PBA Enhances APP Proteolysis Figure 7. PBA stimulates secretase-mediated APP cleavage. The NAG cells were treated with titrated concentrations of PBA for 24 hours. PBA elicited elevated levels of Gal4-reporter reporter transactivation in a concentration dependent fashion (p,0.0001; student t-test 0 versus 10 mM PBA) (A). PBA effects upon APPGV16, PS1 and Nicastrin expression levels were examined in the NAG cells 24 hours post- treatment. APP-Gal4VP16 levels increased slightly across the PBA titration. The CTFGV16 proteolytic fragment, whose molecular weight corresponds to the a-cleavage product of APPGV16, increased substantially across the PBA titration (B, top blot). In contrast, no increases in Nicastrin or PS1 levels were observed (B, middle and bottom blots). CTFGV16 increased in NAG cells treated with PBA and DAPT. Co-treatment with DAPT and PBA increased CTFGV16 protein levels in an additive manner, consistent with PBA stimulating CTF-GV16 production (C). doi:10.1371/journal.pone.0009135.g007 induced reporter activity with PBA treatment, in the absence of any exogenously transfected APP, may be due to promotion of AICD production from endogenously expressed APP. Consistent with previous data showing thapsigargin induced decreases in APPGV16 cleavage, thapsigargin treatment of the APP/Fe65- Gal4 transfected cells resulted in a small, but statistically significant (p,0.001, two-way ANOVA), decrease in Fe65-Gal4 nuclear signaling relative to both untreated and PBA treated cells (Figure 10B). Figure 8. PBA stimulation of APPGV16 requires secretase processing. In order to determine whether PBA stimulation of APPGV16 is dependent upon proteolytic processing, NAG cells were treated with titrated levels of PBA in the presence or absence of specific secretase inhibitor:. 20 mM GM6001 (broad-spectrum metalloprotease inhibitor), 50 mM TAPI-II (a-secretase inhibitor), or 20 mM DAPT (c- secretase inhibitor). PBA stimulated a statistically significant 9-fold increase in APP proteolysis (p,0.0001; student t-test). However, co- treatment with GM6001 or TAPI-II decreased the fold-stimulation by approximately half (p,0.0001; two-way ANOVA). DAPT significantly reduced the levels of activity and eliminated the stimulatory effect of PBA (p,0.0001; two-way ANOVA). The b-secretase contribution to PBA enhanced APP proteolysis was examined using two different concen- trations of beta-secretase inhibitor (BSI) IV (B). PBA stimulated an 8–9 fold increase in normalized Gal4-luciferase activity. BSI IV decreased Gal4-luciferase activity by less than 25% (p,0.01; student t-test) (B). These data suggest that a-secretase plays a more significant role than b-secretase in PBA enhanced APP proteolysis. doi:10.1371/journal.pone.0009135.g008 In order to validate the stimulatory effect of PBA upon APP nuclear signaling, the APP-Gal4 [39] transcriptional activator was transfected into na¨ıve N2a cells, along with Fe65, the Gal4- luciferase reporter, and the bGal normalization vector. Untagged Fe65 was co-transfected with APP-Gal4 at a 1:1 vector ratio. To ensure that the reporter system was in a sensitive range, a low (50 ng) and high (500 ng) concentration of APP-Gal4 and Fe65 were used. At 24 hours post-transfection the cells were either left untreated, or stimulated with 5 mM PBA, for an additional 24 hours. In both low and high vector concentrations, PBA stimulated an approximate 2-fold increase in normalized reporter activity (Figure 10C). Interestingly, both reporter systems detected commensurate levels of increased APP nuclear signaling with PBA treatment, an approximate two-fold increase, which is consider- ably lower than the PBA mediated increase in AICD production. PLoS ONE | www.plosone.org 10 February 2010 | Volume 5 | Issue 2 | e9135