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.

原始摘要：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能中间神经元背侧迁移到达皮质并经历活动依赖性 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以及激发/抑制失衡，这被认为是 是各种疾病的基础，例如精神分裂症、自闭症谱系障碍和癫痫症。 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 人类的皮质回路仍然未知。这主要是由于患者组织缺乏 可用于功能研究。针对这一点，我们最近开发了3D体外 前脑发育平台，称为前脑组装体，其中区域特定的前脑 来自人类诱导多能干细胞 (hiPSC) 的培养物进行功能性组装。 使用这个平台，我们表明 GABA 能中间神经元迁移并整合 谷氨酸能神经元形成皮质群，表现出谷氨酸能和 GABA 能 突触活动。当我们调查中间神经元中差异表达的基因时 在皮质网络中迁移后，我们鉴定了TCF4，一种基本的环-螺旋-环转录因子， 可能表明在中间神经元功能成熟中发挥作用。本着这个想法，几 TCF4 变异体已在临床上常见的不同疾病中被发现 与中间神经元功能障碍有关，例如精神分裂症、自闭症谱系障碍、 智力障碍和癫痫性脑病。 TCF4 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伙伴，可以 在胎儿大脑发育的不同阶段发挥不同的作用。因此，TCF4剂量为 认为在开发过程中受到严格监管。据推测，该程度由 每个 TCF4 变体对其剂量的影响与特定的临床结果相关，尽管 这尚未在人体中进行彻底的测试。该提案的目标是了解 不同的 TCF4 变异影响 TCF4 调控网络并导致 早期皮质回路组装过程中人类中间神经元的分子和细胞缺陷 前脑组装体。在K99阶段，我建议生成并表征hiPSC 使用 CRISPR/Cas9 基因编辑技术携带各种与疾病相关的 TCF4 突变的品系 通过在 Porteus 实验室的培训。然后我将从这些细胞系中生成前脑组合体 并询问迁移、内在特性、突触整合和功能是否 通过训练，皮质中间神经元的连接在皮质整体中被破坏 于格纳德实验室。在独立R00阶段，我将研究分子缺陷TCF4- 与目标 1 和 2 中发现的缺陷相关的相关基因网络并探索药理学 救援实验的目标。