Figure 12. PBA specific effects upon APP proteolytic stimula- tion. In order to determine whether all small molecule chaperones (SMC) stimulate APP proteolysis, three different SMC were titrated onto NAG cells: PBA, TUDCA and DMSO. The concentration range for each titration was based on preceding experiments assaying the toxic levels of each compound. PBA stimulated a 9.3-fold increase in normalized reporter activity, which was statistically significant (p,0.0001, student t- test) (A). TUDCA elicited a statistically significant 1.35-fold increase in APPGV16 proteolysis (p,0.0001, student t-test) (B). DMSO treatment PBA Enhances APP Proteolysis resulted in a smaller 1.29-fold increase, which was also statistically significant (p,0.003, student t-test) (C). All measures of statistical significance employed end-point analysis in which the untreated cells were compared to the final concentration in each titration. Each compound elicited a measureable increase in APPGV16 proteolysis, yet PBA stimulation was 6.89 times greater than TUDCA and 7.25 times greater than DMSO. doi:10.1371/journal.pone.0009135.g012 with BFA treated cells argues for a substantial ER localization in all three cases. The shift in subcellular localization could directly impact the association of APPGV16 with the mature secretases— arguing that ER stress responses may repress one or more stages of APP proteolytic processing. The small molecular chaperone PBA alleviates ER stress and UPR signaling [35,37]. Consistent with ER stress induction playing a primary role in the attenuation of APPGV16 proteolysis observed with all three stress inducing pharmacological agents— PBA rectifies the repression induced by tunicamycin and thapsigargin (Figure 5A). The putative mechanism of small molecular chaperones is the promotion of protein folding by decreasing the energy barrier between conformation states as the protein folds into its cognate conformation [25]. Consequently, PBA may rectify some portion of protein folding impaired by treatment with either tunicamycin or thapsigargin by facilitating a transition between conformation states in the absence of proper glycosylation or chaperone function. Increased protein folding would release APPGV16, and the cleaving secretase, from the ER retention and degradation processes employed to eliminate misfolded proteins[13]. However, there would be no rescue from stress related processes in cells treated with BFA, as ER localization is accomplished by targeting trafficking mechanisms which promotes Golgi dissociation[33]. The critical role of trafficking in APP proteolysis is highlighted by the complete lack of a- and b-cleavage products in the BFA treated cells (Figure 5B)—supporting other work demonstrating that secretase mediated cleavage occurs later in the biogenic pathway [9]. PBA mediated rectification of subcellular trafficking following tunica- mycin and thapsigargin treatment (Figure 6), supports the direct relationship between APP trafficking and proteolytic regulation under ER stress conditions. Intriguingly, PBA promotes APPGV16 trafficking (Figure 6) and dramatically stimulates secretase mediated APPGV16 cleavage (Figure 7) in the absence of any induced stress. The robust stimulatory effect of PBA upon APP proteolysis suggests that PBA may promote changes in the levels of APPGV16, the cleaving secretases, or their functional interaction. Yet, the change in APPGV16 proteolysis does not appear to be due to global changes in protein levels, as there was only a slight increase in APPGV16 and no changes in either Nicastrin or PS1 protein levels across the PBA titration (Figure 7B). The changes in APPGV16 protein levels observed are insufficient to account for the shift in reporter output—however, the CTFGV16 proteolytic species correspond- ing to the a-secretase cleavage product increased considerably across the PBA titration (Figure 7B). These data argue that overall protein synthetic rates are not the mechanism, but rather changes in the functional interactions between APP and the cleaving secretases are responsible for the proteolytic stimulation. This interpretation is consistent with the observation that PBA stimulates CTFGV16 production in the presence of DAPT (a well known c-secretase inhibitor)—suggesting that PBA promotes an active association between APPGV16 and either the a- or b- secretases. This does not preclude PBA mediated stimulation of c- secretase cleavage, as shifts in the subcellular localization of PLoS ONE | www.plosone.org 14 February 2010 | Volume 5 | Issue 2 | e9135