Axonal spheroids (dystrophic neurites) are commonly found around amyloid deposits in Alzheimertextquoterights disease (AD). They impair electrical conduction, disrupt neural circuits, and correlate with AD severity. Despite their significance, the mechanisms underlying spheroid formation remain unknown. To address this, we developed a proximity labeling proteomics approach to uncover the proteome of spheroids in human postmortem and mouse brains. Additionally, we established a human iPSC-derived AD model allowing mechanistic investigation of spheroid pathology and optical electrophysiology. This approach revealed the subcellular molecular architecture of spheroids and identified abnormalities in key biological processes, including protein turnover, cytoskeleton dynamics, and lipid transport. Notably, the PI3K/AKT/mTOR pathway, which regulates these processes, was activated within spheroids. Furthermore, phosphorylated mTOR levels in spheroids strongly correlated with AD severity in humans. Importantly, inhibition of mTOR in iPSC-derived neurons and in mice ameliorated spheroid pathology. Altogether, our study provides a multidisciplinary toolkit for investigating mechanisms and novel targets for axonal pathology in neurodegeneration.Competing Interest StatementThe authors have declared no competing interest.