A Parkinson's Disease-relevant Mitochondrial and Neuronal Morphology High-throughput Screening Assay in LUHMES Cells
A Parkinson's Disease-relevant Mitochondrial and Neuronal Morphology High-throughput Screening Assay in LUHMES Cells
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DOI：
10.21769/bioprotoc.3881
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发表时间：
2021-01-05
期刊：
影响因子：
0.8
通讯作者：
Mortiboys, Heather
中科院分区：
文献类型：
--
作者：
Leah, Tom;Vazquez-Villasenor, Irina;Mortiboys, Heather
帕金森病是一种破坏性的神经退行性疾病，影响2-3%的65岁以上的人口。目前还没有改善疾病的治疗方法。帕金森病的主要病理特征之一是线粒体功能障碍，并且许多工作旨在鉴定可以恢复被破坏的线粒体生理学的治疗性化合物。然而，在疾病相关模型中对线粒体功能障碍进行建模，适合于筛选大型化合物库以获得改善效果，这是一个相当大的挑战。原代患者来源的细胞、SHSY-5 Y细胞和帕金森病的体内模型已被广泛用于研究帕金森病中线粒体功能障碍的贡献。事实上，许多研究已经利用LUHMES细胞来研究帕金森病，然而，LUHMES细胞先前尚未用作PD相关线粒体功能障碍的化合物筛选模型，尽管与其他常用模型相比具有若干优点，例如快速分化和高均匀性（例如，与iPSC衍生的神经元相反），以及与能够分化成高度表达特征性标志物的多巴胺能样神经元的人中脑组织相关的生理学。在先前产生GFP（+）-LUHMES细胞以模拟代谢功能障碍之后，我们报告了使用GFP（+）-LUHMES细胞在PD相关线粒体功能障碍的恢复模型中进行高通量化合物筛选的该方案。该方案描述了通过评估一系列线粒体和神经元形态学参数，使用稳健且可再现的毒素诱导的GFP（+）-LUHMES细胞模型进行高通量化合物筛选。我们还提供了数据和统计分析的详细说明，包括Z分数的示例计算，以评估独立实验的统计效应大小。
Parkinson's disease is a devastating neurodegenerative disorder affecting 2-3% of the population over 65 years of age. There is currently no disease-modifying treatment. One of the predominant pathological features of Parkinson's disease is mitochondrial dysfunction, and much work has aimed to identify therapeutic compounds which can restore the disrupted mitochondrial physiology. However, modelling mitochondrial dysfunction in a disease-relevant model, suitable for screening large compound libraries for ameliorative effects, represents a considerable challenge. Primary patient derived cells, SHSY-5Y cells and in vivo models of Parkinson's disease have been utilized extensively to study the contribution of mitochondrial dysfunction in Parkinson's. Indeed many studies have utilized LUHMES cells to study Parkinson's disease, however LUHMES cells have not been used as a compound screening model for PD-associated mitochondrial dysfunction previously, despite possessing several advantages compared to other frequently used models, such as rapid differentiation and high uniformity (e.g., in contrast to iPSC-derived neurons), and relevant physiology as human mesencephalic tissue capable of differentiating into dopaminergic-like neurons that highly express characteristic markers. After previously generating GFP(+)-LUHMES cells to model metabolic dysfunction, we report this protocol using GFP(+)-LUHMES cells for high-throughput compound screening in a restoration model of PD-associated mitochondrial dysfunction. This protocol describes the use of a robust and reproducible toxin-induced GFP(+)-LUHMES cell model for high throughput compound screening by assessing a range of mitochondrial and neuronal morphological parameters. We also provide detailed instructions for data and statistical analysis, including example calculations of Z'-score to assess statistical effect size across independent experiments.