Regulation of TrkB signaling by 5-HT: role of transglutaminase 2

描述（由申请人提供）：5-羟色胺（5-HT）和脑源性神经营养因子（BDNF）是两种信号分子，在神经回路的发育和可塑性中发挥重要的调节作用，已知神经回路在精神疾病中发生改变 包括抑郁症BDNF信号传导在抑郁行为中的作用通过研究证实，研究显示在死后脑以及抑郁受试者的外周组织中BDNF及其受体TrkB的水平降低。此外，细胞外5-HT的增加已被证明可以增强啮齿动物中的BDNF信号传导。然而，5-HT调节TrkB信号传导的机制知之甚少。总体目标是确定转氨酶2（TG 2）在5-HT调节TrkB信号传导中的作用。TG 2是一种钙依赖性酶，在蛋白质的翻译后修饰中起重要作用。TG 2在5-HT转酰胺至Rac 1中的作用最近在皮质神经元中被报道。Rac 1是参与信号转导途径的小GTP酶Rho家族的成员，已知可促进TrkB内吞作用。我们推测，增加TG 2依赖性转酰胺5-HT Rac 1抑制TrkB信号与抑郁表型的小鼠。我们提供的初步证据表明，神经元TG 2过表达（TG 2 +/+）的小鼠表现出抑郁表型，额叶皮质中Rac 1蛋白水平降低。我们发现减少TrkB信号，Hap 1（亨廷顿蛋白相关蛋白参与胞吞TrkB的细胞内运输）水平和其与TrkB在TG 2 +/+小鼠的额叶皮层，以及在长期5-HT处理的原代皮层神经元。我们还将研究Hap 1在介导Rac 1依赖性TrkB内吞作用中的作用。我们发现，抑制TG 2活性减弱5-HT诱导的Rac 1降解和5-HT与Rac 1的关联，但增加Hap 1和TrkB在皮层神经元中的关联。此外，TG 2蛋白的增加，但TrkB，Rac 1和Hap 1水平的下降，被发现在前额叶皮层的自杀受试者。这些数据表明Rac 1和Hap 1在TG 2的下游发挥作用，并参与将5-HT与TrkB信号传导连接。具体目标是通过使用药理学和遗传学方法在体外和体内模型中对TG 2进行设计，以提供一系列全面的研究，旨在研究TG 2和Hap 1在5-HT的TrkB信号传导中的作用。提出了三个具体目标。具体目标1将检验5-HT的TG 2依赖性转酰胺作用诱导小鼠抑郁表型的假设。具体目标2将检验以下假设：受损的TrkB信号传导有助于在TG 2过表达小鼠中观察到的抑郁表型。具体目标3将检验Hap 1介导Rac 1依赖性受损TrkB内吞作用的假设。鉴于TrkB在神经可塑性中的重要作用，通过5-HT识别TrkB的新调节机制可能为开发抑郁症和相关精神疾病的新疗法提供途径。

DESCRIPTION (provided by applicant): Serotonin (5-HT) and Brain derived neurotrophic factor (BDNF) are two signaling molecules that play important regulatory roles in the development and plasticity of neural circuits that are known to be altered in psychiatric disorders including depression. The role of BDNF signaling in depressive behavior is substantiated by studies showing decreased levels of BDNF and its receptor, TrkB in postmortem brain as well as peripheral tissues of depressed subjects. In addition, increases in extracellular 5-HT have been shown to enhance BDNF signaling in rodents. However, the mechanism by which 5-HT regulates TrkB signaling is poorly understood. The overall goal is to determine the role of transglutaminase 2 (TG2) in regulation of TrkB signaling by 5-HT. TG2 is a calcium-dependent enzyme that plays an important role in posttranslational modification of proteins. The role of TG2 in transamidation of 5-HT to Rac1 has recently been reported in cortical neurons. Rac1 is a member of the Rho family of small GTPases involved in signal transduction pathways and is known to promote TrkB endocytosis. We hypothesize that increased TG2-dependent transamidation of 5-HT to Rac1 inhibits TrkB signaling with depressive phenotype in mice. We provide initial evidence demonstrating that mice with neuronal TG2 overexpression (TG2+/+) show depressive phenotype with reduced Rac1 protein levels in the frontal cortex. We found reductions in TrkB signaling, Hap1 (a huntingtin associated protein involved in intracellular trafficking of endocytic TrkB) levels and its association with TrkB in frontal cortex of TG2+/+ mice as well as in long-term 5-HT-treated primary cortical neurons. We will also investigate the role of Hap1 in mediating Rac1-dependent TrkB endocytosis. We found that inhibition of TG2 activity attenuates 5-HT-induced Rac1 degradation and association of 5-HT with Rac1, but increases the association of Hap1 and TrkB in cortical neurons. Moreover, increases in TG2 protein, but decreases in TrkB, Rac1 and Hap1 levels, were found in prefrontal cortex of suicide subjects. These data suggest that Rac1 and Hap1 function downstream of TG2 and are involved in linking 5-HT to TrkB signaling. The specific aims are designed by using pharmacological and genetic approaches for TG2 in in vitro and in vivo models to provide a comprehensive series of studies aimed at examining the role of TG2 and Hap1 in TrkB signaling by 5-HT. Three Specific Aims are proposed. The Specific Aim 1 will test the hypothesis that that TG2-dependent transamidation of 5-HT induces depressive phenotype in mice. The Specific Aim 2 will test the hypothesis that that impaired TrkB signaling contributes to the depressive phenotype seen in TG2 overexpressed mice. The Specific Aim 3 will test the hypothesis that Hap1 mediates Rac1-dependent impaired TrkB endocytosis. Given the important role of TrkB in neuroplasticity, identifying novel regulatory mechanisms of TrkB by 5-HT may provide avenues to develop newer therapeutics for depression and related psychiatric disorders.