Endoplasmic Reticulum Stress And APP Processing: Implications for Alzheimer's disease

1. ABSTRACT Alzheimer’s disease (AD) poses a health problem of pandemic proportions due to the fact that the causes and underlying mechanisms of the disease are still incompletely defined and pre-symptomatic diagnosis is not yet in routine clinical use. Amyloid Precursor Protein (APP) is best known as the precursor molecule whose processing generates toxic β-amyloid (Aβ), a 37 to 49 amino acid peptide that is the primary component of amyloid plaques that accumulate in the brains of AD patients. The accumulation of misfolded proteins (e.g. mutant or damaged proteins) triggers cellular stress responses that protect cells against the toxic buildup of such proteins. However, prolonged stress due to the buildup of these toxic proteins triggers biochemical signaling pathways that ultimately leads to specific death pathways. Past studies have shown that chronic endoplasmic reticulum (ER) stress is a fundamental pathological event in AD. Therefore, it was of interest to determine whether ER stress triggers (a) abnormal processing of APP, (b) accumulation of toxic pro-AD peptide fragments and (c) abnormal biochemical changes of APP and other downstream signaling molecules that then trigger programmed cell death. We believe that strategies to target and modulate the ER stress signaling pathways could have therapeutic benefits for intervention in AD. As shown in figure 1, in order to test whether 7W-CHO cells are susceptible to ER stress and cell death, cells were treated with different concentrations of thapsigargin and for different periods of time. Exposure of cells to thapsigargin led to a decrease in cell viability in a time and dose-dependent manner (Fig. 1a). A high level of glucose regulated protein (GRP) expression is indicative of ER stress and thapsigargin treatment of 7W-CHO cells resulted in the induction of GRP78 protein (Fig. 1b). The last panel in figure 1b represents endogenous GAPDH protein as a loading control. Endoplasmic Reticulum Stress And APP Processing: Implications for Alzheimer's disease William Howell, Dale E. Bredesen and Rammohan V. Rao Buck Institute for Research on Aging, Novato, CA 3) Effect of Thapsigargin-induced ER stress on APP processing To understand the effect of Thaps-induced ER stress on APP processing, we collected cell extracts prepared from cells treated with thapsigargin and performed immunoblotting using several different APP antisera that were raised against different regions of the APP protein as shown in Figs 3 & 4. 1) Thapsigargin triggers ER stress and cell death in CHO cells. Thapsigargin, an inhibitor of the ER Ca-ATPase, triggers ER stress and induces apoptosis in many cell types. We used thapsigargin to induce ER stress and to investigate its effects on APP processing and APP-mediated downstream signaling events. 7W-CHO (Chinese hamster ovary cells that stably express wild type APP) were treated with thapsigargin at different concentrations and cell extracts prepared. Methods: Quantification of cell death: Assessment of cell death was carried out by pelleting floating and adherent cells after trypsinization. The cell pellet was resuspended in 1X PBS/0.4% Trypan blue and cells were counted using a hemocytometer. Cell death was determined as the percentage of dead cells over the total number of cells. Cell extracts: Briefly, cells were lifted gently, pelleted at 200Xg, and the resulting pellet was washed in 50 ml of ice-cold phosphate-buffered saline. The cell pellet was resuspended in 1x cell-lysis buffer and briefly sonicated. The cell lysate was transferred to an Eppendorf tube and centrifuged for 30 min at 16,000Xg (4 °C). The clear supernatant was removed. and either was used immediately or stored in aliquots at -84 °C. Electrophoresis: SDS-Polyacylamide gel electrophoresis(SDS-PAGE) of equal amounts of total protein was performed and separated proteins were transferred to polyvinylidene fluoride membranes (PVDF) for Western blot analysis. Membranes were probed with a 1:500 dilution of anti-GRP78 antibody. The membranes were incubated in a horseradish peroxidase-coupled secondary antibody for 1 h followed by enhanced chemiluminescence detection of the proteins with Hyperfilm ECL detection (Amersham Pharmacia Biotech, Arlington Heights, IL) METHODS, RESULTS, AND DISCUSSION Figure 3 Figure 4 Figure 5 APP- NT: The antibody is developed in rabbit using a synthetic peptide NVQNGKWDSDPSGTK corresponding to the N-terminal region of human APP695 (amino acids 46-60). APP-NT recognizes all forms of APP (95-100 kDa) and APP cleavage products (60 kDa) by immunoblotting. APP-CT: APP-C-Terminal recognizes human, mouse and rat APP695, APP751 and APP770 (95-100 kDa) by immunoblotting. APP-CT antibody is produced in rabbit using KMQQNGYENPTYKFFEQMQN corresponding to the C-terminal of human APP695 (amino acids 676-695) as an immunogen APP-4G8: This antibody is reactive to amino acid residues 17-24 of beta amyloid. The epitope lies within amino acids 18-22 of beta amyloid (VFFAE). APP-6E10: This antibody is reactive to amino acid residues 1-16 of beta amyloid. The epitope lies within amino acids 3-8 of beta amyloid (EFRHDS). APP-3E9: The N-terminal mouse monoclonal antibody is developed to a synthetic peptide LEVPTDGNAGLLAEPQIAMFC corresponding to amino acids 18-38 of human APP. APP-3E9 recognizes all forms of APP (95-100 kDa) by immunoblotting. As shown in Fig 5a & 5b, thapsigargin treatment resulted in a time dependent decrease in APP protein expression. While we did not observe the presence of any smaller APP products with most of the antibodies, the 4G8 antibody (which recognizes the c-terminal region of the Ab peptide fragment) detected the presence of a ~70Kd APP fragment only in the thapsigargin treated samples. a WB: APP 110 INTRODUCTION anti-APP- NT 80 Alzheimer’s is a neurodegenerative disease that affects approximately 5.2 million people in the U.S. alone. It is estimated that in 2025, 7.1 million people will be afflicted with Alzheimer’s disease. In 2013, the estimated cost for care of Alzheimer’s patients will be about $203 billion in the U.S. So far, there is no known cure or effective treatment for AD patients, which is further complicated by the fact that the underlying causes and mechanisms are undefined (1). Amyloid precursor protein (APP) is an integral membrane protein that is present in many tissues and is concentrated in synapses of neurons. Proteolysis of this protein can form the Ab peptide, a 37-49 amino acid peptide. Several mutations outside the Aβ region associated with familial Alzheimer's have been found to dramatically increase production of Aβ. This causes a toxic accumulation of Ab that is seen in AD patients in the form of plaques. It is believed that this accumulation of Ab is destructive to synapses. However, the Bredesen Lab has shown that APP functions as a molecular switch. Proteolysis at the b, g, and caspase sites lead to the formation of four toxic pro-AD peptides called sAPPb, Ab, J casp, and C31, which mediate neurite retraction and programmed cell death. Cleavage at the a site by a-secretase produces the anti-AD peptide sAPPa and the inhibitor of APP g-site cleavage, aCTF, as shown in the figure below. The decision between these two proteolytic pathways is governed by several factors that could lead either to susceptibility or resistance to AD (1). Past studies have shown that chronic endoplasmic reticulum (ER) stress is a fundamental pathological event in AD and many other neurodegenerative disorders that feature abnormal protein aggregates. Chronic ER stress associated with toxic accumulation of these aggregates results in the release and activation of several pro-death signaling molecules that then trigger programmed cell death (2). ER stress markers together with pro-death molecules have been found in post-mortem samples from patients affected with AD as well in cellular and animal models of these disorders. Therefore it was of interest to determine whether ER stress impacts APP proteolysis including the pro-AD and anti-AD peptide balance differentially, and, if so, by what mechanism. Our hypothesis is that ER stress mediates pro-AD effects that include (a) a shift of APP processing in favor of the pro-AD peptides, (b) down regulation of the anti-AD peptides, (c) an alteration in downstream signaling resulting in the release of pro death molecules and neural cell death. 110 anti-APP- CT Figure 1 Thaps (0.5mM) - 12h 24h 36h a b WB: APP 120 80 anti-APP-6E10 b 100 110 WB: GRP 78 80 60 60 40 20 anti-APP-3E9 110 0 Thaps, 0.1mM Thaps 0.5mM control Thaps 1mM 50 GABDH anti-APP-4G8 30 110 Thaps (0.5mM) - 12h 24h 36h Thaps (0.5mM) - 12h 24h 36h 2) Prolonged treatment of cells with thapsigargin triggers APP processing and reduces sAPPa/sAPPb ratio Methods: sAPP and sAPP Assay. sAPPα and sAPPβ secreted into the cellular media were determined with the AlphaLISA, sAPPα, and sAPPβ immunoassay research kits (PerkinElmer) according to the manufacturer's protocol with some modifications. The standards, blanks, and media were diluted with the buffer provided in the kit and added to the plate. During the first incubation step, the analyte was captured either by an antibody recognizing the α-secretase cleavage site at sAPPα C-terminus (clone 2B3) or the sAPPβ C-terminus, and by a second biotin-labeled antibody specific to the N-terminal part of sAPP (common for both sAPPα and sAPPβ). In the second incubation step, the biotinylated anti-analyte antibody was bound to the streptavidin-coated donor beads. At the end of this reaction, the plates were read on an EnSpire Alpha 2390 multilabel plate reader equipped with the AlphaScreen module. Electrophoresis: SDS-Polyacylamide gel electrophoresis(SDS-PAGE) of equal amounts of total protein was performed and separated proteins were transferred to polyvinylidene fluoride membranes (PVDF) for Western blot analysis as mentioned above. ★Thapsigargin, an inhibitor of the ER Ca-ATPase triggers ER stress and ER stress-induced cell death in 7W-CHO cells that stably express wild-type APP ★Thapsigargin treatment reduces sAPPa/sAPPβ ratio ★Thapsigargin triggers abnormal processing of APP. While protein expression is reduced considerably with time of treatment, we noticed the appearance of a APP cleavage product (~70kDa) only in thapsigargin treated cells. CONCLUSIONS FUTURE DIRECTIONS 1) Determine whether thapsigargin-induced APP processing can be blocked by suitable inhibitors 2) Determine whether ER stress promotes increased APP and Tau phosphorylation 3) Identify drug candidates that will potentially block ER stress and ER stress associated pro-AD processing of APP 2) Prolonged treatment of cells with thapsigargin reduces sAPPα/sAPPβ ratio As shown in figure 2, we assessed the levels of sAPPα and sAPPb after thapsigargin treatment of 7W-CHO cells. Short term thapsigargin treatment increased sAPPα/sAPPβ ratio. However, prolonged treatment reduced the sAPPα/sAPPβ ratio (fig. 2) suggesting that while a short term treatment with thapsigargin may actually be protective to cells as evidenced by a higher sAPPα/sAPPβ ratio, prolonged treatment may be deleterious to the cell due to a reduction in the sAPPα levels and a concomitant decrease in the sAPPα/sAPPβ ratio. Figure 2 SPECIFIC AIMS 35 30 • We have the following specific aims: • Aim 1: Determine the effects of ER stress on APP processing • Aim 2: Determine whether ER stress promotes pro-AD processing of APP through decreased sAPPa and increased aAPPb or Ab. • Aim 3: Determine whether ER stress promotes increased APP and Tau phosphorylation. 25 REFERENCES sAPPα/sAPPβ absolute units/mg protein 20 1. Bredesen DE (2009) Neurodegeneration in Alzheimer's disease: caspases and synaptic element interdependence. Mol Neurodegener 4:27 2. Bredesen DE, Rao RV, & Mehlen P (2006) Cell death in the nervous system. Nature 443(7113):796-802. 15 10 5 0 Thaps, 0.1mM Thaps, 0.5mM Thaps, 1mM control