In the NAG cells treated with thapsigargin, there is an inverse relationship between APP proteolysis and the activation of UPR pathways. Gal4-reporter activity decreased with increasing concentrations of thapsigargin (Figure 2, top). Conversely, thapsigargin induced phospho-PERK, phospho-eIF2a and activa- tion of p38 and JNK (Figure 2) between 0.1 mM and 0.5 mM. Maximal stimulation of each UPR pathway occurs by 0.5 mM thapsigargin. Concordantly, thapsigargin maximally inhibits APP proteolysis by 0.5 mM, suggesting that repression of APP proteolysis occurs coordinately with the induction of ER stress. Maximal repression of Gal4-reporter activity by thapsigargin results in a decrease of 63 percent, less than observed with DAPT, suggesting that ER stress conditions permit low levels of c- secretase mediated APPGV16 proteolysis. Low level stress also attenuates c-secretase mediated APP proteolysis, as the addition of 0.1 mM thapsigargin, which correlates with slight increases in the UPR markers, decreases Gal4-reporter activity. Similarly, tunicamycin treatment induced ER stress and decreased c-secretase mediated APP proteolysis (Figure 3). Tunicamycin inhibited 59 percent of Gal4-reporter activity at the highest titrated dose. Thapsigargin and tunicamycin inhibit APP proteolysis to a similar degree, suggesting that a common mechanism may be involved. Additionally, Gal4-reporter output decreases in a linear inverse relationship with the appearance of UPR markers. The concentration of tunicamycin which induces detectable levels of phospho-PERK and phospho-eIF2a (1 mg/ml) inhibits c-secretase mediated APP proteolysis by approximately 50 percent. Repression of APP proteolysis and stimulation of UPR signaling are both maximal at 5 mg/ml tunicamycin. The tight coupling of APPGV16 proteolytic repression and ER stress induction, following tunicamycin treatment, supports a direct relationship between ER stress and APP proteolysis. Unlike thapsigargin and tunicamycin, the dose-response curve for both UPR activation and APP proteolytic repression is abrupt and non-linear with BFA treatment (Figure 4). Maximal repression of APPGV16 cleavage occurs concordantly with activation of the UPR markers. APPGV16 protein levels remain even, or increase slightly, with BFA treatment—demonstrating that change in APPGV16 protein expression is not responsible for the repressive effects observed. The inverse relationship between APP proteolysis and UPR activation subsequent to BFA treatment is consistent with the effects of tunicamycin and thapsigargin. However, BFA more potently inhibits APPGV16 proteolysis than either thapsi- gargin or tunicamycin—decreasing Gal4-reporter activity by 88 percent. The degree of proteolytic repression with BFA is similar to that observed with DAPT, demonstrating the essential role of ER to Golgi trafficking upon secretase mediated APP processing. All three secretases (a,b, and c) undergo glycosylation mediated maturation through the secretory pathway. As APP proteolytic processing is a sequential process, the repression of any individual secretase would inhibit release of the AICD. Consequently, we examined the effects of ER stress induction upon two other proteolytic fragments directly: C99 and the AICD. DNA vectors encoding each protein fragment fused to Gal4VP16 were transfected into na¨ıve N2a cells. The transfected cells were treated with tunicamycin, thapsigargin or BFA. Consistent with previous reports showing that c-secretase activity requires trafficking into later compartment of the secretory pathway [9], all three ER stress inducing compounds block c-secretase mediated cleavage of C99GV16 to varying degrees. Thapsigargin and BFA induced statistically significant levels of APP proteolytic repression (Figure S2). Confirming that ER stress induction does not influence the genetic reporter system, there is no significant or systematic shifts in C50GV16 (the c-secretase cleaved AICD-GV16 portion of PBA Enhances APP Proteolysis APPGV16) driven Gal4-reporter activity in the presence of the stress inducing compounds. Small Molecular Chaperone PBA Results in Restored AICD Production under Stress Conditions Small molecular chaperones are a class of compounds which facilitate protein folding and subsequent trafficking through the secretory pathway [25]. Numerous reports confirm that PBA, one member of the small molecular chaperone class, relieves ER stress and UPR signaling [35,36,37]. Consequently, we employed PBA to test whether small molecular chaperones could rectify APP proteolysis under ER stress conditions. NAG cells were grown in the presence or absence of all three ER stress inducing compounds and treated with titrated levels of PBA from 0 to 10 mM for 24 hours. As previously observed, the addition of thapsigargin, tunicamycin or BFA induced a statistically significant decrease in the levels of c-secretase mediated APP cleavage (Figure 5A). Co- treatment with PBA eliminated the repression of APP proteolysis associated with thapsigargin and tunicamycin treatment (Figure 5A), increasing Gal4-reporter activity above basal levels by 10 mM. However, PBA had no effect upon BFA induced decreases in c-secretase mediated APPGV16 processing. Since BFA treatment results in Golgi dissolution, and eliminates the possibility of trafficking through the secretory pathway, the lack of any PBA effect in BFA treated cells strongly suggests a role for PBA in stimulating trafficking under stress conditions. Neither the decrease in APPGV16 proteolysis brought about by the ER stress inducing compounds, nor the PBA mediated increase in APPGV16 cleavage, is due to changes in APPGV16 protein levels, as there is no correlation observed between expression level and Gal4-reporter activity (Figure 5B). The a-, b-, and c-secretases undergo maturation by proteolysis and glycosylation as they travel through the secretory pathway [9,16,17,22]. Consequently, the activity of all three secretases could be inhibited under ER stress inducing conditions due to diminished trafficking through the secretory pathway. In order to assess whether the activity of all three classes of secretases are inhibited under stress conditions, we examined the levels of the CTF-GV16 species in the various treatment conditions. Consistent with the interpretation that ER stress induces inhibition of the c- secretase complex, elevated levels of CTF-GV16 were observed following treatment with both thapsigargin and tunicamycin (Figure 5B). This suggests that thapsigargin and tunicamycin more significantly impact c-secretase processing than either a- or b-secretase processing, but does not eliminate the possibility that stress induction may attenuate a- or b-secretases to a lesser degree. Interestingly, BFA elicits dramatic increases in the quantity of holoAPPGV16 and completely attenuates production of the CTF- GV16 species—suggesting that BFA inhibits APPGV16 processing at multiple levels (Figure 5B). BFA may block the maturation of all three secretases, by trapping them in the ER, and preventing their activation in subsequent components of the secretory pathway. Consistently, as PBA enhances APPGV16 cleavage, the level of CTF-GV16 decreases. However, some caution is necessary in interpreting these results as PBA may increase all three classes of secretase activity concomitantly. In order to assess whether PBA induced APP proteolysis correlates with decreases in ER stress, PBA was titrated onto tunicamycin treated cells. As previously shown, both phospho- JNK and phospho-PERK levels increase with tunicamycin treatment (Figure 5C). However, co-treatment with PBA decreases both phospho-JNK and phospho-PERK levels (Figure 5C). This suggests that PBA mediated rectification of APPGV16 proteolysis occurs concurrently with decreases in UPR signaling. The PLoS ONE | www.plosone.org 6 February 2010 | Volume 5 | Issue 2 | e9135