DEVELOPMENT OF A NOVEL HIGH-THROUGHPUT SCREENING METHOD FOR DRUG TARGET DISCOVERY IN SCHIZOPHRENIA BY MEANS OF UTILIZING CO-CULTURED HIPSC-DERIVED CORTICAL NEURONS AND MICROGLIA

Schizophrenia is a highly heritable psychiatric disorder affecting about 1% of the global population, characterized by grey matter loss, impaired network connectivity, and reduced synapse density. Genetic studies highlight the complement component 4 (C4) locus as a major risk factor, with higher C4A activity correlating with excessive synaptic pruning, most consistently observed in the context of microglial involvement.
To investigate this mechanism, we use a human in vitro co-culture of hiPSC-derived cortical neurons and microglia. Overexpression of C4A in neurons revealed enhanced microglial activation and synapse engulfment, leading to pronounced synaptic deficits.
Building on this foundation, we are advancing the assay into a high-throughput compatible format using the Revvity Opera Phenix Plus High-Content Screening system combined with the explorer G3 workstation. A key step in this transition has been the implementation of a new guided differentiation protocol aimed at reducing experimental time through the rapid and reliable generation of homogeneous cortical neuronal cultures. Ongoing work also includes the development of a cryopreservation protocol for microglia to establish a cell bank that facilitates rapid and reproducible large-scale applications. In parallel, an automated analysis pipeline has been developed within the Harmony system for quantitative assessment of synaptic pruning, enabling efficient analysis of large-scale datasets. The pipeline also incorporates automated detection and classification of neuronal and microglial nuclei without the need for a microglial marker, thereby enhancing assay robustness and efficiency. Together, these improvements enable systematic and scalable testing of candidate compounds, providing a powerful platform for discovering potential drugs.