Investigating how signaling via adhesion GPCR Latrophilins regulates synapse formation and specificity in the hippocampus

项目总结： 大脑的功能需要组装和维持不同的突触亚型，形成刻板的神经回路。然而，神经回路中输入特异性突触形成的基本细胞和分子机制仍然知之甚少。我们在K99期的研究揭示了粘附性GPCRLatrophins(Lphns)在海马兴奋性突触特异性中的重要作用。我们最近利用救援性实验发现，Lphn2/3在海马输入特异性兴奋性突触形成中的作用需要G蛋白和/或u-arrestin偶联。在平行研究中，我们发现区块化的突触后cAMP信号是兴奋性突触形成的显著驱动因素。范德比尔特大学R00阶段的工作将集中在研究哺乳动物中枢神经系统突触上的G蛋白和u-arrestin的细胞内信号机制以及其他粘附性GPCRs。此外，我们还将确定未研究的粘附性GPCRs在神经回路中的功能，并定义大脑中孤儿粘附性GPCRs的细胞外配体。我们将开发新的分子工具，包括病毒介导的纳米体靶向信号调节器，以探索区块化突触信号通路在神经回路中输入特异性兴奋性、抑制性和神经调制性突触形成中的作用。我们还将开发新的实时超分辨率风暴和共聚焦成像技术，以实时成像突触的形成、维护和消除。我们希望这些研究将揭示哺乳动物中枢神经系统中不同突触如何建立神经回路的基本原理，并阐明大脑中未被充分研究的粘附性GPCRs的功能和信号机制。

PROJECT SUMMARY: Brain functions require the assembly and maintenance of distinct synaptic subtypes into stereotyped neural circuits. However, the fundamental cellular and molecular mechanisms underlying input-specific synapse formation in neural circuits remain poorly understood. Our studies during the K99 phase revealed an important role of the adhesion GPCR Latrophilins (Lphns) in hippocampal excitatory synaptic specificity. We recently found using rescue experiments that the role of Lphn2/3 in hippocampal input-specific excitatory synapse formation requires G-protein and/or u-Arrestin coupling. In parallel studies, we found that compartmentalized postsynaptic cAMP signaling is a prominent driver of excitatory synapse formation. Efforts during the R00 phase at Vanderbilt University will be focused on examining the G-protein and u-Arrestin intracellular signaling mechanisms of Lphns and other adhesion GPCRs at synapses in the mammalian central nervous system. Furthermore, we will determine the functions of unstudied adhesion GPCRs in neural circuits, and define the extracellular ligands of orphan adhesion GPCRs in the brain. We will develop novel molecular tools including viral-mediated nanobody targeting of signaling modulators to probe the roles of compartmentalized synaptic signaling pathways in input-specific excitatory, inhibitory and neuromodulatory synapse formation in neural circuits. We will also develop novel live super-resolution STORM and confocal imaging techniques to image synapse formation, maintenance and elimination in real-time. We expect these studies to reveal fundamental principles of how neural circuits are established by diverse synapses in the mammalian central nervous system, and elucidate the functions and signaling mechanisms of the understudied adhesion GPCRs in the brain.