APP-CTFβ/C99 oligomers drive synaptic vesicle tethering through C-terminal interactions

Proteolytic processing of the amyloid precursor protein (APP) generates a 99-amino acid precursor, β-carboxyl-terminal fragments (APP-CTFβ or C99). Upon γ-secretase inhibition, APP-CTFβ accumulates and induces synaptic defects, resulting in neuronal hyperactivity. However, mechanistic insights in the critical role of APP-CTFβ has not been completely elucidated. Here, we show that in primary neurons expressing human APP-CTFβ (C99) variants, acute γ-secretase inhibition selectively increases evoked synaptic vesicle release in cells, whereas deletion of the C-terminus abolishes this effect. Using single-molecule approaches and reconstituted membrane systems, we demonstrate that accumulation of APP-CTFβ promotes its oligomerisation. In particular, APP-CTFβ oligomers augment synaptic vesicle tethering via their C-terminal domain. This effect is driven by the interaction with synaptic vesicle proteins, independent of the YENPTY binding motif. Additionally, APP-CTFβ oligomers were associated with alterations in membrane lipid organization. Together, our findings identify APP-CTFβ oligomerization as a constitutional gain-of-function mechanism that enhances presynaptic vesicle tethering and release, providing mechanistic insight into how altered APP processing regulates synaptic activity.