Distal effects of botulinum neurotoxins

肉毒杆菌神经毒素的远端效应

• 批准号: 8582046
• 负责人: Edwin R Chapman
• 金额: $ 18万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-01 至 2015-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8582046
• 关键词: Address; Axon; Biological Assay; Biological Models; Bontoxilysin; Botulinum Toxin Type A; Botulism; Cells; Cellular biology; Cleaved cell; Clinical; Clostridial Neurotoxin; Cytosol; Data; Dendrites; Devices; Diffusion; Disease; Distal; Dystonia; Endocytosis; Exocytosis; Family; Goals; Hand; Human; Imagery; Industry; Injection of therapeutic agent; Ipsilateral; Lateral; Light; Mediating; Medical; Microfluidic Microchips; Microtubules; Modeling; Monitor; Motor Cortex; Movement; Nerve; Neuraxis; Neuromuscular Junction; Neurons; Pain; Pathway interactions; Patients; Peptide Hydrolases; Peripheral; Pharmaceutical Preparations; Proteins; Quantum Dots; Recycling; Relaxation; Research Personnel; S-nitro-N-acetylpenicillamine; Serotyping; Site; Skeletal Muscle; System; Testing; Tetanus; Tetanus Toxin; Therapeutic; Toxin; Toxin Conjugates; Vesicle; Work; Wrist; axoplasm; base; clinical effect; holotoxins; in vivo; mouse model; neuronal cell body; neurotransmitter release; novel; particle; patient safety; prevent; public health relevance; reconstitution; research study; retrograde transport; tetanospasmin; transcytosis; uptake; vesicle-associated membrane protein

DESCRIPTION (provided by applicant): The clostridial neurotoxins (CNTs) comprise a family of eight related toxins: tetanus (TeNT) and seven botulinum neurotoxins (BoNT/A-G), which cause the diseases tetanus and botulism, respectively. BoNT/A and BoNT/B are also used clinically to treat a wide range of serious medical conditions, including dystonia and pain; this represents a two billion dollar per year industry. The therapeutic action of the BoNT/A and B has traditionally been thought to involve the local - at the site of injection - inhibition of neurotransmitter release from neurons (by acting as proteases that selectively cleave SNARE proteins); in the case of dystonia, this presumably results in relaxation of skeletal muscles. However, a new hypothesis posits that in addition to having local effects at the site of injection (i.e. at the neuromuscular junction), BoNT/A can also undergo retrograde transport, away from the site of uptake (nerve terminals in the periphery), transcytosis from the axonal to the somatodendritic compartment, release from the latter compartment, and re-uptake into the nerve terminals of upstream, connected neurons in the central nervous system (CNS), where it exerts some of its medicinal effects. Whether any other BoNTs have distal effects is an issue that has not been explored. The goal of this proposal is to directly determine whether any of the BoNTs (A-G) do in fact undergo retrograde transport, transcytosis, release and re-uptake into upstream, connected neurons in a catalytically active form. In Aim 1 we will determine whether toxins that are added to the cis macrochannel of a compartmentalized microfluidic device (which contains only axons), are transported to, and act within, the trans macrochannel of the device (which contains axons, dendrites, and cell bodies). Strikingly, our preliminary data indicate that many of the BoNTs, including BoNT/A and B, do in fact undergo retrograde transport to the trans macrochannel in an active form. This work will include single particle tracking of toxins conjugated to quantum dots (Qdots) to directly visualize, and quantitatively analyze, transport. In Aim 2 we will address the question of whether the toxins undergo transcytosis, release, and re-uptake to act on neurons upstream of the 'primary' neurons that mediated the initial entry step. Release and re-uptake of the toxins would occur via vesicular carriers, and the fusion/recycling of these carriers can be blocked, in principle, using a CNT distinct from the toxi under study. For example, preliminary data indicate that prior cleavage of the SNARE synaptobrevin, in the trans macrochannel, with TeNT, prevents BoNT/A from cleaving its SNARE substrate, SNAP-25, within the trans macrochannel (after BoNT/A was initially taken up in the cis macrochannel). These data directly demonstrate that BoNT/A acts on neurons upstream of the 'primary' neuron that mediated initial entry. By conducting these experiments with all of the CNTs, we will determine which toxins have only local actions, and which toxins have previously undetected distal actions. This work will shed new light regarding the mechanism of action of these agents.

描述（由申请人提供）：梭菌神经毒素（CNT）包括八种相关毒素家族：破伤风（TeNT）和七种肉毒杆菌神经毒素（BoNT/A-G），它们分别引起破伤风和肉毒杆菌中毒。 BoNT/A 和 BoNT/B 还在临床上用于治疗多种严重的疾病，包括肌张力障碍和疼痛；这代表着每年价值 20 亿美元的产业。传统上认为 BoNT/A 和 B 的治疗作用涉及在注射部位局部抑制神经元释放神经递质（通过充当选择性裂解 SNARE 蛋白的蛋白酶）；在肌张力障碍的情况下，这可能会导致骨骼肌松弛。然而，一个新的假设认为，除了在注射部位（即神经肌肉接头处）产生局部作用外，BoNT/A 还可以逆行转运，远离摄取部位（外周神经末梢），从轴突转胞吞到体树突室，从后一室释放，并重新摄取到神经末梢。 中枢神经系统（CNS）中上游相连的神经元，在那里它发挥一些药用作用。其他 BoNT 是否具有远端作用是一个尚未探讨的问题。 该提案的目标是直接确定是否有任何 BoNT (A-G) 实际上以催化活性形式经历逆行转运、转胞吞作用、释放和重新摄取到上游、连接的神经元中。在目标 1 中，我们将确定添加到区室化微流体装置（仅包含轴突）的顺式大通道中的毒素是否被运输到装置的反式大通道（包含轴突、树突和细胞体）并在其内起作用。引人注目的是，我们的初步数据表明，许多 BoNT，包括 BoNT/A 和 B，实际上确实以活性形式逆行转运至反式大通道。这项工作将包括对与量子点 (Qdot) 结合的毒素进行单粒子跟踪，以直接可视化和定量分析运输。在目标 2 中，我们将解决毒素是否经历转胞吞作用、释放和再摄取以作用于介导初始进入步骤的“初级”神经元上游神经元的问题。毒素的释放和重新摄取将通过囊泡载体发生，原则上可以使用与所研究的毒素不同的碳纳米管来阻止这些载体的融合/回收。例如，初步数据表明，在反式大通道中用 TeNT 预先切割 SNARE 突触短蛋白，可防止 BoNT/A 在反式大通道内切割其 SNARE 底物 SNAP-25（在 BoNT/A 最初被顺式大通道吸收之后）。这些数据直接证明 BoNT/A 作用于介导初始进入的“初级”神经元上游的神经元。通过对所有碳纳米管进行这些实验，我们将确定哪些毒素仅具有局部作用，哪些毒素具有以前未检测到的远端作用。这项工作将为这些药物的作用机制提供新的线索。