Distal effects of botulinum neurotoxins

描述（由申请人提供）：梭状菌神经毒素（CNTs）由8种相关毒素组成：破伤风（TeNT）和7种肉毒杆菌神经毒素（BoNT/ a - g），它们分别引起破伤风和肉毒中毒。BoNT/A和BoNT/B在临床上也用于治疗各种严重的疾病，包括肌张力障碍和疼痛；这代表着一个每年20亿美元的产业。BoNT/A和B的治疗作用传统上被认为涉及局部-在注射部位-抑制神经元的神经递质释放（通过作为选择性切割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，一种新的假设认为，除了在注射部位（即神经肌肉接点）有局部作用外，BoNT/ a还可以逆行运输，远离摄取部位（外周神经末梢），从轴突胞吞到体突室，从后者释放，再被摄取到中枢神经系统（CNS）上游连接神经元的神经末梢。在那里它发挥了一些药用作用。是否有其他BoNTs具有远端效应是一个尚未探讨的问题。这项提议的目标是直接确定是否有任何的bont （a - g）确实以催化活性的形式逆行运输、胞吞、释放和再摄取到上游连接的神经元。在目的1中，我们将确定添加到区隔化微流体装置（仅包含轴突）的顺式大通道中的毒素是否被运输到该装置（包含轴突、树突和细胞体）的反式大通道中并在其中起作用。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运输到跨宏观通道。这项工作将包括单粒子跟踪毒素共轭量子点（Qdots），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过胞吞、释放和再摄取来作用于介导初始进入步骤的“初级”神经元上游的神经元的问题。毒素的释放和再吸收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/再循环。例如，初步数据表明，在跨大通道中，先用TeNT切割SNARE突触brevin，可以阻止BoNT/A在跨大通道中切割其SNARE底物SNAP-25（在BoNT/A最初在顺式大通道中被吸收之后）。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。

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.