Cellular and Molecular Analysis of the Schizophrenia and Autism Spectrum Disorder gene Transcription Factor 4 (TCF4)

精神分裂症和自闭症谱系障碍基因转录因子 4 (TCF4) 的细胞和分子分析

• 批准号: 9344688
• 负责人: BRADY J MAHER
• 金额: $ 45.65万
• 依托单位: LIEBER INSTITUTE, INC.
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-02 至 2020-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9344688
• 关键词: 18q; Alleles; Alpha Cell; Animal Model; Architecture; Behavioral; Bioinformatics; Biological Models; Biological Process; Birth; Brain; CRISPR/Cas technology; Cell model; Cells; Cellular biology; Chronic; Clinical; Cognitive; Cognitive deficits; Complex; Data; Development; Disease; Electrophysiology (science); Electroporation; Etiology; Experimental Designs; Frequencies; Functional disorder; Future; Gene Expression; Gene Targeting; Genes; Genetic; Genetic Risk; Genetic Transcription; Genotype; Goals; Image; Image Analysis; Ion Channel; Medial; Mediating; Mental disorders; Modeling; Molecular; Molecular Analysis; Molecular Biology; Molecular Profiling; Mus; Mutation; Neurodevelopmental Disorder; Neurons; Outcome; Pathologic; Pathway interactions; Pharmacology; Phenocopy; Phenotype; Physiological; Physiology; Platelet Factor 4; Population; Prefrontal Cortex; Process; Proteins; Public Health; Pyramidal Cells; Rattus; Research; Risk; Rodent; Schizophrenia; Slice; Syndrome; Techniques; Time; Transcript; autism spectrum disorder; base; cell type; chromatin immunoprecipitation; design; design and construction; differential expression; disorder risk; experimental study; foot; gene function; genetic risk factor; in utero; in vivo; insight; knock-down; mouse model; neurodevelopment; neuronal excitability; neuropsychiatric disorder; novel; novel therapeutics; overexpression; programs; public health relevance; relating to nervous system; risk variant; small hairpin RNA; therapeutic development; therapeutic target; therapy design; therapy development; transcription factor; transcriptome; transcriptome sequencing; treatment strategy

PROJECT SUMMARY: Neurodevelopmental disorders including Schizophrenia and Autism spectrum disorders (ASD) 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 propose to use two model systems, a cell autonomous model in which expression of the schizophrenia and ASD gene TCF4 (transcription factor 4) is manipulated using in utero electroporation and a mouse model that has a constitutive germline truncation of one TCF4 allele that models Pitt-Hopkins syndrome (PTHS). We will characterize the resulting neuronal phenotypes using brain slice electrophysiology, cell biology, and confocal imaging. Identified 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. In Aim1, we hypothesize that TCF4 transcriptionally regulates intrinsic neuronal excitability and therefore suppression of TCF4 expression will result in abnormal neuronal physiology relevant to PTHS. Our preliminary data suggest that in utero knockdown of TCF4 in layer 2/3 pyramidal cells of the PFC results in abnormal intrinsic excitability and ectopic spike-frequency adaptation. We show the cellular mechanisms of these phenotypes are associated with an increase in the afterhyperpolarization (AHP). Using a novel molecular profiling technique (iTRAP) we have identified two candidate ion channels that are regulated by TCF4 and may underlie cognitive phenotypes observed in PTHS. To validate these target genes, we propose pharmacological rescue and molecular phenocopy experiments. In Aim 2, we propose to use a mouse model of PTHS. Our preliminary data indicates PFC layer 2/3 neurons from TCF4+/tr mice show similar intrinsic excitability deficits to what we observe when TCF4 is knockdown using shRNA/Crispr constructs. We propose cellular and molecular experiments to identify the mechanisms underlying this phenotype with the future goal of using pharmacology to rescue behavioral deficits in these mice. In Aim 3, we propose to identify how the neural transcriptome is altered across development in PTHS mouse model. We hypothesize that identifying molecular consequences of TCF4+/tr on the neural transcriptome will provide mechanistic and pathological insight about PTHS and potentially other idiopathic ASDs. Together, these Aims are designed to identify therapeutic targets for treatment of PTHS.

项目摘要：包括精神分裂症和自闭症在内的神经发育障碍 疾病(ASD)是一种慢性疾病，具有相对未知的病因和病理生理学。近期 在人群水平上理解这些疾病的遗传结构方面的进展导致了 许多遗传风险因素的识别。然而，在大多数情况下，风险的分子机制和 识别的基因的相关功能未知，因此识别治疗靶点 仍然很困难。在我们的提案中，我们概述了确定治疗靶点的路线图 并提供了初步数据，表明我们的方法是有价值的。我们建议使用 两个模型系统，一个细胞自主模型，其中精神分裂症和ASD基因TCF4的表达 (转录因子4)是利用宫内电穿孔和具有构成成分的小鼠模型来操纵的 皮特-霍普金斯综合征(PTHS)模型的一个TCF4等位基因的胚系截断。我们将描述 使用脑切片电生理学、细胞生物学和共聚焦成像得出的神经元表型。 已确定的表型将作为潜在的病理生理和治疗的发展进行评估 治疗将基于我们对相关分子机制的新兴理解。 在Aim1中，我们假设TCF4转录调节固有的神经元兴奋性，因此 抑制TCF4的表达将导致与PTHS相关的神经元生理异常。我们的 初步数据表明，在宫内敲除PFC第2/3层锥体细胞中的TCF4会导致 异常的内在兴奋性和异位的尖峰频率适应。我们展示了它的细胞机制 这些表型与后超极化(AHP)增加有关。用一本小说 分子图谱技术(Itrap)我们已经确定了两个候选离子通道，它们受 TCF4，并可能是PTHS认知表型的基础。为了验证这些靶基因，我们建议 药理抢救和分子表型实验。在目标2中，我们建议使用一个小鼠模型 是PTHS的。我们的初步数据显示，来自TCF4+/tr小鼠的PFC层2/3神经元表现出类似的内在特征 当使用shRNA/Crispr结构敲除TCF4时，我们观察到的兴奋性存在缺陷。我们建议 细胞和分子实验，以确定这种表型的潜在机制，并为未来的目标 使用药理学来挽救这些小鼠的行为缺陷。在目标3中，我们建议确定 在PTHS小鼠模型中，神经转录组在发育过程中发生变化。我们假设辨认 TCF4+/TR对神经转录组的分子后果将提供机制和病理 对PTHS和其他潜在的特发性ASD的洞察。总而言之，这些目标旨在确定 PTHS的治疗靶点。