Oleocanthal-rich extra-virgin olive oil enhances donepezil effect by reducing amyloid-β load and related toxicity in a mouse model of Alzheimer's disease

Abstract

Previous evidence suggested that extra-virgin olive oil (EVOO) is linked to attenuating amyloid-β (Aβ) pathology and improving cognitive function in Alzheimer's disease (AD) mouse models. In addition, we recently reported the beneficial effect of oleocanthal, a phenolic compound in EVOO, against AD pathology. Currently, medications available to target AD pathology are limited. Donepezil is an acetylcholine esterase inhibitor approved for use for all AD stages. Donepezil has been reported to have limited Aβ-targeting mechanisms beside its acetylcholine esterase inhibition. The aim of this study was to investigate the consumption of EVOO rich with oleocanthal (hereafter EVOO) as a medical food on enhancing the effect of donepezil on attenuating Aβ load and related toxicity in 5xFAD mouse model of AD. Our results showed that EVOO consumption in combination with donepezil significantly reduced Aβ load and related pathological changes. Reduced Aβ load could be explained, at least in part, by enhancing Aβ clearance pathways including blood-brain barrier (BBB) clearance and enzymatic degradation, and shifting amyloid precursor protein processing toward the nonamyloidogenic pathway. Furthermore, EVOO combination with donepezil up-regulated synaptic proteins, enhanced BBB tightness and reduced neuroinflammation associated with Aβ pathology. In conclusion, EVOO consumption as a medical food combined with donepezil offers an effective therapeutic approach by enhancing the noncholinergic mechanisms of donepezil and by providing additional mechanisms to attenuate Aβ-related pathology in AD patients.

Keywords: Alzheimer’s disease; Amyloid-β; Blood–brain barrier; Donepezil; EVOO; Oleocanthal.

Fig. 1

EVOO consumption and donepezil treatment significantly reduced Aβ burden in the hippocampus of 5xFAD mice. (A–D) Representative hippocampus sections stained with 6E10 (green) antibody against Aβ to detect total Aβ load and DAPI (blue) to stain nuclei; (A) control group, (B) EVOO group, (C) Donepezil group, (D) EVOO-donepezil group. (E) Total Aβ optical density quantification of A–D images. Two-way ANOVA demonstrated significant main effects of EVOO (F_(1,40)=103.1, P<0.0001) and donepezil (F_(1,40)=11.03, P=0.003) without significant interaction (F_(1,40)=3.076, P=0.94). (F–I) Representative hippocampus sections stained with Anti-Aβo (green) antibody to detect Aβo load and DAPI (blue) to stain nuclei; (F) control group, (G) EVOO group, (H) Donepezil group, (I) EVOO-donepezil group. (J) Aβo optical density quantification of F–I images. Two-way ANOVA demonstrated significant main effect of EVOO (F_(1,40)=50.7, P<0.0001) and donepezil (F_(1,40)=12.26, P=0.003) with significant interaction (F_(1,40)=6.74, P= 0.019). Soluble (K) Aβ₄₂ levels (with significant main effects of EVOO (F_(1,40)=307.3, P<0.0001) and donepezil ((F_(1,40)=26.9, P<0.0001), and significant interaction ((F_(1,40)=9.22, P= 0.0065), and (L) Aβ₄₀ levels (with significant main effects of EVOO (F_(1,40)=66.2, P<0.0001) and donepezil ((F_(1,40)=19.22, P=0.0002), and significant interaction (F_(1,40)=14.54, P=0.001) as determined by ELISA. Data are presented as mean ± SEM of 6 mice/sex/treatment. ^**P<0.01 and ^***P<0.001 statistically significant as determined by Bonferroni’s post hoc test.

Fig. 2

EVOO consumption and donepezil treatment modulated APP processing in the brains of 5xFAD mice without altering total APP. Representative blots and densitometry analysis of full-length APP, sAPPα, and sAPPβ in mice brains homogenates from EVOO group, donepezil (Donp) group, and their combination demonstrated either treatment or their combination has no effect on APP levels, but the two-way ANOVA demonstrated significant main effects of EVOO (F_(1,20)=64.3, P<0.0001) and donepezil (F_(1,20)=7.13, P=0.017) with no significant interaction (F_(1,20)= 0.087, P= 0.771) on sAPPα, and significant main effects of EVOO (F_(1,20)=44.2, P<0.0001) and donepezil (F_(1,20)=6.59, P=0.021) with no significant interaction (F_(1,20)= 0.434, P= 0.519) on sAPPβ. Data are presented as mean ± SEM of 6 mice in each group.

Fig. 3

EVOO consumption and donepezil treatment significantly induced ABCA1 through the activation of PPARγ in the brains of 5xFAD mice. Representative blots and densitometry analysis of ABCA1, ApoE, PPAR-γ, RXR, and LXR in mice brains homogenates from EVOO group, donepezil (Donp) group, and their combination. The two-way ANOVA demonstrated significant main effects of EVOO (F_(1,20)=30.92, P<0.0001) and donepezil (F_(1,20)=41.44, P<0.0001) with no significant interaction (F_(1,20)= 1.102, P=0.312) on ABCA1, and significant main effects of EVOO (F_(1,20)=15.61, P=0.001) and donepezil (F_(1,20)=22.35, P=0.0002) with no significant interaction (F_(1,20)= 0.051, P=0.825) on PPARγ. The treatments have no significant effect on ApoE, RXR and LXR levels. Data are presented as mean ± SEM of 6 mice in each group. ^*P<0.05, ^**P<0.01 and ^***P<0.001 statistically significant as determined by Bonferroni’s post hoc test.

Fig. 4

EVOO consumption and donepezil treatment up-regulated neuro-synaptic involved proteins in the brains of 5xFAD mice. Representative blots and densitometry analysis of GLT1, PSD-95, and SNAP-25 in mice brains homogenates from EVOO group, donepezil (Donp) group, and their combination demonstrated significant main effects of EVOO (F_(1,20)=32.98, P<0.0001) and donepezil (F_(1,20)=4.82, P=0.047) with no significant interaction (F_(1,20)= 0.549, P=0.472) on GLT1; significant main effects of EVOO (F_(1,20)=6.96, P=0.0195) and donepezil (F_(1,20)=26.75, P=0.0001) with no significant interaction (F_(1,20)= 0.001, P=0.974) on PSD-95; and significant main effect of donepezil (F_(1,20)=44.43, P<0.0001) with no significant main effect of EVOO (F_(1,20)=4.31, P=0.054) and no significant interaction (F_(1,20)= 1.26, P=0.279) on SNAP-25 as determined by two-way ANOVA. Data are presented as mean ± SEM of 6 mice in each group. ^**P<0.01 and ^***P<0.001 statistically significant as determined by Bonferroni’s post hoc test.

Fig. 5

EVOO consumption combined with donepezil treatment significantly up-regulated BBB-Aβ clearance proteins LRP1 and P-gp, the brain capillary tight junction protein claudin-5, reduced IgG extravasation, and induced the expression of the Aβ degrading enzyme NEP in the brains of 5xFAD mice. (A) Representative blots and densitometry analysis of LRP1, P-gp, and claudin-5 in brain capillaries of mice from EVOO group, donepezil (Donp) group, and their combination. The two-way ANOVA analysis demonstrated significant main effects of EVOO (F_(1,20)=24.98, P=0.0002) and donepezil (F_(1,20)=4.95, P=0.043) with no significant interaction (F_(1,20)= 1.198, P=0.292) on LRP1, significant main effects of EVOO (F_(1,20)=101.2, P<0.0001) and donepezil (F_(1,20)=7.86, P=0.014) with no significant interaction (F_(1,20)= 3.02, P=0.104) on P-gp, and significant main effects of EVOO (F_(1,20)=27.31, P=0.0002) and donepezil (F_(1,20)=6.25, P=0.028) with no significant interaction (F_(1,20)= 0.328, P=0.578) on claudin-5. (B) Representative brain sections stained with Anti-IgG (green) antibody to detect IgG extravasation and anti-collagen (red) antibody to detect capillaries; the two-way ANOVA analysis of the their optical density quantitation demonstrated significant main effects of EVOO (F_(1,20)=29.84, P<0.0001) but not donepezil (F_(1,16)=3.78, P=0.066) with no significant interaction (F_(1,20)= 0.928, P=0.347) on IgG. (C) Representative blots and densitometry analysis of IDE and NEP n mice brains homogenates from EVOO group, donepezil (Donp) group, and their combination demonstrated no significant main effects of EVOO or donepezil on IDE, but on NEP there was significant main effects of EVOO (F_(1,20)=17.59, P=0.0011) and donepezil (F_(1,20)=27.18, P=0.0002) with no significant interaction (F_(1,20)= 1.056, P=0.323). Data are presented as mean ± SEM of 6 mice in each group. ^*P<0.05 and ^**P<0.01 statistically significant as determined by Bonferroni’s post hoc test.

Fig. 6

EVOO consumption combined with donepezil treatment reduced neuroinflammation in 5xFAD mice. Neuroinflammation was assessed by measuring antioxidant capacity, IL-1β levels, and astrocytes activation monitored by GFAP intensity and astrocytes morphology. (A) Total antioxidant capacity, and (B) IL-1β levels in mice brains homogenates from EVOO group, donepezil (Donp) group, and their combination. The two-way ANOVA analysis demonstrated significant main effect of EVOO (F_(1,20)=30.08, P=0.0006) but not donepezil (F_(1,20)=0.025, P=0.878) with no significant interaction (F_(1,20)=0.161, P=0.698) on total antioxidant capacity, and significant main effect of EVOO (F_(1,20)=31.90, P=0.0001) and donepezil (F_(1,20)=7.09, P=0.021) with no significant interaction (F_(1,20)=0.117, P=0.738) on IL-1β. (C–F) Representative brain sections stained with Anti-GFAP (red) antibody to detect activated astrocytes and 6E10 (green) antibody to detect Aβ in (C) control group, (D) EVOO group, (E) Donepezil group, and (F) EVOO-donepezil group. (G) GFAP optical density quantification analyzed by two-way ANOVA demonstrated significant main effects of EVOO (F_(1,20)=74.60, P<0.00016) and donepezil (F_(1,20)=5.74, P=0.025) with no significant interaction (F_(1,20)=3.09, P=0.092). Data are presented as mean ± SEM of 6 mice in each group. ^**P<0.01 statistically significant as determined by Bonferroni’s post hoctest.

Fig. 7

EVOO consumption didn’t interfere with donepezil acetylcholine esterase inhibitory effect in the brains of 5xFAD mice. Two-way ANOVA analysis demonstrated no significant main effect of EVOO (F_(1,20)=0.317, P=0.584) but significant main effect of donepezil on acetylcholine levels (F_(1,20)=27.36, P=0.0002) with no significant interaction (F_(1,20)=0.011, P=0.918). Data are presented as mean ± SEM of 6 mice in each group. ^**P<0.01 statistically significant as determined by Bonferroni’s post hoc test.