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

描述(申请人提供)：5-羟色胺(5-羟色胺)和脑源性神经营养因子(BDNF)是两个信号分子，在神经回路的发育和可塑性中发挥重要调节作用，已知在精神障碍中改变 包括抑郁症。研究表明，抑郁受试者死后大脑和周围组织中BDNF及其受体TrkB水平降低，证实了BDNF信号在抑郁行为中的作用。此外，细胞外5-羟色胺的增加也被证明增强了啮齿类动物的BDNF信号。然而，5-羟色胺调节TrkB信号转导的机制却知之甚少。总的目标是确定转谷氨酰胺酶2(TG2)在5-羟色胺对TrkB信号的调节中的作用。TG2是一种钙依赖酶，在蛋白质的翻译后修饰中起着重要作用。最近在大脑皮层神经元中，TG2在5-羟色胺转酰胺化为rac1中的作用被报道。Rac1是Rho家族中的一员，参与信号转导途径，促进TrkB内吞作用。我们假设，增加TG2依赖的5-羟色胺转胺化为rac1抑制了小鼠抑郁表型的TrkB信号。我们提供的初步证据表明，神经元TG2过度表达(TG2+/+)的小鼠表现出抑郁的表型，额叶皮质中的rac1蛋白水平降低。我们发现在TG2+/+小鼠的额叶皮质以及长期5-HT处理的原代皮质神经元中，TrkB信号、Hap1(一种参与细胞内TrkB细胞内运输的亨廷顿蛋白相关蛋白)水平以及它与TrkB的相关性都降低了。我们还将研究Hap1在介导依赖于rac1的TrkB内吞作用中的作用。我们发现，抑制TG2活性可减弱5-羟色胺诱导的rac1降解以及5-羟色胺与rac1的联系，但增加Hap1和TrkB在皮质神经元中的联系。此外，在自杀者的前额叶皮质中，TG2蛋白表达增加，TrkB、rac1和Hap1水平降低。这些数据表明，Rac1和Hap1在TG2下游发挥作用，并参与连接5-HT和TrkB信号。通过在体外和体内模型中使用TG2的药理学和遗传学方法来设计特定的目标，以提供一系列全面的研究，旨在研究TG2和Hap1在5-羟色胺介导的TrkB信号转导中的作用。提出了三个具体目标。具体目标1将测试TG2依赖的5-羟色胺转胺化导致小鼠抑郁表型的假设。具体目标2将检验这样的假设，即TrkB信号受损导致TG2过度表达的小鼠出现抑郁表型。具体目标3将检验Hap1介导依赖于rac1的TrkB内吞作用的假说。鉴于TrkB在神经可塑性中的重要作用，通过5-羟色胺识别TrkB的新调节机制可能为开发治疗抑郁症和相关精神障碍的新疗法提供途径。

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.