TETANUS AND BOTULINUM NEUROTOXINS AND NEURONAL CELL BIOLOGY

梭菌神经毒素（破伤风和七种血清型肉毒杆菌神经毒素）通过切割突触囊泡融合中涉及的特定蛋白质来阻断囊泡神经递质的释放。这些毒素是许多神经系统疾病（包括脑瘫）的重要治疗药物，也是了解神经递质释放、膜运输和蛋白质分选、运输和靶向的宝贵工具。我们研究了突触前行动的毒素使用生化和形态学技术在原代细胞培养的胚胎小鼠脊髓。我们已经发现，药物伏马菌素B1，它阻止神经节苷脂的合成，保护神经元免受肉毒杆菌神经毒素A（BoNT A）的作用，并添加外源性神经节苷脂恢复毒素的作用。 在这方面，GQ 1b和GT 1b大致相当，GD 1b的效果较差。 这些发现表明，神经元膜神经节苷脂是将BoNT A递送到神经元胞质溶胶所必需的。 BoNT的A和E血清型都切割神经元蛋白SNAP-25。 虽然随后的神经递质释放的阻断与BoNT E-损伤的SNAP-25的量一致，但释放的阻断几乎是从BoNT A-损伤的SNAP-25的水平预期的两倍。 我们已经获得的证据表明，SNAP-25的BoNT A裂解产物作为完整SNAP-25的竞争性抑制剂，并在生理浓度的钙下阻断完整神经元中的突触囊泡融合。 这一发现与BoNT A相对于BoNT E的更大效力一致。

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.