Distal effects of botulinum neurotoxins

描述(申请人提供)：梭状芽胞杆菌神经毒素(CNT)由八种相关毒素组成：破伤风(TANT)和七种肉毒神经毒素(BONT/A-G)，分别导致破伤风和肉毒中毒。BONT/A和BONT/B还被用于临床治疗各种严重的疾病，包括肌张力障碍和疼痛；这代表着每年20亿美元的行业。传统上认为，BONT/A和B的治疗作用包括在注射部位局部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，这可能导致骨骼肌松弛。然而，一个新的假说认为，除了在注射部位(即在神经肌肉接头)有局部作用外，BoNT/A还可以逆行运输，离开摄取部位(周围的神经末梢)，从轴突向躯体树突室跨细胞，从后者释放，然后重新摄取到中枢神经系统(CNS)中上游连接的神经元的神经末梢，在那里发挥一些药效。是否有任何其他BoNTs具有远端影响是一个尚未探索的问题。这项提议的目的是直接确定是否有任何BoNTs(A-G)确实经历了逆行运输、跨细胞、释放和以催化活性形式重新摄取到上游连接的神经元。在目标1中，我们将确定添加到分区化微流控设备(只包含轴突)顺式大通道的毒素是否被运输到设备的跨大通道(包含轴突、树突和细胞体)并在其内起作用。值得注意的是，我们的初步数据表明，许多BONT，包括BONT/A和B，实际上确实以活跃的形式逆行运输到跨大通道。这项工作将包括对与量子点(Qdots)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跨细胞作用、释放和重新摄取的问题，以作用于参与初始进入步骤的“初级”神经元上游的神经元。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CNT来阻止这些载体的融合/再循环。例如，初步数据表明，在跨宏通道中，SNARE Synaptobrevin在TANET中的先前切割，阻止了BONT/A在跨宏通道内(在BONT/A最初被顺式宏通道占据之后)切割其SNAP-25。这些数据直接证明，BONT/A作用于“初级”神经元上游的神经元，该神经元介导了最初的进入。通过对所有碳纳米管进行这些实验，我们将确定哪些毒素只具有局部作用，哪些毒素具有以前未被检测到的远端作用。这项工作将为了解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.