Phenylbutyric Acid Rescues Endoplasmic Reticulum Stress-Induced Suppression of APP Proteolysis and Prevents Apoptosis in Neuronal Cells Jesse C. Wiley1*, James S. Meabon 2 , Harald Frankowski2, Elise A. Smith1, Leslayann C. Schecterson 2 , Mark Bothwell2, Warren C. Ladiges1 1 Department of Comparative Medicine, University of Washington, Seattle, Washington, United States of America, 2 Department of Physiology and Biophysics, University of Washington, Seattle, Washington, United States of America Abstract Background: The familial and sporadic forms of Alzheimer’s disease (AD) have an identical pathology with a severe disparity in the time of onset [1]. The pathological similarity suggests that epigenetic processes may phenocopy the Familial Alzheimer’s disease (FAD) mutations within sporadic AD. Numerous groups have demonstrated that FAD mutations in presenilin result in ‘loss of function’ of c-secretase mediated APP cleavage [2,3,4,5]. Accordingly, ER stress is prominent within the pathologically impacted brain regions in AD patients [6] and is reported to inhibit APP trafficking through the secretory pathway [7,8]. As the maturation of APP and the cleaving secretases requires trafficking through the secretory pathway [9,10,11], we hypothesized that ER stress may block trafficking requisite for normal levels of APP cleavage and that the small molecular chaperone 4-phenylbutyrate (PBA) may rescue the proteolytic deficit. Methodology/Principal Findings: The APP-Gal4VP16/Gal4-reporter screen was stably incorporated into neuroblastoma cells in order to assay c-secretase mediated APP proteolysis under normal and pharmacologically induced ER stress conditions. Three unrelated pharmacological agents (tunicamycin, thapsigargin and brefeldin A) all repressed APP proteolysis in parallel with activation of unfolded protein response (UPR) signaling—a biochemical marker of ER stress. Co- treatment of the c-secretase reporter cells with PBA blocked the repressive effects of tunicamycin and thapsigargin upon APP proteolysis, UPR activation, and apoptosis. In unstressed cells, PBA stimulated c-secretase mediated cleavage of APP by 8–10 fold, in the absence of any significant effects upon amyloid production, by promoting APP trafficking through the secretory pathway and the stimulation of the non-pathogenic a/c-cleavage. Conclusions/Significance: ER stress represses c-secretase mediated APP proteolysis, which replicates some of the proteolytic deficits associated with the FAD mutations. The small molecular chaperone PBA can reverse ER stress induced effects upon APP proteolysis, trafficking and cellular viability. Pharmaceutical agents, such as PBA, that stimulate a/c- cleavage of APP by modifying intracellular trafficking should be explored as AD therapeutics. Citation: Wiley JC, Meabon JS, Frankowski H, Smith EA, Schecterson LC, et al. (2010) Phenylbutyric Acid Rescues Endoplasmic Reticulum Stress-Induced Suppression of APP Proteolysis and Prevents Apoptosis in Neuronal Cells. PLoS ONE 5(2): e9135. doi:10.1371/journal.pone.0009135 Editor: Colin Combs, University of North Dakota, United States of America Received October 9, 2009; Accepted January 19, 2010; Published February 9, 2010 Copyright: 2010 Wiley et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was funded by a New Investigator Award from the Alzheimer’s Association (NIRG-07-59865). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: jwiley@u.washington.edu Introduction The aggregation of misfolded proteins in early compartments of the secretory pathway occurs in many neurodegenerative diseases, including Alzheimer’s disease (AD) [recently reviewed [12]]. One of the primary intracellular sites in which misfolded proteins accumulate is the endoplasmic reticulum (ER). Improp- erly folded proteins generally fail to traffic out of the ER, as they are retained by the resident chaperone-mediated ER quality control mechanisms [13,14], when the rate of protein synthesis exceeds the capacity of the ER to direct proper folding of the de novo proteins. Under these circumstances, elevated levels of unfolded proteins result in a phenomenon known as ER stress, which initiates a set of events known as the unfolded protein response (UPR). UPR signaling is a multifaceted cascade designed to either restore ER homeostasis or terminate the cell [13,14]. Consequently, there is transcriptional up-regulation of specific chaperones, transcription factors, and a regulated interruption of some classes of translation. The identification of active UPR signaling in AD patients, specifically within the pathologically affected brain regions, suggests that ER stress contributes to the pathological progression in AD [15]. Recent reports show that beta-amyloid precursor protein (APP) traffick- ing through the secretory pathway is impaired under ER stress conditions, suggesting that APP is retained within the ER, or early components of the secretory pathway [7,8]. The link between AD and ER stress likely involves trafficking defects, as altered trafficking of APP and the cleaving secretases would be predicted to directly impact APP cleavage—a central component of AD pathogenesis. PLoS ONE | www.plosone.org 1 February 2010 | Volume 5 | Issue 2 | e9135