Mechanisms of Neuromuscular Repair by Schwann Cells

描述（由申请人提供）：完整的运动神经末梢响应于麻痹或部分去神经而发芽，并与失活的肌纤维重新建立功能性突触连接，从而确保维持肌肉神经支配和力量。神经肌肉可塑性的这一显著特征已被深入研究，这既是因为它对我们理解突触形成和维持至关重要，也是因为它为促进神经损伤的修复提供了潜在的途径。然而，这种类型的神经可塑性的机制知之甚少。我们和其他人最近表明，覆盖神经末梢的终末雪旺细胞（tSC）在这个过程中发挥着核心作用，通过延长触发末梢发芽的过程，并将这些芽引导到不活跃的肌纤维。我们现在提出鉴定激活静止的tSC以延长过程的分子和激活的tSC用于引发发芽的分子。目的1将测试减少的神经递质释放和随后的毒蕈碱信号传导失活激活tSC的假设。将使用该毒蕈碱信号传导在体内的药理学抑制和毒蕈碱信号传导缺陷的敲除小鼠系的分析。目的2将评估tSC在介导生长因子诱导的发芽中的作用。我们将通过外源性应用IGFs和CNTF（两种已知会引发发芽的生长因子）来测试这一点，以确定这些药物是否激活tSC以延长过程。然后，我们将检查CNTF -/-小鼠，其神经末梢显然不会响应于部分去神经支配或肉毒杆菌毒素而发芽，以确定它们的tSC是否在对正常发芽刺激的反应中有缺陷。在目标3中，我们将检验NCAM和N-钙粘蛋白（在活化的tSC中上调的两种细胞粘附分子（CAM））引起发芽的假设。我们将利用弹道转染技术在体内静止的tSC中选择性地过表达这些CAM，并检查终末发芽的发生率。第二，我们将转基因表达CAM的成纤维细胞移植到完整的终板上，以确定这些成纤维细胞是否获得诱导终末发芽的能力。通过这项工作，我们试图进一步确定雪旺细胞在反应性发芽中的作用，并获得新的见解与终端发芽之间和细胞内信号。

DESCRIPTION (provided by applicant): Intact motor nerve terminals sprout in response to paralysis or partial denervation and re-establish functional synaptic connections with inactive muscle fibers, thus insuring the maintenance of muscle innervation and strength. This remarkable feature of neuromuscular plasticity has been studied intensively, both because it is essential to our understanding of synapse formation and maintenance and because it provides a potential avenue for promoting repair of nerve injuries. However, the mechanisms underlying this type of neural plasticity are poorly understood. We and others have recently shown that the terminal Schwann cells (tSCs) that cap the nerve terminal play a central role in this process, by extending processes that trigger terminals to sprout and that guide these sprouts to inactive muscle fibers. We now propose to identify the molecules that activate quiescent tSCs to extend processes and the molecules that activated tSCs use to elicit sprouting. Aim 1 will test the hypothesis that reduced neurotransmitter release, and subsequent inactivation of muscarinic signaling, activates tSCs. Pharmacological inhibition of this muscarinic signaling in vivo and analyses of knockout mouse lines that are defective in muscarinic signaling will be used. Aim 2 will assess the role of tSCs in mediating growth factor induced sprouting. We will test this by exogenous application of IGFs and CNTF, two growth factors known to elicit sprouting, to determine whether these agents activate tSCs to extend processes. We will then examine CNTF -/- mice, whose nerve terminals apparently do not sprout in response to partial denervation or botulinum toxin, to determine whether their tSCs are defective in their response to normal sprouting stimuli. In Aim 3, we will test the hypothesis that NCAM and N-cadherin, the two cell adhesion molecules (CAMs) upregulated in activated tSCs, elicit sprouting. We will exploit the ballistic transfection technique to overexpress these CAMs selectively in quiescent tSCs in vivo, and examine the incidence of terminal sprouting. Secondly, we will transplant fibroblasts genetically modified to express the CAMs to intact endplates, to determine whether these fibroblasts acquire the ability to induce terminal sprouting. Through this work, we attempt to further define the role of Schwann cells in the reactive sprouting and to gain new insights into the inter- and intracellular signaling associated with terminal sprouting.