The central question of this work is whether ER stress alters secretase mediated APP proteolysis. In order to address this question, we employed pharmacological treatment of the NAG cells, using agents known to alter protein processing within, or trafficking from, the ER. All pharmacological means of inducing ER stress result in the accumulation of misfolded proteins in the ER by distinct mechanisms. Our objective was to assess whether the general effects of ER stress alter APP processing; consequently, three different pharmacological stress inducing compounds are employed throughout this work—tunicamycin, thapsigargin, and BFA. Tunicamycin directly inhibits N-linked glycosylation of ER proteins, while thapsigargin targets SERCA-mediated calcium reuptake in the ER. Both of these approaches directly impact protein folding, as glycosylation-state promotes the transition from one conformational state to another—guiding the protein towards its native conformation 
. Similarly, blocking calcium reuptake into the ER impairs chaperone function—as many of the ER chaperones use calcium as a co-factor, and consequently depend on intralumenal calcium to direct proper protein folding 
. BFA directly targets the Arf-GTPase, and hence impairs ER to Golgi trafficking and promotes dissociation of the Golgi 
Our initial hypothesis purported that ER stress induced changes in trafficking would attenuate the levels of secretase mediated APP processing. In almost direct correspondence to UPR activation (–, blots) there was a partial arrest in APP proteolysis (–, bar graphs). As ER stress induction regulates both cellular transcriptional and translational responses, it could modify the reporter output associated with this assay system. However, there was no effect of ER stress induction upon βGal normalization or APPGV16 protein levels. Additionally, transiently transfected AICD-GV16, corresponding to the γ-secretase mediated APPGV16 cleavage product, produced stable activation of the Gal4-reporter when treated with thapsigargin, tunicamycin or BFA (Figure S2
). Consequently, the decrement in APPGV16 driven reporter activity with ER stress induction, represents a decrease in proteolytic liberation of the AICD-GV16 moiety. Consistent with other work 
, we find that induction of ER stress restricts APPGV16 localization to early components of the secretory pathway. As BFA blocks the trafficking of transmembrane proteins out of the ER, the similarity in APPGV16 localization pattern in tunicamycin and thapsigargin treated cells 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 
. 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 (). 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 
. 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
. 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
. The critical role of trafficking in APP proteolysis is highlighted by the complete lack of α- and β-cleavage products in the BFA treated cells ()—supporting other work demonstrating that secretase mediated cleavage occurs later in the biogenic pathway 
. PBA mediated rectification of subcellular trafficking following tunicamycin and thapsigargin treatment (), supports the direct relationship between APP trafficking and proteolytic regulation under ER stress conditions.
Intriguingly, PBA promotes APPGV16 trafficking () and dramatically stimulates secretase mediated APPGV16 cleavage () 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 (). The changes in APPGV16 protein levels observed are insufficient to account for the shift in reporter output—however, the CTFGV16 proteolytic species corresponding to the α-secretase cleavage product increased considerably across the PBA titration (). 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 γ-secretase inhibitor)—suggesting that PBA promotes an active association between APPGV16 and either the α- or β-secretases. This does not preclude PBA mediated stimulation of γ-secretase cleavage, as shifts in the subcellular localization of APPGV16 could alter proteolysis in the absence of change in γ-secretase protein levels.
PBA stimulation of α-secretase mediated APP cleavage is supported by experiments targeting either α- or β-secretase with selective inhibitors. Coordinate treatment with α-secretase inhibitors substantially repressed PBA mediated stimulation of APPGV16 cleavage (). The contribution of β-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 β-secretase inhibitor BSI IV resulted in a relatively small decrease in the overall stimulation observed (). However, as the pathogenic Aβ42 form of amyloid is produced at considerably lower levels than Aβ40, relatively small changes in β/γ 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 (). These data suggest that PBA mediated stimulation of APP processing occurs predominantly through α/γ-cleavage, and does not significantly impact amyloidogenic processing. This is consistent with other work which suggests that under normal circumstances α- and β-secretase processing is not competitive 
. Additionally, the NAG cells express sufficient levels of APPGV16 that competition for substrate between the α- and β-secretases may not occur. Furthermore, blocking γ-secretase activity with DAPT eliminates the PBA mediated enhancement of APPGV16 cleavage, demonstrating 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 γ-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 γ-secretase mediated APP cleavage 
. Numerous genes are implicated as APP/Fe65 regulatory targets including APP 
, neprilysin 
, KAI1 
, GSK-3β 
and others. While there is some contention about the validity and significance of these putative gene targets 
, 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 
; the other assay employs the Gal4 binding domain fused to the carboxy-terminus of APP695 
. 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 (). 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 α/γ-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 
. Consequently, we 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 (). In stressed cells, PBA prevented apoptosis in response to all three pharmacological treatments (). While these results are consistent with the anti-apoptotic effects of PBA in other systems 
, 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 
, which promotes neuronal survival 
. 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) 
. Neither DMSO nor TUDCA stimulated APPGV16 proteolysis analogously to PBA (). 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 
. Despite the cognitive rescue, the amyloid plaque pathology was not ameliorated. However, initiating PBA treatment 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 α/γ and β/γ 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 α/γ-cleavage may drive down the levels of APP available to β-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, β-secretase mediated processing of APP decreases, amyloid plaque formation is lessened, and the cognitive capacities of the dual transgenic animals is improved 
. Conversely, over-expression of the dominant negative ADAM10 leads to an exacerbation of the AD phenotype 
. Taken together, these data suggest that the balance of α- and β-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 α/γ-cleavage is consistent with the growing body of evidence pointing to a loss of function associated with the genetically heritable Familial Alzheimer's disease (FAD) mutations 
. Herein, the FAD mutations in PS1 are associated with a decrement in total amyloid levels 
and a decrease in AICD production 
. As noted, the loss in γ-secretase proteolytic function may be mechanistically coupled with the increased production of the pathogenic Aβ42 amyloid species 
. Pathogenic amyloid production may induce ER stress 
, providing a mechanistic link between one of the cardinal features of AD and the manifestation of ER stress in AD patients. Deficits in proteolytic degradation and protein quality control observed in AD patients may promote protein aggregation 
, and subsequent ER stress induction, which could decrease γ-secretase mediated processing of APP in sporadic AD patients. In this context, the disruption of de novo protein maturation and trafficking in the ER may promote stress and UPR activation, and replicate the loss of function component of AD pathogenesis. The capacity of PBA to counteract ER stress and promote protein trafficking through the secretory pathway, along with PBA mediated stimulation of α/γ-cleavage, strongly supports the investigation into the therapeutic potential of PBA for the treatment of AD.