Amyloid deposits in Alzheimer’s disease (AD) are surrounded by large numbers of plaque-associated axonal spheroids (PAAS). PAAS disrupt axonal electrical conduction and neuronal network function, and correlate with AD severity. However, the mechanisms that govern their formation remain unknown. To uncover the molecular architecture of PAAS, we applied proximity labeling proteomics of spheroids in human AD postmortem brains and mice. We then implemented a human iPSC-derived AD model recapitulating PAAS pathology for mechanistic studies. Using this strategy, we uncovered hundreds of previously unknown PAAS-enriched proteins and signaling pathways, including PI3K/AKT/mTOR. Phosphorylated mTOR was highly enriched in PAAS and strongly correlated with disease severity in humans. Importantly, pharmacological mTOR inhibition in iPSC-derived human neurons or AAV-mediated knockdown in mice, led to a marked reduction of PAAS pathology. Altogether, our study provides a novel platform to examine mechanisms of axonal pathology in neurodegeneration and to evaluate the therapeutic potential of novel targets.