GABA/Glutamate Neuron Differentiation Imbalance and Increased AKT/mTOR Signaling in CNTNAP2-/- Cerebral Organoids

Kleanthi Chalkiadaki; Elpida Statoulla; Maria Zafeiri; Georgia Voudouri; Theoklitos Amvrosiadis; Alexandra Typou; Niki Theodoridou; Dimitrios Moschovas; Apostolos Avgeropoulos; Martina Samiotaki; John O Mason; Christos G Gkogkas

doi:10.1016/j.bpsgos.2024.100413

GABA/Glutamate Neuron Differentiation Imbalance and Increased AKT/mTOR Signaling in CNTNAP2^-/- Cerebral Organoids

Biol Psychiatry Glob Open Sci. 2024 Nov 8;5(1):100413. doi: 10.1016/j.bpsgos.2024.100413. eCollection 2025 Jan.

Affiliations

¹ Biomedical Research Institute, Foundation for Research and Technology-Hellas, University Campus, Ioannina, Greece.
² Centre for Discovery Brain Sciences, University of Edinburgh, Edinburgh, United Kingdom.
³ Department of Materials Science Engineering, University of Ioannina, Ioannina, Greece.
⁴ Biomedical Sciences Research Center "Alexander Fleming", Vari, Greece.
⁵ Simons Initiative for the Developing Brain, University of Edinburgh, Edinburgh, United Kingdom.

Abstract

Background: The polygenic nature of autism spectrum disorder (ASD) requires the identification of converging genetic pathways during early development to elucidate its complexity and varied manifestations.

Methods: We developed a human cerebral organoid model from induced pluripotent stem cells with targeted genome editing to abolish protein expression of the CNTNAP2 ASD risk gene.

Results: CNTNAP2^-/- cerebral organoids displayed accelerated cell cycle, ventricular zone disorganization, and increased cortical folding. Proteomic analysis revealed disruptions in glutamatergic/GABAergic (gamma-aminobutyric acidergic) synaptic pathways and neurodevelopment, and transcriptomic analysis revealed differentially expressed genes belonging to inhibitory neuron-related gene networks. Interestingly, there was a weak correlation between the 2 datasets, suggesting nuanced translational control mechanisms. Along these lines, we found upregulated AKT/mTOR (mechanistic target of rapamycin) signaling in CNTNAP2^-/- organoids. Spatial transcriptomic analysis of CNTNAP2^-/- ventricular-like zones demonstrated pervasive changes in gene expression, implicating upregulation of cell cycle regulation, synaptic, and glutamatergic/GABAergic pathways. We noted significant overlap of all day-30 organoid omics datasets differentially expressed genes from idiopathic ASD (macrocephaly) induced pluripotent stem cell-derived telencephalic organoids, where FOXG1 was upregulated. Moreover, we detected increased GAD1-expressing and decreased TBR1-expressing cells, suggesting altered GABAergic/glutamatergic neuron development.

Conclusions: These findings potentially highlight a shared mechanism in the early cortical development of various forms of ASD, further elucidate the role of CNTNAP2 in ASD pathophysiology and cortical development, and pave the way for targeted therapies that use cerebral organoids as preclinical models.

Keywords: AKT/mTOR; Autism; CNTNAP2; Cerebral organoids; GABA/glutamate; Neurodevelopment.

Plain language summary

Chalkiadaki et al. developed a human cerebral organoid model by using genome-edited induced pluripotent stem cells to study autism spectrum disorder (ASD). The model, which lacks the CNTNAP2 gene associated with ASD, showed abnormal brain development, including disrupted neuron differentiation and increased signaling in pathways linked to neurodevelopment. These findings suggest that CNTNAP2 plays a critical role in early brain formation and may contribute to ASD pathophysiology, thus providing a platform for exploring potential therapies. The study highlights the importance of CNTNAP2 in neuron development and its impact on cortical structure in ASD.