Interplay between serotonergic and L1-mediated signaling in regulation of neuronal morphology and functions under physiological and pathological conditions

应激相关疾病与神经元粘附分子L1的功能紊乱有关。L1介导的神经元迁移，发展和再生的基础上的同/异嗜性L1的相互作用似乎是依赖于介绍L1无论是作为一个膜结合的形式或作为一个蛋白水解片段。在我们的初步实验中，我们已经确定了神经元L1作为基质金属蛋白酶-9（MMP-9）的蛋白水解底物，并证明了MMP-9裂解产生的L1片段参与了解离海马神经元的棘形成。结合L1可通过磷酸化肌动蛋白结合蛋白cofilin启动神经突起生长的观察，这些数据表明MMP-9介导的L1蛋白水解可能参与了L1启动的调节神经元肌动蛋白重排的信号通路。神经递质5-羟色胺（5-HT）参与神经元轴突生长、生长锥运动和突触发生等多种生理和病理功能的调节。5-羟色胺通过激活多种5-HT受体发挥作用，因此在本项目中，我们将重点关注5-HT 4受体（5-HT 4 R）。在哺乳动物大脑中，这种受体有助于调节学习和长期记忆，并参与各种中枢和外周疾病，包括神经退行性疾病和抑郁症。在我们的初步结果中，我们已经表明，5-HT 4 R的刺激诱导释放的酶活性MMP-9在海马神经元，其中5-HT 4 R和L1是紧密共定位在突触。这些结果表明，5-HT 4 R可能调节L1脱落的MMP-9依赖的方式，从而调节同/异嗜性的相互作用和旁分泌功能的L1。我们以前已经表明，5-HT 4 R刺激的结果在小GTdR RhoA的激活，导致细胞变圆和神经突回缩。在我们的初步实验中，我们也证明了这种作用可以通过cofilin的磷酸化介导。因此，cofilin可能是5-HT 4 R和L1共同的下游效应子，这表明5-HT 4 R、MMP-9和L1属于参与神经元形态调控的相同信号模块。为此，我们将结合联合收割机生化，分子和细胞的方法与先进的福斯特共振能量转移（FRET）技术和定量分子显微镜。通过这些研究，我们希望揭示5-羟色胺调节神经元网络形成和可塑性的新的分子机制，并希望为包括抑郁症在内的应激相关疾病的药物治疗提供新的靶点。

Stress-related disorders are shown to be associated with functional disturbance of neuronal adhesion molecule L1. The L1-mediated neuronal migration, development, and regeneration based on homo/ heterophilic L1 interactions seems to be depended on presentation of L1 either as a membrane-bound form or as a proteolytic fragments. In our preliminary experiments we have identified the neuronal L1 as a proteolytic substrate for the matrix metalloproteinase-9 (MMP-9) and demonstrated that L1 fragments generated by MMP-9 cleavage are involved in spine formation in dissociated hippocampal neurons. Together with the observation that the L1 can initiate neurite outgrowth via phosphorylation of actin-binding protein cofilin, this data suggests that MMP-9-mediated proteolysis of L1 might be involved in L1-initiated signaling pathway regulating actin rearrangement in neurons.Serotonergic system has been identified as crucially important to the pathophysiology and the treatment of mood disorders. Neurotransmitter serotonin (5-HT) regulates a wide range of physiological and pathological functions, and is involved in neurite outgrowth, growth cone motility and synaptogenesis. Serotonin operates via activation of multiple 5-HT receptors, whereby in this project we will focus on the 5-HT4 receptor (5-HT4R). In the mammalian brain this receptor contributes to regulation of learning and long term memory and is involved in various central and peripheral disorders, including neurodegenerative disease and depression. In our preliminary results we have shown that stimulation of 5-HT4R induces the release of enzymatically active MMP-9 in hippocampal neurons, where 5-HT4R and L1 are tightly co-localized at the synapses. These results demonstrate that 5-HT4R might regulate L1 shedding in an MMP-9-dependent manner, thereby modulating the homo/heterophilic interaction and paracrine function of L1. We have previously shown that 5-HT4R stimulation results in activation of the small GTPase RhoA, leading to cell rounding and neurite retraction. In our preliminary experiments we have also demonstrated that this effect can be mediated by phosphorylation of cofilin. Thus, cofilin may represent a common downstream effector for both 5-HT4R and L1 suggesting that 5-HT4R, MMP-9 and L1 belong to the same signaling module involved in regulation of neuronal morphology.Within this proposal we will study molecular mechanisms of 5-HT4R-mediated L1 processing as well as the functional implication of L1 fragments for neural development and survival. For that we will combine biochemical, molecular and cellular approaches with advanced Förster resonance energy transfer (FRET) techniques and quantitative molecular microscopy. From these investigations we expect to reveal a novel molecular mechanisms by which serotonin can regulate formation and plasticity of neuronal networks, and hope to provide new targets for pharmacological treatment of stress-associated disorders, including depression.