TETANUS AND BOTULINUM NEUROTOXINS AND NEURONAL CELL BIOLOGY

梭状芽胞杆菌神经毒素(破伤风和七种血清类型的肉毒杆菌神经毒素)每一种都通过裂解与突触小泡融合有关的特定蛋白来阻断小泡神经递质的释放。这些毒素是包括脑瘫在内的许多神经疾病的重要治疗剂，是了解神经递质释放、膜运输以及蛋白质分类、运输和靶向的宝贵工具。在原代培养的胎鼠脊髓细胞中，我们用生化和吗啡技术研究了毒素的突触前作用。我们发现，使用药物伏马菌素B1来阻断神经节苷脂的合成，神经元膜神经节苷脂是破伤风毒素内化所必需的，是帐篷受体的关键成分。对突触小泡膜再循环的研究表明，BANT和肉毒杆菌神经毒素A(BoNT A)均可阻断小泡的胞吐作用，小泡内吞作用可在BoNT A中驱动，但不能在被TANT阻断的终末驱动，并且膜的内吞作用是钙依赖的。这些发现表明，突触小泡膜的储存库位于神经末梢的质膜上，而BoNT A不干扰小囊膜的提取。最后，A型和E型的BONT作用于相同的蛋白底物SNAP-25，但体内培养的神经元在暴露于BONT A后恢复功能的速度比暴露于E的慢。我们已经证明，BONT A后突触阻断的持续时间是由于其催化活性的持续而不是SNAP-25翻转的失败。-破伤风、肉毒杆菌、突触小泡循环、胞吐、内吞、神经节苷脂、受体、脊髓细胞培养

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 morphololgic techniques in primary cell cultures of fetal mouse spinal cord.We have found, using the drug fumonisin B1 to block ganglioside synthesis, that neuronal membrane gangliosides are required for tetanus toxin (TeNT) internalization and are a critical component of the TeNT receptor. Studies of synaptic vesicle membrane recycling demonstrate that TeNT and botulinum neurotoxin A (BoNT A) both block vesicle exocytosis, that vesicle endocytosis can be driven in BoNT A- but not in TeNT- blocked terminals, and that the membrane endocytosis is calcium-dependent. These findings suggest that a reservoir of synaptic vesicle membrane resides on the plasma membrane of the nerve terminal and that BoNT A does not interfere with vesicle membrane retrieval. Finally, both A and E serotypes of BoNT act on the same protein substrate SNAP-25, but neurons in cell culture as in vivo regain function more slowly after exposure to BoNT A than to E. We have shown that the prolonged duration of synaptic blockade after BoNT A is due to persistence of its catalytic activity rather than a failure of SNAP-25 turnover. - tetanus, botulinum, synaptic vesicle recycling, exocytosis, endocytosis, gangliosides, receptor, spinal cord cell cultures