动力蛋白重链对帕金森病模型细胞α-突触核蛋白自噬性降解的调控机制

Regulating mechanism of dynein heavy chain on autophagic degradation ofα-synuclein in Parkinson disease model cells

目的探讨干扰或过表达动力蛋白重链(dynein heavy chain,Dynhc)基因后,帕金森病(Parkinson disease,PD)模型细胞中α-突触核蛋白的自噬溶酶体途径(autophagy-lysosome pathway,ALP)降解机制。方法按实验要求将SH-SY5Y细胞分为对照组、PD组、Dynhc干扰组、Dynhc过表达组、Dynhc干扰+雷帕霉素组。用Western blot检测细胞中Dynhc、α-突触核蛋白、微管相关蛋白1轻链3(microtubule-associated protein l light chain 3,LC3)、溶酶体相关膜蛋白2(lysosome-associated membrane protein 2,LAMP2)、微管蛋白、动力蛋白激活蛋白p150、驱动蛋白KIF5B的蛋白表达水平;流式细胞仪检测细胞凋亡水平;免疫共聚焦镜观察微管蛋白结构以及LC3与LAMP的共定位情况。采用SPSS 23.0软件进行统计分析,多组间比较采用单因素方差分析,进一步两两比较采用LSD-t检验。结果5组细胞α-突触核蛋白、自噬标记蛋白、微管及微管相关蛋白表达均差异有统计学意义(均P<0.001)。PD组Dynhc、α-突触核蛋白、LC3、LAMP2、p150、KIF5B的蛋白表达水平均高于对照组(均P<0.05);Dynhc干扰组Dynhc、LAMP2、微管蛋白、p150的蛋白表达水平均低于PD组(均P<0.05),α-突触核蛋白、LC3、KIF5B的蛋白表达水平均高于PD组(均P<0.05);Dynhc过表达组α-突触核蛋白、LC3、KIF5B的蛋白表达水平均低于PD组(均P<0.05),Dynhc、LAMP2、p150的蛋白表达水平高于PD组(均P<0.05);Dynhc干扰+雷帕霉素组LC3的蛋白表达水平高于Dynhc干扰组(P<0.05),Dynhc、α-突触核蛋白、LAMP2、微管蛋白、p150、KIF5B的蛋白表达水平与Dynhc干扰组比较差异无统计学意义(均P>0.05)。与对照组比,PD组细胞凋亡水平提高[(12.77±1.66)%,(7.64±1.45)%],微管形态不变,自噬体与溶酶体融合较多;与PD组比,Dynhc干扰组细胞凋亡水平[(18.45±1.91)%]提高,微管形态稀疏,自噬体与溶酶体融合较少;与PD组比,Dynhc过表达组细胞凋亡水平[(9.95±1.56)%]降低,微管结构无明显改变,自噬体与溶酶体融合较多;与Dynhc干扰组比,Dynhc干扰+雷帕霉素组细胞凋亡水平[(19.05±2.46)%]、微管形态、自噬体与溶酶体融合无明显改变。结论Dynhc可以提高细胞自噬溶酶体途径降解功能,并通过增加α-突触核蛋白降解以及维持微管结构完整使细胞凋亡减少。

Objective To explore the regulatory mechanism ofα-synuclein in the degradation of autophagy-lysosome pathway(ALP)in Parkinson disease(PD)model cells after interference or overexpression of dynein heavy chain(Dynhc)gene.Methods SH-SY5Y cells were divided into control group,PD group,Dynhc interference group,Dynhc overexpression group,and Dynhc interference+rapamycin group according to experimental requirements.Using Western blot to detect Dynhc,α-synuclein,microtubule-associated protein l light chain 3(LC3),lysosome-associated membrane protein 2(LAMP2),tubulin,dynein activator protein p150,and kinesin KIF5B.Flow cytometry was used to detect the level of cell apoptosis.Immunoconfocal microscopy was used to observe the structure of tubulin and the co-localization of LC3 and LAMP.SPSS 23.0 software was used for statistical analysis.One-way ANOVA was used for inter group comparisons,and further pairwise comparisons were conducted by LSD-t test.Results There were statistically significant differences in the expression ofα-synuclein,autophagy-related proteins,microtubules,and microtubule-related proteins among cells in the 5 groups(all P<0.001).The protein expression levels of Dynhc,α-synuclein,LC3,LAMP2,p150,and KIF5B in the PD group were higher than those in the control group(all P<0.05).The protein levels of Dynhc,LAMP2,tubulin and p150 in the Dynhc interference group were lower than those in the PD group(all P<0.05),while the protein levels ofα-synuclein,LC3 and KIF5B were higher than those in the PD group(all P<0.05).The protein levels ofα-synuclein,LC3,and KIF5B in the Dynhc overexpression group were lower than those in the PD group(all P<0.05),while the protein levels of Dynhc,LAMP2 and p150 were higher than those in the PD group(all P<0.05).The protein level of LC3 in the Dynhc interference+rapamycin group was higher than that in the Dynhc interference group(P<0.05).There were no statistically significant differences in the protein levels of Dynhc,α-synuclein,LAMP2,microtubule protein,p150 and KIF5B compared to the Dynhc interference group(all P>0.05).Compared with the control group,the cell apoptosis rate in PD group increased((12.77±1.66)%,(7.64±1.45)%),the microtubule morphology remained unchanged,and autophagosomes fused more with lysosomes.Compared with the PD group,the cell apoptosis rate of Dynhc overexpression group decreased,and there was no significant change in microtubule structure,and there was more fusion between autophagosomes and lysosomes.Compared with the PD group,the cell apoptosis rat of Dynhc interference group increased((18.45±1.91)%),and the microtubule morphology was sparse,and there was less fusion between autophagosomes and lysosomes.Compared with the PD group,the Dynhc overexpression group showed a decrease in cell apoptosis rate((9.95±1.56)%),no significant changes in microtubule structure,and more fusion between autophagosomes and lysosomes.Compared with the Dynhc interference group,the Dynhc interference+rapamycin group showed no significant changes in cell apoptosis rate((19.05±2.46)%),microtubule morphology,and fusion of autophagosomes and lysosomes.Conclusion Dynhc can reduce cell apoptosis by enhancing cell ALP function,increasing the degradation ofα-synuclein and maintaining of microtubule structure integrity.