Transgenic Mouse Model to Address Heterogeneity in Autism Spectrum Disorders

描述（由申请人提供）：突触5-羟色胺（5-HT）失活的主要决定因素是抗抑郁药敏感性5-HT转运蛋白（SERT，SLC 6A 4）。SERT启动子中常见的多态性影响SERT表达，并与焦虑、强迫症和自闭症有关。SERT也受到强大的翻译后调节机制的影响。最近，炎性细胞因子，包括IL 12和TNF-1，已被证明能增强神经末梢制剂中的SERT活性，这种调节可以通过全身性细胞因子升高在体内重现。这些发现提高了SERT可能是基因/环境相互作用的汇聚点的可能性，这些相互作用共同改变5-HT信号传导并提高疾病风险。由于SERT在脑发育早期表达，因此最感兴趣的是青少年发病的5-HT相关疾病。在这方面，5-HT信号传导中断和免疫功能中断都被认为是自闭症风险的决定因素。最近，我们在自闭症受试者的高度保守的氨基酸位置上鉴定了多个功能性SERT变体。最常见的SERT变体Gly 56 Ala导致1）增强的hSERT催化活性，2）升高的hSERT基础磷酸化，和3）缺乏对多种SERT翻译后调节途径（包括由炎性细胞因子激活的p38 MAPK途径）的响应性。重要的是，Ala 56 hSERT变体与两种描述良好的自闭症特征，僵硬强迫行为和感觉厌恶相关。我们已经成功地用SERT Ala 56“敲入”构建体靶向129 S6小鼠胚胎干细胞，并获得了生殖系传递。SERT Ala 56小鼠表现出全血高胆固醇血症、对5 HT 2A/2C激动剂刺激的高反应性以及社会和感觉行为改变的证据。在我们目前的项目中，我们提出了一个为期2年的计划，以1）确定母体、幼年和成年SSRIs给药减弱129 S6 SERTAla 56小鼠改变的行为和药物反应的能力，2）使用速度同源方法将56 Ala等位基因置于C57 Bl/6 J背景上，从而扩大Ala 56小鼠的可测试行为库，和3）建立C57 BL/6 J背景下的SERT Ala 56脑制备物对体外PKG/p38 MAPK刺激和体内天然免疫系统刺激的敏感性。这些研究将增强我们对改变SERT活性和调节对自闭症异质性的贡献的理解，并为治疗干预的临床前评估提供新的平台。 公共卫生相关性：导致血清素转运蛋白（SERT）功能亢进的罕见功能性基因变异与自闭症谱系特征、僵硬强迫行为和感觉厌恶的特定子集相关。我们已经产生了一种新的转基因小鼠模型，表达这些SERT变体中最常见的Ala 56。基于表达Ala 56 SERT的小鼠表现出多种行为和生化表型的令人兴奋的初步证据，我们提出了一个为期两年的项目，以扩展我们的研究，并剖析SERT功能亢进在体内的影响。

DESCRIPTION (provided by applicant): The principal determinant of synaptic serotonin (5-hydroxytryptamine; 5-HT) inactivation is the antidepressant-sensitive 5-HT transporter (SERT, SLC6A4). Common polymorphisms in the SERT promoter impact SERT expression and have been associated with anxiety, obsessive- compulsive disorder and autism. SERT is also subject to powerful, posttranslational regulatory mechanisms. Recently, inflammatory cytokines, including IL12 and TNF-1, have been shown to enhance SERT activity in nerve terminal preparations, regulation that can be recapitulated in vivo by systemic cytokine elevation. These findings raise the possibility that SERT may be a convergence point for gene/environment interactions that conspire to alter 5-HT signaling and elevate disease risk. Because SERT is expressed early in brain development, of greatest interest are 5-HT linked disorders of juvenile onset. In this regard, both disrupted 5-HT signaling and disrupted immune function have been advanced as determinants of autism risk. Recently, we identified multiple, functional SERT variants at highly conserved amino acid positions in autism subjects. The most common SERT variant, Gly56Ala, leads to 1) enhanced hSERT catalytic activity, 2) elevated hSERT basal phosphorylation, and 3) a lack of responsiveness to multiple SERT posttranslational regulatory pathways including p38 MAPK pathways activated by inflammatory cytokines. Importantly, the Ala56 hSERT variant was associated with two well-described autism traits, rigid-compulsive behavior and sensory aversion. We have successfully targeted 129S6 mouse embryonic stem cells with a SERT Ala56 "knock-in" construct and obtained germ-line transmission. The SERT Ala56 mice display whole blood hyperserotonemia, hyper-responsiveness to 5HT2A/2C agonist stimulation, and evidence of alterations in social and sensory behaviors. In our current project, we propose a 2-year program to 1) determine the ability of maternal, juvenile and adult SSRIs administration to attenuate the altered behavior and drug responses of 129S6 SERTAla56 mice, 2) to place the 56Ala allele on a C57Bl/6J background using speed-congenic approaches and thereby expand the testable behavioral repertoire of the Ala56 mice, and 3) to establish the sensitivity of SERT Ala56 brain preparations on the C57BL/6J background to in vitro PKG/p38 MAPK stimulation and in vivo stimulation of the native immune system. These studies will enhance our understanding of the contributions of altered SERT activity and regulation to autism heterogeneity and provide a novel platform for the preclinical evaluation of therapeutic interventions. PUBLIC HEALTH RELEVANCE: Rare, functional gene variants that lead to hyperfunction of the serotonin transporter (SERT) have been associated with a specific subset of autism spectrum traits, rigid-compulsive behaviors and sensory aversion. We have produced a novel, transgenic mouse model expressing the most common of these SERT variants, Ala56. Based on exciting preliminary evidence that mice expressing Ala56 SERT exhibit multiple behavioral and biochemical phenotypes, we propose a two-year project to extend our studies and dissect the impact of SERT hyperfunction in vivo.