Tetanus & Botulinum Neurotoxins & Neuronal Cell Biology

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。 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。在细胞培养物中，通过钾刺激大大增强了BoNT向脊髓神经元的摄取，这暗示了作为摄取隔室的再循环突触囊泡。毒素穿过囊泡膜进入神经元胞质溶胶的运动需要低的囊泡内pH值。康卡霉素A抑制空泡ATP酶，防止囊泡酸化。该药物将毒素捕获在囊泡内，允许研究毒素穿过囊泡膜的同步易位。用康卡那霉素预处理培养物可防止两种BoNT切割SNAP-25。在毒素暴露后15分钟内加入康卡那霉素，完全消除了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的易位。这表明BoNT/E运输比BoNT/A更快地超过pH敏感步骤。当在毒素暴露后40分钟加入时，康卡那霉素没有作用，表明这两种毒素的易位在此时完成。这项研究表明，突触囊泡回收介导BoNT/A和/E的吸收，并且这两种毒素血清型在吸收后的运输方式不同。我们还监测了突触囊泡的内吞和胞吐的定量动力学测量的突触染色和褪色的荧光FM染料。在染料存在下，在5分钟的钾去极化期间，用FM 2 -10对BoNT A封闭的和未处理的培养物进行染色。BoNT A处理的培养物比对照少摄取约50%的FM染料，在脱色时仅损失约50%，并且显示出显著不同的脱色动力学。目前使用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，特别是如由BoNT A修饰的。

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.