Lrrk2 R1628P转基因小鼠尿液外泌体诱导帕金森病样病理变化

Study on Parkinson′s disease-like pathology induced by Lrrk2 R1628P transgenic mouse′s urine exosomes

目的:探讨Lrrk2 R1628P转基因小鼠尿液外泌体能否诱导帕金森病(PD)样病理改变。方法:提取野生型(WT)和Lrrk2 R1628P转基因小鼠尿液外泌体,使用免疫印迹法检测尿液外泌体标记物TSG101和CD81,Y216位点磷酸化糖原合成酶激酶-3β(pGSK-3β-216)表达水平。在C57小鼠纹状体中分别注射WT小鼠(C57-WT组,n=8)和Lrrk2 R1628P小鼠尿液外泌体(C57-Lrrk2组,n=8)。注射前3天,注射后1、3、6个月,利用平衡木、挂线、转棒和旷场实验对小鼠进行行为学分析,免疫组化法分析脑组织中磷酸化α-突触核蛋白(pα-syn)的沉积情况,免疫印迹法检测脑组织中pα-syn的表达。结果:Lrrk2 R1628P小鼠尿液外泌体TSG101和CD81表达水平与WT小鼠比较,差异无统计学意义(P>0.05),pGSK-3β-216表达水平高于WT小鼠(P<0.001)。C57-Lrrk2组小鼠出现PD样行为学表型(P<0.001),皮层、纹状体和黑质中有pα-syn沉积,脑组织中pα-syn表达水平为(0.44±0.06),C57-WT组则无表达(P<0.001)。结论:小鼠纹状体中注射Lrrk2 R1628P转基因小鼠的尿液外泌体能够诱导PD样病理改变。

Aim:To observe the Parkinson′s disease(PD)-like pathology induced by injection of Lrrk2 R1628P transgenic mouse urine exosomes into the striatum.Methods:Urine exosomes were extracted from wild type(WT)and Lrrk2 R1628P mice,and the expressions of urine exosome markers TSG101 and CD81,and phosphorylated glycogen synthetase kinase-3βat site Y216(pGSK-3β-216)were detected by Western blot.The urine exosomes of WT mice(C57-WT group)and Lrrk2 R1628P mice(C57-Lrrk2 group)were injected into the striatum of C57 mice(8 in each group),and the behavioral function was analyzed by beam working test,hanging-wire grip test,rotarod test and open field test before injection,and 1 month,3 months,6 months after injection.The deposition of phosphorylatedα-syn(pα-syn)in the brain was analyzed by immunohistochemical staining,and the expression level of pα-syn in the brain was evaluated by Western blot.Results:Compared with WT mice,the expressions of TSG101 and CD81 in urine exosomes of Lrrk2 R1628P mice were not significantly different(P>0.05),but the expression of pGSK-3β-216 was higher(P<0.001).In C57-Lrrk2 group,PD-like behavioral phenotype was observed(P<0.001),pα-syn was deposited in the cortex,striatum and substantia nigra,and the expression of pα-syn in the brain was(0.44±0.06),while none in C57-WT group(P<0.001).Conclusion:PD-like pathology can be induced by injecting urinary exosomes of Lrrk2 R1628P transgenic mice into the striatum.