Figure 4: AEP interacts with APP in the endolysosomal system.

(a,b) AEP and APP distribution in the subcellular fractions. Brain samples from 5-month-old WT (a) and age-matched 5XFAD (b) mice were homogenated and fractionated on a discontinuous sucrose gradient. The fractions were analysed by western blotting (WB) for AEP, APP fragments, BACE1, EEA1 (endosome marker), GGA3 (trans-Golgi network maker) and LAMP1 (lysosome marker). The relative amount of AEP, EEA1 and LAMP1 in each fraction was quantified (mean±s.e.m. of three independent experiments, t-test, *P<0.01 compared with WT mice). (c) APP co-localizes with AEP and EEA1. Primary neuronal cultures (DIV 12) were treated with 2 or 20 μM of pre-aggregated Aβ for 24 h, followed by immunostaining with various antibodies including anti-AEP, anti-APP or anti-EEA1. Aβ treatment increased the co-localization of APP and AEP in primary cortical neurons (left panels). APP co-localized with the endosomal marker EEA1 as well (right panels). Shown are the representative figures of two independent experiments. Scale bar, 10 μm. (d) Aβ treatment elicits AEP activation in neurons in a dose-dependent manner. (e) Internalization assay showing the effect of 20 μM Aβ on APP endocytosis. (f) Co-immunoprecipitation of APP and AEP in WT and 5XFAD mouse brain. AEP in mouse brain lysates was immunoprecipitated with anti-AEP antibody and analysed by immunoblotting with anti-APP antibody. MW, molecular weight.