Development of a Scalable, High-Throughput-Compatible Assay to Detect Tau Aggregates Using iPSC-Derived Cortical Neurons Maintained in a Three-Dimensional Culture Format
Development of a Scalable, High-Throughput-Compatible Assay to Detect Tau Aggregates Using iPSC-Derived Cortical Neurons Maintained in a Three-Dimensional Culture Format
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DOI：
10.1177/1087057116638029
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发表时间：
2016-09-01
影响因子：
--
通讯作者：
Cabrera-Socorro, A.
中科院分区：
文献类型：
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作者：
Medda, X.;Mertens, L.;Cabrera-Socorro, A.
Tau聚集是与阿尔茨海默病（AD）的进展最相关的病理学标志。由过度磷酸化的tau形成的神经元缠结（NFT）的存在导致神经元功能障碍和损失，并且与在AD患者中观察到的认知下降直接相关。靶向β-淀粉样蛋白病理学的有限成功强化了阻断tau磷酸化、聚集和/或扩散作为治疗AD的替代治疗切入点的假设。新疗法的鉴定需要能够在体外环境中再现病理学的关键特征的疾病相关和可扩展的测定。在这里，我们使用诱导多能干细胞（iPSC）作为人类皮层神经元的几乎无限的来源，以开发与高通量筛选（HTS）兼容的强大且可扩展的tau聚集模型。我们将细胞培养条件缩小到384孔板格式，并使用Matrigel引入额外的物理保护以防止细胞脱离，从而降低剪切应力并更好地重现病理条件。我们用AlphaLISA技术补充了该测定，以高通量兼容的形式检测tau聚集体。该测定在用户之间是可重复的，并且与不同的市售iPSC系一起工作，代表了用于鉴定针对tau蛋白病（包括AD）的新型治疗的高度翻译工具。
Tau aggregation is the pathological hallmark that best correlates with the progression of Alzheimer's disease (AD). The presence of neurofibrillary tangles (NFTs), formed of hyperphosphorylated tau, leads to neuronal dysfunction and loss, and is directly associated with the cognitive decline observed in AD patients. The limited success in targeting -amyloid pathologies has reinforced the hypothesis of blocking tau phosphorylation, aggregation, and/or spreading as alternative therapeutic entry points to treat AD. Identification of novel therapies requires disease-relevant and scalable assays capable of reproducing key features of the pathology in an in vitro setting. Here we use induced pluripotent stem cells (iPSCs) as a virtually unlimited source of human cortical neurons to develop a robust and scalable tau aggregation model compatible with high-throughput screening (HTS). We downscaled cell culture conditions to 384-well plate format and used Matrigel to introduce an extra physical protection against cell detachment that reduces shearing stress and better recapitulates pathological conditions. We complemented the assay with AlphaLISA technology for the detection of tau aggregates in a high-throughput-compatible format. The assay is reproducible across users and works with different commercially available iPSC lines, representing a highly translational tool for the identification of novel treatments against tauopathies, including AD.