Antioxidant Compound, Oxyresveratrol, Inhibits APP Production through the AMPK/ULK1/mTOR-Mediated Autophagy Pathway in Mouse Cortical Astrocytes

Abstract

Oxyresveratrol (OxyR), a well-known polyphenolic phytoalexin, possesses a wide range of pharmacological and biological properties, comprising antioxidant, anti-inflammatory, free radical scavenging, anti-cancer, and neuroprotective activities. Autophagy is a cellular self-degradation system that removes aggregated or misfolded intracellular components via the autophagosome-lysosomal pathway. Astrocyte accumulation is one of the earliest neuropathological changes in Alzheimer's disease (AD), and amyloid precursor protein (APP) is the hallmark of AD. OxyR could affect APP modulation via the autophagy pathway. Here, we have reported that OxyR promotes autophagy signaling and attenuates APP production in primary cortical astrocytes based on immunofluorescence and immunoblotting assay results. Co-treatment with the late-stage autophagy inhibitor chloroquine (CQ) and OxyR caused significantly higher microtubule-associated protein light chain 3 (LC3)-II protein levels and LC3 puncta counts, demonstrating that OxyR stimulated autophagic flux. We also found that OxyR significantly reduced the levels of the autophagy substrate p62/SQSTM1, and p62 levels were significantly augmented by co-treatment with OxyR and CQ, because of the impaired deficiency of p62 in autolysosome. Likewise, pretreatment with the autophagy inhibitor, 3-methyladenine (3-MA), resulted in significantly fewer OxyR-induced LC3 puncta and lower LC3-II expression, suggesting that OxyR-mediated autophagy was dependent on the class III PI3-kinase pathway. In contrast, OxyR caused significantly lower LC3-II protein expression when pretreated with compound C, an AMP-activated protein kinase (AMPK) inhibitor, indicating that AMPK signaling regulated the OxyR-induced autophagic pathway. Additionally, co-treatment with OxyR with rapamycin intended to inhibit the mammalian target of rapamycin (mTOR) caused significantly lower levels of phospho-S6 ribosomal protein (pS6) and higher LC3-II expression, implying that OxyR-mediated autophagy was dependent on the mTOR pathway. Conversely, OxyR treatment significantly upregulated unc-51-like autophagy activating kinase 1 (ULK1) expression, and ULK1 small interfering RNAs (siRNA) caused significantly lower OxyR-induced LC3 puncta counts and LC3-II expression, indicating that ULK1 was essential for initiating OxyR-induced autophagy. However, we found that OxyR treatment astrocytes significantly increased the expression of lysosome-associated membrane protein 1 (LAMP1). Finally, we established a stress-induced APP production model using corticosterone (CORT) in cortical astrocytes, which produced significantly more APP than the equivalent using dexamethasone (DEX). In our experiment we found that CORT-induced APP production was significantly attenuated by OxyR through the autophagy pathway. Therefore, our study reveals that OxyR regulates AMPK/ULK1/mTOR-dependent autophagy induction and APP reduction in mouse cortical astrocytes.

Keywords: AMPK-ULK1; LC3 puncta; amyloid precursor protein (APP); autophagy; mTOR; oxyresveratrol.

Figure 1

OxyR treatment induced autophagic flux in cortical astrocytes and neurons. (A,E) Before harvest, cells were treated with 10 µM CQ for 2 h. Images were acquired using a confocal microscope. (B,F) Numbers of LC3 puncta were counted and analyzed using one-way ANOVA (n = 3). (C,G) Before harvest, CQ treatment for 2 h was performed and representative LC3 and p62 levels were determined by Western blotting. (D,H) Statistically significant differences were determined by one-way ANOVA (n = 3). Data are presented as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns: non-significant. OxyR, oxyresveratrol; CQ, chloroquine; GFAP, glial fibrillary acidic protein; LC3, microtubule-associated protein light chain 3; DAPI, the nuclear marker 4′,6-diamidino-2-phenylindole.

Figure 2

The autophagy inhibitor 3-MA blocked OxyR-mediated autophagy in cortical astrocytes and neurons. (A,E) Astrocytes and neurons were pretreated with 3-MA (2.5 mM) for 3 h, and LC3 puncta were visualized by immunofluorescence staining with anti-GFAP (green), anti-MAP2 (red), and anti-LC3 (red) antibodies under a confocal microscope. (B,F) LC3 puncta formation was determined from at least 3 randomly selected independent areas on each slide; the puncta of at least 5 cells were counted. (C,G) Astrocytes and neurons were pretreated with 3-MA for 3 h; OxyR treatment included incubation with 10 µM OxyR for 24 h. Representative LC3 levels were determined by immunoblotting analysis. (D,H) Western blot band intensities were measured by using ImageJ software, and each expression was normalized to that of β-actin. Statistical analysis was performed by one-way ANOVA (n = 3). Data are presented as mean ± SEM. ** p < 0.01, *** p < 0.001, **** p < 0.0001. OxyR, oxyresveratrol; 3-MA, 3-methyladenine; GFAP, glial fibrillary acidic protein; MAP2, microtubule-associated protein 2; DAPI, the nuclear marker 4′,6-diamidino-2-phenylindole; LC3, microtubule-associated protein light chain 3.

Figure 3

OxyR-activated autophagy via the AMPK)-mTOR pathway in cortical astrocytes. (A) Astrocytes were pretreated with compound C (10 µM) for 1 h in the presence or absence of 10 µM OxyR for a further 24 h. pAMPK, AMPK, phospho-S6 ribosomal protein (Ser240/244), S6 ribosomal protein, and LC3 expression levels were determined by performing immunoblotting. (B–D) Densitometry analyses of the represented proteins were performed using ImageJ, and individual expression levels were normalized to those of β-actin. Statistical analysis was accomplished by one-way ANOVA. (E) Cells were pretreated with rapamycin for 30 min before OxyR (10 µM) treatment for 24 h. Phospho-S6, S6, and LC3 levels were measured by performing immunoblotting. (F,G) All data were derived from one-way ANOVA. Data are presented as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns: non-significant. OxyR, oxyresveratrol; C.C, compound C (AMPK inhibitor); AMPK, AMP-activated protein kinase; pAMPK, phosphorylated AMPK; LC3, microtubule-associated protein light chain 3; Rapa, rapamycin; S6, S6 ribosomal protein; pS6, phosphorylated S6.

Figure 4

OxyR-mediated autophagy initiated via the ULK1-dependent pathway in cortical astrocytes. (A) Astrocytes were cultured in the presence or absence of OxyR (10 µM) for 24 h. ULK1, pULK1 (ser757), and pULK1 (ser555) expression levels were examined by Western blotting. (B) Densitometry analysis was performed by using ImageJ software. Statistical analysis was accomplished by conducting one-way ANOVA. (C) Control siRNA and ULK1 siRNA were transfected for 48 h and incubated with 10 µM of OxyR for 24 h. Astrocytes were fixed and stained with Alexa Fluor-conjugated anti-GFAP mouse mAb (green) and Alexa Fluor-conjugated anti-LC3 antibody (red). Images were acquired using a confocal microscope. (D) LC3 puncta were counted in three independent, randomly selected areas, and at least 5 cells were counted. (E) Representative ULK1 and LC3 expression levels were determined by Western blotting. (F,G) Densitometry and statistical analysis were performed using ImageJ and one-way ANOVA, respectively. Data are presented as mean ± SEM. *** p < 0.001, **** p < 0.0001. OxyR, oxyresveratrol; ULK1, unc-51-like autophagy activating kinase 1; p-ULK1, phosphorylated ULK1; siRNA, small interfering RNA; GFAP, glial fibrillary acidic protein; LC3, microtubule-associated protein light chain 3; DAPI, the nuclear marker 4′,6-diamidino-2-phenylindole.

Figure 5

OxyR promoted autophagosome–lysosome fusion in astrocytes. (A) After 24 h of OxyR treatment, LysoTracker Green-HCK-123 was added and the culture was maintained at 37 °C for 2 h before fixation. (B) From each slide, 3 random areas were selected and the number of LC3 puncta containing LysoTracker was counted in each and analyzed using one-way ANOVA. (C) LAMP1 expression levels were quantified using Western blotting. (D) Statistical significance was revealed by one-way ANOVA. Data are presented as mean ± SEM. *** p < 0.001. OxyR, oxyresveratrol; LC3, microtubule-associated protein light chain 3; DAPI, the nuclear marker 4′,6-diamidino-2-phenylindole; LAMP1, lysosomal-associated membrane protein 1.

Figure 6

OxyR reduced APP production through the autophagy pathway. (A,B) Astrocytes were treated with 10 µM of CORT and DEX for 48 h. APP expression was determined by Western blotting. The intensity of the APP signal was measured by using ImageJ. (C,D) After conducting CORT and DEX treatments for 48 h, 10 µM CQ was added 2 h before harvest. The presence of LC3 was determined by Western blotting. (n = 3). (E) Astrocytes were treated with CORT for 48 h, and then with 10 µM OxyR for 24 h. CQ treatment (10 µM) was performed 2 h before cell harvest. The presence of APP and LC3 was determined by Western blotting. (F,G) Statistical analysis was performed. Data are presented as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns: non-significant. CON, control; CORT, corticosterone; DEX, dexamethasone; APP, amyloid precursor protein; CQ, chloroquine; LC3, microtubule-associated protein light chain 3.

Figure 7

Model of OxyR-mediated APP reduction and autophagy induction in cortical astrocytes. OxyR initiates autophagy via stimulation of the AMPK/ULK1/mTOR pathway to activate phagophore formation and subsequently autophagosome maturation. CORT-initiated APP expression is followed by APP engulfment by autophagosomes. The lysosomal protein LAMP1 supports the binding of lysosomes with autophagosomes. Finally, APP is degraded by the autophagosome-lysosomal pathway, and the released nutrients and metabolites are recycled. AMPK, AMP-activated protein kinase; mTOR, mammalian target of rapamycin; ULK1, Unc-51-like autophagy activating kinase 1; APP, amyloid precursor protein; ClassIII PI3K, class III PI3-kinase; 3-MA, 3-methyladenine; LAMP1, lysosomal-associated membrane protein 1.