帕金森病患者血浆增强α-突触核蛋白细胞毒性及其机制研究
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摘要
目的研究帕金森病(Parkinson's disease,PD)患者血浆对α-突触核蛋白(α-synuclein,α-Syn)细胞毒性的影响及其机制。方法将重组人α-Syn加入PD和对照血浆中,采用免疫印迹法及酶联免疫吸附(enzyme linked immunosorbent assay,ELISA)法鉴定α-Syn寡聚体和磷酸化形成情况。在原代神经培养基中加入终浓度为5μmol/L的α-Syn单体及寡聚体,同时添加30%(体积分数) PD血浆、正常人血浆和胎牛血浆,孵育14 d后,MTT及流式细胞技术观察细胞死亡情况。酶活力试剂盒检测各组原代神经元中蛋白磷酸酶2A(protein phosphatase 2A,PP2A)酶活力。结果PD患者血浆较正常受试者(normal subject,NS)血浆可促使更多的α-Syn磷酸化和寡聚化。与NS血浆相比,添加到原代神经元培养物中的PD血浆导致更多细胞在细胞外α-Syn存在下死亡,同时伴有PP2A活力降低。结论PD患者血浆中可能通过降低PP2A酶活力使α-Syn发生磷酸化,导致其聚集并进一步使得原代培养神经元死亡。
Objective To investigate the effect of plasma of Parkinson's disease( PD) on the cytotoxicity of !-synuclein( !-Syn) and its mechanism. Methods Recombinant human !-Syn was added to PD and normal subject plasma. Western blotting and ELISA method were used to identify !-Syn oligomers and phosphorylation. Add α-Syn monomer and oligomer at a final concentration of 5 "M to primary neuronal medium with 30% PD plasma,normal subject plasma and fetal bovine plasma,after 14 days of incubation,MTT and flow cytometry were used to observe the cell death. Enzyme activity kit was used to detect the activity of protein phosphatase 2 A( PP2 A)enzyme in primary neurons of each group. Results Plasma in PD patients induced more α-Syn phosphorylation and oligomerization than normal subjects( NS) plasma. Compared to NS plasma,PD plasma added to primary neuronal cultures resulted in more cells dying in the presence of extracellular !-Syn,accompanied by decreased protein phosphatase 2 A activity. Conclusion The plasma of PD patients may phosphorylate !-Syn by decreasing the activity of PP2 A enzyme,leading to aggregation and further death of primary cultured neurons.
Objective To investigate the effect of plasma of Parkinson's disease( PD) on the cytotoxicity of !-synuclein( !-Syn) and its mechanism. Methods Recombinant human !-Syn was added to PD and normal subject plasma. Western blotting and ELISA method were used to identify !-Syn oligomers and phosphorylation. Add α-Syn monomer and oligomer at a final concentration of 5 "M to primary neuronal medium with 30% PD plasma,normal subject plasma and fetal bovine plasma,after 14 days of incubation,MTT and flow cytometry were used to observe the cell death. Enzyme activity kit was used to detect the activity of protein phosphatase 2 A( PP2 A)enzyme in primary neurons of each group. Results Plasma in PD patients induced more α-Syn phosphorylation and oligomerization than normal subjects( NS) plasma. Compared to NS plasma,PD plasma added to primary neuronal cultures resulted in more cells dying in the presence of extracellular !-Syn,accompanied by decreased protein phosphatase 2 A activity. Conclusion The plasma of PD patients may phosphorylate !-Syn by decreasing the activity of PP2 A enzyme,leading to aggregation and further death of primary cultured neurons.
引文
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[11] Oueslati A. Implication ofα-synuclein phosphorylation at S129 in synucleinopathies:what have we learned in the last decade[J]. J Parkinsons Dis,2016,6(1):39-51.
[12] Emamzadeh F. Role of apolipoproteins andα-Synuclein in parkinson's disease[J]. J Mol Neurosci,2017,62(3):344-355.
[2] Braak H,Ghebremedhin E,Rub U,et al. Stages in the development of parkinson's disease-related pathology[J].Cell Tissue Res,2004,318(1):121-134.
[3] Paleologou K E,Kragh C L,Mann D M,et al. Detection of elevated levels of soluble alpha-synuclein oligomers in postmortem brain extracts from patients with dementia with Lewy bodies[J]. Brain,2009,132(Pt 4):1093-1101.
[4] Michele P,Celine G,Alexander M,et al. A natural product inhibits the initiation ofα-synuclein aggregation and suppresses its toxicity[J]. Proc Natl Acad Sci U S A,2017,114(6):e1009-e1017.
[5] Lin C H,Yang S Y,Horng H E,et al. Plasmaα-synuclein predicts cognitive decline in parkinson's disease[J]. J Neurol Neurosurg Psychiatry,2017,88(10):818-824.
[6] Zhang Z T,Kang S S,Liu X,et al. Asparagine endopeptidase cleavesα-synuclein and mediates pathologic activities in Parkinson's disease[J]. Nat Struct Mol Biol,2017,24(8):632-642.
[7]李雁,殷娟娟,李昕,等.α-突触核蛋白寡聚体对多巴胺能神经细胞线粒体膜电位的影响[J].中国现代神经疾病杂志,2008,8(1):26-31.
[8] Abdelkarim H,Marshall M S,Scesa G,et al.α-Synuclein interacts directly but reversibly with psychosine:implications forα-synucleinopathies[J]. Sci Rep,2018,8(1):12462.
[9] Han M,Nagele E,DeMarshall C,et al. Diagnosis of parkinson's disease based on disease-specific autoantibody profiles in human sera[J]. PLoS One, 2012, 7(2):e32383.
[10] Chen H,O'Reilly E J,Schwarzschild M A,et al. Peripheral inflammatory biomarkers and risk of parkinson's disease[J]. Am J Epidemiol,2008,167(1):90-95.
[11] Oueslati A. Implication ofα-synuclein phosphorylation at S129 in synucleinopathies:what have we learned in the last decade[J]. J Parkinsons Dis,2016,6(1):39-51.
[12] Emamzadeh F. Role of apolipoproteins andα-Synuclein in parkinson's disease[J]. J Mol Neurosci,2017,62(3):344-355.