Gs signaling in synaptic development and function

描述（由申请人提供）：神经系统的功能取决于发育过程中神经元和靶标之间复杂有序的突触连接的构建。在果蝇幼虫神经肌肉连接处（NMJ）和许多其他突触，神经活动通过Ca2+调节腺苷酸环化酶（AC）来调节cAMP水平，导致亲同质细胞粘附分子（如FASII）水平的降低，这些分子通过抑制突触生长起作用；因此，通过活动介导的突触cAMP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ACs是重要的巧合检测器，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ACs。为了验证通过s的受体信号传导在突触生长中起作用的观点，我们利用了所有受体介导的途径或ACs的激活都需要Gs复合物（Gsa）的a亚基这一事实。与Gsa信号的作用一致，我们已经证明Gsa蛋白集中在生长的突触钮扣中，编码Gsa的基因突变抑制神经元树突和钮扣形成，导致在行为和生理水平上分析的感觉运动过程的缺陷。此外，Gsa的限制性表达表明，Gsa通路可能参与成熟突触生长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，依赖于gsa的AC活性调节在突触生长和可塑性的过程中起着重要作用。为了进一步验证这一假设，本提案侧重于以下三个具体目标：1。dgs突变产生的NMJ表型的综合评价。为了以更高的分辨率研究次形dgs突变体中NMJ的形成，我们将仔细量化这些突变在em水平上产生的超微结构缺陷，并使用电生理学方法来确定突触形态的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，即次形dgs突变导致Gsa信号减弱，而不是消除，我们将检查Gsa C端其他残基突变产生的表型，并通过生化方法直接评估通过单个突变Gsa的受体介导信号。Gsa信号通过遗传上位作用在NMJ形成中的作用的测试模型。这一目标的目的是通过检查通过遗传上位性特定突变定义的过程之间的功能关系来测试我们修订的NMJ形成模型。我们的重点将放在已经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了证明（例如，影响神经元活动、cAMP、细胞粘附的突变）。我们将利用这些研究来精确定义每个组织（神经元和肌肉）中腺苷酸环化酶的受体依赖性激活在突触连接的建立和生长中的作用。鉴定参与NMJ形成过程中由Gsa激活的通路的分子。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的gsa调节的分子。因此，本研究的目的是通过使用果蝇可能的遗传相互作用策略，确定在NMJ形成过程中特异性操作的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.