein increased in the early and middle stage but decreased in the late stage in the cerebral cortex of ALS mice compared with wide-type mice. The levels of Atg4 a mRNA increased in the striatum of ALS mice in the early, middle and late stage, however, the levels of Atg4 a protein increased in the early and middle stage but decreased in the late stage. It was found that Atg4 a was expressed with neurons in the cerebral cortex and striatum by double immunofluorescence staining in the middle stage. Compared with wild-type mice, the Atg4 a immunoreactivities in the cerebral cortex and striatum of ALS mice were enhanced.Conclusions The abnormal expression of Atg4 a is closely related to the pathogenesis of SOD1-G93 A mutant ALS transgenic mice.
引文
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2 Morello G,Spampinato AG,Cavallaro S.Neuroinflammation and ALS:transcriptomic insights into molecular disease mechanisms and therapeutic targets[J].Mediators Inflamm,2017;2017:7070469.
3 Lei H,Dirren E,Poitry-Yamate C,et al.Evolution of the neurochemical profiles in the G93A-SOD1 mouse model of amyotrophic lateral sclerosis[J].J Cereb Blood Flow Metab,2018;2018:271678X18756499.
4 Chen Y,Liu H,Guan Y,et al.The altered autophagy mediated by TFEB in animal and cell Models of amyotrophic lateral sclerosis[J].Am J Transl Res,2015;7(9):1574-87.
5 周风华,刘焕彩,陈燕春,等.自噬相关基因Atg4B及LC3-Ⅱ在肌萎缩侧索硬化症转基因鼠海马中的表达及意义[J].中国老年学杂志,2015;35(12):3216-8.
6 刘永新,管英俊,陈燕春,等.肌萎缩侧索硬化症转基因鼠脊髓中自噬相关基因ATG5表达降低[J].中国组织化学与细胞化学杂志,2016;25(2):107-11.
7 Maruyama T,Noda NN.Autophagy-regulating protease Atg4:structure,function,regulation and inhibition[J].J Antibiot (Tokyo),2018;71(1):72-8.
8 Liu PF,Tsai KL,Hsu CJ,et al.Drug repurposing screening identifies tioconazole as an ATG4 inhibitor that suppresses autophagy and sensitizes cancer cells to chemotherapy[J].Theranostics,2018;8(3):830-45.
9 Yang SW,Ping YF,Jiang YX,et al.Atg4a promotes tumor metastasis by inducing the epithelial-mesenchymal transition and stem-like properties in gastric cells[J].Oncotarget,2016;7(26):39279-92.
10 Chen G,Zhou B,Zhu H,et al.White matter volume loss in amyotrophic lateral sclerosis:a meta-analysis of voxel-based morphometry studies[J].Prog Neuropsychopharmacol Biol Psychiatry,2018;83:110-7.
11 Sepers MD,Smith-Dijak A,Le Due J,et al.Endocannabinoid-specific impairment in synaptic plasticity in striatum of Huntington′s disease mouse model[J].J Neurosci,2018;38(3):544-54.
12 王炳蔚,杨晓宁,张辰雨,等.纹状体神经通路与运动调控[J].生理科学进展,2016;47(4):241-8.
13 Proenca CC,Stoehr N,Bernhard M,et al.Atg4b-dependent autophagic flux alleviates Huntington′s disease progression[J].PLoS One,2013;8(7):e68357.
14 Vidoni C,Secomandi E,Castiglioni A,et al.Resveratrol protects neuronal-like cells expressing mutant Huntingtin from dopamine toxicity by rescuing ATG4-mediated autophagos-ome formation[J].Neurochem Int,2018;117:174-87.