CSF1R T567M mutation induces microglial dysfunction and synaptic impairment in patient iPSC-derived cerebral organoids of CSF1R-related disorder.

CSF1R-related disorder (CSF1R-RD) is a rare autosomal dominant neurodegenerative disease characterized by cognitive decline, motor dysfunction, psychiatric symptoms, and white matter abnormalities. It is caused by mutations in the CSF1R gene. Despite the identification of many pathogenic CSF1R variants, the molecular mechanisms behind neuropathogenesis in CSF1R-RD remain poorly understood due to the lack of disease modeling. This study focuses on a novel CSF1R mutation, T567M, located outside the tyrosine kinase domain, whose pathogenic impact has not been characterized. To gain molecular insights into the pathogenic mechanisms of the CSF1R-T567M mutation, we established an induced pluripotent stem cell (iPSC) model system consisting of mutant (CSF1R-MT) and CRISPR/Cas9-corrected isogenic control lines. Using these iPSCs, we generated iPSC-derived microglia (iMGL) and cerebral organoids (COs). Through RNA sequencing, we identified altered genes and pathways involved in neuroinflammation in MT iMGL. We then investigated microglial migration, phagocytosis, cytokine profiling, neurodevelopment, and synaptic function in iMGL and iMGL-CO co-culture to study the role of T567M mutation in CSF1R-RD. Our research revealed that the CSF1R-MT caused haploinsufficiency of CSF1R, reducing autophosphorylation of CSF1R at Tyr546 and activating autophagy. CSF1R-MT iMGL induced neuroinflammation, increased phagocytosis, and impaired migration. Transcriptomic analysis showed upregulation of immune activity and downregulation of synaptic function. Additionally, CSF1R-MT promoted proliferation, inhibited neural differentiation and maturation, and caused neurodevelopmental defects in COs. Whole-cell patch-clamp recordings indicated impaired synaptic function in CSF1R-MT COs. Furthermore, CSF1R-MT microglia impaired synaptic protein expression when co-cultured with CSF1R-MT COs. Collectively, our study provides detailed mechanistic insights into the pathogenesis driven by the CSF1R-T567M mutation, highlighting the critical role of CSF1R signaling in neural homeostasis. This isogenic iPSC model serves as a valuable platform for probing mutation-specific mechanisms and future therapeutic screening.