Neuritogenic Actions of Botulinum Neurotoxin A on Cultured Motor Neurons
Neuritogenic Actions of Botulinum Neurotoxin A on Cultured Motor Neurons
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DOI：
10.1124/jpet.108.147744
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发表时间：
2009-07-01
影响因子：
3.5
通讯作者：
Yan, Xiuzhen
中科院分区：
文献类型：
--
作者：
Coffield, Julie A.;Yan, Xiuzhen
肉毒杆菌神经毒素 (BoNT) 是一种极其有效的神经肌肉毒物，通过可溶性 N-乙基马来酰亚胺敏感因子附着蛋白受体 (SNARE) 蛋白裂解来抑制神经递质释放。 BoNT 血清型 A (BoNT/A) 在极低剂量下消除局部递质释放的能力已得到充分表征。在当前的研究中，我们研究了 BoNT/A 诱导神经生长（有时称为发芽）的鲜为人知的特征。这种现象通常被认为是对 BoNT/A 麻痹作用的继发反应，并且尚未研究可能引发这种发芽的其他潜在因素。或者，我们假设 BoNT/A 通过突触前受体激活诱导出芽，这独立于其对可溶性 N-乙基马来酰亚胺敏感因子附着受体 (SNARE) 25 kDa 突触体相关蛋白 (SNAP-25) 的已知细胞内作用。为了测试这一点，使用富含从胚胎小鼠脊髓分离的运动神经元的原代培养物检查了 BoNT/A 应用对神经突生长的影响。在该系统中，BoNT/A 在浓度低至 0.01 nM 时即可有效刺激神经突发生。 BoNT/A 暴露的神经炎形成作用是浓度依赖性的，并被小麦凝集素（一种已知的 BoNT 竞争性拮抗剂）拮抗。用分离的 BoNT/A 结合域观察到类似的结果，表明神经突发生可以仅由 BoNT/A 的结合作用启动。此外，BoNT/A 是否存在 SNAP-25 裂解并不是 BoNT/A 诱导的神经突发生的决定因素。总的来说，这些结果表明 BoNT/A 与运动神经元膜的结合通过尚未确定的细胞内途径激活神经突发生，与其已知的对囊泡释放的作用无关。
Botulinum neurotoxins (BoNTs) are extremely potent neuromuscular poisons that act through soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein cleavage to inhibit neurotransmitter release. The ability of BoNT serotype A (BoNT/A) to eliminate localized transmitter release at extremely low doses is well characterized. In the current study, we investigated the less understood characteristic of BoNT/A to induce nerve outgrowth, sometimes referred to as sprouting. This phenomenon is generally considered a secondary response to the paralytic actions of BoNT/A, and other potential factors that may initiate this sprouting have not been investigated. Alternatively, we hypothesized that BoNT/A induces sprouting through presynaptic receptor activation that is independent of its known intracellular actions on the soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) synaptosomal associated protein of 25 kDa (SNAP-25). To test this, the effects of BoNT/A application on neurite outgrowth were examined using primary cultures enriched with motor neurons isolated from embryonic mouse spinal cord. In this system, BoNT/A potently stimulated neuritogenesis at concentrations as low as 0.01 nM. The neuritogenic effects of BoNT/A exposure were concentration dependent and antagonized by Triticum vulgaris lectin, a known competitive antagonist of BoNT. Similar results were observed with the isolated BoNT/A binding domain, revealing that neuritogenesis could be initiated solely by the binding actions of BoNT/A. In addition, the presence or absence of SNAP-25 cleavage by BoNT/A was not a determinant factor in BoNT/A-induced neuritogenesis. Collectively, these results suggest that binding of BoNT/A to the motor neuronal membrane activates neuritogenesis through as yet undetermined intracellular pathway(s), independent of its known action on vesicular release.