TETANUS AND BOTULINUM NEUROTOXINS AND NEURONAL CELL BIOLOGY

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）通过切割与突触囊泡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。我们发现抑制神经节苷脂合成的伏马菌素B1可以保护神经元免受肉毒杆菌神经毒素A （BoNT A）的作用，并且外源性神经节苷脂的添加可以恢复毒素的作用。在这方面，GQ1b和GT1b大致相当，GD1b效果较差。这些发现表明神经元膜神经节苷是BoNT A传递到神经元细胞质所必需的。A和E血清型BoNT都能切割神经元蛋白SNAP-25。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的预期水平的两倍。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更强的效力是一致的。

Clostridial neurotoxins (tetanus and seven serotypes of botulinum neurotoxin) each block vesicular neurotransmitter release by cleaving specific proteins implicated in synaptic vesicle fusion. 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 have studied 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, protects neurons against the action of botulinum neurotoxin A (BoNT A) and that addition of exogenous gangliosides restores toxin action. In this regard, GQ1b and GT1b are approximately equivalent and GD1b is less effective. These findings indicate that neuronal membrane gangliosides are required for delivery of BoNT A to the neuronal cytosol. Both A and E serotypes of BoNT cleave the neuronal protein SNAP-25. 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.