Investigating the role of TCF4 in human interneuron function and dysfunction

项目总结/摘要 胎儿皮层发育过程中皮层回路的形成涉及到海马能神经元的组装， GABA能中间神经元。在它们的特化之后，GABA能中间神经元向背侧迁移以到达皮质， 经历活性依赖性成熟并整合到突触能回路中。这一过程的遗传干扰可以 导致早期皮层回路错误布线和兴奋/抑制失衡，这被认为是各种 例如精神分裂症、自闭症谱系障碍和癫痫。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，我们最近开发了一个3D体外平台， 前脑发育，称为前脑组装体，其中区域特异性前脑培养物来自人类 诱导多能干细胞（hiPSC）被功能性组装。利用这个平台，我们发现GABA能 中间神经元向海马能神经元迁移并与之整合，形成表现出 谷氨酸能和GABA能突触活性。当我们调查中间神经元的差异表达基因时， 迁移到皮层网络中，我们鉴定了TCF 4，一种基本的环-螺旋-环转录因子，可能表明TCF 4是一种在大脑皮层网络中迁移的转录因子。 中间神经元功能成熟的作用。根据这一想法，已经在临床上鉴定了几种TCF 4变体。 经常与中间神经元功能障碍相关的不同疾病，如精神分裂症、自闭症谱系 疾病、智力残疾和癫痫性脑病。TCF 4是一个主要的转录中心，通过其细胞- 由细胞内钙离子水平调节的类型特异性二聚化伴侣，可以在不同的阶段承担不同的作用。 胎儿大脑发育因此，TCF 4剂量被认为在发育过程中受到严格调控。已经 假设每种TCF 4变体影响其剂量的程度与特定的临床结果相关， 尽管这还没有在人类身上进行彻底的测试。本提案的目标是了解 不同的TCF 4变体影响TCF 4调节网络，并导致人类中间神经元的分子和细胞缺陷 在前脑组装体中组装早期皮层回路的过程中在K99阶段，我建议生成和 使用CRISPR/Cas9基因编辑通过以下方式表征携带各种疾病相关TCF 4突变的hiPSC系： 在Porteus实验室接受培训然后，我将从这些线生成前脑组装体，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实验室的训练，在独立R 00阶段，我将研究分子缺陷 与目标1和2中发现的缺陷相关的TCF 4相关基因网络，并探索补救的药理学靶点 实验在斯坦福大学接受帕斯卡、胡格纳德和波特斯博士的全面培训， 所需的技能，以进一步追求这条线的研究作为一个独立的调查员。这些努力将导致机械化 深入了解一个主要的生物途径，这是潜在的共享在各种精神疾病。

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.