1 National Center for Natural Products Research, Research Institute of Pharmaceutical Sciences, University of Mississippi, University, MS, USA.
2 Department of BioMolecular Sciences, School of Pharmacy, University of Mississippi, University MS, USA.
SLAS Discov. 2017 Jul;22(6):667-675. doi: 10.1177/2472555217698677. Epub 2017 Mar 17.
Neurotrophic assays are phenotypic methods to identify molecules that stimulate differentiation of neuronal cells. Bioactive small molecules with neurotrophic actions hold great promise as therapeutic agents for the treatment of neurodegenerative diseases and neuronal injuries by virtue of their ability to stimulate neuritic outgrowth. A combined in vitro method, which measures neurotrophic activity and cytotoxicity in a single assay, has been described. This assay, performed in 96-well microplates with PC12 and Neuroscreen-1 (NS-1; a subclone of PC12) cells, is a simple tool for identification of new neurotrophic agents. Stimulation of neurite outgrowth was measured with NIS software by analysis of digital cell images as multiple parameters, namely, mean neurite length, neurite length/cell, nodes/cell, and number of neurites/cell. The assay has been standardized and validated with dose-response analysis for nerve growth factor (NGF) and mechanism-based inhibitors of NGF-induced neurite outgrowth, namely, SU6656 (an Src family kinase inhibitor) and PD98059 (a MEK inhibitor). The assay has been successfully applied for screening natural and synthetic compound libraries for cytotoxicity and neurotrophic activity. Screening of a set of harmala alkaloids identified harmine as a potential neurotrophic molecule that significantly stimulated NGF-induced neurite outgrowth in the NS-1 cells. Important advantages of this method are its simplicity and determination of cytotoxicity and neurotrophic activity in a single assay. This assay may be suitable for primary and cultured neuronal cells.
神经营养测定法是一种表型方法，可用于鉴定刺激神经元细胞分化的分子。具有神经营养作用的生物活性小分子有望成为治疗神经退行性疾病和神经元损伤的治疗剂，因为它们能够刺激神经突生长。已经描述了一种将神经营养活性和细胞毒性在单一测定中进行测量的组合体外方法。该测定法在 PC12 和 Neuroscreen-1（NS-1；PC12 的亚克隆）细胞的 96 孔微孔板中进行，是鉴定新的神经营养剂的简单工具。通过对数字细胞图像进行分析，以多个参数（即平均神经突长度、神经突长度/细胞、节点/细胞和神经突/细胞数量）来测量神经突生长的刺激。该测定法已经通过对神经生长因子（NGF）和 NGF 诱导的神经突生长的基于机制的抑制剂（即 Src 家族激酶抑制剂 SU6656 和 MEK 抑制剂 PD98059）的剂量反应分析进行了标准化和验证。该测定法已成功应用于筛选天然和合成化合物文库的细胞毒性和神经营养活性。一组哈尔明碱的筛选鉴定出哈尔明是一种潜在的神经营养分子，可显著刺激 NS-1 细胞中 NGF 诱导的神经突生长。该方法的重要优点是其简单性和在单一测定中测定细胞毒性和神经营养活性的能力。该测定法可能适用于原代和培养的神经元细胞。
