Divergent toxicity mechanisms of amyloid-beta aggregates arising from a single aggregation reaction.

Amyloid-β 1-42 (Aβ42) aggregation is among the earliest pathological signs in Alzheimer's disease (AD). Here, we characterized Aβ42 species at several aggregation stages at the single-molecule level and examined their toxicity in murine organotypic brain slices, where we observed a stage-dependent recapitulation of multiple aspects of the cellular phase of AD. Aggregates formed during the lag phase of the Aβ42 aggregation elevated neuronal baseline Ca2+ levels and impaired long-term potentiation (LTP), while promoting microglial homeostatic exit and transition to disease-associated microglia (DAM) state. In contrast, aggregates enriched during the growth phase downregulated homeostatic microglial markers and induced TLR4-mediated microglial activation, cytokine production, and complement activation, leading to synaptic engulfment and severe disruption of neuronal activity. Together, these findings reveal that structurally distinct Aβ42 aggregate species engage different cellular and molecular pathways. This framework advances mechanistic understanding of amyloid toxicity in neurodegeneration and could inform the design of combination therapeutic strategies.