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.
(Public Health Relevance Statement) Assembly of inhibitory and excitatory neurons into early networks represents a critical period in cortical development during which early principles of circuit formation and function are established. This elaborate process, which involves inhibitory neurons migrating long distances to eventually integrate with excitatory neurons, is often associated with various neuropsychiatric disorders; however, the basis of how disease-causing mutations affect discreet steps of this process in humans remains unknown. This proposal aims to investigate, using a stem-cell-based model of human cortical development, how distinct mutations in the gene TCF4, each of which has been implicated in distinct disorders such as schizophrenia, intellectual disability and epilepsy, affect the assembly of early human cortical networks. The long-term goal is to attain a comprehensive understanding of disease biology surrounding atypical cortical networks in patients with discrete TCF4 mutations and eventually identify new drug targets that are potentially shared across various disorders.
