Gs signaling in synaptic development and function

描述(由申请人提供)：神经系统的功能依赖于发育过程中神经元和目标之间复杂、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，神经活动通过腺酰环化酶(AC)的钙调节cAMP水平，导致FASII等同嗜性细胞黏附分子水平降低，这些分子通过抑制突触生长而发挥作用；因此，通过活性介导的突触cAMP增加下调FASII是突触结构扩张所必需的。然而，钙调节的急性冠脉综合征是重要的符合探测器，它通过异源三聚体G蛋白Gs将神经元钙的增加与跨膜受体的激活结合在一起，刺激急性冠脉综合征。为了验证通过S的受体信号在突触生长中发挥作用的想法，我们利用了这样一个事实，即所有受体介导的通路或激活的ACS都需要Gs复合体的a亚单位(GSA)。与GSA信号的作用一致，我们已经证明了GSA蛋白集中在生长的突触末端，编码GSA的基因的突变抑制了神经元的树枝和突触的形成，导致在行为和生理水平上的感觉-运动过程的缺陷。此外，GSA的限制性表达表明，GSA通路可能参与了突触前和突触后细胞之间的相互作用，这是成熟突触生长和发育所必需的。这些和其他初步结果表明，依赖于GSA的AC活性调节在突触生长和可塑性的过程中发挥着重要作用。为了进一步验证这一假说，这一建议集中在以下三个具体目标上：1.DGS突变所产生的NMJ表型的综合评估。为了以更高的分辨率研究亚型DGS突变体中NMJ的形成，我们将在电磁水平上仔细量化这些突变所产生的超微结构缺陷，并使用电生理方法来确定突触形态改变是否伴随着突触传递的改变。此外，为了验证我们的工作假设，即亚型DGS突变会导致通过GSA的信号减弱，但不会被消除，我们将检查GSA C末端额外残基突变所产生的表型，并通过生化方法直接评估通过单个突变体GSA的受体介导的信号。GSA信号通过遗传上位性在NMJ形成中作用的测试模型。这个目标的目的是通过检查由特定突变通过遗传上位性定义的过程之间的功能关系来检验我们修订的NMJ形成模型。我们的重点将放在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，影响神经元活动、cAMP、细胞黏附的突变)。我们将利用这些研究，在每个组织(神经元和肌肉)的背景下，精确地确定受体依赖的腺酰环化酶通过GSA激活在幼虫NMJ.3建立和生长突触连接中的作用。确定在NMJ形成过程中参与GSA激活的途径的分子。果蝇NMJ的一个主要优势是可以应用强大的遗传学方法来识别参与GSA调节突触可塑性的分子。因此，这一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识别在NMJ形成过程中特定操作的GSA激活途径的参与者。

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.