Genetic Models of Serotonin Transporter Regulation Linked to Mental Disorders

描述(申请人提供)：5-羟色胺能传递改变和突触前5-羟色胺(5-羟色胺，5-羟色胺)转运体(SERT)的表达长期以来与精神疾病有关，包括抑郁、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SSRI，已成功地用于治疗精神障碍。Pi的团队、合作者和同事的研究已经确定了通过激酶/磷酸酶调节SERT的信号机制。值得注意的是，人类SERT编码突变体Gly56Ala、Ile425Val突变与强迫症、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活性调节的发现表明，这种形式的调节在SERT的正常生理中是重要的，SERT的调节失调可能会影响由于5-HT信号受损而引起的疾病的风险。目前还没有转基因动物模型来测试SERT磷酸化改变是否会导致自闭症、强迫症和其他精神疾病的行为改变。在这个R21提案中，认识到风险与回报的使命，我们建议检验这样一个假设，即模仿结构性SERT PKG-磷酸化的转基因小鼠模型显示5-羟色胺转运表型增加，而PKG介导的上调和磷酸化丢失，拯救与强迫症和阿斯伯格综合征中发现的Ile425Val突变相似的行为表型。在特定的目的1中，我们建议构建携带Thr276Asp突变的靶向载体，通过将突变插入到小鼠SERT基因组序列中来模拟与强迫症和Asperger综合征相关的PKG磷酸化和Ile425Val突变，以产生SERT转基因SERT小鼠。特异性目标2将验证Thr276Asp和Ile425Val SERT小鼠的SERT调节和磷酸化，并阐明在敲除小鼠中拯救的重要的5-羟色胺相关行为表型。然而，超出这一建议范围的未来研究将利用Thr276Asp和/或Ile425Val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给药和其他治疗药物的反应。因此，这些独特的小鼠模型的产生为探索激酶介导的SERT调节通路提供了创新的工具，这些调节通路是在疾病相关的人类SERT变体中发现的扰乱正常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5-羟色胺相关基因调控网络，并开发有效的治疗精神障碍的药理药物。 公共卫生相关性：人类SERT编码突变体Gly56Ala、Ile425Val突变与强迫症、自闭症和其他精神障碍的关联，以及这些突变体改变SERT活性的PKG/p38 MAPK调节的发现表明，这种形式的调节在SERT的正常生理中是重要的。拟议中的研究将产生敲入小鼠模型，将能够识别与精神障碍有关的神经元网络，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单胺异常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.