RBFOX1 Dysfunction Unlocks APOE4 ‐Associated Microglial Genesis and Exacerbates Alzheimer's Pathology in Human Cerebral Organoids

ABSTRACT Alzheimer's disease (AD) pathogenesis is strongly influenced by APOE4, though how cooperative genetic factors modulate this relationship remains unclear. While genomic studies have tentatively linked RBFOX1 to AD susceptibility, its functional synergy with APOE4 has never been experimentally defined. We engineered APOE3 or APOE4 isogenic human cerebral organoids with CRISPR/Cas9‐mediated RBFOX1 knockout. Remarkably, RBFOX1 depletion selectively triggered robust microglial generation exclusively in APOE4 organoids. Time‐course gene expression revealed that this APOE4‐specific effect correlated with prolonged mesodermal priming during early embryoid body differentiation, creating a permissive niche for microglial lineage specification. The emergent microglia exhibited pronounced neurotoxic phenotypes, including pro‐inflammatory factor secretion, synaptic architecture remodeling, and lipid droplet accumulation in organoids. These changes coincided with aggravated Tau hyperphosphorylation and electrophysiological abnormalities, collectively mirroring multifaceted AD pathology. Our findings establish RBFOX1 as a potential AD protective factor, a critical suppressor of APOE4‐glia crosstalk, and demonstrate that its loss unleashes a microglia‐mediated neurodegenerative cascade. By developing cerebral organoids with autonomous microglial networks, we present a platform capable of modeling genotype‐dependent neuron‐glia interactions in AD, opening new avenues for mechanistic and therapeutic exploration.