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

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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.