Gs signaling in synaptic development and function

描述（由申请人提供）：神经系统功能取决于发育过程中神经元和靶点之间复杂有序的突触连接的构建。在果蝇幼虫神经肌肉接头（NMJ）和许多其他突触，神经活动通过腺苷酸环化酶（AC）的Ca 2+调节来调节cAMP水平，导致通过抑制突触生长起作用的嗜同性细胞粘附分子（如FASII）水平降低;因此，通过突触cAMP活性介导的增加来下调FASII对于突触的结构扩张是必要的。然而，Ca+2调节的AC是重要的重合检测器，将神经元Ca+2的增加与跨膜受体的激活结合起来，通过异源三聚体G蛋白Gs刺激AC。为了验证通过s的受体信号传导在突触生长中起作用的想法，我们利用了所有受体介导的通路或AC的激活都需要Gs复合物（Gsa）的a亚基的事实。与Gsa信号传导的作用一致，我们已经表明，Gsa蛋白集中在不断增长的突触终扣中，并且编码Gsa的基因中的突变抑制神经元树枝化和终扣形成，导致在行为和生理水平上测定的感觉运动过程中的缺陷。此外，Gsa的限制性表达表明，Gsa途径可能参与成熟突触的生长和发育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，GSA依赖的AC活动的调节过程中起着重要的作用，突触生长和可塑性的基础。为了进一步验证这一假设，本建议侧重于以下三个具体目标：1。由dgs突变产生的NMJ表型的综合评估为了以更高的分辨率研究亚纯型dgs突变体中NMJ的形成，我们将仔细量化由这些突变在E.M.水平和使用电生理方法来确定改变的突触形态是否伴随着改变的突触传递。此外，为了检验我们的工作假设，即亚型dgs突变导致减弱，但不消除，通过Gsa的信号传导，我们将检查由Gsa C末端中的额外残基突变产生的表型，并通过生物化学方法直接评估通过单个突变体Gsa的受体介导的信号传导。通过遗传上位性对Gsa信号在NMJ形成中的作用的测试模型本研究的目的是通过检查通过遗传上位性由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。我们的重点将放在已用于开发现有模型的突变上，因为这种方法的实用性已经得到证明（例如，影响神经元活性、cAMP、细胞粘附的突变）。我们将使用这些研究来精确地定义在幼虫NMJ的突触连接的建立和生长中的每个组织（神经元和肌肉）的上下文中通过Gsa的腺苷酸环化酶的受体依赖性激活的作用。识别参与NMJ形成过程中Gsa激活途径的分子果蝇NMJ的一个主要优势是，强大的遗传方法可以应用于识别参与突触可塑性Gsa调节的分子。因此，这个目标的目的是确定参与GSA激活的途径，特别是在NMJ形成过程中，通过使用可能在果蝇的遗传相互作用策略。

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.