Alzheimer’s disease linked amyloid beta 1‐42 exerts product feedback inhibition on gamma‐secretase impairing downstream signaling cascades

Abstract

AbstractBackgroundΓ‐secretases are proteolytic switches at the membrane regulating multiple signaling cascades. Their dysfunction, resulting in enhanced generation of longer amyloid β (Aβ) peptides from the amyloid precursor protein (APP), leads to neurodegeneration in the context of Alzheimer’s disease (AD), while their inhibition causes neurodegenerative phenotypes in mice and cognitive worsening in AD patients treated with γ‐secretase inhibitors. The accumulation of Aβ in the brain is the earliest pathological hallmark of AD. Based on the proven affinity of Aβ peptides for γ‐secretases, we hypothesized that elevations in Aβ levels would promote an inhibitory‐feedback mechanism on γ‐secretases and impair downstream cell signaling events.MethodWe conducted rigorous kinetic analyses of γ‐secretase activity in the presence of a series of Aβ and p3 peptides, by quantifying the levels of intracellular domains generated from different γ‐secretase substrates in cell‐free assays. In addition, we determined the effects of these peptides on endogenous γ‐secretase activity in living neurons, using a ratiometric FRET‐based reporter and western blot analysis of the levels of immediate γ‐secretase substrates. Furthermore, we assessed the impact of Aβ and p3 peptides on γ‐secretase‐mediated, p75‐ and TrkA‐dependent downstream signaling via immunostaining for an apoptotic marker: cleaved caspase 3. Finally, we evaluated the impact of Aβ peptides on APP processing in synaptosome fractions derived from mouse brains.ResultOur analyses showed that human Aβ42 inhibited γ‐secretase activity and accordingly caused accumulation of unprocessed γ‐secretase substrates in neuronal cells, i.e. CTFs of APP, p75 and pan‐cadherin. Remarkably, neither murine Aβ42 nor human p3 (17‐42) peptides exerted the inhibition. In TrkA signaling deficient PC12 cells and basal forebrain cholinergic neurons, Aβ1‐42‐mediated inhibition of γ‐secretase led to the accumulation of unprocessed p75‐CTFs and potentiated p75‐dependent cell death, mimicking the effects of γ‐secretase inhibitors.ConclusionWe demonstrate that the pathologically relevant human Aβ1‐42 exerts product feedback inhibition on γ‐secretases, leading to dysregulation of downstream cellular signaling. These findings provide a novel conceptual framework for investigations of Aβ toxicity in the context of γ‐secretase‐dependent homeostatic signaling and raise the possibility that Aβ42‐mediated inactivation of these enzymes contributes to AD development.