Genetic approaches to neurotrophin signaling

描述(申请人提供)：这项建议旨在确定神经营养素在发育中和成人神经系统中调节突触结构和功能中通过Trk受体发出信号的作用。将以小鼠神经肌肉突触为模型系统。由神经肌肉连接、突触周围雪旺细胞、突触前运动神经元终末和突触后肌肉纤维组成的细胞类型均表达神经营养因子和Trks，提示该突触的神经营养因子信号是多方向的，涉及所有三种细胞类型。同样，中枢神经系统神经元表达多种神经营养因子和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NT4/5)主要在突触后表达，在乙酰胆碱受体(AChR)富集区及其周围的肌纤维膜上表达，而TrkC亚型定位于突触周围的雪旺细胞，TrkA不定位于神经肌肉连接。通过腺病毒介导的截短的、非信号形式的TrkB(TrkB.t1)的过度表达，下调肌肉纤维中的TrkB信号，导致突触后AChR富集区的拆除。相反，初步结果显示，TrkC通过这些细胞调节突起的延长，这些细胞在轴突萌发和再神经支配中发挥重要作用。这些观察结果导致假设，通过TrkB或TrkC受体发出信号的配体交换在突触成熟和维持中发挥着不同的功能作用。虽然腺病毒方法是有用的，并得到另一项拨款的支持，以确定这些信号分子在突触中的作用，但选择性地从神经肌肉突触的一种细胞类型中删除神经营养素或Trk基因将是必不可少的。由于小鼠的许多相关突变会导致围产期死亡，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，NT3)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特征来分析。这些实验结果将使我们对神经营养素和Trk介导的信号在发育中和成年突触中的功能作用(S)有了新的见解，并扩大了我们对前向和逆行突触信号在外周和中枢神经系统中的相对作用的理解。

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.