Acute ER stress regulates amyloid precursor protein processing through ubiquitin-dependent degradation

Abstract

Beta-amyloid (Aβ), a major pathological hallmark of Alzheimer's disease (AD), is derived from amyloid precursor protein (APP) through sequential cleavage by β-secretase and γ-secretase enzymes. APP is an integral membrane protein, and plays a key role in the pathogenesis of AD; however, the biological function of APP is still unclear. The present study shows that APP is rapidly degraded by the ubiquitin-proteasome system (UPS) in the CHO cell line in response to endoplasmic reticulum (ER) stress, such as calcium ionophore, A23187, induced calcium influx. Increased levels of intracellular calcium by A23187 induces polyubiquitination of APP, causing its degradation. A23187-induced reduction of APP is prevented by the proteasome inhibitor MG132. Furthermore, an increase in levels of the endoplasmic reticulum-associated degradation (ERAD) marker, E3 ubiquitin ligase HRD1, proteasome activity, and decreased levels of the deubiquitinating enzyme USP25 were observed during ER stress. In addition, we found that APP interacts with USP25. These findings suggest that acute ER stress induces degradation of full-length APP via the ubiquitin-proteasome proteolytic pathway.

Figure 1. Effects of increased intracellular calcium levels on APP processing.

7w-PSML cells were treated with A23187 (1 μM) for 12 h. (A), Cellular lysates are blotted for full-length APP (22C11 antibody). Loading control; β-actin. Conditioned media were blotted for sAPPα (6E10 antibody) and sAPPβ. (B), Quantification of sAPPα (6E10 antibody) and sAPPβ. (C), Aβ40 and Aβ42 measured in conditioned media. (D), 7w-PSML cells were incubated for various times with A23187 (0.5 μM or 1 μM). (E, F). Treatment with A23187 did not affect cell viability. Cell viability was measured by the MTT assay and calcein-AM assay. (G), Total APP mRNA level is unchanged in HEK cells treated with A23187 for 12 h. Statistical significance was tested by unpaired t-test (n = 4, ***P < 0.001 versus control group). All the gels were run under the same experimental conditions. Full-length images are presented in the supplementary information.

Figure 2. ER stress induces APP degradation.

(A), 7w-PSML cells pretreated with MG132 or vehicle for 30 min were exposed to A23187 for 12 h. Cells were harvested for immunoblotting with anti-22C11 (APP) and anti-β-actin. The bottom panel shows a representative western blot, and the top panel shows the quantification based on densitometry. Statistical significance was analyzed by one-way ANOVA followed by a Tukey's multiple-comparison test. (n = 4, **P < 0.01 versus control group; ^##P < 0.01 versus A23187-treated group) (B, C), Pretreatment with MG132, a proteasome inhibitor, protects the APP protein from ER stress-mediated degradation. 7w-PSML cells were pre-treated with MG132 for 30 min followed by treatment with thapsigargin (B) or tunicamycin (C) for 12 h, and harvested for western blot analysis to analyze expression levels of APP (22C11). (D), 7w-PSML cells were pretreated for 60 min with BAPTA/AM (5 μM) before a 12 h treatment with A23187 (1 μM). Chelation of the intracellular calcium with BAPTA prevented degradation of APP. Statistical significance was analyzed by one-way ANOVA followed by a Tukey's multiple-comparison test (n = 5, ***P < 0.001 and *P < 0.05). All the gels were run under the same experimental conditions. For each experiment, APP level was quantified by densitometry and normalized to β-actin loading control. Full-length images are presented in the supplementary information.

Figure 3. APP is preferentially degraded through the proteasome pathway in response to ER-stress.

(A), CHO cells were pretreated with 10 μM MG132, 1 mM 3MA, 20 mM NH₄Cl, or 10 nM Bafilomycin (Baf) for 30 min and later treated with A23187 (1 μM) for 12 h. Representative western blots show APP (22C11) and β-actin expression. (B), Quantification of APP. Statistical significance was tested by one-way ANOVA followed by a Tukey's multiple comparison test (n = 5; *P < 0.05, **P < 0.01). All the gels were run under the same experimental conditions. For each experiment, APP level was quantified by densitometry and normalized to β-actin loading control. Full-length images are presented in the supplementary information.

Figure 4. Polyubiquitination of APP by intracellular calcium overload.

(A), CHO cells were treated with A23187 (1 μM) for 12 h. Proteasome activity measured using a 20S proteasome activity kit (APT280; Millipore). (B), CHO cells were co-transfected with HA-tagged ubiquitin (Ub-HA) and Flag-tagged APP (Flag-APP). After 24 h, CHO cells were pre-incubated with MG132 or vehicle for 30 min followed by incubation with A23187 for 12 h, and equivalent lysates were immunoprecipitated for Flag (APP) and blotted with antibodies to HA for HA-ubiquitin. Statistical significance was tested by one-way ANOVA followed by unpaired t-test (n = 4. *P < 0.05 versus vehicle). Full-length images are presented in the supplementary information.

Figure 5. ER stress regulates APP stability via ER-associated protein degradation pathway.

(A), CHO cells were treated with A23187 or tunicamycin for 12 h. Cells were then harvested for immunoblotting with anti-22C11 (APP), anti-USP25, anti-HRD1, anti-Bip, and anti-β-actin antibodies. (B), CHO cells pre-incubated with MG132 or vehicle for 30 min were exposed to A23187 for 12 h. Whole-cell extract of CHO cells was subjected to immunoprecipitation (IP) with anti-USP25 antibody and then immunoblotted with anti-22C11 (APP), anti-USP25, anti-HRD1, and anti-β-actin antibodies. (C), CHO cells were treated with A23187 for 12 h after transfection with USP25. The total cell lysates were analyzed by western blotting with the antibodies. For each experiment, APP level was quantified by densitometry and normalized to β-actin loading control. Statistical significance was analyzed by one-way ANOVA followed by a Tukey's multiple-comparison test. (n = 3. *P < 0.05 versus vehicle). All the gels were run under the same experimental conditions. Full-length images are presented in the supplementary information.

Figure 6. Proteasome inhibition induces the accumulation of APP in the ER under ER-stress conditions.

CHO cells pre-treated with proteasome inhibitor, lactacystin, or vehicle for 30 min were exposed to A23187 for 12 h. Cells were fixed and stained with antibodies against APP (22C11) and calnexin, followed by secondary antibodies conjugated with Alexa 594 and Alexa 488, respectively. Scale bar; 20 μm.