Investigating the role of TCF4 in human interneuron function and dysfunction

项目摘要/摘要 胎儿皮质发育期间皮质回路的形成涉及谷氨酸能神经元和 GABA能中间神经元。在指定后，GABA能中间神经元向背侧迁移，到达皮质和 经历依赖活动的成熟和整合到谷氨酸能回路。这一过程的遗传扰动可以 导致早期大脑皮层回路的错误连接和兴奋/抑制失衡，这被认为是各种 精神分裂症、自闭症谱系障碍和癫痫等障碍。疾病的神经生物学基础- 相关的基因变异影响人类早期皮质回路的组装仍不清楚。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，称为前脑集合体，其中特定区域的前脑培养来自人类 诱导多能干细胞(HiPSCs)是一种功能性组装细胞。利用这个平台，我们展示了GABA能 中间神经元向谷氨酸能神经元迁移并与之整合，形成皮质团簇，表现为 谷氨酸和氨基丁酸能突触活动。当我们调查中间神经元中差异表达的基因时 在大脑皮层网络中迁移，我们确定了TCF4，一个基本的环-螺旋-环转录因子，可能表明 在神经元间功能成熟中的作用。根据这一想法，临床上已经发现了几种TCF4变异体 经常与神经元间功能障碍相关的独特疾病，如精神分裂症、自闭症 精神障碍、智力残疾和癫痫脑病。TCF4是一个主要的转录枢纽，通过它的细胞- 受细胞内钙水平调节的特定类型的二聚化伙伴，可以在不同的阶段扮演不同的角色 胎儿大脑发育。因此，TCF4的剂量被认为在发育过程中受到严格控制。一直以来 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， 尽管这还没有在人体上进行彻底的测试。这项提议的目标是了解通过什么机制 不同的TCF4变异体影响TCF4调节网络，并导致人中间神经元分子和细胞缺陷 在前脑集合体的早期皮质回路的组装过程中。在K99阶段，我建议生成和 通过CRISPR/Cas9基因编辑鉴定具有各种疾病相关TCF4突变的HiPSC系 在波特乌斯实验室接受训练。然后我会根据这些线条生成前脑集合体，并询问迁徙是否， 皮质中间神经元的固有特性、突触整合和功能连接受到破坏 通过在Huguenard实验室接受培训，合唱团。在独立的R00阶段，我将调查分子缺陷 与目标1和目标2中发现的缺陷相关的TCF4相关基因网络和探索拯救的药理靶点 实验。在斯坦福大学接受帕斯卡、胡格纳德和波特乌斯博士的全面培训将为我提供 作为一名独立调查员，进一步从事这一领域研究所需的技能。这些努力将导致机械化 对一条可能在各种精神障碍中共享的主要生物学途径的见解。

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.