Investigating the role of TCF4 in human interneuron function and dysfunction

研究 TCF4 在人类中间神经元功能和功能障碍中的作用

• 批准号: 9903457
• 负责人: Fikri Birey
• 金额: $ 8.84万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-04-01 至 2022-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9903457
• 关键词: 3-Dimensional; Affect; Biological; Biological Assay; Biology; Brain; Brain Diseases; CRISPR/Cas technology; Calcium; Cell Line; Cells; Clinical; Clustered Regularly Interspaced Short Palindromic Repeats; Data; Development; Dimerization; Disease; Dorsal; Electrophysiology (science); Epilepsy; Exhibits; Forebrain Development; Functional disorder; Future; Genes; Genetic; Genetic Predisposition to Disease; Genetic Transcription; Genome; Glutamates; Goals; Human; In Vitro; Intellectual functioning disability; Interneuron function; Interneurons; Intervention; Lead; Mediating; Medical Genetics; Mental disorders; Methods; Modeling; Modification; Molecular; Mutation; Neurobiology; Neurons; Outcome; Pathogenicity; Pathologic; Pathway interactions; Patients; Pharmaceutical Preparations; Pharmacology; Phase; Phenotype; Physiological; Pregnancy; Process; Property; Prosencephalon; Radial; Research; Research Personnel; Role; Schizophrenia; Slice; Surveys; Synapses; System; TCF7L2 gene; Testing; Tissues; Training; Universities; Variant; autism spectrum disorder; base; cell type; critical period; design; differential expression; disease phenotype; disease-causing mutation; dosage; epileptic encephalopathies; excitatory neuron; experimental study; fetal; genetic variant; human model; induced pluripotent stem cell; inhibitory neuron; insight; laboratory experience; migration; multiphoton imaging; neuropsychiatric disorder; new therapeutic target; novel strategies; optogenetics; programs; public health relevance; response; skills; stem cells; transcription factor; transcriptome sequencing; transcriptomics

Project Summary / Abstract Formation of cortical circuits during fetal cortical development involves the assembly of glutamatergic neurons and GABAergic interneurons. After their specification, GABAergic interneurons migrate dorsally to reach the cortex and undergo activity-dependent maturation and integration into glutamatergic circuits. Genetic perturbations of this process can lead to miswiring of early cortical circuits and to excitation/inhibition imbalance which is thought to underlie various disorders such as schizophrenia, autism spectrum disorders and epilepsies. The neurobiological basis of how disease- associated gene variants affect the assembly of early cortical circuits in humans remain unknown. This is mainly due to the lack of patient tissue available for functional studies. In response to this, we have recently developed a 3D in vitro platform of forebrain development, termed forebrain Assembloids, where region-specific forebrain cultures derived from human induced pluripotent stem cells (hiPSCs) are functionally assembled. Using this platform, we showed that GABAergic interneurons migrate towards and integrate with glutamatergic neurons forming cortical ensembles that exhibits glutamatergic and GABAergic synaptic activity. When we surveyed for differentially expressed genes in interneurons that migrated in the cortical network, we identified TCF4, a basic loop-helix-loop transcription factor, potentially indicating a role in interneuron functional maturation. In line with this idea, several TCF4 variants have been identified across clinically distinct disorders that have been frequently associated with interneuron dysfunction, such as schizophrenia, autism spectrum disorders, intellectual disability and epileptic encephalopathies. TCF4 is a major transcriptional hub that, through its cell- type-specific dimerization partners regulated by intracellular calcium levels, can assume different roles at various stages of fetal brain development. As such, TCF4 dosage is thought to be tightly regulated during development. It has been hypothesized that the degree by which each TCF4 variants affects its dosage is correlated with specific clinical outcomes, although this has not yet been thoroughly tested in humans. The goal of this proposal is to understand mechanisms by which distinct TCF4 variants affect the TCF4 regulatory network and lead to molecular and cellular deficits in human interneurons during assembly of early cortical circuits in the forebrain Assembloids. During the K99 phase, I propose to generate and characterize hiPSC lines harboring various disease-associated TCF4 mutations using CRISPR/Cas9 gene editing through training in the Porteus lab. I will then generate forebrain Assembloids from these lines and interrogate whether migration, intrinsic properties, synaptic integration and functional connectivity of cortical interneurons are disrupted in cortical ensembles, through training in the Huguenard lab. During the independent R00 phase, I will investigate molecular deficits TCF4-related gene networks related to deficits uncovered in Aim 1 and 2 and explore pharmacological targets for rescue experiments. Comprehensive training with Drs. Pasca, Huguenard and Porteus at Stanford University will provide me with the skills required to further pursue this line of research as an independent investigator. These efforts will lead to mechanistic insights into a major biological pathway that is potentially shared across a diverse array of psychiatric disorders.

项目概要/摘要 胎儿皮质发育过程中皮质回路的形成涉及谷氨酸能神经元的组装和 GABA能中间神经元。在其规范化后，GABA能中间神经元向背侧迁移到达皮质并 经历活性依赖性成熟并整合到谷氨酸能回路中。这个过程的遗传扰动可以 导致早期皮质回路的错误接线和兴奋/抑制失衡，这被认为是各种疾病的基础 精神分裂症、自闭症谱系障碍和癫痫等疾病。疾病如何发生的神经生物学基础 影响人类早期皮质回路组装的相关基因变异仍然未知。这主要是由于 缺乏可用于功能研究的患者组织。针对这一点，我们最近开发了3D体外平台 前脑发育的过程，称为前脑组装体，其中特定区域的前脑文化源自人类 诱导多能干细胞 (hiPSC) 进行功能性组装。使用这个平台，我们证明了 GABAergic 中间神经元向谷氨酸能神经元迁移并与谷氨酸能神经元整合，形成皮质群，表现出 谷氨酸能和 GABA 能突触活性。当我们调查中间神经元中差异表达的基因时 在皮质网络中迁移后，我们发现了 TCF4，一种基本的环-螺旋-环转录因子，可能表明 间神经元功能成熟中的作用。根据这一想法，临床上已鉴定出多种 TCF4 变体 经常与中间神经元功能障碍相关的独特疾病，例如精神分裂症、自闭症谱系 疾病、智力障碍和癫痫性脑病。 TCF4 是一个主要的转录中心，通过其细胞- 受细胞内钙水平调节的类型特异性二聚化伙伴，可以在不同阶段发挥不同的作用 胎儿大脑发育。因此，TCF4 剂量被认为在开发过程中受到严格控制。它一直 假设每个 TCF4 变体影响其剂量的程度与特定的临床结果相关， 尽管这尚未在人类身上进行彻底的测试。该提案的目标是了解机制 不同的 TCF4 变异影响 TCF4 调节网络并导致人类中间神经元的分子和细胞缺陷 在前脑组装体中早期皮质回路的组装过程中。在K99阶段，我建议生成并 使用 CRISPR/Cas9 基因编辑来表征含有各种疾病相关 TCF4 突变的 hiPSC 系 在 Porteus 实验室进行培训。然后我将从这些细胞系中生成前脑组装体并询问是否迁移， 皮质中间神经元的内在特性、突触整合和功能连接在皮质中被破坏 通过在 Huguenard 实验室的培训来完成合奏。在独立R00阶段，我将调查分子缺陷 TCF4相关基因网络与目标1和2中发现的缺陷相关，并探索救援的药理学靶点 实验。与博士进行全面培训。斯坦福大学的 Pasca、Huguenard 和 Porteus 将为我提供 作为独立调查员进一步从事这一研究领域所需的技能。这些努力将导致机械化 对多种精神疾病可能共有的主要生物学途径的见解。