Generation of Alpha-Synuclein Preformed Fibrils from Monomers and Use In Vivo
Generation of Alpha-Synuclein Preformed Fibrils from Monomers and Use In Vivo
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DOI：
10.3791/59758
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发表时间：
2019-06-01
影响因子：
1.2
通讯作者：
Sortwell, Caryl E.
中科院分区：
文献类型：
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作者：
Patterson, Joseph R.;Polinski, Nicole K.;Sortwell, Caryl E.
突触核蛋白病的体内α α-突触核蛋白预形成的原纤维（α-syn PFF）模型的使用在旨在对帕金森病突触核蛋白病和黑质纹状体变性建模的研究人员中越来越受欢迎。α-syn PFF生成和体内应用的标准化是至关重要的，以确保一致的、稳健的α-syn病理学。在这里，我们提出了一个详细的协议，从单体α-syn，原纤化后的质量控制步骤，并建议参数成功的神经外科注射α-syn PFF到大鼠或小鼠的原纤维的产生。从单体α-syn开始，在最佳缓冲液条件、浓度和温度下振荡的同时，在7天的孵育期内发生原纤化。原纤化后的质量控制是通过沉淀试验通过可造粒原纤的存在，用硫磺素T试验在原纤中形成淀粉样蛋白构象，和原纤的电子显微镜可视化来评估的。虽然使用这些测定法的成功验证是成功所必需的，但它们不足以保证PFF将在神经元中接种α-syn包涵体，因为每个PFF批次的这种聚集活性应在细胞培养物或中试动物队列中进行测试。在使用之前，PFF必须在精确标准化的条件下进行超声处理，然后使用电子显微镜或动态光散射进行检查，以确认原纤维长度在最佳尺寸范围内，平均长度为50 nm。然后可以将PFF添加到细胞培养基中或用于动物。通过免疫染色磷酸化α-syn（psyn;丝氨酸129）可检测的病理学分别在细胞培养和啮齿动物模型中几天或几周后明显。
Use of the in vivo alpha alpha-synuclein preformed fibril (alpha-syn PFF) model of synucleinopathy is gaining popularity among researchers aiming to model Parkinson's disease synucleinopathy and nigrostriatal degeneration. The standardization of alpha-syn PFF generation and in vivo application is critical in order to ensure consistent, robust alpha-syn pathology. Here, we present a detailed protocol for the generation of fibrils from monomeric alpha-syn, post-fibrilization quality control steps, and suggested parameters for successful neurosurgical injection of alpha-syn PFFs into rats or mice. Starting with monomeric alpha-syn, fibrilization occurs over a 7-day incubation period while shaking at optimal buffer conditions, concentration, and temperature. Post-fibrilization quality control is assessed by the presence of pelletable fibrils via sedimentation assay, the formation of amyloid conformation in the fibrils with a thioflavin T assay, and electron microscopic visualization of the fibrils. Whereas successful validation using these assays is necessary for success, they are not sufficient to guarantee PFFs will seed alpha-syn inclusions in neurons, as such aggregation activity of each PFF batch should be tested in cell culture or in pilot animal cohorts. Prior to use, PFFs must be sonicated under precisely standardized conditions, followed by examination using electron microscopy or dynamic light scattering to confirm fibril lengths are within optimal size range, with an average length of 50 nm. PFFs can then be added to cell culture media or used in animals. Pathology detectable by immunostaining phosphorylated alpha-syn (psyn; serine 129) is apparent days or weeks later in cell culture and rodent models,respectively.