Oleocanthal ameliorates amyloid-β oligomers' toxicity on astrocytes and neuronal cells: In vitro studies

Abstract

Extra-virgin olive oil (EVOO) has several health promoting effects. Evidence have shown that EVOO attenuates the pathology of amyloid-β (Aβ) and improves cognitive function in experimental animal models, suggesting it's potential to protect and reduce the risk of developing Alzheimer's disease (AD). Available studies have linked this beneficial effect to oleocanthal, one of the active components in EVOO. The effect of oleocanthal against AD pathology has been linked to its ability to attenuate Aβ and tau aggregation in vitro, and enhance Aβ clearance from the brains of wild-type and AD transgenic mice in vivo. However, the ability of oleocanthal to alter the toxic effect of Aβ on brain parenchymal cells is unknown. In the current study, we investigated oleocanthal effect on modulating Aβ oligomers (Aβo) pathological events in neurons and astrocytes. Our findings demonstrated oleocanthal prevented Aβo-induced synaptic proteins, SNAP-25 and PSD-95, down-regulation in neurons, and attenuated Aβo-induced inflammation, glutamine transporter (GLT1) and glucose transporter (GLUT1) down-regulation in astrocytes. Aβo-induced inflammation was characterized by interleukin-6 (IL-6) increase and glial fibrillary acidic protein (GFAP) upregulation that were reduced by oleocanthal. In conclusion, this study provides further evidence to support the protective effect of EVOO-derived phenolic secoiridoid oleocanthal against AD pathology.

Keywords: amyloid-β; astrocytes; neuroinflammation; neurons; oleocanthal.

Figure 1

Astrocytes showed linear and rapid uptake of both Aβm and Aβo. (A) Concentration-in cellular uptake of Aβm, (C) time-dependent increase in cellular uptake of Aβm, and (E) time-dependent decrease in Aβm levels in media. (B) Concentration-dependent increase in cellular uptake of Aβo, (D) time-dependent increase in cellular uptake of Aβo, and (F) time-dependent decrease in Aβo levels in media. After 3 and 7 days treatment with Aβo, oleocanthal or combination, Aβm (200 nM) uptake study was initiated for 15 min, (G) effect of treatments on Aβm levels in astrocytes media (3 days treatment), (H) effect of treatments on Aβm levels in cell lysate (3 days treatment), (I) effect of treatments on Aβm levels in astrocytes media (7 days treatment), and (J) effect of treatments on Aβm levels in cell lysate (7 days treatment). Values are normalized to the control. Data is presented as mean ± SD, n= 3 independent experiments.

Figure 2

(A) Representative Western blots and densitometry analysis of IDE, NAP and LRP1 in CCF-STTG1 cells after 3 and 7 days treatment with Aβo, oleocanthal, or combination. None of the treatments altered these proteins expression. (B) Representative Western blots and densitometry analysis of ABCA1 in CCF-STTG1 cells after 3 and 7 days treatment with Aβo, oleocanthal, or combination. ABCA1 was significantly up-regulated by Aβo treatment for 3 and 7 days; oleocanthal addition didn’t significantly alter ABCA1 expression. Data is presented as mean ± SD (*P<0.05, **P<0.01, ***P<0.001), n= 3 independent experiments.

Figure 3

After 3 (A) and 7 days (B) treatment with Aβo, oleocanthal, or combination, ACM was collected for IL-6 measurement using ELISA. Oleocanthal reduced the baseline release of IL-6 and attenuated Aβo-induced secretion of IL-6; values were normalized to the control. The relative expression of astrocytes’ GFAP with the same treatments and duration as above, was determined using Western blot analysis. (C) Representative Western blots and densitometry analysis of GFAP showed significant up-regulation by the 3 days exposure to Aβo; oleocanthal addition attenuated Aβo induced GFAP up-regulation. (D) Representative Western blots and densitometry analysis of GFAP showed a significant up-regulation by the 7 days exposure to Aβo; oleocanthal addition attenuated Aβo induced GFAP up-regulation. Data is presented as mean ± SD (*P<0.05, **P<0.01, ***P<0.001), n= 3 independent experiments.

Figure 4

Representative Western blots and densitometry analysis of GLT1 and GLUT1 expressions in astrocytes treated for 7 days with Aβo, oleocanthal, or combination showed that Aβo significantly down-regulated both proteins that were rectified by oleocanthal treatment. Data is presented as mean ± SD (*P<0.05, **P<0.01), n= 3 independent experiments.

Figure 5

(A) Representative Western blots and densitometry analysis of GLT1, PSD-95, and SNAP-25 following 7 days treatment with Aβo, oleocanthal, or combination. Aβo significantly reduced GLT1 and PSD-95 expressions that were rectified by oleocanthal to levels above their baseline expressions; SNAP-25 was not significantly altered by the 7 days exposure to Aβo. (B) Representative Western blots and densitometry analysis of APP, sAPPα, and sAPPβ; 7 days exposure to Aβo significantly increased APP, sAPPα, and sAPPβ levels that were not modulated by oleocanthal. LRP1 was not significantly altered by the treatments. Data is presented as mean ± SD (*P<0.05, **P<0.01), n = 3 independent experiments.

Figure 6

Representative Western blots and densitometry analysis of GLT1, PSD-95, and SNAP-25 in SH-SY5Y after 3 and 7 days treatment with Aβo, oleocanthal, or combination. None of the treatments altered these proteins expression in SH-SY5Y cells. Data is presented as mean ± SD, n= 3 independent experiments.

Figure 7

The relative expression of neuronal synaptic proteins GLT1, PSD-95, and SNAP-25 were compared in SH-SY5Y-APP and non-transfected SH-SY5Y cells using Western blot. Densitometry analysis of GLT1, PSD-95, and SNAP-25 revealed significantly lower expressions in SH-SY5Y-APP cells compared to SH-SY5Y cells. Data is presented as mean ± SD (***P<0.001), n= 3 independent experiments.

Figure 8

The relative expression of neuronal synaptic proteins GLT1, PSD-95, and SNAP-25 was measured after 3 days of ACM addition to SH-SY5Y and SH-SY5Y-APP cells using Western blot analysis. (A) Representative Western blots and densitometry analysis showed ACM from Aβo and combination treated astrocytes to significantly increase GLT1, PSD-95, and SNAP-25 expressions in SH-SY5Y cells. Oleocanthal treatment has no effect on their expression levels. (B) Representative Western blots and densitometry analysis showed ACM from Aβo, oleocanthal and the combination treated astrocytes have no effect on GLT1 and PSD-95 expressions in SH-SY5Y-APP cells. Only the expression of SNAP-25 was significantly increased by ACM from Aβo and combination treatments. Data is presented as mean ± SD (**P<0.01, ***P<0.001), n= 3 independent experiments.