Embryonic stem cell-derived neurons are a novel, highly sensitive tissue culture platform for botulinum research
Embryonic stem cell-derived neurons are a novel, highly sensitive tissue culture platform for botulinum research
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DOI：
10.1016/j.bbrc.2010.12.132
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发表时间：
2011-02-04
影响因子：
3.1
通讯作者：
Mesngon, Mariano
中科院分区：
文献类型：
--
作者：
McNutt, Patrick;Celver, Jeremy;Mesngon, Mariano
没有药物治疗来挽救肉毒杆菌神经毒素（BoNT）介导的神经肌肉信号传导的麻痹。在某种程度上，这种失败可归因于缺乏基于神经元的细胞培养模型系统，其允许详细阐明BoNT发病机制的基础，但仍与现代细胞和分子方法相容。我们已经开发了一种方法，从悬浮培养的小鼠胚胎干细胞（ES）中获得高度富集的神经元。假设ES细胞衍生的神经元（ESNs）可能包括一个新的平台，以研究神经毒性的BoNT，我们评估了ESNs的敏感性BoNT/A和BoNT/E使用分子和功能测定。ESNs表达神经元特异性蛋白，发育突触，并在去极化条件下以钙依赖性方式释放谷氨酸。它们表达BoNT底物SNARE蛋白SNAP 25、VAMP 2和突触融合蛋白，并且用BoNT/A和BoNT/E全毒素处理导致SNAP 25在24小时内蛋白水解，EC 50分别为0.81和68.6 pM。BoNT/A中毒导致钾诱导的钙依赖性谷氨酸释放的功能抑制。ESN在接种后长达90天内保持活力并对中毒敏感，从而能够进行纵向筛选，探索突触阻滞持续存在的毒素特异性机制。有证据表明，衍生神经元是一种新型的生物相关模型系统，它将原代神经元的逼真性与连续细胞系的遗传易处理性和可扩展性相结合，因此应该显着加速BoNT研究和药物发现，同时显着减少动物使用。爱思唯尔公司出版
There are no pharmacological treatments to rescue botulinum neurotoxin (BoNT)-mediated paralysis of neuromuscular signaling. In part, this failure can be attributed to the lack of a cell culture model system that is neuron-based, allowing detailed elucidation of the mechanisms underlying BoNT pathogenesis, yet still compatible with modern cellular and molecular approaches. We have developed a method to derive highly enriched, glutamatergic neurons from suspension-cultured murine embryonic stem (ES) cells. Hypothesizing that ES cell-derived neurons (ESNs) might comprise a novel platform to investigate the neurotoxicology of BoNTs, we evaluated the susceptibility of ESNs to BoNT/A and BoNT/E using molecular and functional assays. ESNs express neuron-specific proteins, develop synapses and release glutamate in a calcium-dependent manner under depolarizing conditions. They express the BoNT substrate SNARE proteins SNAP25, VAMP2 and syntaxin, and treatment with BoNT/A and BoNT/E holotoxin results in proteolysis of SNAP25 within 24 h with EC50s of 0.81 and 68.6 pM, respectively. Intoxication with BoNT/A results in the functional inhibition of potassium-induced, calcium-dependent glutamate release. ESNs remain viable and susceptible to intoxication for up to 90 days after plating, enabling longitudinal screens exploring toxin-specific mechanisms underlying persistence of synaptic blockade. The evidence suggests that derived neurons are a novel, biologically relevant model system that combines the verisimilitude of primary neurons with the genetic tractability and scalable expansion of a continuous cell line, and thus should significantly accelerate BoNT research and drug discovery while dramatically decreasing animal use. Published by Elsevier Inc.