REGULATION OF SYNAPTIC DEVELOPMENT AND FUNCTION BY NEUROTROPHIC FACTORS

这项研究的目的是了解它们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、C及其下游信号分子。考察……的作用 神经营养因子在突触形成中，我们将产生DNA结构 编码融合蛋白，其中不同元素的成分 突触(释放区、突触小泡)，如VAMP、突触素I、 和钙通道β亚基与绿色荧光蛋白融合 (GFP)。这些干细胞将被注射到非洲爪哇胚胎细胞中，其后代 包括视网膜神经节细胞。“标记”视网膜异体植入术 从这些胚胎到新鲜胚胎，视网膜轴突和突触的发育将是 在视顶盖进行研究。神经营养因子对突触形成的影响 而BDNF受体TrkB的主要负性结构将是 学习。在分化中重要的信号通路将是 通过注入TrkA结构确定，这些结构在与 单独的下游信号分子及其决定作用 外源性神经生长因子。在将GFP嵌合体插入到有缺陷的 腺病毒，感染小鼠视网膜将允许检查突触 正常和神经营养素缺陷突变小鼠的发育。第二 该项目将寻求了解神经营养因子的分子基础 其他人在非洲爪哇神经肌肉组织观察到的胞吐作用的调节 连接和Schaeffer侧支CA1突触分布在海马区.我们 将确定NGF应用对突触传递的影响 在注射TrkA前体后，非洲爪哇NMJ。向下游解剖 信号通路，TrkA受体在特异性激活中的缺陷 信令路径将在相同的模型中使用。为了进一步描绘 一条途径，下游显性负向结构的影响 信号分子，如PI-3激酶和ras，将被检测。努力 将这一分析扩展到生物化学 利用PC12细胞表达具有信号转导的受体的顺应性系统 神经营养因子应用对血管紧张素转运蛋白表达的影响 与胞吐装置相关的蛋白质的磷酸化将 可以进行检查，并可以评估功能后果。

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.