APPGV16 could alter proteolysis in the absence of change in c- secretase protein levels. PBA stimulation of a-secretase mediated APP cleavage is supported by experiments targeting either a- or b-secretase with selective inhibitors. Coordinate treatment with a-secretase inhib- itors substantially repressed PBA mediated stimulation of APPGV16 cleavage (Figure 8A). The contribution of b-secretase to the PBA mediated stimulation of APPGV16 cleavage appears to be relatively minor—as the coordinate treatment of NAG cells with PBA and the b-secretase inhibitor BSI IV resulted in a relatively small decrease in the overall stimulation observed (Figure 8B). However, as the pathogenic Ab42 form of amyloid is produced at considerably lower levels than Ab40, relatively small changes in b/c cleavage could result in relatively large shifts in amyloid ratios. Consequently, we examined the levels of each secreted amyloid species in direct comparison to reporter output. Consistent with the inhibitor studies, PBA had little effect upon the biogenesis of either amyloid species (Figure 9). These data suggest that PBA mediated stimulation of APP processing occurs predominantly through a/c-cleavage, and does not significantly impact amyloidogenic processing. This is consistent with other work which suggests that under normal circumstances a- and b- secretase processing is not competitive [43]. Additionally, the NAG cells express sufficient levels of APPGV16 that competition for substrate between the a- and b-secretases may not occur. Furthermore, blocking c-secretase activity with DAPT eliminates the PBA mediated enhancement of APPGV16 cleavage, demon- strating that the PBA mediated increases in reporter activity, are due to changes in the proteolytic processing of APPGV16. Unlike amyloid secretion assays, the APPGV16/Gal4-reporter system interrogates the levels of AICD released into the intracellular compartment subsequent to c-secretase mediated cleavage. The APP intracellular domain (AICD) forms a complex with Fe65, which is reported to traffic to the nucleus and activate gene expression following c-secretase mediated APP cleavage [38,39]. Numerous genes are implicated as APP/Fe65 regulatory targets including APP [44], neprilysin [45], KAI1 [38], GSK-3b [41] and others. While there is some contention about the validity and significance of these putative gene targets [46], we sought to assess whether PBA treatment or ER stress induction alters AICD/Fe65 nuclear signaling using recombinant reporter assays. Two separate assays were used: one in which Fe65 is fused to the Gal4 binding domain and co-transfected with wild-type human APP695 [47]; the other assay employs the Gal4 binding domain fused to the carboxy-terminus of APP695 [39]. Both assay systems demonstrated a significant increase in AICD nuclear signaling following PBA treatment, while stress induction by thapsigargin induced a repression in nuclear signaling (Figure 10A-C). Interestingly, PBA stimulation of Gal4-reporter activity via the transactivation potential of the AICD/Fe65 complex is far smaller than observed within the NAG cell proteolytic assay. This may be due to the far weaker transactivation capacity of the AICD/Fe65 complex relative to the potent GV16 transactivation domain. However, ER stress induction and PBA treatment act qualitatively similar upon AICD production (assayed with the APPGV16/ Gal4-reporter assay) and AICD/Fe65 mediated Gal4-reporter activity—suggesting that repression of AICD production in ER stress and facilitation of AICD production with PBA treatment are both likely to alter AICD mediated gene expression. One of the issues associated with pursuing a/c-cleavage promoting agents as therapeutics in AD is the notion that either the AICD or the AICD/Fe65 complex may promote apoptosis. Numerous groups have reported neurotoxic effects associated with over-expression of the AICD [41,42,48,49,50]. Consequently, we PBA Enhances APP Proteolysis examined the effects of prolonged PBA treatment in stressed and unstressed NAG cells. PBA treatment had no effects upon unstressed cells—suggesting that the elevated levels of AICD induced by PBA is insufficient to promote apoptosis (Figure 11A and B). In stressed cells, PBA prevented apoptosis in response to all three pharmacological treatments (Figure 11A and B). While these results are consistent with the anti-apoptotic effects of PBA in other systems [35,51], it was surprising that PBA could overcome the apoptotic effects of BFA—as PBA has no effect upon the BFA induced repression of APP proteolysis. The butyrate short-chain fatty acid moiety of PBA inhibits histone deacetylation (HDAC) activity [52], which promotes neuronal survival [53]. The HDAC inhibitory capacity of PBA may contribute to its anti-apoptotic activity, potentially accounting for the apoptotic rescue observed in the BFA treated cells. The capacity of PBA to function as both a small molecule chaperone and an HDAC inhibitor may represent a convergence of biological activities that vests it with a unique therapeutic potential. Irrespective of the mode action underlying the anti-apoptotic effects of PBA, these data demonstrate that stimulation of AICD production alone is not guaranteed to induce apoptosis. The potential multiplicity of biological roles of PBA led us to examine the effects of other members of the small molecular chaperone family upon APP proteolytic processing in the NAG cells. Two other commonly studied small molecules with reported chaperone-like function are taurine-conjugated ursodeoxycholic acid (TUDCA) and dimethylsulfoxide (DMSO) [13,25,37]. Neither DMSO nor TUDCA stimulated APPGV16 proteolysis analogously to PBA (Figure 12). The significance of these data is unclear—yet, one argument is that generic chaperone function is not sufficient to stimulate APP proteolysis, at least in the absence of ER stress inducing conditions. Alternatively, there may be some target specificity within the small molecular chaperone family through which different members are more effective in promoting the folding and trafficking of certain classes or types of proteins. The plausibility of PBA providing therapeutic potential to AD patients is supported by a recent study in which the cognitive capacity of AD transgenic mice is rescued by transient PBA treatment [54]. Despite the cognitive rescue, the amyloid plaque pathology was not ameliorated. However, initiating PBA treat- ment in late stage pathological progression would not be expected to alleviate amyloid deposition as the plaques form prior to PBA treatment. Earlier administration of PBA may alter the relative abundance of a/c and b/c cleavage of APP. While we observe little effect of PBA upon amyloid biogenesis in vitro, PBA may have different effects in vivo than we observed within the NAG cells—most notably, the increase in a/c-cleavage may drive down the levels of APP available to b-secretase. The lack of competition we observe within the NAG cells may be due to the profound over- expression of the APPGV16 substrate. In AD transgenic models in which ADAM10 is over-expressed, b-secretase mediated process- ing of APP decreases, amyloid plaque formation is lessened, and the cognitive capacities of the dual transgenic animals is improved [55]. Conversely, over-expression of the dominant negative ADAM10 leads to an exacerbation of the AD phenotype [56]. Taken together, these data suggest that the balance of a- and b- secretase mediated APP proteolysis may be a critical factor in determining the pathogenic progression in AD. Our group is currently examining the potential therapeutic effects of PBA in other AD transgenic mouse models, in which PBA administration begins prior to the pathological onset, and is administered across its anticipated progression. The rationale for pursuing APP proteolytic stimulation via a/c- cleavage is consistent with the growing body of evidence pointing PLoS ONE | www.plosone.org 15 February 2010 | Volume 5 | Issue 2 | e9135