Tetanus & Botulinum Neurotoxins & 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)每种都通过切割与突触前膜融合的突触小泡所涉及的特定蛋白来阻断泡状神经递质的释放。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。我们在原代培养的胎鼠脊髓细胞中，用生化和形态学技术研究了毒素的突触前作用。 A型和E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要长得多，这表明这些毒素的运输存在差异。在细胞培养中，脊髓神经元对BONT的摄取被钾刺激大大增强，这意味着循环突触小泡作为摄取间隔。毒素穿过囊泡膜进入神经元胞浆需要较低的囊泡内pH。刀豆素A抑制空泡-ATPase，防止囊泡酸化。这种药物将毒素捕获在小泡内，从而可以研究毒素在小泡膜上的同步转移。用刀豆素预处理培养物可防止SNAP-25被两种BoNTs切割。在毒素暴露后15分钟内加入刀豆素培养物，可完全取消BoNT/A的作用，但即使在毒素暴露后立即加入，也不能阻止BoNT/E的移位。这表明，BoNT/E转运比BoNT/A更快地超过pH敏感的步骤。当在毒素暴露40分钟后添加刀豆素时，没有影响，表明此时两种毒素的转运都已完成。这项研究表明，突触小泡再循环介导了BoNT/A和/E的摄取，并且这两种毒素血清型在摄取后被不同的转运。我们还通过突触染色和荧光FM染料染色的定量动力学测量来监测突触小泡的内吞和胞吐作用。在有染料存在的钾去极化5分钟期间，用FM2-10对BotA阻断和未处理的培养物进行染色。Bont A处理的培养物比对照少使用约50%的FM染料，仅损失约50%的脱色，并显示出明显不同的脱色动力学。目前使用电子显微镜的研究目的是确定突触小泡外排和内吞之间的关系，特别是被BoNT A修饰的突触小泡外排和内吞的关系。

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. 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. Uptake of BoNT into spinal cord neurons in cell culture is enhanced greatly by potassium stimulation, implicating recycling synaptic vesicles as the uptake compartment. Movement of toxin across the vesicle membrane into the neuronal cytosol requires low intravesicular pH. Concanamycin A inhibits vacuolar-ATPase, preventing vesicle acidification. This drug traps toxin within vesicles allowing study of the synchronized translocation of toxin across the vesicle membrane. Pretreating cultures with concanamycin prevents cleavage of SNAP-25 by both BoNTs. Addition to cultures of concanamycin at times up to 15 min after toxin exposure completely abrogated BoNT/A action but did not prevent translocation of BoNT/E even when added immediately after toxin exposure. This suggests that BoNT/E trafficking proceeds beyond a pH-sensitive step more rapidly than BoNT/A. Concanamycin has no effect when added 40 min after toxin exposure, indicating that translocation of both toxins is complete by this time. This study suggests that synaptic vesicle recycling mediates uptake of BoNT/A and /E, and that these two toxin serotypes are trafficked differently after uptake. We also have monitored synaptic vesicle endocytosis and exocytosis by the quantitative kinetic measurement of synaptic staining and destaining with fluorescent FM dyes. Both BoNT A-blocked and untreated cultures are stained with FM2-10 during 5 min of potassium depolarization in the presence of dye. BoNT A treated cultures, which take up about 50% less FM dye than controls, lose only about 50% on destaining and show markedly different destaining kinetics. Current studies using electron microscopy are aimed at defining the relationship between synaptic vesicle exo- and endocytosis particularly as modified by BoNT A.