Genetic approaches to neurotrophin signaling

描述（由申请人提供）：本提案旨在确定神经营养因子在发育和成人神经系统中通过Trk受体发出信号调节突触结构和功能的作用。小鼠神经肌肉突触将被用作模型系统。细胞类型，包括神经肌肉接头，突触周围的施万细胞，突触前运动神经元末梢，和突触后肌纤维，每一个表达的神经营养因子和Trks的补充，这表明在这个突触神经营养因子信号是多方向的，涉及所有三种细胞类型。类似地，CNS神经元表达几种神经营养因子和Trks，但每个信号通路在突触成熟和维持中的相对作用尚不清楚。以前的工作和我们实验室的初步结果表明，TrkB亚型（结合神经营养因子BDNF和NT 4/5）主要在突触后表达，在乙酰胆碱受体（AChR）丰富的区域内和周围的肌纤维膜中，而TrkC亚型定位于突触周的许旺细胞，TrkA不定位于神经肌肉接头。通过腺病毒介导的TrkB截短的非信号形式（TrkB.t1）的过度表达下调肌纤维中的TrkB信号，诱导突触后AChR丰富区域的拆除。相比之下，初步结果表明，TrkC调节这些细胞在轴突发芽和神经再支配中发挥重要作用的过程的延伸。这些观察结果导致的假设，通过TrkB或TrkC受体信号的配体的交换在突触成熟和维持中发挥功能上不同的作用。虽然腺病毒方法是有用的，并得到另一项资助的支持，以确定这些信号分子在突触中的作用，但必须选择性地删除神经肌肉突触中一种细胞类型的神经营养因子或Trk基因。由于小鼠中的许多相关突变导致围产期致死，我们将使用cre介导的重组以空间和时间控制的方式从感兴趣的细胞中删除神经营养因子或Trks。神经营养因子（BDNF，NT 3）或TrkB缺失对神经肌肉突触结构和功能的影响将通过体内成像，免疫染色，共聚焦显微镜和突触强度的电生理表征进行分析。这些实验的结果将提供新的见解神经营养因子和Trk介导的信号传导在发育和成年突触的功能作用，并扩展我们的理解的相对作用，在外周神经系统以及中枢神经系统中的突触和逆行信号传导。

DESCRIPTION (provided by applicant): This proposal is aimed at determining the role of neurotrophins that signal through Trk receptors in modulating synapse structure and function in the developing and adult nervous system. Mouse neuromuscular synapses will be used as a model system. The cell types that comprise neuromuscular junctions, the perisynaptic Schwann cells, presynaptic motor neuron terminals, and postsynaptic muscle fibers, each express a complement of neurotrophins and Trks, suggesting that neurotrophin signaling at this synapse is multi-directional and involves all three cell types. Similarly, CNS neurons express several neurotrophins and Trks, but the relative roles of each signaling pathway in synaptic maturation and maintenance are unclear. Previous work and preliminary results from our lab showed that TrkB isoforms (which bind the neurotrophins BDNF and NT4/5) are expressed primarily postsynaptically, in the muscle fiber membrane in and around acetylcholine receptor (AChR) rich regions, while TrkC isoforms are localized to perisynaptic Schwann cells and TrkA is not localized to neuromuscular junctions. Down-regulation of TrkB signaling in muscle fibers, via adenovirus-mediated over-expression of a truncated, non-signaling form of TrkB (TrkB.t1), induced the dismantling of postsynaptic AChR rich regions. In contrast, preliminary results show that TrkC modulates the extension of processes by these cells that play important roles in axon sprouting and reinnervation. These observations lead to the hypothesis that exchange of ligands that signal through TrkB or TrkC receptors play functionally distinct roles in synaptic maturation and maintenance. While adenoviral methods are useful, and are the supported by another grant, to determine the role of these signaling molecules at synapses, it will be essential to selectively delete neurotrophin or Trk genes from one of the cell types at neuromuscular synapses. Because many of the relevant mutations in mice result in perinatal lethality, we will use cre-mediated recombination to delete neurotrophins or Trks from cells of interest in a spatially and temporally controlled fashion. The effect of neurotrophin (BDNF, NT3) or TrkB deletion on neuromuscular synaptic structure and function will be analyzed with in vivo imaging, immunostaining, confocal microscopy and electrophysiological characterization of synaptic strength. The results of these experiments will provide new insights into the functional role(s) of neurotrophin and Trk-mediated signaling at developing and adult synapses, and extend our understanding of the relative roles of antero- and retrograde synaptic signaling in the peripheral as well as central nervous system.