Gs signaling in synaptic development and function

突触发育和功能中的 Gs 信号传导

• 批准号: 6750167
• 负责人: MICHAEL A FORTE
• 金额: $ 28.69万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-08-01 至 2007-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6750167
• 关键词: Drosophilidae; G protein; adenylate cyclase; biological signal transduction; cyclic AMP; dopamine; electron microscopy; electrophysiology; enzyme activity; gene interaction; gene mutation; genetic screening; genetically modified animals; neural plasticity; neural transmission; neurogenetics; neuromuscular junction; neurophysiology; octopamine; phenotype; receptor; receptor expression; synaptogenesis; transfection

DESCRIPTION (provided by applicant): Nervous system function depends on the construction of complex, ordered synaptic connections among neurons and targets during development. At the Drosophila larval neuromuscular junction (NMJ), and many other synapses, neural activity regulates cAMP levels through Ca2+ regulation of adenylyl cyclases (AC), leading to reductions in the levels of the homophilic cell adhesion molecules like FASII that act by restraining synaptic growth; thus down regulation of FASII through the activity-mediated increase in synaptic cAMP is necessary for structural expansion of the synapse. However, Ca+2 regulated ACs are important coincidence detectors, integrating increases in neuronal Ca+2 with the activation of transmembrane receptors coupled to the stimulation of ACs through the heterotrimeric G protein, Gs. To test the idea that receptor signaling through s plays a role in synaptic growth, we have taken advantage of the fact that all receptor-mediated pathways or activation of ACs require the a subunit of the Gs complex (Gsa). Consistent with a role for Gsa signaling, we have shown that the Gsa protein is concentrated in growing synaptic boutons and that mutations in the gene encoding Gsa inhibit neuronal arborization and bouton formation, leading to deficits in sensory-motor processes as assayed on both a behavioral and physiological level. Furthermore, restricted expression of Gsa indicates that Gsa pathways are likely involved in the reciprocal interactions between pre- and postsynaptic cells required for the growth and development of mature synapses. These and other preliminary results suggest that Gsa-dependent regulation of AC activity plays an important role during processes that underlie synaptic growth and plasticity. To further test this hypothesis, this proposal focuses on the following three specific aims:1. Comprehensive Assessment of the NMJ Phenotypes Generated by dgs Mutations.In order to investigate the formation of NMJ in hypomorphic dgs mutants at higher resolution, we will carefully quantify ultrastructural defects generated by these mutations at the E.M. level and use of electrophysiological approaches to determine if altered synaptic morphology is accompanied by altered synaptic transmission. In addition, to test our working hypothesis that hypomorphic dgs mutations result in attenuated, but not eliminated, signaling through Gsa, we will examine phenotypes generated by mutations in additional residues in the C terminus of Gsa and directly assess receptor-mediated signaling though individual mutant Gsa by biochemical approaches.2. Test Models of the Role of Gsa Signaling in NMJ Formation through Genetic Epistasis.The object of this aim is to test our revised model of NMJ formation by examining the functional relationship between processes defined by specific mutations through genetic epistasis. Our focus will be on mutations which have been used to develop existing models, since the utility of this approach has already been demonstrated (e.g., mutations affecting neuronal activity, cAMP, cell adhesion). We will use these studies to precisely define the role of receptor-dependent activation of adenylyl cyclase through Gsa within the context of each tissue (neurons and muscle) in the establishment and growth of synaptic connections at the larval NMJ.3. Identify Molecules that Participate in Pathways Activated By Gsa during NMJ Formation.A major advantage of the Drosophila NMJ is that powerful genetic approaches can be applied in the identification of molecules that participate in Gsa-regulation of synaptic plasticity. Thus, the goal of this aim is to identify participants in the Gsa-activated pathway operating specifically during NMJ formation through the use of the genetic interaction strategies possible in Drosophila.

描述（由申请人提供）：神经系统功能取决于在发育过程中神经元和目标之间复杂的，有序的突触连接的构建。在果蝇幼虫神经肌肉连接（NMJ）以及许多其他突触中，神经活动通过Ca2+调节腺苷酸环酶（AC）调节cAMP水平，从而导致均质细胞粘附分子（如FASII）的水平降低，例如FASII，该水平通过限制了Synaptic Grownper cartion Synaptic Exprient synaptic;因此，通过活性介导的突触cAMP的增加对FASII的下降是突触的结构扩张所必需的。然而，Ca+2受调节的AC是重要的巧合探测器，将神经元Ca+2的增加与通过异构三聚体G蛋白GS刺激的跨膜受体的激活相结合。为了测试通过S通过S的受体信号在突触生长中起作用的想法，我们利用了这样一个事实，即所有受体介导的途径或AC的激活都需要GS复合物（GSA）的亚基。与GSA信号传导的作用一致，我们已经表明，GSA蛋白集中在生长的突触胸子上，并且编码GSA的基因中的突变抑制了神经元一半型和胸子形成，从而导致在行为和生理学水平上分析了感觉运动过程中的缺陷。此外，GSA的受限表达表明，GSA途径可能参与成熟突触的生长和发展所需的突触前和突触后细胞之间的相互作用。这些和其他初步结果表明，AC活性的GSA依赖性调节在构成突触生长和可塑性的过程中起着重要作用。为了进一步检验这一假设，该提案重点介绍以下三个特定目的：1。 Comprehensive Assessment of the NMJ Phenotypes Generated by dgs Mutations.In order to investigate the formation of NMJ in hypomorphic dgs mutants at higher resolution, we will carefully quantify ultrastructural defects generated by these mutations at the E.M. level and use of electrophysiological approaches to determine if altered synaptic morphology is accompanied by altered synaptic transmission.此外，为了检验我们的工作假设，即肌dgs突变导致通过GSA发出的信号导致但未消除，我们将检查GSA C末端中其他残基中突变产生的表型，并直接通过生物化学方法来评估受体介导的信号传导，虽然受体介导的信号传导。2。 GSA信号在NMJ形成中的作用通过遗传上的测试模型。该目的的目的是通过检查通过特定突变通过遗传性遗传的特定突变定义的过程之间的功能关系来测试我们的NMJ形成的修订模型。我们的重点将放在用于开发现有模型的突变上，因为已经证明了这种方法的实用性（例如，影响神经元活动的突变，cAMP，细胞粘附）。我们将使用这些研究来精确地定义了在每个组织（神经元和肌肉）在幼虫NMJ.3处的突触连接的建立和生长中，在每个组织（神经元和肌肉）的背景下，通过GSA激活受体依赖性的激活。确定参与NMJ形成过程中GSA激活的途径的分子。果蝇NMJ的主要优势是，可以将强大的遗传方法应用于鉴定参与GSA的突触可变性的分子。因此，该目标的目的是通过使用果蝇中可能的遗传相互作用策略来确定在NMJ形成期间专门运行的GSA激活途径中的参与者。