Electrophysiological and Morphological Characterization of Human iPSC-Derived Neurons with a Schizophrenia-Associated SETD1A Mutation

Abstract

Schizophrenia is a severe and highly heritable psychiatric disorder, and rare high-risk genetic variants provide important opportunities to investigate its underlying mechanisms. One such gene harbouring these rare variants is SETD1A, with heterozygous loss-of-function mutations causing haploinsufficiency and conferring a substantially increased risk of schizophrenia, alongside a broader neurodevelopmental phenotype. This study investigated the effects of a patient-relevant SETD1A loss-of-function mutation on axonal morphology and network-level electrophysiological activity in human induced pluripotent stem cell (hiPSC)-derived neurons.

SETD1A mutant and isogenic control hiPSC lines were differentiated into neurogenin-2 (Ngn2)-induced neurons and co-cultured with astrocytes. Electrophysiological activity was assessed using high-density microelectrode arrays (HD-MEAs), while axonal phenotypes were investigated using microfluidic microtunnel devices. In parallel, the Ngn2 differentiation protocol was optimized to improve culture robustness.

SETD1A haploinsufficiency was associated with reduced spontaneous
neuronal activity and altered burst organization without alterations in temporal firing regularity, whereas no detectable difference in distal axonal occupancy was observed between SETD1A haploinsufficient and isogenic control cultures. The differentiation protocol was improved by incorporation of a replating step, resulting in improved culture homogeneity, long-term stability, and electrophysiological activity, with the strongest effects observed in later-stage network-burst organization. Furthermore, the microfluidic platform was established as a tool for studying axonal phenotypes in human neurons, although complete compartmentalization was not achieved.

Together, these findings extend the electrophysiological characterization of SETD1A haploinsufficiency using HD-MEAs and provide an initial assessment of distal axonal occupancy in a human neuronal model. More broadly, the study provides insights into neuronal phenotypes associated with SETD1A haploinsufficiency, a schizophrenia-relevant genetic model, while highlighting methodological considerations important for human neuronal disease modelling.