Distal effects of botulinum neurotoxins

描述（由申请人提供）：梭菌神经毒素（CNT）包括一个由八种相关毒素组成的家族：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也在临床上用于治疗广泛的严重医学病症，包括肌张力障碍和疼痛;这代表了每年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-抑制神经递质从神经元释放（通过充当选择性切割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，一种新的假说认为，除了在注射部位产生局部效应外，（即在神经肌肉接头处），BoNT/A也可以经历逆行转运，远离摄取位点（外周的神经末梢），从轴突到体树突隔室的转胞吞，从后者隔室释放，并重新摄取到上游的神经末梢，在中枢神经系统（CNS）中连接神经元，在那里发挥其一些药用作用。是否有任何其他BoNT具有远端效应是一个尚未探索的问题。 该提议的目标是直接确定任何BoNT（A-G）是否实际上以催化活性形式经历逆行运输、转胞吞、释放和再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（其仅包含轴突）的顺式大通道中的毒素是否被运输到装置（其包含轴突、树突和细胞体）的反式大通道并在其中起作用。引人注目的是，我们的初步数据表明，许多BoNT，包括BoNT/A和B，实际上经历逆行转运到反式大通道的活性形式。这项工作将包括单粒子跟踪与量子点（Qdots）结合的毒素，以直接可视化和定量分析运输。在目标2中，我们将解决的问题，是否毒素进行转胞吞，释放和再摄取的“初级”神经元，介导的初始进入步骤的神经元上游的作用。毒素的释放和再摄取将通过囊泡载体发生，并且原则上可以使用与所研究的毒素不同的CNT来阻断这些载体的融合/再循环。例如，初步数据表明，在反式大通道中，先用TeNT切割SNARE突触泡蛋白，会阻止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）。这些数据直接表明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CNT进行这些实验，我们将确定哪些毒素仅具有局部作用，哪些毒素具有先前未检测到的远端作用。这项工作将揭示新的光有关的机制，这些代理商的行动。

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.