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首页> 外文期刊>Biochemistry >Uptake of Botulinum Neurotoxin into Cultured Neurons
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Uptake of Botulinum Neurotoxin into Cultured Neurons

机译：肉毒杆菌神经毒素被吸收到培养的神经元中

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Botulinum neurotoxins (BoNTs) act within the synaptic terminal to block neurotransmitter release.The toxin enters the neuron by binding to neuronal membrane receptor(s),being taken up into an endosome-like compartment,and penetrating the endosome membrane via a pH-dependent translocation process.Once within the synaptic cytoplasm,BoNT serotypes A and E cleave separate sites on the C-terminus of the neuronal protein SNAP-25,one of the SNARE proteins required for synaptic vesicle fusion.In this study,we measured the effect of brief toxin exposure on SNAP-25 proteolysis in neuronal cell cultures as an indicator of toxin translocation.The results indicate that (1) uptake of both BoNT-A and-E is enhanced with synaptic activity induced by K+ depolarization in the presence of Ca~(2+) and (2) translocation of BoNT-A from the acidic endosomal compartment is slow relative to that of BoNT-E.Polyclonal antisera against each toxin protect cells when applied with the toxin during stimulation but has no effect when added immediately after toxin exposure,indicating that toxin endocytosis occurs with synaptic activity.Both serotypes cleave SNAP-25 at concentrations between 50 pM and 4 nM.IC_(50) values for SNAP-25 cleavage are approximately 0.5 nM for both serotypes.Inhibition of the pH-dependent translocation process by pretreating cultures with concanamycin A (Con A) prevents cleavage of SNAP-25 with IC5o values of approx=25 nM.Addition of Con A at times up to 15 min after toxin exposure abrogated BoNT-A action;however,addition of Con A after 40 min was no longer protective.In contrast,Con A inhibited,but did not prevent,translocation of BoNT-E even when added immediately after toxin exposure,indicating that pH-dependent translocation of BoNT-E is rapid relative to that of BoNT-A.This study demonstrates that uptake of both BoNT -A and approx=E is enhanced with synaptic activity and that translocation of the toxin catalytic moiety into the cytosol occurs at different rates for these two serotypes.
机译：肉毒杆菌神经毒素（BoNTs）在突触末端内起作用以阻止神经递质的释放。毒素通过与神经元膜受体结合进入神经元，并被吸收到一个内体样腔室中，并通过pH依赖性物质穿透内体膜在突触细胞质中，BoNT血清型A和E分别在神经元蛋白SNAP-25的C末端切割不同的位点，SNAP-25是突触小泡融合所需的一种SNARE蛋白。在本研究中，我们测量了结果表明，（1）Ca +存在下K +去极化诱导的突触活性增强了（1）BoNT-A和-E的摄取均增加了神经元细胞培养物中SNAP-25蛋白水解的毒素暴露。相对于BoNT-E而言，（2+）和（2）的BoNT-A从酸性内体区室的转移较慢。针对每种毒素的多克隆抗血清在刺激期间与毒素一起使用时可保护细胞，但具有接触毒素后立即添加无影响，表明毒素发生内吞并具有突触活性。两种血清型均以50 pM和4 nM的浓度裂解SNAP-25.SNAP-25裂解的IC_（50）值均为两种血清型约0.5 nM通过用刀豆素A（Con A）预处理培养物抑制pH依赖的转运过程，可防止SNAP-25裂解，IC50值约为25 nM。在接触毒素后最多15分钟的时间添加Con A会废除BoNT-一个作用;但是，在40分钟后添加Con A不再具有保护作用。相反，即使在毒素暴露后立即添加，Con A也抑制但没有阻止BoNT-E的转位，表明BoNT-E的pH依赖性转位-E相对于BoNT-A而言是快速的。这项研究表明BoNT -A和近似= E的摄取都随着突触活性而增强，并且毒素催化部分向细胞质中的转运以不同的速率发生。两种血清型。

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