Tetanus And Botulinum Neurotoxins And Neuronal Cell Biol

梭状芽胞杆菌神经毒素（破伤风和7种血清型肉毒杆菌神经毒素）通过切割与突触前膜融合有关的特定蛋白质来阻断囊泡神经递质释放。这些毒素是包括脑瘫在内的许多神经系统疾病的重要治疗剂，也是了解神经递质释放、膜运输、蛋白质分类、运输和靶向的宝贵工具。我们利用生物化学和形态学技术在胎鼠脊髓原代细胞培养中研究了毒素的突触前作用。我们发现药物伏马菌素B1可以阻断神经节苷脂的合成并消耗神经节苷脂的膜，从而保护神经元免受肉毒杆菌神经毒素A （BoNT A）的作用。将外源性神经节苷脂添加到神经节苷脂耗尽的神经元中可恢复毒素作用。在这方面，GQ1b和GT1b大致相当，GD1b效果较差。高浓度的神经节苷能增强毒素效应，甚至超过对照培养。这些发现表明，神经元膜神经节苷是将BoNT a传递到神经元细胞质所需的受体的关键成分。A和E血清型BoNT都能切割神经元蛋白SNAP-25，尽管BoNT A在突触末端保持催化活性的时间更长，这表明这些毒素的运输是不同的。我们评估了内体pH值对BoNT的A和E向细胞质转移的影响。阻断内体酸化的抗生素巴菲霉素A1对培养物进行滴定，结果显示BoNT a比BoNT e更依赖于pH值，这一特性可能是这两种毒素在神经元内不同运输的主要因素。此外，尽管随后对神经递质释放的阻断与BoNT e损伤SNAP-25的量一致，但释放的阻断几乎是BoNT a损伤SNAP-25水平预期的两倍。我们已经获得证据表明，SNAP-25的BoNT A切割产物作为完整SNAP-25的竞争性抑制剂，并在生理钙浓度下阻断完整神经元的突触囊泡融合。这一发现与BoNT A相对于BoNT e的更大效力相一致。目前使用荧光和电子显微镜的研究旨在确定突触囊泡外显和内吞作用之间的关系，特别是BoNT A修饰的内吞作用。

Clostridial neurotoxins (tetanus and seven serotypes of botulinum neurotoxin) each block vesicular neurotransmitter release by cleaving specific proteins implicated in synaptic vesicle fusion with the presynaptic membrane. These toxins are important therapeutic agents for a number of neurologic disorders including cerebral palsy and are valuable tools for understanding neurotransmitter release, membrane trafficking, and protein sorting, transport, and targeting. We study the presynaptic action of the toxins using biochemical and morphologic techniques in primary cell cultures of fetal mouse spinal cord. We have found that the drug fumonisin B1, which blocks ganglioside synthesis and depletes membranes of gangliosides, protects neurons against the action of botulinum neurotoxin A (BoNT A). Addition of exogenous gangliosides to ganglioside-depleted neurons restores toxin action. In this regard, GQ1b and GT1b are approximately equivalent and GD1b is less effective. High concentrations of gangliosides enhance toxin effect even beyond that in control cultures. These findings indicate that neuronal membrane gangliosides are a critical component of the receptor required for delivery of BoNT A to the neuronal cytosol. Both A and E serotypes of BoNT cleave the neuronal protein SNAP-25, although BoNT A remains catalytically active within the synaptic terminal for a much longer time, suggesting differential trafficking of these toxins. We assessed the effect of endosomal pH on the translocation of BoNT's A and E to the cytosol. Titrating cultures with the antibiotic bafilomycin A1, which blocks acidification of endosomes, shows a greater dependence of BoNT A on pH than of BoNT E. This characteristic may be a primary factor underlying distinct trafficking of these two toxins within neurons. Furthermore, whereas the ensuing blockade of neurotransmitter release is consistent with the amount of BoNT E-damaged SNAP-25, block of release is almost twice as great as that expected from the levels of BoNT A-damaged SNAP-25. We have obtained evidence that the BoNT A cleavage product of SNAP-25 acts as a competitive inhibitor of intact SNAP-25, and blocks synaptic vesicle fusion in intact neurons at physiological concentrations of calcium. This finding is consistent with the greater potency of BoNT A relative to BoNT E. Current studies using fluorescence and electron microscopy are aimed at defining the relationship between synaptic vesicle exo- and endocytosis particularly as modified by BoNT A.