Tetanus And Botulinum Neurotoxins And Neuronal Cell Biol

梭菌神经毒素（破伤风和肉毒杆菌神经毒素的七种血清型）通过切割参与突触囊泡与突触前膜融合的特异性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胎鼠脊髓。我们已经发现，药物伏马菌素B1，它阻止神经节苷脂的合成和消耗神经节苷脂的膜，保护神经元免受肉毒杆菌神经毒素A（BoNT A）的作用。向神经节苷脂耗竭的神经元中加入外源性神经节苷脂可恢复毒素的作用。在这方面，GQ 1b和GT 1b大致相当，GD 1b的效果较差。高浓度的神经节苷脂增强毒素作用，甚至超过对照培养物。这些发现表明神经元膜神经节苷脂是将BoNT A递送至神经元胞质溶胶所需的受体的关键组分。BoNT的A和E血清型都切割神经元蛋白SNAP-25，尽管BoNT A在突触末端内保持催化活性更长的时间，表明这些毒素的差异运输。我们评估了内体pH值对BoNT的A和E易位到胞质溶胶的影响。用抗生素巴弗洛霉素A1滴定培养物，其阻断内体的酸化，显示BoNT A比BoNT E对pH的依赖性更大。这种特性可能是神经元内这两种毒素不同运输的主要因素。此外，尽管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。目前使用荧光和电子显微镜的研究旨在确定突触囊泡胞吞作用和胞吞作用之间的关系，特别是被BoNT A修饰的突触囊泡胞吞作用和胞吞作用之间的关系。

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.