Genetic Models of Serotonin Transporter Regulation Linked to Mental Disorders

描述（由申请人提供）：长期以来，改变的多巴胺能传递和突触前5-羟色胺（5-羟色胺，5-HT）转运蛋白（SERT）表达与精神疾病相关，包括抑郁症、自杀、自闭症、强迫症、冲动性暴力。事实上，阻断SERT的药物，如三环类抗抑郁药和SSRIs，已成功用于治疗精神疾病。PI的团队，合作者和同事的研究已经确定了激酶/磷酸酶调节SERT的信号机制。值得注意的是，人SERT编码变体Gly 56 Ala、Ile 425 Val突变与OCD、自闭症和其他精神障碍的关联，以及这些突变改变PKG/p38 MAPK对SERT活性的调节的发现表明，这种形式的调节在SERT的正常生理学中是重要的，并且SERT的失调可能影响归因于受损的5-HT信号传导的疾病的风险。目前还没有转基因动物模型可用于测试改变的SERT磷酸化是否是自闭症，强迫症和其他精神疾病中发现的行为改变的原因。在这个R21建议中，认识到风险与回报的使命，我们建议测试的假设，即模拟组成性SERT PKG-磷酸化的转基因小鼠模型显示5-HT转运表型的获得与PKG介导的上调和磷酸化的损失拯救行为表型，平行Ile 425 Val突变体在强迫症和Alfreger综合征中确定。在具体目标1中，我们提出构建携带Thr 276 Asp突变的靶向载体，其模拟与OCD和Alfreger综合征相关的PKG磷酸化和Ile 425 Val突变，通过将突变插入小鼠SERT基因组序列来产生SERT转基因SERT小鼠。具体目标2将验证Thr 276 Asp和Ile 425 Val SERT小鼠中的SERT调节和磷酸化，并阐明敲入小鼠中拯救的重要5-HT相关行为表型。然而，超出本提案范围的未来研究将利用Thr 276 Asp和/或Ile 425 Val敲入SERT小鼠来分析神经化学、行为和基因表达谱，并测量对急性和慢性体内SSRI给药和其他治疗剂的反应。因此，这些独特的小鼠模型的产生提供了用于探索激酶介导的SERT调节途径的创新工具，所述激酶介导的SERT调节途径是在与疾病相关的人SERT变体中发现的破坏正常SERT功能的设定点。此外，这些SERT转基因小鼠将有助于未来的研究，探索可能与精神障碍有关的阿托伐他汀相关的基因调控网络，并开发治疗精神障碍的有效药物。 公共卫生相关性：人类SERT编码变体Gly 56 Ala、Ile 425 Val突变与OCD、自闭症和其他精神疾病的关联，以及这些突变改变PKG/p38 MAPK对SERT活性的调节的发现表明，这种形式的调节在SERT的正常生理学中是重要的。拟议的研究产生一个敲入小鼠模型将能够识别与精神障碍相关的神经元网络，并有助于开发有效的药理学药物，用于治疗精神障碍和其他由异常单胺传递引起的疾病状态。

DESCRIPTION (provided by applicant): Altered serotonergic transmission and presynaptic serotonin (5-hydroxytryptamine, 5-HT) transporter (SERT) expression have long been associated with psychiatric disorders including depression, suicide, autism, OCD, impulsive violence. Indeed, the drugs that block SERT such as tricyclic antidepressants and SSRIs are successfully used for the treatment of mental disorders. Studies from PI's group, collaborators and colleagues have identified signaling mechanisms of SERT regulation by kinases/phosphatases. Remarkably, the association of human SERT coding variants Gly56Ala, Ile425Val mutation with OCD, autism and other psychiatric disorders, and the discovery that these mutations alter PKG/p38 MAPK regulation of SERT activity suggest that this form of regulation is important in the normal physiology of SERT and dysregulation of SERT may influence risk for disorders attributed to compromised 5-HT signaling. Currently no transgenic animal models are available to test whether altered SERT phosphorylation is causative for altered behavior found in autism, OCD and other psychiatric disorders. In this R21 proposal, recognizing the risk versus reward mission, we propose to test the hypothesis that transgenic mouse models mimicking constitutive SERT PKG-phosphorylation show gain of 5-HT transport phenotype with loss of PKG-mediated upregulation and phosphorylation rescuing behavioral phenotypes that parallel Ile425Val mutant identified in OCD and Asperger syndrome. In Specific Aim 1, we propose to construct targeting vectors carrying Thr276Asp mutation that mimic PKG-phosphorylation and Ile425Val mutation associated with OCD and Asperger syndrome by inserting the mutations into mouse SERT genomic sequences to generate SERT transgenic SERT mice. Specific Aim 2 will validate SERT regulation and phosphorylation in Thr276Asp and Ile425Val SERT mice, and elucidate important 5-HT related behavioral phenotypes rescued in the knock-in mice. However, future studies that are beyond the scope of this proposal, will utilize Thr276Asp and/or Ile425Val knock-in SERT mice to analyze neurochemical, behavioral and gene expression profiles, and to measure responses to acute and chronic in vivo SSRI administrations and other therapeutic agents. Thus, generation of these unique mouse models provide innovative tools for exploring the kinase mediated SERT regulatory pathways that are set points in disrupting normal SERT function found in disease-linked human SERT variants. In addition, these SERT transgenic mice will aid future studies exploring serotonin-related gene regulatory network that may be linked to mental disorders and in the development of effective pharmacological agents for the treatment of mental disorders. PUBLIC HEALTH RELEVANCE: The association of human SERT coding variants Gly56Ala, Ile425Val mutation with OCD, autism and other psychiatric disorders, and the discovery that these mutants alter PKG/p38 MAPK regulation of SERT activity suggest that this form of regulation is important in the normal physiology of SERT. The proposed research to generate a knock-in mouse model will enable to identify the neuronal network linked to mental disorders and aid in the development of effective pharmacological agents for the treatment of mental disorders and other disease states resulting from aberrant monoamine transmission.