【作者】 张旭；

【导师】 李林；

【作者基本信息】 首都医科大学 ， 药理学， 2017， 硕士

【摘要】 目的α-突触核蛋白(a-synuclein,α-syn)在多种神经退行性疾病的发病机制中起着重要作用,这些疾病包括帕金森病、路易小体痴呆、阿尔茨海默病、多系统萎缩等,又统称为突触核蛋白病。聚集的α-Syn是路易小体的重要组成部分,而且突变的α-syn或多倍体α-syn可导致家族性帕金森病。但目前这些疾病都缺乏有效的治疗药物。二苯乙烯苷(2,3,5,4’-tetrahydroxy-stilbene glucoside,TSG)是中药何首乌的主要有效成分和标志成分。我们的前期研究发现TSG能够抑制APP转基因阿尔茨海默病小鼠模型海马和大脑皮层α-syn过表达和聚集,抑制老年小鼠纹状体和海马α-syn过表达和聚集。本研究的目的是在A53T突变型α-syn转基因小鼠模型和α-syn基因转染神经细胞模型上,进一步研究TSG对α-syn过表达和聚集的影响及其在翻译后修饰和蛋白降解方面的作用机制。方法1.应用6月龄A53T突变型α-syn转基因小鼠,灌胃给予TSG 6个月或9个月。应用爬杆试验和筑巢试验检测小鼠的运动能力,Y迷宫试验检测小鼠的记忆能力;应用免疫组化方法观察小鼠脑内酪氨酸羟化酶(TH)标记的DA能神经元、Iba-1标记的小胶质细胞;用Western blot法检测小鼠脑中α-syn单体和寡聚体、磷酸化a-syn、Parkin、Beclin1、Atg、LC3的表达。2.采用α-syn基因转染的SH-SY5Y细胞,与TSG孵育24 h后,换含小剂量鱼藤酮的培养液继续孵育24 h。应用CCK8检测细胞活性;免疫荧光双标法检测α-syn的表达;Western blot法检测α-syn、磷酸化α-syn、磷酸化PP2A的表达。结果1.12月龄和15月龄a53tα-syn转基因小鼠在爬杆试验中的潜伏期延长,筑巢行为评分减低,y迷宫自发性交替反应减低;tsg灌胃给药6个月或9个月能够明显缩短12月龄和15月龄模型小鼠在爬杆试验中的潜伏期,增高筑巢行为评分和y迷宫自发性交替反应,表明tsg能够改善α-syn转基因小鼠的运动功能和记忆能力。2.15月龄a53tα-syn转基因模型组小鼠黑质致密部th标记的da能神经元数量减少,在黑质和纹状体部位iba-1标记的小胶质细胞数量增高。tsg灌胃给药9个月能够增加模型小鼠黑质致密部da能神经元数量,减少黑质和纹状体部位小胶质细胞数量,表明tsg具有神经保护作用。3.15月龄a53tα-syn转基因小鼠大脑皮层α-syn单体和寡聚体的表达增高;tsg能够减低模型小鼠大脑皮层α-syn单体和寡聚体的表达水平,表明tsg对α-syn转基因小鼠脑内α-syn的过表达和聚集有抑制作用。4.在作用机制方面,我们发现15月龄a53tα-syn转基因模型组小鼠纹状体、大脑皮层中的α-syn在ser129位点的磷酸化水平增高;tsg能够降低模型小鼠纹状体和大脑皮层中α-syn的磷酸化水平(ser129位点),提示tsg能够改善α-syn的翻译后修饰,这可能是tsg抑制α-syn聚集的作用机制之一。5.tsg能够增高15月龄a53tα-syn转基因小鼠大脑皮层及小脑中parkin蛋白、atg5和lc3表达;提示tsg可能通过上调泛素-蛋白酶体系统(usp)和自噬途径从而增强α-syn的降解,这可能是tsg抑制α-syn过表达和聚集的另一个作用机制。6、在体外实验中,tsg能够抑制α-syn基因转染sh-sy5y细胞内α-syn过表达,降低鱼藤酮复合α-syn基因转染细胞模型中α-syn在ser129位点的磷酸化水平和pp2a的磷酸化水平,提示tsg可能通过下调pp2a的磷酸化引起pp2a活性增高,这可能是TSG抑制α-syn磷酸化的作用机制之一。结论TSG灌胃给药能够改善15月龄A53Tα-syn转基因小鼠的运动能力和记忆功能,增高黑质多巴胺能神经元数量,抑制黑质和纹状体小胶质细胞激活,抑制脑内α-syn过表达和聚集;其作用机制可能涉及通过下调PP2A磷酸化而抑制α-syn的磷酸化,通过上调泛素-蛋白酶体系统和自噬途径而增强α-syn的降解。结果提示TSG可能有利于防治突触核蛋白相关的神经退行性疾病。更多还原

【Abstract】 Objectiveα-Synuclein(α-syn)plays an important role in the pathogenesis of a variety of neurogenerative diseases,including Parkinson’s disease(PD),dementia with Lewy bodies(DLB),Alzheimer’s disease(AD),multiple system atrophy and so on,which are collectively termed synucleinopathies Aggregated α-syn is the primary component of Lewy bodies,and mutations or multiplications in the α-syn gene result in familial PD.However,these diseases currently lack effective therapeutic drugs.2,3,5,4’-Tetrahydroxy-stilbene glucoside(TSG)is the main active and symbolic component of Polygonum multiflorum.Our previous studies found that TSG inhibited the overexpression and aggregation of a-syn in the hippocampus and cerebral cortex of APP transgenic AD mice and in the striatum and hippocampus of aged mice.The aim of the present study was to further investigate the effects of TSG on a-syn over-expression and aggregation and its mechanisms related to the modification after translation and protein degradation by using A53 T mutant a-syn transgenic mice and a-syn transfected nerve cells.Methods1.6-month-old A53 T mutant a-syn transgenic mice were intragastrically administered with TSG for 6 or 9 months.The movement ability was measured by pole test and nest building test,and the memory function was detected by Y-maze test.The immunohistochemical method was used to detect tyrosine hydroxylase(TH)-labeled dopaminegic neurons and Iba-1-labeled microglia in the brain of mice.The western blotting was used to measure the expression of a-syn monomer and oligomers,phosphorylated a-syn,Parkin,Beclin1,Atg and LC3 in the brain tissue.2.α-Syn-transfected SH-SY5 Y cells were incubated with TSG for 24 h,then with the medium containing low dose of rotenone for 24 h.The cell viability was measured by CCK8 test.The expression of a-syn,phosphorylated-a-syn and phosphorylated-PP2 A was detected by western blotting and immunofluorescence double labeling methods.Results1.The latency prolonged in the pole test the score of nest building decreased and the alternation declined in Y-maze test in A53 T α-syn transgenic mice at the age of 12 and 15 months.Intragastrical administration of TSG for 6 or 9 months significantly shortened the latency in the pole test,increased the score of nest building and elevated the alternation in Y-maze test in 12-or 15-month old model mice.The results indicated that TSG improved the motor function and memory ability in A53 T α-syn transgenic mice.2.The number of TH-labeled dopaminergic neurons declined in the substantia nigra compacta,and the number of Iba-1-labeled microglia increased in the substantia nigra and striatum of A53 T α-syn transgenic mice at the age of 15 months.Intragastrical administration of TSG for 9 months significantly elevated the number of dopaminergic neurons in the substantia nigra compacta,and decreased the number of microglia in the substantia nigra and striatum of α-syn transgenic mice,demonstrating that TSG had neuroprotective effect.3.The expression of a-syn monomer and oligomers increased in the cerebral cortex of 15-month-old A53 T α-syn transgenic mice.TSG treatment decreased the expression of a-syn monomer and oligomers in the cerebral cortex of model mice,indicating that TSG exerted inhibition on the overexpression and aggregation of a-syn in the brain of α-syn transgenic mice.4.In the mechanism study,we found that the expression of phosphorylated α-syn(at Ser129 site)elevated in the cerebral cortex and striatum of A53 T a-syn transgenic mice at the age of 15 months.TSG treatment reduced the expression of phosphorylated α-syn at Ser129 in both cortex and striatum of model mice,suggesting that TSG may suppress the aggregation of α-syn through regulating its post-translational modification.5.Administration of TSG increased the protein expression of Parkin,Atg5 and LC3 in the cerebral cortex and cerebellum of A53 T a-syn transgenic mice at the age of 15 months.The results suggest that TSG may enhance the degradation of excessive and aggregated α-syn through upregulating ubiquitin-proteasome system(UPS)and autopsy pathway.6.In vitro study showed that incubation of TSG for 24 h decreased the expression of α-syn,phosphorylated α-syn at Ser129 site and phosphorylated PP2 A in rotenone conbining α-syn-transfected SH-SY5 Y cells.The results suggest that TSG may elevate PP2 A activity via declining the phosphorylation of PP2 A,thus decrease the phosphorylation of α-syn.ConclusionIntragastrical administration of TSG ameliorated motor function and memory ability,increased the number of dopaminergic neurons and inhibited the activation of microglia,and decreased α-syn over-expression and aggregation in the brain of 15-month-old A53 T α-syn transgenic mice.The action mechanisms of TSG may be related to its down-regulating the phosphorylation of α-syn and PP2 A,as well as enhancing the degradation of α-syn through upregulating UPS and autopsy pathway.It is suggested that TSG may be beneficial to the pvevention and treatment of synuclein-related neurodegenerative diseases.更多还原