Er Safak, Hlushchuk Irena, Airavaara Mikko, Chmielarz Piotr, Domanskyi Andrii
Institute of Biotechnology, HiLIFE, University of Helsinki.
Neuroscience Center, HiLIFE, University of Helsinki.
J Vis Exp. 2020 Aug 16(162). doi: 10.3791/61118.
The goal of this protocol is to establish a robust and reproducible model of α-synuclein accumulation in primary dopamine neurons. Combined with immunostaining and unbiased automated image analysis, this model allows for the analysis of the effects of drugs and genetic manipulations on α-synuclein aggregation in neuronal cultures. Primary midbrain cultures provide a reliable source of bona fide embryonic dopamine neurons. In this protocol, the hallmark histopathology of Parkinson's disease, Lewy bodies (LB), is mimicked by the addition of α-synuclein pre-formed fibrils (PFFs) directly to neuronal culture media. Accumulation of endogenous phosphorylated α-synuclein in the soma of dopamine neurons is detected by immunostaining already at 7 days after the PFF addition. In vitro cell culture conditions are also suitable for the application and evaluation of treatments preventing α-synuclein accumulation, such as small molecule drugs and neurotrophic factors, as well as lentivirus vectors for genetic manipulation (e.g., with CRISPR/Cas9). Culturing the neurons in 96 well plates increases the robustness and power of the experimental setups. At the end of the experiment, the cells are fixed with paraformaldehyde for immunocytochemistry and fluorescence microscopy imaging. Multispectral fluorescence images are obtained via automated microscopy of 96 well plates. These data are quantified (e.g., counting the number of phospho-α-synuclein-containing dopamine neurons per well) with the use of free software that provides a platform for unbiased high-content phenotype analysis. PFF-induced modeling of phosphorylated α-synuclein accumulation in primary dopamine neurons provides a reliable tool to study the underlying mechanisms mediating formation and elimination of α-synuclein inclusions, with the opportunity for high-throughput drug screening and cellular phenotype analysis.
本实验方案的目标是在原代多巴胺神经元中建立一个强大且可重复的α-突触核蛋白积累模型。结合免疫染色和无偏倚的自动图像分析，该模型能够分析药物和基因操作对神经元培养物中α-突触核蛋白聚集的影响。中脑原代培养物提供了真正的胚胎多巴胺神经元的可靠来源。在本实验方案中，通过直接向神经元培养基中添加α-突触核蛋白预形成纤维（PFFs）来模拟帕金森病的标志性组织病理学特征——路易小体（LB）。在添加PFFs后7天，通过免疫染色即可检测到多巴胺神经元胞体中内源性磷酸化α-突触核蛋白的积累。体外细胞培养条件也适用于预防α-突触核蛋白积累的治疗方法的应用和评估，如小分子药物和神经营养因子，以及用于基因操作的慢病毒载体（例如，使用CRISPR/Cas9）。将神经元培养在96孔板中可提高实验设置的稳健性和效能。在实验结束时，用多聚甲醛固定细胞用于免疫细胞化学和荧光显微镜成像。通过96孔板的自动显微镜获得多光谱荧光图像。这些数据使用免费软件进行量化（例如，计算每孔中含磷酸化α-突触核蛋白的多巴胺神经元数量），该软件提供了一个用于无偏倚高内涵表型分析的平台。PFF诱导的原代多巴胺神经元中磷酸化α-突触核蛋白积累模型为研究介导α-突触核蛋白包涵体形成和消除过程的潜在机制提供了一个可靠工具，有机会进行高通量药物筛选和细胞表型分析。
