concomitant increase in APP proteolysis and decrease in UPR markers, argues that stimulation of APP proteolysis occurs due to changes in ER stress related processes following PBA treatment. PBA Promotes APPGV16 Trafficking Previous reports suggest that ER stress induction may alter APP trafficking [7,8]. APPGV16 localization was examined in the NAG cells under non-stress inducing conditions, or in the presence of thapsigargin, tunicamycin or BFA. In the untreated cells, APPGV16 localized throughout the cell, with limited peri-nuclear aggregation, consistent with trafficking through the early secretory pathway. Treatment with all three stress-inducing agents promot- ed accumulation in intracellular sites that probably represent ER localization. In cells treated with BFA, the peri-nuclear aggrega- tion was more robust, with no APPGV16 observed beyond the peri-nuclear region (Figure S1). In order to assess whether the small molecular chaperone PBA rectifies trafficking under ER stress-inducing conditions, NAG cells were treated with each of the ER stress-inducing pharmacological agents in the presence or absence of PBA. The cells were examined by thin-section laser scanning confocal microscopy. In the untreated cells, APPGV16 distributed ubiquitously throughout the cell, as previously observed. With increasing concentrations of PBA, the localization pattern shifted toward the outer region of the cell, consistent with a predominantly plasma membrane localization by 5 mM PBA (Figure 6, top row). The NAG cells treated with either tunicamycin or thapsigargin had restricted APPGV16 localization, accumulat- ing in the peri-nuclear region, consistent with the data in Figure S1. However, in the presence of PBA, the localization of APPGV16 is more evenly distributed throughout the cell, appearing similar to basal conditions by 5 mM PBA (Figure 6, second and third rows). Discordantly, in the NAG cells treated with BFA, APPGV16 localized to the peri-nuclear region, and was unaffected by PBA (Figure 6, bottom row). These data suggest that there is a strong correlation between APPGV16 localization and proteolytic processing in the different treatment conditions. PBA Stimulates APP Proteolysis The PBA mediated stimulation of APP proteolytic processing under ER stress conditions, and the changes in subcellular localization, suggests that PBA may promote c-secretase mediated cleavage of APP under basal conditions. In order to assess what effects PBA has upon APP cleavage in non-stress related conditions, PBA was titrated onto NAG cells in the absence of ER stress inducing compounds. PBA treatment stimulated a dose- dependent increase in APP proteolysis, reaching a near 8-fold increase by 10 mM (Figure 7A). Higher concentrations of PBA elicited no additional increase in Gal4-reporter activity (data not shown). The PBA induced stimulation of c-secretase mediated APPGV16 proteolysis was observed in three separate experiments, each demonstrating a similar magnitude of effect. There was no effect of PBA upon constitutive bGal expression, ruling out non- specific transcriptional effects contributing to the observed stimulation. In parallel experiments, gradual titrations of PBA were performed to assess the effects of PBA upon APPGV16 and mature c-secretase levels. Across the PBA titration, APPGV16 protein levels increased, but not proportionally to the observed increases in Gal4-reporter output (Figure 7B). Additionally, the levels of CTF-GV16 rose appreciably with PBA treatment, suggesting that PBA may foster APP processing at multiple stages. Specifically, based upon molecular weight PBA appears to increase the a-secretase derived CTF-GV16 species. In order to determine the effects of PBA on c-secretase, we examined PS1 and nicastrin PBA Enhances APP Proteolysis levels. We observed no effect of PBA upon PS1 or nicastrin levels across the titrated concentrations (Figure 7B). Consequently, the stimulatory effects of PBA are not due to changes in the raw protein levels of the core c-secretase components. Furthermore, PBA stimulated the accumulation of the CTF-GV16 proteolytic product in an additive manner with DAPT—wherein both PBA and DAPT elicit elevated levels of CTF-GV16, yet co-treatment of the NAG cells with both DAPT and PBA promoted increases in CTF-GV16 additively (Figure 7C). The additive effect suggests that PBA promotes APPGV16 cleavage by either a- or b-secretase. a- and c-Secretase Mediated APP Cleavage Is Required for PBA Stimulation In order to directly address which secretases are involved in PBA mediated stimulation of APP processing, the NAG cells were co-treated with PBA and pharmacological secretase inhibitors. In Figure 8A, NAG cells were treated with titrated levels of PBA in the presence or absence of either a-secretase inhibitors (GM6001 and TAPI-II) or high levels of the c-secretase inhibitor DAPT. Consistent with previous observations, PBA alone stimulated dramatic changes in Gal4-luciferase output. However, treatment with either of the a-secretase inhibitors blocked approximately 50 percent of observed reporter output. Further, the PBA titration had no appreciable effect upon NAG cells pretreated with 20 mM DAPT (twice the concentration used in Figure 1). This strongly supports the requisite involvement of both the a- and c-secretases in the PBA mediated stimulation of APP proteolysis. To examine the potential effects of PBA on b-secretase processing of APP, NAG cells were treated with PBA in the presence or absence of a b-secretase inhibitor (BSI IV). The data is normalized to fold- induction to quantify the observed effect. The application of either of two concentrations of BSI IV resulted in only a fractional repression of APPGV16 processing—approximately a 25 percent decrease from that observed in the absence of the b-secretase inhibitor (Figure 8B). These data indicate that PBA may impact both a- and b-secretase processing of APPGV16—with the stimulation of a-secretase contributing dominantly to the stimu- latory effects observed with PBA. PBA Stimulates AICD Production in the Absence of Increased Amyloid Biogenesis The previous studies suggest that PBA has a minimal effect upon b-secretase mediated APP cleavage. However, to test the effects of PBA upon b-secretase processing directly, we performed parallel experiments examining AICD-GV16 induced Gal4- reporter activity and Ab40 and Ab42 production. The media was drawn off of the cells and employed in a classic sandwich ELISA procedure to measure the levels of Ab40 and Ab42 secreted during the PBA treatment period. After the media is drawn off and employed in the ELISA, the cells were washed, lysed and used to measure the levels of c-secretase mediated AICD production by the reporter assays already discussed. By combining these two assays, we directly compared the effects of PBA on amyloid biogenesis and AICD generation. Separate sets of NAG cells were employed for the Ab40 and Ab42 assays, and accordingly, there are two separate assays for PBA effects upon AICD production. Consistent with previous experiments, PBA stimulated AICD production approximately 8–10 fold (Figure 9A and B). However, PBA had only minimal effects upon amyloid production. There is an approximately 32% increase in Ab40 levels (142 pg/ml to 188 pg/ml) (Figure 9C) and a 35% decrease in Ab42 levels (35.6 pg/ml to 22.8 pg/ml) (Figure 9D). The R2 value for each amyloid standard curve was at least 0.96 in all PLoS ONE | www.plosone.org 8 February 2010 | Volume 5 | Issue 2 | e9135