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: 10596187
• 负责人: Fikri Birey
• 金额: $ 24.9万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-04-01 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10596187
• 关键词: 3-Dimensional; Affect; Biological; 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; Migration Assay; Modeling; Modification; Molecular; Mutation; Neurobiology; Neurons; Outcome; Pathogenicity; Pathologic; Pathway interactions; Patients; Pharmaceutical Preparations; Phase; Phenotype; Pregnancy; Process; Property; Prosencephalon; Radial; Research; Research Personnel; Role; Schizophrenia; Slice; Specific qualifier value; Surveys; Synapses; TCF7L2 gene; Techniques; Testing; Tissues; Training; Universities; Variant; autism spectrum disorder; cell type; critical period; design; differential expression; disease phenotype; disease-causing mutation; dosage; epileptic encephalopathies; excitatory neuron; experimental study; fetal; gene network; genetic variant; human model; induced pluripotent stem cell; inhibitory neuron; insight; laboratory experience; microphysiology system; migration; multiphoton imaging; myocyte-specific enhancer-binding-factor 2C; neuropsychiatric disorder; new therapeutic target; novel strategies; optogenetics; pharmacologic; programs; public health relevance; response; skills; stem cell model; 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.

项目摘要 /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。规格后，GABA能中间神经元在背侧迁移以达到皮层，​​并 经历活动依赖性成熟并整合到谷氨酸能电路中。这个过程的遗传扰动可以 导致早期皮质电路的偏离和激发/抑制失衡，这被认为是各种各样的 精神分裂症，自闭症谱系障碍和癫痫病等疾病。疾病如何的神经生物学基础 相关的基因变异会影响人类早期皮质回路的组装仍然未知。这主要是由于 缺乏用于功能研究的患者组织。为此，我们最近开发了一个3D体外平台 前脑发育，称为前脑组合，其中特定于地区的前脑文化源自人类 诱导的多能干细胞（HIPSC）在功能上组装。使用此平台，我们证明了Gabaergic 中间神经元向向谷氨酸能神经元迁移并与形成表现出的皮质集合的神经元整合 谷氨酸能和GABA能突触活动。当我们调查中的中间神经元中差异表达的基因时 在皮质网络中迁移，我们确定了TCF4，这是一种基本的环路 - 环转录因子，可能表明 在中间功能成熟中的作用。与这个想法一致，在临床上已经确定了几种TCF4变体 经常与中间神经元功能障碍有关的不同疾病，例如精神分裂症，自闭症谱系 疾病，智力残疾和癫痫性脑病。 TCF4是一个主要的转录枢纽，通过其细胞 - 受细胞内钙水平调节的类型特异性二聚化伴侣可以在各个阶段承担不同的作用 胎儿脑发育。因此，TCF4剂量被认为在开发过程中受到严格调节。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局相关， 尽管尚未在人类中对此进行彻底的测试。该提议的目的是了解机制 不同的TCF4变体影响TCF4调节网络，并导致人类中间神经元的分子和细胞缺陷 在前脑组合物中的早期皮质回路组装过程中。在K99阶段，我建议生成和 通过CRISPR/CAS9基因编辑具有各种疾病相关的TCF4突变的HIPSC系 在Porteus实验室进行培训。然后，我将从这些线中生成前脑组合物，并询问是否迁移，是否迁移 皮质中间神经元的内在特性，突触整合和功能连通性在皮质中破坏 合奏，通过Huguenard Lab的培训。在独立的R00阶段，我将研究分子缺陷 与AIM 1和2中发现的与赤字有关的与TCF4相关的基因网络，并探索药理学靶标的抢救 实验。与博士的全面培训。帕斯卡（Pasca），休格纳德（Huguenard）和斯坦福大学（Porteus） 作为独立研究者，进一步追求这一研究所需的技能。这些努力将导致机械 洞悉主要生物学途径，该途径可能会在各种各样的精神疾病中共享。