Functional analysis of the Schizophrenia and Autism Spectrum Disorder gene TCF4 i

描述（由申请人提供）：包括精神分裂症和自闭症在内的神经发育障碍是慢性和衰弱的，病因和病理生理学相对未知。最近在人群水平上对这些疾病的遗传结构的了解取得了进展，从而确定了许多遗传风险因素。然而，在大多数情况下，风险的分子机制和已鉴定基因的相关功能尚不清楚，因此确定治疗靶点仍然很困难。在我们的建议中，我们概述了确定精神疾病治疗靶点的路线图，并提供了初步数据，表明我们的方法有可取之处。我们将首先通过在子宫内电穿孔发育中的大鼠前额皮质（PFC），通过基因操纵精神分裂症和自闭症谱系障碍基因TCF4的表达，创建一个可测试的风险细胞模型。然后，我们将利用急性脑切片电生理学、细胞生物学和Ca2+成像来表征由此产生的神经元表型。这些表型将被评估为潜在的病理生理学，治疗方法的发展将基于我们对相关分子机制的新兴理解，并通过分子和药理救援或表型复制验证这些机制。在Aim1中，我们假设TCF4转录调节离子通道基因的表达，这些基因是正常神经元生理所必需的，特别是通道和/或Ca2+传感器是后超极化（AHP）的基础。我们的初步数据表明，子宫内PFC第2/3层锥体细胞中TCF4的敲低会导致异常的内在兴奋性和异位的尖峰频率适应。我们表明这些表型的机制与AHP的增加有关，并通过减少Ca2+内流而获救。我们建议进行实验，以更具体地确定负责的机制。在Aim 2中，我们发现TCF4的过表达导致神经元迁移缺陷和发育中的pfc皮层异常微柱的形成。我们提供的初步数据表明，这些表型可能与异常的Eph/ephrin信号传导有关，我们假设加速的神经元迁移将增强内在兴奋性的发展，从而破坏神经元融入周围回路的能力。在Aim 3中，我们提供了来自精神分裂症患者和对照组的死后大脑的新的RNA测序数据，确定了TCF4的一个特定的5'外显子，该外显子通过诊断差异表达，与TCF4中的GWAS阳性snp相关，并且是单一TCF4亚型（TCF4H）所特有的。我们建议通过改变子宫内TCF4H的表达，将这些新信息应用到我们的路线图中，以更有效地模拟精神分裂症的风险，并为细胞模型提供额外的有效性。我们认为这些创新的方法将使我们能够为这些疾病的病因学和病理生理学提供重要的见解，并最终为新的治疗方法打开大门。

DESCRIPTION (provided by applicant): Neurodevelopmental disorders including Schizophrenia and Autism are chronic and debilitating, with relatively unknown etiology and pathophysiology. Recent progress towards understanding the genetic architecture of these disorders at the population level has led to the identification of many genetic risk factors. However, in most cases the molecular mechanism of risk and the relevant functions of the identified genes are not known and therefore identifying therapeutic targets remains difficult. In our proposal we have outlined a roadmap for the identification of therapeutic targets for psychiatric disorders and provide preliminary data that suggests our approach has merit. We will start by creating a testable cellular model of risk by genetically manipulating the expression of the schizophrenia and autism spectrum disorder gene TCF4 using in utero electroporation of the developing rat prefrontal cortex (PFC). We will then characterize the resulting neuronal phenotypes using acute brain slice electrophysiology, cell biology, and Ca2+ imaging. Such phenotypes will be evaluated as potentially pathophysiological and the development of therapeutic treatments will be based on our emerging understanding of the molecular mechanism responsible and by validating these mechanisms with molecular and pharmacological rescue or phenocopying. In Aim1, we hypothesize that TCF4 transcriptionally regulates the expression of ion channels genes that are necessary for normal neuronal physiology and in particular channels and/or Ca2+ sensors that underlie the afterhyperpolarization (AHP). Our preliminary data suggest that in utero knockdown of TCF4 in PFC layer 2/3 pyramidal cells results in abnormal intrinsic excitability and ectopic spike-frequency adaptation. We show the mechanisms of these phenotypes are associated with an increase in the AHP and are rescued by decreasing Ca2+ influx. We propose experiments to more specifically identify the mechanisms responsible. In Aim 2, we show that over-expression of TCF4 results in neuronal migration defects and the formation of abnormal cortical microcolumns in the developing PFC. We provide preliminary data that suggests these phenotypes may involve abnormal Eph/ephrin signaling and we hypothesize that accelerated neuronal migration will augment the development of intrinsic excitability and consequently disrupt the neuron's ability to integrate into the surrounding circuit. In Aim 3, we provide novel RNA sequencing data from postmortem brains of schizophrenia patients and controls that identifies a specific 5' exon of TCF4 that is differentially expressed by diagnosis, associated wit GWAS positive SNPs in TCF4, and is unique to a single TCF4 isoform (TCF4H). We propose to apply this new information to our roadmap by altering TCF4H expression in utero to more effectively model schizophrenia risk and provide additional validity to cellular models. We think these innovative approaches will enable us to provide significant insights into the etiology and pathophysiology of these disorders and will ultimately open doors to novel therapeutic treatments.