APP and the cleaving secretases traffic and mature through the secretory pathway. The activity of the secretases is dependent upon their glycosylation and proteolytic maturation, which occurs in later components of the secretory pathway, most notably within the Golgi[16,17]. The cleavage of APP by a-secretase is mediated by members of the metalloproteinase family [18], with TACE and ADAM10 being the predominant members [19]. TACE and ADAM10 are activated by the furin-mediated proteolytic release of their regulatory domains, which occurs in the Golgi complex [16]. The pro-amyloidogenic processing of APP is initiated by b- secretase cleavage in the extracellular domain by the atypical aspartyl protease BACE [20]. The proteolytic maturation of BACE also requires processing within the Golgi complex [21,22]. Furthermore, the complex formation and activation of the heterotetrameric c-secretase complex requires maturation in the Golgi [23,24], in which presenilin-1 (PS1) and Nicastrin are both glycosylated and the holo-form of presenilin is cleaved into the amino- and carboxy-terminal fragments (NTF and CTF respec- tively) required for c-secretase activity [9]. Consequently, inducing a state of ER stress, which can subsequently halt the passage of proteins through the ER, could directly impact the secretase mediated proteolytic processing of APP at multiple levels. The possibility of ER stress playing a role in AD pathogenesis is particularly interesting in light of the discovery of bioactive and bioavailable small molecules which act like chaperones in promoting protein folding and subsequent trafficking through the secretory pathway. These small molecules are referred to as small molecular chaperones as they are believed to decrease the energy barrier between the intermittent transition-states that occur as proteins fold into their native conformation [25]. Small molecular chaperones were originally developed to promote cystic fibrosis transmembrane conductance regulator (CFTR) trafficking, and one member of the family, phenylbutyric acid (PBA) is already FDA approved for treatment of urea cycle disorder. PBA, and other members of the small molecule chaperone family, consis- tently decrease the levels of UPR signaling, supporting the protective effects of these compounds against ER stress [26]. Significantly, PBA is also blood-brain barrier permeable [27], making it an exciting candidate therapeutic for neuropathological conditions. We hypothesized that induction of ER stress would decrease the secretase-mediated generation of the APP intracellular domain (AICD) associated with APP nuclear signaling. However, the AICD is difficult to quantitate by standard biochemical means owing to its small size and its remarkable instability [28]. Consequently, in order to test this hypothesis, we generated a strain of stably transformed N2a neuroblastoma cells containing the Gal4-luciferaseEGFP c-secretase reporter system we recently engineered. In this genetic reporter system, c-secretase mediated cleavage of APP fused in frame to the yeast-viral hybrid transcription factor Gal4-VP16 (referred to as APPGV16) activates the Gal4-luciferaseEGFP reporter gene. Using this system we demonstrate that pharmacological agents that induce ER stress consistently decrease c-secretase mediated cleavage of APP. Furthermore, we show that co-treatment with the small molecule chaperone PBA rectifies the proteolytic deficit, stimulates AICD production, promotes trafficking and protects against ER stress induced apoptosis. Materials and Methods Chemicals and Antibodies The selection reagents zeomycin (Invitrogen) and G418 (Cellgro) were used at 200 mg/ml and 500 mg/ml respectively. PBA Enhances APP Proteolysis Tunicamycin (Calbiochem) was dissolved in DMSO at a stock concentration 10 mg/ml. Thapsigargin (Sigma) was dissolved in DMSO at 0.5 mM concentration. Brefeldin A (MP Biomedicals) was suspended in DMSO at 10 mg/ml. The phenylbutyric acid (4-PBA, Calbiochem) was dissolved in filtered sterile water at a 1M stock concentration. The VP16 antibody (Sigma) used for subcellular localization of APPGV16 was diluted at 1:500 for immunohistochemistry. The donkey anti-rabbit Alexa 546 (Mo- lecular Probes) antibody secondary was employed with the VP16 antibody in subcellular localization studies at a 1:5000 dilution. The eIF2a (Cell Signaling), phoshpo-eIF2a (Cell Signaling), and phospho-PERK antibody (Cell Signaling) were diluted for western blot at 1:1000. The Gal4 antibody (Calbiochem) used for identification of APPGV16 protein and its proteolytic fragments by western blotting was diluted at 1:500. The phospho-JNK and phospho-p38 antibodies (Cell Signaling) were both used at 1:1000. Luciferin (Biosynth) was reconstituted at 1 mM for the luciferase assays. Galacton (Tropix) and Emerald Amp (Tropix) were used for the b-galactosidase normalization assays. Vector Construction The tricistronic reporter construct was developed by fusing pieces from numerous different expression vectors. The Gal4- luciferase vector (pFRluc, Stratagene) was used as an initial template for construction of the reporter vector. Primers were designed against the Gal4-luciferase operon in which the SalI- BglII restriction site sequences were added to the 59 end of the 59 primer. The StyI site was added to the 59 end of the 39 primer against Gal4-luciferase and the stop codon was deleted. High fidelity PCR amplification of the Gal4-luciferase sequence containing the new restriction sites was completed, and the SalI- StyI Gal4-luciferase fragment was cloned into the SalI-XbaI sites in CMV-EGFP. CMV-EGFP was generated by amplifying the EGFP sequence from pEGFP (Clontech) and inserting XbaI at the 59 end and XmaI at the 39 end of the coding sequence. This was cloned into the XbaI-XmaI sites in GFPxlt (provided by Dr Randall Moon), following site-directed mutagenesis to insert XmaI in between the 39 end of the GFP coding sequence and the polyA sequence. Subsequently, a BglII site was inserted 39 to the polyA sequence for later subcloning events. The CMV promoter in CMV-EGFP was deleted by inserting Gal4-luciferase into the vector, as the SalI site in CMV-EGFP was 59 to the CMV promoter. The cloning of Gal4-luciferase in frame with EGFP resulted in the luciferase-EGFP fusion reporter driven exclusively by the Gal4 promoter. In order to normalize the Gal4- transactivation data, a constitutively expressing nuclear targeted b-galactosidase cassette was inserted into the reporter vector. The pNeoZTK2 murine gene targeting vector (provided by Dr Richard Palmiter) was used as a template. PCR primers were designed against the NLS-bGal portion of the targeting vector, with a NheI site incorporated into the 59 portion of the 59 primer and a HindIII site added to the 59 end of the 39 primer. The NLS- bGal coding region was amplified by high fidelity PCR, and the NheI-HindIII fragment was subcloned into pcDNA3.1Zeo (Invitrogen). The CMV promoter was removed and replaced by inserting the elongation factor 1a promoter amplified off of the pCEFL expression vector with NruI and NheI sites contained in the 59 and 39 primers respectively. This resulted in the generation of EF1-NLS-bGal. Two BglII sites were identified and mapped in EF1-NLS-bGal. The one adjacent to the EF1-NLS-bGal coding region was eliminated by linearizing the plasmid through a partial digest and blunt ending the vector and religating the blunt ends. The second BglII site was used to subclone the BglII-Gal4- luciferaseEGFP-BglII fragment containing the associated polyA PLoS ONE | www.plosone.org 2 February 2010 | Volume 5 | Issue 2 | e9135