REGULATION OF SYNAPTIC DEVELOPMENT AND FUNCTION BY NEUROTROPHIC FACTORS

本研究的目的是了解在调节突触中的作用 神经营养因子及其受体trkA的形成、功能和可塑性， B和C及其下游信号分子。审查的作用 神经营养因子在突触形成中的作用，我们将产生DNA构建体 编码融合蛋白，在该融合蛋白中， 突触（释放区，突触囊泡），如VAMP，突触蛋白I， 和Ca通道β亚基与绿色荧光蛋白融合 （GFP）。这些将被注入非洲爪蟾胚胎细胞， 包括视网膜神经节细胞。移植“标记”视网膜原基后 从这些到新鲜的胚胎，视网膜轴突和突触的发育将是 在视顶盖中进行研究。神经营养素对突触形成的影响 而BDNF受体trkB的显性负性结构将被 研究了在分化中重要的信号通路将是 通过注射在与以下物质的相互作用中有缺陷的trkA构建体来测定： 单个下游信号传导分子和确定 外源性NGF在将GFP嵌合体插入到缺陷的 腺病毒，感染鼠视网膜将允许检查突触 在正常和神经营养因子缺陷突变小鼠中的发育。第二 该项目将寻求了解神经营养因子的分子基础 调节胞吐作用，其他人在非洲爪蟾神经肌肉 连接和Schaeffer侧支CA 1突触。我们 将确定神经生长因子应用对突触传递的影响， 注射trkA前体后的非洲爪蟾NMJ。去解剖下游 信号通路，trkA受体在特定的 信号通路将用于相同的模型中。进一步界定 一个途径，下游显性负结构的影响 将检测信号分子，如PI-3激酶和ras。努力 将使这种分析扩展到生物化学 使用表达具有信号传导的受体的PC 12细胞的可操作系统 神经营养素应用对神经细胞的影响 与胞吐器相关的蛋白质的磷酸化将 可以检查和评估功能后果。

The goal of this research is to understand the roles in regulating synapse formation, function and plasticity of neurotrophins, their receptors trkA, B, and C and their downstream signaling molecules. To examine the role of neurotrophins in synapse formation, we will generate DNA constructs encoding fusion proteins in which constituents of different elements of the synapse (release zone, synaptic vesicle), such as VAMP, synapsin I, and the Ca channel beta subunit are fused to Green Fluorescent Protein (GFP). These will be injected into Xenopus embryo cells whose descendents include retinal ganglion cells. After grafting of "tagged" retinal anlage from these to fresh embryos, retinal axon and synapse development will be studied in the optic tectum. Effects on synapse formation of neurotrophins and dominant negative constructs of the BDNF receptor trkB will be studied. Signaling pathways important in differentiation will be determined by injecting trkA constructs defective in interactions with individual downstream signaling molecules and determining effects of exogenous NGF. After insertion of the GFP chimeras into a defective Adenovirus, infections of murine retina will permit examination of synapse development in normal and neurotrophin-deficient mutant mice. The second project will seek to understand the molecular bases of neurotrophin regulation of exocytosis, observed by others at the Xenopus neuromuscular junction and Schaeffer collateral CA 1 synapses in the hippocampus. We will determine effects of NGF application on synaptic transmission at the Xenopus NMJ after injection of precursors with trkA. To dissect downstream signaling pathways, trkA receptors defective in activation of specific signalling pathways will be used in the same model. To further delineate a pathway, effects of dominant negative constructs of downstream signalling molecules, such PI-3 kinase and ras, will be examined. Efforts will be made to extend this analysis will be extended to a biochemically amenable system using PC12 cells expressing receptors with signalling pathway deficiencies where effects of neurotrophin application on phosphorylation of proteins associated with the exocytotic apparatus will be examined and functional consequences can be assessed.