Dynamic regulation of small Rho GTPases via serotonin receptors in neurons: Effects on the cytoskeleton, neuronal morphology and functions

肌动蛋白骨架的重组是调控神经元形态和运动的关键步骤之一。神经元肌动蛋白细胞骨架的动态变化是由Rho家族的小gtpase控制的，包括RhoA、Rac1和Cdc42。Rho gtpase也被认为是树突和脊柱结构可塑性的重要调节因子，并且是新突触生长所需的初始分子级联反应的一部分。多项研究表明，Rac1和Cdc42是促进神经突生长和树突丝状足形成的正调节因子，而RhoA的激活诱导应激纤维形成，导致神经突缩回。尽管Rho GTPases的重要性已被广泛接受，但神经元中调控Rho GTPases活性的信号通路尚未完全表征。我们最近发现5-羟色胺受体5-HT4可以通过异源三聚体G13蛋白激活RhoA，刺激5-HT4受体可引起RhoA介导的神经突收缩。我们还发现Cdc42是5-HT7受体和G12蛋白的一种新的下游效应物。刺激5-HT7/G12信号通路促进神经突生长，促进突触发生，增强海马神经元突触活性。然而，负责这些相反的信号通路之间协调（相互作用）作用的分子机制仍然知之甚少。因此，本项目的中心目标是从分子上确定5-HT4和5-HT7受体调节小gtpase的空间排列和活性依赖性再分布导致细胞骨架重组的精确机制。我们将首先确定5-HT4/G13/RhoA和5-HT7/G12/Cdc42信号传导模块的下游效应物，然后定量评估其活性，重点关注肌动蛋白细胞骨架的重组。此外，我们将使用基于fret的生物传感器分析Cdc42和RhoA的时空激活谱。这些实验将为我们提供有关复杂信号网络中这些gtpase之间功能串扰的详细信息。该项目的第二个主要目标是阐明受体介导的细胞骨架重组在神经元形态、树突棘形成、突触发生和突触可塑性调节中的作用。这些研究将在小鼠海马神经元的原代和器官型培养中进行。5-HT4/G13信号的功能作用也将在体内进行测试。此外，我们将以兴奋性毒性诱导的神经元损伤为模型，分析5-HT7/G12通路对神经元保护和再生的贡献。这些实验将为我们提供关于5-HT7/G12信号作为神经保护和神经再生剂可能的治疗作用的信息。

Reorganization of actin cytoskeleton is one of the critical steps in regulation of neuronal morphology and motility. Dynamic changes of actin cytoskeleton in neurons are controlled by small GTPases of the Rho family, including RhoA, Rac1 and Cdc42. Rho GTPases have also emerged as important regulators for dendrite and spine structural plasticity, and are a part of the initial molecular cascade required for the growth of new synapses. Multiple studies suggest that Rac1 and Cdc42 are positive regulators promoting neurite outgrowth and formation of dendritic filopodia, while the activation of RhoA induces stress fibre formation, leading to neurite retraction. Although the importance of Rho GTPases is widely accepted, the signalling pathways regulating Rho GTPases activity in neurons are not fully characterized. We have recently shown that the serotonin receptor 5-HT4 can activate RhoA via the heterotrimeric G13 protein, and that stimulation of 5-HT4 receptors causes RhoA-mediated neurite retraction. We also identified Cdc42 as a novel down-stream effector of the 5-HT7 receptor and G12 protein. Stimulation of the 5-HT7/G12 signalling pathway facilitates neurite outgrowth, promotes synaptogenesis and enhances synaptic activity in hippocampal neurons. However, molecular mechanisms responsible for the coordinated (inter-)action between these opposite acting signalling pathways remain poorly understood. Thus, the central goal of the present project is to molecularly define the precise mechanisms through which 5-HT4 and 5-HT7 receptors regulate spatial arrangement and activity-dependent redistribution of small GTPases leading to the cytoskeleton reorganization. We will start by determining down-stream effectors for 5-HT4/G13/RhoA and 5-HT7/G12/Cdc42 signalling modules, followed by the quantitative evaluation of their activities with the focus on the reorganization of actin cytoskeleton. Furthermore, we will analyse the spatio-temporal activation profiles of Cdc42 and RhoA using FRET-based biosensors. These experiments will provide us with detailed information about the functional cross-talk between these GTPases within complex signalling networks. The second major goal of this project is to elucidate the role of receptor-mediated cytoskeleton reorganization for modulation of neuronal morphology, formation of dendritic spines, synaptogenesis and synaptic plasticity. These studies will be carried out in primary and organotypic cultures of hippocampal neurons from mice. The functional role of the 5-HT4/G13 signalling will also be tested in vivo. In addition, we will analyse the contribution of the 5-HT7/G12 pathway to neuronal protection and regeneration using excitotoxicity-induced neuronal damage as a model. These experiments will provide us with information about the possible therapeutic role of 5-HT7/G12 signalling as a neuroprotective and neuroregenerative agent.