Increased Foxo3a nuclear translocation and activity is an early neuronal response to βγ-secretase mediated processing of the Amyloid-β-Protein Precursor Protein: Utility of an AβPP-GAL4 reporter assay

Sequential cleavage of the amyloid-β protein precursor (AβPP) by BACE1 (β-secretase) followed by the γ-secretase complex, is strongly implicated in Alzheimer’s Disease (AD) but the initial cellular responses to these cleavage events are not fully defined. β-secretase-mediated AβPP processing yields an extracellular domain (sAβPPβ) and a C-terminal fragment of APP of 99 amino acids (C99). Subsequent cleavage by γ-secretase produces amyloid-β (Aβ) and an APP intracellular domain (AICD). A cellular screen based on the generation of AICD from an AβPP-Gal4 fusion protein was adapted by introducing familial AD (FAD) mutations into the AβPP sequence and linking the assay to Gal4-UAS driven luciferase and GFP expression, to identify responses immediately downstream of AβPP processing in neurons with a focus on the transcription factor Foxo3a which has been implicated in neurodegeneration. The K670N/M671L, E682K, E693G, V717I FAD mutations and the A673T protective mutation, were introduced into the AβPP sequence by site directed mutagenesis. When expressed in mouse cortical neurons AβPP-Gal4-UAS driven luciferase and GFP expression was substantially reduced by γ-secretase inhibitors, lowered by β-secretase inhibitors and enhanced by α-secretase inhibitors suggesting that AICD is a product of the β-secretase pathway. AβPP-Gal4-UAS driven GFP expression was exploited to identify individual neurons undergoing amyloidogenic AβPP processing, revealing increased nuclear localization of Foxo3a and enhanced Foxo3a-mediated transcription downstream of AICD production. Foxo3a translocation was not driven by AICD directly but correlated with reduced Akt phosphorylation. Collectively this suggests that βγ-secretase-mediated AβPP processing couples to Foxo3a which could be an early neuronal signaling response in AD.