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: 10348034
• 负责人: Fikri Birey
• 金额: $ 2.6万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-02-09 至 2021-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10348034
• 关键词: 3-Dimensional; Affect; Biology; Brain; CRISPR/Cas technology; Calcium; Cell Line; Clinical; Development; Dimerization; Disease; Dorsal; Epilepsy; Exhibits; Forebrain Development; Functional disorder; Genes; Genetic; Genetic Transcription; Glutamates; Goals; Human; In Vitro; Intellectual functioning disability; Interneuron function; Interneurons; Lead; Molecular; Mutation; Neurobiology; Neurons; Outcome; Patients; Pharmacology; Phase; Process; Property; Prosencephalon; Role; Schizophrenia; Surveys; Synapses; TCF7L2 gene; Testing; Tissues; Training; Variant; autism spectrum disorder; cell type; critical period; differential expression; disease-causing mutation; dosage; epileptic encephalopathies; excitatory neuron; experimental study; fetal; genetic variant; human model; induced pluripotent stem cell; inhibitory neuron; migration; neuropsychiatric disorder; new therapeutic target; response; stem cell model; transcription factor

Original 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 remains 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.

在胎儿皮层发育过程中皮层回路的形成涉及 谷氨酸能神经元和GABA能中间神经元的组装。在他们的规范之后， GABA能中间神经元向背侧迁移到达皮质，并经历活动依赖性的 成熟并整合到神经元能回路中。这一过程的遗传干扰可以 导致早期皮层回路的错误连接和兴奋/抑制失衡， 导致各种疾病，如精神分裂症，自闭症谱系障碍和癫痫。 疾病相关基因变异如何影响早期神经元组装的神经生物学基础 人类的皮层回路仍然未知。这主要是由于缺乏患者组织 可用于功能研究。针对这一点，我们最近开发了一种3D体外 前脑发育的平台，称为前脑组装体，其中区域特异性前脑 功能性组装源自人诱导多能干细胞（hiPSC）的培养物。 使用这个平台，我们发现GABA能中间神经元迁移并整合到 形成表现出多巴胺能和GABA能的皮质集合体的多巴胺能神经元 突触活动。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， 可能表明在中间神经元功能成熟中的作用。根据这一想法，一些 TCF 4变体已在临床上不同的疾病中被鉴定，这些疾病经常被 与中间神经元功能障碍相关，例如精神分裂症，自闭症谱系障碍， 智力残疾和癫痫性脑病。TCF 4是一个主要的转录中心， 通过其受细胞内钙水平调节的细胞类型特异性二聚化伴侣， 在胎儿大脑发育的不同阶段扮演不同的角色。因此，TCF 4剂量为 被认为在发育过程中受到严格的调控。据推测， 每种TCF 4变体影响其剂量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。本提案的目的是了解 不同的TCF 4变体影响TCF 4调节网络并导致 人类中间神经元在早期皮质回路组装过程中的分子和细胞缺陷 前脑组装体在K99阶段，我建议生成并表征hiPSC 使用CRISPR/Cas9基因编辑携带各种疾病相关TCF 4突变的细胞系 通过在波特乌斯实验室的训练然后我将从这些线条中生成前脑神经胶质瘤 并询问是否迁移，内在属性，突触整合和功能 皮质中间神经元的连接性在皮质系综中被破坏， Huguenard实验室。在独立R 00阶段，我将研究TCF 4- 目标1和目标2中发现的缺陷相关的基因网络，并探索药理学 救援实验的目标。