PBA Enhances APP Proteolysis Figure 6. PBA stimulates trafficking out of the intracellular organelles. NAG cells were untreated, or treated with thapsigargin (0.25 mM), tunicamycin (5 mg/ml), or brefeldin A (5 mg/ml) in the presence or absence of PBA for 24 hours. The cells were stained with the VP16 antibody (red) and counterstained with Hoescht (blue) to localize the nuclei. Confocal imaging was performed to examine protein localization. In untreated cells, APPGV16 localized throughout the cytosol with slight peri-nuclear aggregations observed, consistent with the ER localization of de novo membrane proteins. PBA treatment promoted migration of APPGV16 toward the plasma membrane (top row). In tunicamycin, thapsigargin and brefeldin A treated cells, APPGV16 localized to the ER-like perinuclear region (left column, lower three images). Upon the addition of 1 mM or 5 mM PBA, APPGV16 localization shifted away from the nucleus in tunicamycin and thapsigargin treated NAG cells. In contrast, PBA had no effect upon the localization of APPGV16 in the brefeldin A treated cells. doi:10.1371/journal.pone.0009135.g006 assays considered. All conditions were set-up and assayed in triplicate. The changes in Ab40 and Ab42 concentration across the PBA titration were not statistically significant at the p,0.05 level by either two-way ANOVA analysis nor by t-test compar- isons of the high and low points of the curve. Repeated experiments consistently confirmed no significant effect of PBA on amyloid secretion levels. These data support a model in which PBA stimulates APP proteolysis through the promotion of a-/c- cleavage. PBA Promotes AICD/FE65 Nuclear Signaling Numerous genes may be regulated by APP nuclear signaling, in which the AICD, liberated from the membrane by c-secretase cleavage, associates with Fe65 and travels to the nucleus to activate transcription [38,39]. In order to address whether PBA or ER stress impact APP regulated gene expression, we employed two different Gal4 systems designed to assay AICD/Fe65 mediated reporter activation. The primary study employed Fe65-Gal4 [40] and HA-tagged APP, which were co-transfected at a 1:1 ratio into na¨ıve N2a cells with the Gal4-reporter and the EF1-bGal normalization vector. Subsequently, the transfected cells were treated with titrated concentrations of PBA for 24 hours and reporter activity was measured. PBA stimulated an approximate 250% increase in Fe65-Gal4 nuclear signaling (Figure 10A). In order to optimize the Fe65: APP ratio, na¨ıve N2a cells were transfected with 100 ng/well Fe65-Gal4 and the quantity of APP expression vector was incrementally increased across transfection conditions. The cells transfected with titrated quantities of the APP expression vector were subject to either thapsigargin treatment, to induce ER stress, or PBA, to stimulate AICD production, or left untreated as the control. In all three treatment conditions, maximal activation of the Fe65-Gal4 reporter is observed when the two expression vectors are transfected at a 1:1 ratio, which occurs at 100 ng/well of the APP expression vector (Figure 10B). Additionally, PBA stimulates Fe65-Gal4 signaling at statistically significant levels (p,0.0001; two-way ANOVA) above both the untreated and thapsigargin treated conditions across the APP vector titration (Figure 10B). The elevated level of Fe65-Gal4 PLoS ONE | www.plosone.org 9 February 2010 | Volume 5 | Issue 2 | e9135