Abstract INTRODUCTION Neuronal degeneration and immune cell activation occur early in Alzheimer's disease (AD), but the responsible molecules remain undetermined. While exogenous amyloid beta oligomers (AβOs) induce neuronal death and gliosis, the role of endogenous AβOs is less defined. METHODS Brain sections from 1‐ to 12‐month‐old 5xFAD mice were immunolabeled for AβOs, activated glia, phosphorylated transactive response DNA‐binding protein 43 kDa (pTDP‐43), and other AD markers. Neuropathology was analyzed following 60‐day oral treatment with NU‐9, a small‐molecule AβO inhibitor. RESULTS By 8 weeks, AβOs accumulated in the subiculum alongside early reactive astrocytes and activated microglia. Clinical‐stage antibody ACU193 detected AβOs in early‐stage degenerating neurons, while NU4‐labeled denser deposits in late‐stage degenerating neurons. ACU193 + AβOs accumulated on reactive astrocyte surfaces, which also contained pTDP‐43, and later emerged inside activated microglia. NU‐9 reduced astrocyte‐associated ACU193 + AβOs, pTDP‐43, and markedly diminished glial fibrillary acidic protein. DISCUSSION These findings demonstrate in vivo efficacy of NU‐9 and support targeting ACU193 + AβOs to mitigate AD progression. Highlights

ACU193 + AβOs accumulated as puncta in neurons at an early stage of degeneration, while NU4 + AβOs appeared as dense deposits only in late‐stage degenerating neurons.

The onset and progression of ACU193 + AβOs paralleled activated microglia and reactive astrocytes.

ACU193 + AβOs significantly increased on reactive astrocyte surfaces, as NU4 + AβOs accumulated in halos around Thio‐S + plaque cores.

In older mice, the ACU193 signal decreased on astrocytes and was found inside activated microglia.

Sixty‐day oral NU‐9 treatment significantly reduced astrocyte ACU193 + AβOs and markedly decreased reactive astrogliosis.