Adler M, Keller J E, Sheridan R E, Deshpande S S
Neurotoxicology Branch, Pharmacology Division, US Army Medical Research Institute of Chemical Defense, Aberdeen Proving Ground, MD 21010-5400, USA.
Toxicon. 2001 Feb-Mar;39(2-3):233-43. doi: 10.1016/s0041-0101(00)00120-3.
Botulinum neurotoxin serotypes A (BoNT/A) and E (BoNT/E) inhibit neurotransmitter release from peripheral cholinergic nerve terminals by cleaving different sites on SNAP-25, a protein involved in synaptic vesicle docking and exocytosis. Since recovery from BoNT/A is protracted, but reversal of BoNT/E intoxication is relatively rapid, it was of interest to determine whether sequential exposure to BoNT/A and BoNT/E could provide insight into the factors responsible for persistence of BoNT action. Extensor digitorum longus (EDL) muscles from rats were injected locally with 5 mouse LD(50) units of BoNT/A or 20 mouse LD(50) units of BoNT/E; these doses were selected to produce total paralysis of EDL muscles within 48 hr. Additional groups of rats were injected sequentially with either BoNT/A followed 48 h later by BoNT/E or with BoNT/E followed 48 h later by BoNT/A. Muscle tensions were elicited in situ in response to supramaximal stimulation of the peroneal nerve to monitor recovery from BoNT intoxication. Tensions returned to 53% and 94% of control, respectively, 7 and 15 days after injection of BoNT/E. In contrast, tensions in muscles injected with BoNT/A returned to only 2% and 12% of control at these time points. Preparations injected sequentially with BoNT/A followed by BoNT/E or with BoNT/E followed by BoNT/A exhibited slow recovery times resembling those recorded in the presence of BoNT/A alone. Pronounced atrophy of the EDL muscle was observed in rats injected with BoNT/A or in those receiving serotype combinations in either sequence, whereas no loss of muscle mass was observed in animals treated with BoNT/E alone. Data suggesting that BoNT/E can enter BoNT/A-treated preparations was obtained by findings that 3,4-diaminopyridine, which readily reversed muscle paralysis after BoNT/A exposure, lost this ability within 1 h of BoNT/E addition. Evidence that BoNT/E was able to cleave SNAP-25 at its characteristic site during sequential neurotoxin exposure was demonstrated by western blot analysis of cultured primary cortical neurons. Since the sequential exposure studies indicate that recovery from BoNT intoxication is lengthened by exposure to serotype A, but not shortened by exposure to serotype E, the duration of BoNT/A intoxication appears to be determined predominantly by the intracellular stability of catalytically active BoNT/A light chain.
A型肉毒杆菌神经毒素（BoNT/A）和E型肉毒杆菌神经毒素（BoNT/E）通过切割SNAP-25上的不同位点，抑制外周胆碱能神经末梢的神经递质释放，SNAP-25是一种参与突触小泡对接和胞吐作用的蛋白质。由于从BoNT/A中毒中恢复的过程较为漫长，而BoNT/E中毒的逆转相对较快，因此确定依次暴露于BoNT/A和BoNT/E是否能深入了解导致肉毒杆菌作用持续存在的因素，这一点很有意义。给大鼠的趾长伸肌（EDL）局部注射5个小鼠半数致死量（LD50）单位的BoNT/A或20个小鼠LD50单位的BoNT/E；选择这些剂量是为了在48小时内使EDL肌肉完全麻痹。另外几组大鼠先注射BoNT/A，48小时后再注射BoNT/E，或者先注射BoNT/E，48小时后再注射BoNT/A。通过对腓总神经进行超强刺激，在原位诱发肌肉张力，以监测从肉毒杆菌中毒中恢复的情况。注射BoNT/E后7天和15天，肌肉张力分别恢复到对照值的53%和94%。相比之下，在这些时间点，注射BoNT/A的肌肉中的张力仅恢复到对照值的2%和12%。先注射BoNT/A后注射BoNT/E或先注射BoNT/E后注射BoNT/A的制剂，其恢复时间较慢，类似于仅存在BoNT/A时记录的恢复时间。在注射BoNT/A的大鼠或按任何顺序接受血清型组合的大鼠中，观察到EDL肌肉明显萎缩，而单独用BoNT/E处理的动物未观察到肌肉质量损失。通过以下发现获得了表明BoNT/E可进入经BoNT/A处理的制剂的数据：3,4-二氨基吡啶在BoNT/A暴露后能迅速逆转肌肉麻痹，但在添加BoNT/E后1小时内就失去了这种能力。通过对培养的原代皮层神经元进行蛋白质印迹分析，证明了在依次暴露于神经毒素期间，BoNT/E能够在其特征性位点切割SNAP-25。由于依次暴露研究表明，暴露于A型血清型会延长从肉毒杆菌中毒中恢复的时间，但暴露于E型血清型不会缩短恢复时间，因此BoNT/A中毒的持续时间似乎主要由具有催化活性的BoNT/A轻链的细胞内稳定性决定。
