Tetanus And Botulinum Neurotoxins And Neuronal Cell Biol

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。巴菲霉素A1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。毒素转运在摄取后约25分钟发生，并在90分钟内完成。巴菲罗星在阻断BoNT E的转位方面不如-A有效，这表明在较浅的pH梯度存在的情况下，BoNT E能够跨膜。这种差异可能反映了细胞内毒素转运的根本差异，可能与BoNT A作用时间较长有关。我们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。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. Bafilomycin A1 inhibits vacuolar-ATPase, preventing vesicle acidification. This drug traps toxin within vesicles allowing study of the synchronized translocation of toxin across the vesicle membrane. Toxin translocation occurs about 25 min after uptake and is complete within 90 min. Bafilomycin is less effective at blocking the translocation of BoNT E than of -A, indicating that BoNT E is able to cross the membrane in the presence of a more shallow pH gradient. This difference may reflect a fundamental difference in intracellular toxin trafficking, perhaps relevant to the long duration of action of BoNT A. We 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.