【作者】 李云；

【导师】 宋宁；

【作者基本信息】 青岛大学 ， 生理学， 2021， 硕士

【摘要】 帕金森病(Parkinson’s disease,PD)是一种常见的神经退行性疾病,其典型的病理学特征是黑质致密部多巴胺能神经元的丢失以及残存神经元内路易小体的形成。路易小体的主要成分是错误折叠和聚集的α-突触核蛋白。除了其细胞内分布,α-突触核蛋白在细胞外主要以游离形式或外泌体形式存在。虽然外泌体形式α-突触核蛋白较少,但是外泌体α-突触核蛋白毒性更强,更容易被邻近细胞所摄取。目前认为,α-突触核蛋白的细胞间传播是PD进程呈进行性发展的重要原因。铁是人体所必需的重要微量元素之一。正常生理情况下,多巴胺能神经元内铁维持稳态,但在PD状态下,黑质区和残存的多巴胺能神经元内出现铁沉积。细胞内铁沉积可通过芬顿反应引起氧化应激,还可诱导铁死亡,是神经退变发生的重要机制之一。作为PD的两大重要的神经病理学表现,多巴胺能神经元内铁沉积和α-突触核蛋白聚集存在相互促进关系。然而细胞内铁沉积是否及如何影响α-突触核蛋白的细胞外释放尚未见研究报道。本实验在体外培养的诱导高表达野生型(Wild type,WT)和A53T突变型人源α-突触核蛋白的PC12细胞(WT hα-syn PC12细胞和A53T hα-syn PC12细胞)模型中,应用免疫印迹法检测细胞内α-突触核蛋白的表达变化及聚集。在WT hα-syn PC12细胞中,应用免疫印迹法、纳米颗粒跟踪分析法(Nanoparticle tracking analysis,NTA)检测外泌体数目变化;应用斑点杂交法检测总外泌体中聚集形式的α-突触核蛋白含量;应用酶联免疫吸附试验(Enzyme linked immunosorbent assay,ELISA)观察细胞上清、外泌体中α-突触核蛋白含量的变化;应用铁试剂盒检测细胞裂解液、细胞上清、外泌体中铁浓度的变化。应用免疫印迹法检测细胞内铁死亡标记物谷胱甘肽过氧化物酶4(Glutathione peroxidase 4,GPX4),α-突触核蛋白,小分子GTP酶Rab7、Rab11、Rab35蛋白的表达变化。本实验拟阐明细胞内铁沉积对外泌体α-突触核蛋白释放的影响,并探讨其可能机制。研究结果如下:1.用2μg/m L多西环素(Doxycycline,DOX)诱导PC12细胞高表达WT人源α-突触核蛋白,处理12 h、24 h时α-突触核蛋白表达量与对照组相比均明显上调(P<0.001,P<0.001);与处理12 h组相比,处理24 h组α-突触核蛋白表达量上调更明显(P<0.05)。在后续实验中选择用2μg/m L DOX处理24 h的PC12细胞(WT hα-syn PC12细胞)。用2μg/m L DOX处理24 h诱导PC12细胞高表达A53T突变型人源α-突触核蛋白(A53T hα-syn PC12细胞)。2.用100μmol/L枸橼酸铁铵(Ferric ammonium citrate,FAC)处理WT hα-syn PC12细胞24 h,FAC组Triton X-100可溶性、Triton X-100不可溶性α-突触核蛋白的表达量与对照组相比均明显上调(P<0.001,P<0.05)。在A53T hα-syn PC12细胞中也观察到了FAC处理组Triton X-100可溶性、Triton X-100不可溶性α-突触核蛋白的表达量与对照组相比均明显上调(P<0.001,P<0.01)。结果表明,细胞内高铁可促进WT和A53T型α-突触核蛋白的表达和聚集。3.在WT hα-syn PC12细胞中,分别用10μmol/L铁死亡诱导剂Erastin、铁死亡抑制剂Ferrostatin-1预处理30 min后再分别加入100μmol/L FAC共处理12 h、24 h。处理12 h时,各组GPX4、α-突触核蛋白表达水平均没有变化(P>0.05)。处理24 h时,FAC和Erastin共处理组与对照组相比GPX4表达下调(P<0.05),其它各组均没有变化(P>0.05),提示在铁死亡诱导剂存在时,细胞内铁沉积可诱导明显的铁死亡;FAC组、FAC和Erastin共处理组、FAC和Ferrostatin-1共处理组与对照组相比α-突触核蛋白均表达上调(P<0.05,P<0.001,P<0.05),且FAC和Erastin共处理组α-突触核蛋白表达上调比单独FAC组更明显(P<0.001)。结果表明,在WT hα-syn PC12细胞中,铁死亡可能促进α-突触核蛋白的表达。4.用超速离心法提取外泌体后,用免疫印迹法检测到外泌体标记蛋白Alix表达。用透射电子显微镜(Transmission electron microscopy,TEM)观察到外泌体呈杯状。用NTA检测到外泌体粒径峰值为108.1 nm-118.8 nm,平均粒径为127.8 nm-148.2 nm。5.用100μmol/L FAC处理WT hα-syn PC12细胞24 h后提取外泌体,免疫印迹检测结果显示,与对照组相比,FAC组外泌体标记蛋白Alix、Flotillin-1表达下调(P<0.01,P<0.01);NTA检测结果显示,与对照组相比,FAC组外泌体数目减少(P<0.001),粒径峰值、平均粒径分布均无差异。结果表明,细胞内高铁可减少外泌体释放的数目。6.用100μmol/L FAC处理WT hα-syn PC12细胞24 h后提取外泌体,斑点杂交检测结果显示,与对照组相比,FAC组总外泌体中聚集形式的α-突触核蛋白含量降低(P<0.01)。ELISA试剂盒检测结果显示,与对照组相比,FAC组细胞上清中α-突触核蛋白含量没有变化(P>0.05);单个外泌体中α-突触核蛋白含量升高(P<0.01)。结果表明,细胞内高铁可降低总外泌体中聚集形式的α-突触核蛋白含量,但单个外泌体中α-突触核蛋白的总含量明显升高。7.用100μmol/L FAC处理WT hα-syn PC12细胞24 h后提取外泌体,铁试剂盒检测结果显示,与对照组相比,FAC组细胞裂解液、细胞上清和外泌体中铁含量均升高(P<0.05,P<0.001,P<0.01)。8.无论是在DOX诱导高表达WTα-突触核蛋白的WT hα-syn PC12细胞,还是在未加DOX诱导的PC12细胞中,用100μmol/L FAC处理12 h时,与对照组相比,FAC组Rab7、Rab11蛋白表达水平均没有变化(P>0.05),但Rab35蛋白表达上调(P<0.05,P<0.01);处理24 h时,FAC组Rab7、Rab11、Rab35蛋白表达水平均没有变化(P>0.05)。在WT hα-syn PC12细胞中,分别用10μmol/L Erastin、Ferrostatin-1预处理30 min后再分别加入100μmol/L FAC共处理12 h时,各组Rab7、Rab11蛋白表达水平均没有变化(P>0.05)。FAC组、FAC和Erastin共处理组与对照组相比Rab35蛋白表达均上调(P<0.01,P<0.05),Ferrostatin-1可阻断FAC诱导的Rab35上调(P<0.05)。处理24 h时,FAC和Ferrostatin-1共处理组与对照组、FAC组相比Rab35蛋白表达下调(P<0.001,P<0.001),其它各组Rab7、Rab11、Rab35蛋白表达水平均没有变化(P>0.05)。结果表明,Rab35蛋白的表达调控可能与铁死亡相关,但Rab蛋白的表达变化可能不参与铁调控外泌体的释放。以上结果表明,细胞内铁沉积可促进PC12细胞中细胞内WT和A53T型α-突触核蛋白的表达上调与聚集,并且铁死亡可能促进铁诱导的α-突触核蛋白的表达上调;铁沉积可减少外泌体释放的数量,且减少总外泌体中聚集形式的α-突触核蛋白含量,但同时单个外泌体中α-突触核蛋白及铁含量增加。Rab蛋白的表达变化可能不参与铁调控外泌体的释放。本实验探讨了细胞内铁沉积对外泌体α-突触核蛋白释放的影响并探讨可能机制,为进一步研究α-突触核蛋白的细胞间传播提供了新的实验依据。更多还原

【Abstract】 Parkinson’s disease(PD)is a common neurodegenerative disease characterized by progressive loss of dopaminergic neurons in the substantia nigra compacta and the formation of Lewy bodies in the remaining neurons.The main components of Lewy bodies are misfolded and aggregated α-synuclein.In addition to its intracellular distribution,α-synuclein mainly exists in free form or exosomal form outside the cell.Although the portion of exosomal α-synuclein are low,the exosomal α-synuclein is more toxic and easier to be taken up by neighboring cells.It is currently believed that the cell-to-cell transmission ofα-synuclein is an important contributor in PD progression.Iron is one of the essential trace elements in the body.Under normal physiological conditions,iron metabolism is balanced in dopaminergic neurons,however,iron deposits in the substantia nigra and the remaining dopaminergic neurons in PD.Intracellular iron deposition contributes to oxidative stress through Fenton reaction,as well as induces ferroptosis,which is one of the important mechanisms underlying neurodegeneration.As the two important neuropathological manifestations of PD,iron deposition and α-synuclein aggregation in dopaminergic neurons promote each other.However,whether and how intracellular iron deposition affects the extracellular release of α-synuclein has not been reported yet.In this study,invitro PC12 cells(WT hα-syn PC12 cells and A53 T hα-syn PC12 cells)models highly expressing wild type(WT)and A53 T mutant human α-synuclein were generated.The α-synuclein protein levels and α-synuclein aggregation in cells were detected by western blotting.In WT hα-syn PC12 cells,the number of exosomes were detected by western blotting and nanoparticle tracking analysis(NTA).The dot blot was used to detected the content of aggregated α-synuclein in all exosomes derived from WT hα-syn PC12 cells.The enzyme-linked immunosorbent assay(ELISA)was used to observe the changes in α-synuclein content in the cell supernatant and exosomes derived from WT hα-syn PC12 cells.The iron assay kit was used to detect the iron concentration changes in the cell lysates,cell supernatant,and exosomes derived from WT hα-syn PC12 cells.Western blotting was used to detect the protein levels of ferroptosis marker glutathione peroxidase 4(GPX4),α-synuclein,small molecule GTPases Rab7,Rab11,and Rab35 proteins.We aim to clarify the effect of intracellular iron deposition on the release of exosomal α-synuclein and explore its possible mechanism.The results of the study are as follows:1.Doxycycline(DOX,2 μg/m L)was applied to induce high expression of WT human α-synuclein in PC12 cells.Compared with the control group,the protein levels of α-synuclein were significantly increased at 12 h and 24 h(P<0.001,P<0.001);compared with the 12 h treatment group,the protein levels of α-synuclein in the 24 h treatment group were increased further(P<0.05).Therefore,PC12 cells(WT hα-syn PC12 cells)treated with 2μg/m L DOX for 24 h were used in the following experiments.Treatment with 2 μg/m L DOX for 24 h induced high expression of A53 T mutant human α-synuclein in PC12 cells(A53T hα-syn PC12 cells).2.WT hα-syn PC12 cells were treated with 100 μmol/L ferric ammonium citrate(FAC)for24 h.Compared with the control group,the protein levels of Triton X-100 soluble and Triton X-100 insoluble α-synuclein were both obviously up-regulated in the FAC group(P<0.001,P<0.05).In A53 T hα-syn PC12 cells,it was also observed that the protein levels of Triton X-100 soluble and Triton X-100 insoluble α-synuclein in the FAC group were significantly up-regulated(P<0.001,P<0.01).This suggests that intracellular iron overload promotes expression and aggregation of WT and A53 T α-synuclein.3.WT hα-syn PC12 cells were pretreated with 10 μmol/L ferroptosis inducer Erastin and ferroptosis inhibitor Ferrostatin-1 for 30 min,and then treated with 100 μmol/L FAC for 12 h and 24 h respectively.The protein levels of GPX4 and α-synuclein in each group did not change at 12h(P>0.05).Compared with the control group,the protein levels of GPX4 were decreased in the FAC and Erastin co-treatment group at 24h(P<0.05),and there was no change in the other groups(P>0.05),suggesting that in the presence of ferroptosis inducers,intracellular iron deposition can induce significant ferroptosis.Compared with the control group,the protein levels of α-synuclein were up-regulated in the FAC group,the FAC and Erastin co-treatment group,the FAC and Ferrostatin-1 co-treatment group(P<0.05,P<0.001,P<0.05);compared with the FAC group,the protein levels of α-synuclein in the FAC and Erastin co-treatment group was increased further(P<0.001).This suggests that in WT hα-syn PC12 cells,ferroptosis promotes the expression of α-synuclein.4.After purifying exosomes by ultracentrifugation,the exosomes specific marker Alix was detected by western blotting.The exosomes were observed to be cup-shaped using transmission electron microscopy(TEM).The isolated exosomes were analyzed using NTA to demonstrate that the peak particles size was 108.1 nm-118.8 nm and the average particles size was 127.8 nm-148.2 nm.5.Exosomes were collected from WT hα-syn PC12 cells treated with 100 μmol/L FAC for24 h.Compared with the control group,the protein levels of Alix and Flotillin-1 were significantly decreased in the FAC group(P<0.01,P<0.01).NTA data showed that compared with the control group,the number of exosomes were significantly decreased in the FAC group(P<0.001).There was no difference of the peak particles size and the average particles size distribution.This suggests that intracellular iron overload decreases the number of exosomes.6.Exosomes were collected from WT hα-syn PC12 cells treated with 100 μmol/L FAC for24 h.Dot blot showed that compared with the control group,the content of aggregate α-synuclein were significantly decreased in all exosomes in the FAC group(P<0.01).ELISA data showed that α-synuclein content was unchanged in the supernatant in the FAC group(P>0.05).Compared with the control group,total α-synuclein content was increased in exosomes in the FAC group(P<0.01).This suggests that intracellular iron overload decreases the content of aggregate α-synuclein in all exosomes,but increases the total content of α-synuclein in single exosome.7.Exosomes were collected from WT hα-syn PC12 cells treated with 100 μmol/L FAC for24 h.Iron assay data showed that compared with the control group,iron concentrations in the cell lysates,cell supernatant and exosomes were increased in the FAC group(P<0.05,P<0.001,P<0.01).8.In both WT hα-syn PC12 cells and PC12 cells without WT α-synuclein overexpression,when treated with 100 μmol/L FAC treatment for 12 h up-regulated the protein levels of Rab35(P<0.05,P<0.01),while the protein levels of Rab7 and Rab11 were unchanged(P>0.05).The protein levels of Rab7,Rab11 and Rab35 did not change in cells with FAC treatment for 24 h(P>0.05).In WT hα-syn PC12 cells,when they were pretreated with 10 μmol/L Erastin and Ferrostatin-1 for 30 min prior to 100 μmol/L FAC treatment for12 h,the protein levels of Rab7 and Rab11 in each group did not change(P>0.05),but the protein levels of Rab35 was up-regulated in the FAC group,as well as in FAC and Erastin co-treatment group,compared with the control group(P<0.01,P<0.05);Ferrostatin-1 could prevent iron-induced Rab35 up-regulation(P<0.05).The protein levels of Rab7 and Rab11 in each group did not change with FAC treatment for 24h(P>0.05);compared with the control group and FAC group,the protein levels of Rab35 were down-regulated in the FAC and Ferrostatin-1 co-treatment group(P<0.001,P<0.001),but no changes were observed in the other groups(P>0.05).This suggests that the expression of Rab35 protein might be regulated by ferroptosis,but the regulation of Rab expression might not participate in the release of exosomes from cells with iron overload.The above results indicate that intracellular iron deposition can promote the expression and aggregation of WT and A53 T α-synuclein in PC12 cells and ferroptosis promotes ironinduced α-synuclein expression.Intracellular iron deposition can decrease the numbers of exosomes,decrease the content of α-synuclein aggregates in all exosomes but increase the total content of α-synuclein and iron in single exosome.The regulation of Rab expression might not participate in the release of exosomes from cells with iron overload.This study explored the effects of intracellular iron deposition on the release of exosomal α-synuclein and explored possible underlying mechanisms.It provides a new experimental basis for further research on the intercellular spread of α-synuclein.更多还原