Dynamin Inhibition Blocks Botulinum Neurotoxin Type A Endocytosis in Neurons and Delays Botulism
Dynamin Inhibition Blocks Botulinum Neurotoxin Type A Endocytosis in Neurons and Delays Botulism
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DOI：
10.1074/jbc.m111.283879
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发表时间：
2011-10-14
影响因子：
4.8
通讯作者：
Meunier, Frederic A.
中科院分区：
文献类型：
--
作者：
Harper, Callista B.;Martin, Sally;Meunier, Frederic A.
肉毒杆菌神经毒素（BoNT）是引起麻痹性疾病肉毒中毒的双链细菌蛋白。在结合到胆碱能运动神经末梢的质膜之后，BoNT被内化到内吞隔室中。尽管已经在神经元中表征了几种内吞途径，但突触前神经末梢吸收BoNT的分子机制仍不清楚。在这里，重组BoNT/A重链结合结构域（Hc）被用来解开的内化途径，通过荧光和电子显微镜。BoNT/A-Hc最初以活性依赖性方式进入培养的海马神经元，进入突触囊泡和网格蛋白包被的囊泡，然后也进入内体结构和多泡体。我们发现，用新的有效Dynasore类似物Dyngo-4a（TM）抑制发动蛋白足以消除海马神经元中BoNT/A-Hc内化和BoNT/A诱导的SNAP 25切割。Dyngo-4a还干扰BoNT/A-Hc内化进入运动神经末梢。此外，Dyngo-4a提供了对BoNT/A诱导的大鼠膈肌麻痹的保护。在注射Dyngo-4a的小鼠中观察到肉毒中毒发作的>30%的显著延迟。因此，动力蛋白抑制提供了治疗肉毒杆菌中毒和由共享动力蛋白依赖性摄取机制的病原体引起的其他疾病的治疗途径。
The botulinum neurotoxins (BoNTs) are di-chain bacterial proteins responsible for the paralytic disease botulism. Following binding to the plasma membrane of cholinergic motor nerve terminals, BoNTs are internalized into an endocytic compartment. Although several endocytic pathways have been characterized in neurons, the molecular mechanism underpinning the uptake of BoNTs at the presynaptic nerve terminal is still unclear. Here, a recombinant BoNT/A heavy chain binding domain (Hc) was used to unravel the internalization pathway by fluorescence and electron microscopy. BoNT/A-Hc initially enters cultured hippocampal neurons in an activity-dependent manner into synaptic vesicles and clathrin-coated vesicles before also entering endosomal structures and multivesicular bodies. We found that inhibiting dynamin with the novel potent Dynasore analog, Dyngo-4a (TM), was sufficient to abolish BoNT/A-Hc internalization and BoNT/A-induced SNAP25 cleavage in hippocampal neurons. Dyngo-4a also interfered with BoNT/A-Hc internalization into motor nerve terminals. Furthermore, Dyngo-4a afforded protection against BoNT/A-induced paralysis at the rat hemidiaphragm. A significant delay of >30% in the onset of botulism was observed in mice injected with Dyngo-4a. Dynamin inhibition therefore provides a therapeutic avenue for the treatment of botulism and other diseases caused by pathogens sharing dynamin-dependent uptake mechanisms.