PBA Enhances APP Proteolysis Figure 9. Amyloid biogenesis (Ab40 and Ab42) is unaffected by the PBA mediated stimulation in AICD production. The NAG cells were treated with titrated values of PBA (0, 0.5, 1.5, and 5 mM). A two-part assay measured c-secretase dependent AICD production (A and B) and secreted amyloid biogenesis (C and D) from the same samples. The Gal4-reporter assays were performed with the cell lysate, while the media was assayed for species specific amyloid concentrations. PBA stimulated c-secretase mediated proteolysis approximately ten-fold (A and B) in parallel assays to the ELISA measures for Ab40 (C) and Ab42 (D). Each concentration step in the PBA titration induced a statistically significant increase in Gal4-reporter activity ([PBA] shift: 0 to 0.5 mM, p,0.0003; 0.5 mM to 1.5 mM, p,0.0002; 1.5 mM to 5 mM, p,0.0001; analysis performed with the values in (A)). In contrast, there was no statistically significant change in either Ab40 or Ab42 levels. Ab40 levels increased slightly from 142 picograms/ml to 188 picograms/ml, with a p-value of 0.09. In contrast, Ab42 levels decreased from 35.6 picograms/ml to 22.7 picograms/ml, with a p-value of 0.06. In total, Ab40 levels increased by 32.3 percent and Ab42 levels decreased 36.05 percent—neither change reaching statistical significance. Each assay was performed in triplicate. The standard curves for Ab40 and Ab42 were linear in the tested concentration range with R2.0.96. Consequently, PBA stimulation of APP proteolysis occurs in a non-amyloidogenic manner. doi:10.1371/journal.pone.0009135.g009 These observations support PBA induced stimulation of AICD production leading to elevated APP/Fe65 mediated nuclear signaling. Due to the disparity in level of PBA induced signaling between the APPGV16 assays and the AICD/Fe65 reporter assays, these data support a model in which c-cleavage is not the only regulatory mechanism in APP nuclear signaling. PBA Blocks ER Stress-Induced Apoptosis Prolonged ER stress conditions are known to result in cellular apoptosis [13,14]. Additionally, there are reports suggesting that Fe65/AICD nuclear signaling induces apoptosis [41,42]. Conse- quently, the effects of ER stress induction and PBA stimulated AICD production upon cellular apoptosis were examined in NAG cells. The cells were either untreated or treated with a midrange dose of thapsigargin (0.5 mM), tunicamycin (1 mg/ml) or BFA (1 mg/ml) and co-treated with various concentrations of PBA from 0 to 5 mM. The cells were maintained for 48 hours under these conditions and then two separate assays of apoptosis were performed: a count of detached cells and a count of adherent cells with pycnotic nuclei. The detached cells were taken from three separate plates of treated cells. The numbers represent the totals summed from all sets of cells. In order to assure that there was no experimenter bias in the pycnotic nuclei count, the slides were coded and examined under blind conditions. Intriguingly, by neither assay did PBA alone stimulate any observable levels of apoptosis (Figure 11A and B). Additionally, PBA decreased the levels of apoptosis observed in thapsigargin, tunicamycin and BFA treated cells to levels near those of untreated cells within both assays. These data confirm the low toxicity and counter apoptotic effects of PBA reported by other groups, and suggest that elevation of the APP AICD alone is insufficient to induce measurable level of apoptosis, at least in the presence of PBA. Stimulation of AICD Production Is Not a Consistent Feature of Other Molecular Chaperones In order to assess the generality of molecular chaperone effects upon APP proteolytic processing, comparative titrations were performed with the NAG cells using three different small molecular chaperones: PBA, TUDCA, and DMSO. Consistent with previous experiments, PBA stimulated high levels of AICD- GV16 induced Gal4-reporter activity (Figure 12A). However, the PBA mediated effects upon APP proteolysis were not observed with either TUDCA or DMSO (Figure 12B and 12C). Both TUDCA and DMSO treatment elicited a 20–25% increase in PLoS ONE | www.plosone.org 11 February 2010 | Volume 5 | Issue 2 | e9135