Transgenic Mouse Model to Address Heterogeneity in Autism Spectrum Disorders

描述(申请人提供)：突触5-羟色胺(5-羟色胺；5-羟色胺)失活的主要决定因素是抗抑郁药敏感的5-羟色胺转运体(SERT，SLC6A4)。SERT启动子的常见多态会影响SERT的表达，并与焦虑、强迫症和自闭症有关。SERT还受制于强大的翻译后监管机制。最近，包括IL12和TNF-1在内的炎性细胞因子被证明可以增强神经末梢准备中的SERT活性，这一调节可以在体内通过全身细胞因子的升高来概括。这些发现提出了这样一种可能性，即SERT可能是基因/环境相互作用的汇合点，这些相互作用共同改变了5-羟色胺信号并增加了疾病风险。因为SERT在大脑发育的早期就有表达，所以人们最感兴趣的是青少年发病的5-羟色胺相关障碍。在这方面，5-羟色胺信号转导受阻和免疫功能受阻都被认为是自闭症风险的决定因素。最近，我们在自闭症患者中发现了高度保守的氨基酸位置上的多个功能性SERT变体。最常见的SERT变异体Gly56Ala导致1)hSERT催化活性增强，2)hSERT基础磷酸化水平升高，3)对多种SERT翻译后调节途径缺乏反应性，包括炎性细胞因子激活的p38 MAPK途径。重要的是，Ala56 hSERT变异与自闭症的两个特征有关，这两个特征是僵硬的强迫行为和感觉厌恶。我们成功地将129S6小鼠胚胎干细胞作为靶点，构建了SERT-Ala56“敲入”基因，并获得了胚系传递。SERT Ala56小鼠表现出全血高5-羟色胺血症、对5HT2A/2C激动剂刺激的高反应性，以及社会和感觉行为的改变。在我们目前的项目中，我们提出了一个为期两年的计划，以1)确定母亲、青少年和成年SSRIs给药减轻129S6 SERTAla56小鼠改变的行为和药物反应的能力，2)使用速度同源基因方法将56Ala等位基因放置在C57BL/6J背景上，从而扩大Ala56小鼠的可测试行为谱，以及3)建立C57BL/6J背景下SERT Ala56脑制剂对体外PKG/p38 MAPK刺激和体内本地免疫系统刺激的敏感性。这些研究将加深我们对SERT活性和调控改变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一个新的平台。 公共卫生相关性：导致5-羟色胺转运体(SERT)功能亢进的罕见功能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有关。我们已经创造了一种新型的转基因小鼠模型，表达了这些最常见的SERT变体Ala56。基于令人兴奋的初步证据表明，表达Ala56 SERT的小鼠表现出多种行为和生化表型，我们提出了一个为期两年的项目，以扩大我们的研究并剖析SERT过度功能在体内的影响。

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.