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

应激相关疾病被证明与神经元黏附分子L1的功能障碍有关。L1介导的神经元迁移、发育和再生基于同/异亲和性的L1相互作用，似乎依赖于L1以膜结合形式或以蛋白降解片段的形式呈现。在我们的初步实验中，我们已经确定神经元L1是基质金属蛋白酶-9(MMP9)的蛋白分解底物，并证明了由MMP9裂解产生的L1片段参与了分离的海马神经元的棘突形成。结合L1可以通过肌动蛋白结合蛋白cofilin的磷酸化启动轴突生长的观察，这一数据表明，MMP9介导的L1蛋白分解可能参与了L1启动的调节神经元肌动蛋白重排的信号通路。5-羟色胺能系统在情绪障碍的病理生理学和治疗中起着至关重要的作用。神经递质5-羟色胺(5-HT)调节多种生理和病理功能，参与轴突生长、生长锥运动和突触形成。5-羟色胺通过激活多个5-羟色胺受体发挥作用，因此在本项目中，我们将重点介绍5-HT4受体(5-HT4R)。在哺乳动物的大脑中，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，刺激5-HT4R可以诱导海马神经元释放具有酶活性的基质金属蛋白酶-9，其中5-HT4R和L1紧密地共存于突触。这些结果表明，5-HT4R可能以一种依赖于基质金属蛋白酶-9的方式调节L1的脱落，从而调节L1的同/异嗜性相互作用和旁分泌功能。我们之前已经证明，5-HT4R刺激导致小GTP酶RhoA的激活，导致细胞圆整和轴突回缩。在我们的初步实验中，我们还证明了这种作用可以通过cofilin的磷酸化来介导。因此，Cofilin可能是5-HT4R和L1共同的下游效应因子，提示5-HT4R、MMP9和L1属于参与神经元形态调控的同一信号模块。在这一建议中，我们将研究5-HT4R介导的L1处理的分子机制以及L1片段对神经发育和生存的功能意义。为此，我们将结合生化、分子和细胞方法与先进的Förster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.