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. Author manuscript; available in PMC: 2020 Aug 21.
Published in final edited form as: ACS Chem Neurosci. 2019 Jun 25;10(8):3543–3554. doi: 10.1021/acschemneuro.9b00175

Oleocanthal-Rich Extra-Virgin Olive Oil Restores the Blood–Brain Barrier Function through NLRP3 Inflammasome Inhibition Simultaneously with Autophagy Induction in TgSwDI Mice

Sweilem B Al Rihani †,, Lucy I Darakjian , Amal Kaddoumi †,‡,*
PMCID: PMC6703911  NIHMSID: NIHMS1045909  PMID: 31244050

Abstract

Alzheimer’s disease (AD) is a complex neurodegenerative disorder characterized by multiple hallmarks including extracellular amyloid (Aβ) plaques, neurofibrillary tangles, dysfunctional blood-brain barrier (BBB), neuroinflammation, and impaired autophagy. Thus, novel strategies that target multiple disease pathways would be essential to prevent, halt, or treat the disease. A growing body of evidence including our studies supports a protective effect of oleocanthal (OC) and extra-virgin olive oil (EVOO) at early AD stages before the onset of pathology. In addition, we reported previously that OC and EVOO exhibited such effect by restoring the BBB function; however, the mechanism(s) by which OC and EVOO exert such an effect and whether this effect extends to a later stage of AD remain unknown. In this work, we sought first to test the effect of OC-rich EVOO consumption at an advanced stage of the disease in TgSwDI mice, an AD mouse model, starting at the age of 6 months for 3 months treatment, and then to elucidate the mechanism(s) by which OC-rich EVOO exerts the observed beneficial effect. Overall findings demonstrated that OC-rich EVOO restored the BBB function and reduced AD-associated pathology by reducing neuroinflammation through inhibition of NACHT, LRR, and PYD domain-containing protein 3 (NLRP3) inflammasome and inducing autophagy through activation of AMP-activated protein kinase (AMPK)/Unc-51-like autophagy activating kinase 1 (ULK1) pathway. Thus, diet supplementation with OC-rich EVOO could provide beneficial effect to slow or halt the progression of AD.

Keywords: oleocanthal, extra-virgin olive oil, blood–brain barrier, NLRP3 inflammasome, autophagy, Alzheimer’s disease

Graphical Abstract

graphic file with name nihms-1045909-f0009.jpg

INTRODUCTION

Alzheimer’s disease (AD) is the most common neurodegenerative disease and form of dementia and accounts for 60–80% of all cases worldwide.1 Unfortunately, currently there are no disease modifying treatments for AD. The pathology of this devastating neurodegenerative disease is complex. While the cause of AD is not clear, the cardinal features commonly observed in the brains of AD patients are the deposition of amyloid-β (Aβ) plaques, aggregation of hyperphosphorylated tau protein, abnormal neurites, neuropil threads, synapsis and neuronal loss, astrogliosis, microglial activation, cerebral amyloid angiopathy (CAA), and a disrupted blood−brain barrier (BBB).27

Current evidence suggests that aging, cerebrovascular disruption, or Aβ or tau accumulation can induce BBB dysfunction by altering the neurovascular unit.8,9 Disruption of the BBB results in neuronal damage and reduced Aβ clearance, which would further accumulate Aβ and BBB dysfunction leading to AD progression. Therefore, to maintain healthy BBB function or restore BBB function provides a novel strategy to prevent or treat AD.

A primary risk factor for AD is advanced age.10 While aging itself is not a disease, it is usually accompanied by a low degree of chronic inflammation.11 Therefore, the term inflamm-aging was introduced, which involves a number of factors and processes characterized by complex interactions of several molecular mediators such as inflammatory markers.11,12 Examples of such markers include C-reactive protein and cytokines.11,12 In AD, cerebral neuroinflammation contributes to the disease pathogenesis. Growing evidence has demonstrated increased interleukin-1β (IL-1β) levels, activation of NACHT, LRR, and PYD domain-containing protein 3 (NLRP3) inflammasome in microglia, and subsequent inflammatory events as downstream consequences of Aβ deposition.13,14 The continuous presence of stimulus in the AD brain, such as Aβ, has been recently reported to activate NLRP3 and promote formation of the inflammasome complex, which results in the activation of caspase-1 and production of the pro-inflammatory cytokines IL-1β and IL-18.15,16 These findings suggest that the NLRP3 inflammasome is an important contributor to cerebral neuroinflammation in the AD brain, and its inhibition could provide a therapeutic strategy to prevent, halt or treat AD. In addition, considerable evidence suggests that the dysregulation of autophagy, an essential homeostatic pathway, occurs in the brains of AD animal models and AD patients.1719 Autophagy plays an important role in the clearance of Aβ,20 and the clearance of APP and tau.2123 Thus, autophagy dysregulation could play a key role in the pathology of AD by enhancing the accumulation of Aβ, APP, and tau. Collectively, there is a substantial interest in the discovery of therapeutic inflamma-some and autophagy modulators.

Currently, there is suffcient scientific evidence supporting that chronic exposure to the Mediterranean diet (MedD) is linked to low risk of cognitive impairment and AD.24 Among the key elements of the MedD is the daily consumption of extra-virgin olive oil (EVOO), which provides anti-inflammatory and antioxidant effects,25 in addition to other health benefits.26 Data from our group have shown that EVOO added to the diet of AD model mice at early age and before the pathology onset was able to protect mice from Aβ-related pathology by restoring the BBB function.27,28 EVOO consumption for 6 months starting at one month of age significantly enhanced BBB function, reduced parenchymal Aβ load, reduced Aβ production, enhanced Aβ clearance, and decreased vascular deposits of Aβ and total tau and its phosphorylation in the brains of the AD model TgSwDI mice.29 In addition, available studies have shown that EVOO administration to triple transgenic mice (3xTg) as model of AD, improved memory function, reduced synaptic toxicity, and significantly reduced Aβ deposition by autophagy activation.30 In addition, EVOO increased antioxidants levels, such as glutathione, in mouse brains.25 EVOO contains several phenolic compounds, among which is oleocanthal (OC). When tested in mice, OC restored the BBB function, reduced Aβ brain levels, and reduced inflammation as evident by reduced astrogliosis and brain levels of cytokines.27,28,31,32 These results suggest that OC could largely contributed to EVOO observed and reported benefits. Besides OC, EVOO contains hydroxyoleocanthal (also known as oleacein) and oleuropein aglycone; while studies reporting hydroxyoleocanthal activity are lacking, several studies demonstrated the beneficial effect of oleuropein aglycone against Aβ related pathology when added to mouse diet at 50 mg/kg.33

The potential beneficial effect of OC and EVOO on AD in disease models, especially at the very early stages, have been studied previously by us and others.29,30,32 However, the mechanism(s) by which OC and EVOO rectified the BBB function, and exerted the reported beneficial effect remains unclear; also, whether EVOO effectiveness against AD extends to late stages is unknown. Thus, the purpose of this study was to evaluate the beneficial effect and associated mechanism(s) of OC-rich EVOO consumption at an advanced stage of the disease in TgSwDI mice starting at the age of 6 months for 3 months treatment.

RESULTS

OC-Rich EVOO Improved BBB Tightness in TgSwDI Mouse Brains.

Effect of OC-rich EVOO (hereafter EVOO) on BBB leakiness was evaluated by immunostaining of immunoglobulin-G (IgG) extravasation, which was used as an endogenous BBB permeability marker. As shown in Figure 1, EVOO consumption significantly reduced IgG extravasation by 75% and 31% in the cortex and the hippocampus regions, respectively, when compared to refined olive oil (OO; control) group. These findings are consistent with our previous results.27,29

Figure 1.

Figure 1.

Representative brain sections stained with anti-mouse IgG antibody to detect IgG extravasation (green) and anti-collagen antibody (red) in (A) brain cortex and (B) hippocampus. (C) IgG optical density in mouse cortexes and hippocampi were quantified for IgG extravasation. Data are presented as box-and-whisker plots representing median and interquartile range (IQR), with minimum and maximum values. Statistical analysis was determined by Student’s t test for n = 7 mice/group. **P < 0.01, ***P < 0.001 versus control group. Scale bar, 100 μm.

EVOO Reduces Total Aβ Load and Plaques in TgSwDI Mouse Brains.

As shown in Figure 2, compared to control group, EVOO significantly reduced total 6E10-detected Aβ by 61% and 73% in brain cortex and hippocampus, respectively (Figure 2A). In addition, thioflavin-S positive Aβ plaques staining showed similar pattern with an overall 47% and 79% reduction in cortex and hippocampus regions, respectively (Figure 2B).

Figure 2.

Figure 2.

(A) Representative cortex and hippocampus sections stained with 6E10 (green) antibody against Aβ with corresponding quantification for total Aβ deposition; DAPI (blue) was used to stain nuclei. Closed insets show higher magnification of the hippocampus region. (B) Representative cortex and hippocampus sections stained with Thio-S (green) to detect Aβ plaque load with corresponding quantification of area covered with Aβ plaques; anti-collagen IV (red) was used to stain microvessels. Data are presented as box-and-whisker plots representing median and IQR, with minimum and maximum values. Statistical analysis was determined by Student’s t test for n = 7 mice/group. ***P < 0.001 versus control group. Scale bar, 100 μm.

EVOO Modulates APP Processing, Total Tau, and Its Phosphorylation Expression.

We then assessed EVOO effects on the processing of APP in TgSwDI mice brains by Western blot. As shown in Figure 3, EVOO treatment significantly increased sAPPα levels by 1.4-fold and decreased sAPPβ and BACE1 expressions by ~30%. Interestingly, a significant 40% reduction in the level of total APP was also observed. In addition to APP processing, we sought to confirm EVOO effect on the expression of tau protein and its phosphorylation in TgSwDI mice with advanced disease pathology at 9 months by Western blot. A significant reduction in total tau by 48% and tau phosphorylation at threonine position 231 (p-Tau-Thr231) by 38% was observed.

Figure 3.

Figure 3.

EVOO consumption effect on APP processing, tau, and tau phosphorylation at amino acid residue threonine 231 in TgSwDI mouse brains. Representative blots and densitometry analysis showed a significant increase in sAPPα and reduction in APP, sAPPβ BACE-1 enzyme, total-tau, and phosphorylated-tau(Thr231) levels in brain homogenates of mice that consumed EVOO compared to control group. Data are presented as box-and-whisker plots representing median and IQR, with minimum and maximum values. Statistical analysis was determined by Student’s t test of n = 7 mice/group. **P < 0.01 versus control group.

EVOO Attenuates Brain Neuroinflammation.

Astrocyte inflammatory activation is recognized by increased levels of glial fibrillary acidic protein (GFAP) with elongated shape and thick branches.34 As shown in Figure 4A, in comparison to the control group, EVOO significantly reduced astrocyte activation and ameliorated the astrocyte shape in mouse cortexes and hippocampi. Moreover, EVOO significantly reduced brain levels of the microglial marker ionized calcium binding adaptor molecule 1 (Iba1) as analyzed by immunostaining (Figure 4C), which was further confirmed by immunoblotting demonstrating a 43% reduction in Iba1 (Figure 4D). Reduced microglial activation was also associated with 40% reduction in FEPPA levels, a specific marker for activated microglia, as determined by HPLC analysis, in the brains of mice consuming EVOO when compared to the control group (Figure 4B). In addition, Western blot analysis of metalloproteinase-9 (MMP9) enzyme showed EVOO to significantly reduce MMP9 by 57% compared to control group (Figure 4D).

Figure 4.

Figure 4.

(A) Representative brain sections from 9-month-old TgSwDI mice were colabeled with GFAP antibody (red) to detect activated astrocytes and thioflavin-S (green) to detect Aβ plaques in control and EVOO groups. Hippocampus is seen at higher magnification. Activated astrocytes in cortex are shown with long and thick branches in control group that were significantly reduced by EVOO as demonstrated at higher magnification in the closed inserts. Scale bar, 100 μm. (B) Results from HPLC analysis of FEPPA levels, a specific marker for microglial activation in the brain homogenate of TgSwDI mice, which was significantly reduced in EVOO diet group compared to control (n = 3 mice/group). (C) Representative images from cortex and hippocampal brain sections stained with Iba1 antibody (red) to detect activated microglia and 6E10 antibody (green) to detect total Aβ in control and EVOO groups. Scale bar, 100 μm. (D) Representative blots and densitometry analysis of MMP9, Iba1, and TRPA1 in TgSwDI mouse brain homogenates. (E) Effect of EVOO on IL-1β levels in mouse brain homogenates. (F) Effect of EVOO on IL-10 levels in mouse brain homogenates. For panels D–F, data are presented as box-and-whisker plots representing median and IQR, with minimum and maximum values (n = 7 mice/group). For panel B, data is presented as mean + SEM for n = 3 mice/group. Statistical analysis was determined by Student’s t test. **P < 0.01, and ***P < 0.001 versus control group.

Transient receptor potential ankyrin 1 (TRPA1) is a membrane-associated cation channel that is widely expressed in neuronal and non-neuronal cells.3537 TRPA1 activation has been linked with inflammation.3537 Here and as shown in (Figure 4D), TRPA-1 expression was significantly reduced by EVOO treatment by 46% when compared to control group.

IL-1β and IL-10 were analyzed by ELISA and as demonstrated in Figure 4E,F, EVOO significantly reduced IL-1β brain levels by 75% and increased IL-10 levels by 31%.

EVOO Reduces Oxidative Stress Markers in TgSwDI Mouse Brains.

Carbonyl formation is an important detectable marker of protein oxidation, and protein carbonyls are significantly increased in AD;38 therefore we evaluated the effect of EVOO on total protein carbonyl formation. EVOO consumption significantly reduced protein carbonyl levels by 64% (Figure 5A). In addition, the effect of EVOO consumption on superoxide dismutase (SOD) brain levels was evaluated. SOD is an enzyme that acts as an antioxidant that catalyzes the dismutation of superoxide anion free radical into molecular oxygen and hydrogen peroxide, which is decreased in AD suggesting oxidative stress.39 EVOO significantly increased SOD formation by 79% compared to control group (Figure 5B).

Figure 5.

Figure 5.

(A) Effect of EVOO on protein carbonyl levels in mouse brain homogenates. (B) Effect of EVOO on SOD levels in mouse brain homogenates. Data are presented as box-and-whisker plots representing median and IQR, with minimum and maximum values of n = 7 mice in each group. Statistical analysis was determined by Student’s t test. *P < 0.05 and ***P < 0.001 versus control group.

EVOO Reduces IL-1β Production through the NLRP3 Pathway in TgSwDI Mouse Brains.

The IL-1β upstream inflammasome activation and production pathway was evaluated. Findings revealed that EVOO significantly reduced NLRP3 protein expression by 41%, which was also associated with a significant reduction in the expression levels of procaspase-1 and cleaved caspase-1 p20 and p10 by 31%, 45%, and 28%, respectively. In addition, this effect was also associated with a significant reduction in procaspase-8 by 56% and cleaved caspase-8 p18 by 60% when compared to control diet (Figure 6).

Figure 6.

Figure 6.

EVOO reduced inflammasome formation by modulating NLRP-3/caspase-1 and −8 pathway. Representative blots and densitometry analysis showed a significant reduction in the expression of NLRP3, procaspase-1, cleaved caspase p20 and p10 (Cle-Casp1 p20 and 10), procaspase-8, and cleaved caspase-8 p18 (Cle-Cas8 p18) in brain homogenates of mice that consumed EVOO compared to control group. Data are presented as box-and-whisker plots representing median and IQR, with minimum and maximum values of n = 7 mice in each group. Statistical analysis was determined by Student’s t test. **P < 0.01, and ***P < 0.001 versus control group.

EVOO Induces Autophagy in TgSwDI Mouse Brains.

Recent studies established that autophagy induction plays a key role in the regulation of BACE1 turnover and APP processing and that the autophagy process is downregulated with aging.40 Several autophagy markers affecting either the initiation or the maturation of autophagosomes and autophagic flux were analyzed in this study. As shown in Figure 7, EVOO significantly induced major autophagosome initiation proteins AMP-activated protein kinase (AMPK) (2.2-fold) and Unc-51-like autophagy activating kinase 1 (ULK1) (2.4-fold) expression and their phosphorylation (30–60%). While the increase in beclin-1 levels did not reach a significant level, a significant increase in ATG7 and ATG3 protein levels by 2.4- and 1.7-fold, respectively, was observed without significant alteration of ATG-12/5 level (Figure 7). In addition, the microtubule-associated protein light chain 3 conversion (LC3-I) and the ubiquitin-binding protein p62 were significantly reduced by 22% and 37%, respectively, which was associated with 1.6-fold increase in the level of LC3-II (Figure 7). On the other hand, EVOO consumption has no significant effect on the mTOR pathway or its downstream proteins P70S6K and 4E-BP1 and their phosphorylation (Supporting Information, Figure S1).

Figure 7.

Figure 7.

EVOO induced autophagy through AMPK pathway activation. Representative blots and densitometry analysis of AMPK, phosphorylated-AMPK (pAMPK), ULK1, phosphorylated-ULK1 (pULK1), beclin, ATG 7, ATG 5/12, ATG 3, SQSTM1/p62, LC3 I, and LC3 II in brain homogenates of mice that consumed EVOO compared to control group. Data are presented as box-and-whisker plots representing median and IQR, with minimum and maximum values of n = 7 mice in each group. Statistical analysis was determined by Student’s t test. ns = not significant, *P < 0.05, **P < 0.01, and ***P < 0.001 versus control group.

Effect of EVOO on Neurosynaptic Markers and Morris Water Maze Test Performance.

As shown in Figure 8, Western blot findings demonstrated EVOO significantly increased the expression of three major neurosynaptic markers, PSD-95, synapsin-1, and SNAP-25, by 2.3-, 1.3- and 1.7-fold, respectively, compared to OO enriched-diet (control group). In addition, MWM test was performed to assess EVOO effect on learning and memory on the fourth day of training. The following parameters were analyzed: the time a mouse takes to find the platform (latency, s), swimming distance (cm), and swimming speed (cm/s). As shown in Figure 8BE, EVOO fed mice found the platform faster than vehicle-treated mice(15.5 ± 3.1 vs 43.9 ± 6.0 s, respectively, P < 0.01), and EVOO diet significantly reduced mouse swimming distance to 476.4 ± 97.8 cm from 1124 ± 135.7 cm for the control group. These results suggest that EVOO improved MWM performance. There was no difference between the two groups in swimming speed, thus excluding motor changes as a cause of the observed improvement.

Figure 8.

Figure 8.

(A) Representative blots and densitometry analysis of the neurosynaptic markers SNAP-25, synapsin-1, and PSD-95 in brain homogenates of mice that consumed EVOO compared to control group. Statistical analysis was determined by Student’s t test (n = 7 mice/group).(B) Typical swimming patterns of control and EVOO-treated mice on the fourth day of the Morris water maze test. (C–E) Effect of EVOO-enriched diet on latency, swimming distance, and swimming speed of the mice. EVOO significantly decreased latency and swimming distance compared to the control mice on the fourth day of training. The swimming speed was not significantly different between the groups (n = 5 mice/group). Data are presented as box-and-whisker plots representing median and IQR with minimum and maximum values. ns = not significant, *P <0.05, **P < 0.01, ***P < 0.001 versus control group.

DISCUSSION

Recently, we have reported that OC and EVOO improved BBB integrity and function, increased endothelial cell tightness, upregulated Aβ major transport proteins localized at the endothelial cells of the BBB, and enhanced clearance of Aβ across the BBB.2729,32 The in vivo studies were performed in two mouse models, namely, TgSwDI and 5xFAD mice, used at the age of one month and before the disease starts as a prevention mode.2729,32 In the current study, we evaluated the effect of 3 months consumption of OC-rich EVOO added to the mouse diet, on the BBB function, Aβ-related pathology, and MWM performance in TgSwDI mice at an older age with advanced pathology and investigated potential mechanisms by which OC-rich EVOO rectified BBB function and reduced Aβ related pathology. To the best of our knowledge, findings of this study demonstrated for the first time that the long-term dietary supplementation with OC-rich EVOO significantly reduced inflammasome activation through NLRP3 inhibition and increased autophagy through AMPK−ULK1 pathway activation when compared to mice consuming a refined olive oil-enriched diet. Reduced neuroinflammation and induced autophagy by EVOO could explain, at least in part, the improved BBB function as determined by the significant reduction in IgG extravasation in mouse cortexes and hippocampi. This effect was concomitant to reduced Aβ-load and associated synaptotoxicity, reduced tau hyperphosphorylation, and reduced oxidative stress biomarkers in TgSwDI mice with extensive disease pathology, which collectively enhanced learning.

During aging and in AD, the BBB function has been reported to deteriorate.8,41,42 Neuroinflammation and autophagy dysregulation in aging and AD,19,40,43 have also been linked to BBB disruption.44,45 Thus, their simultaneous restoration could provide an effective approach for functional BBB, healthy aging, and reduced risk of AD or halt in AD progression.

NLRP3 inflammasome activation in microglia and astrocytes has been associated with numerous chronic inflammatory diseases. NLRP3 can detect inflammatory and Aβ aggregates.13,46 The activation of NLRP3 inflammasome by Aβ enhances AD progression by facilitating chronic inflammatory responses,13 which are partly involved in restricting glial function and mediating synaptic dysfunction and cognitive impairment.13 In addition, available studies reported that NLRP3 inflammasome activation associated with IL-1β release contributed to BBB damage and increased BBB permeability and that the knockout of NLRP3 in mice with ischemic stroke reduced BBB damage.47 Therefore, blocking NLRP3 inflammasome activity could effectively interfere with the progression of AD.13,46 NLRP3 inflammasome processes pro-IL-1 to mature and functional IL-1β. In TgSwDI mice, EVOO consumption significantly reduced IL-1β levels in mouse brains compared to that in mice treated with refined olive oil. This effect was accompanied by a significant reduction in the expression of two major caspases responsible for the increased formation of IL-1β, namely, caspase-148 and caspase-8.49 In addition, consequent to NLRP3-inflammasome inhibition by EVOO, a significant reduction in astrogliosis, measured by GFAP, and microglial activation, measured by Iba1 and FEPPA, was also observed. FEPPA is a specific ligand for the translocator protein tryptophan-rich sensory protein (TSPO), which is used as a biomarker to assess glial activation and neuroinflammation by positron emission tomography.50 TSPO is located in the outer mitochondrial membrane of microglia; its expression increases in response to microglial activation, and thus it is considered a valid biomarker for neuroinflammation.51 Our results from FEPPA study demonstrated that EVOO significantly reduced TSPO, which suggests reduced inflammation. A recent study reported a link between NLRP3 inflammasome activation and TSPO upregulation, which is implicated in mitochondrial dysfunction,52 suggesting an additional therapeutic target for EVOO.

Moreover, calcium signaling has been identified as a critical mediator pathway in NLRP3 inflammasome activation where increased levels of intracellular calcium were associated with NLRP3 inflammasome activation.53 In the brain, TRPA1 receptor is expressed in primary sensory neurons and in nonneuronal cells mainly in astrocytes.35,37 TRPA1 plays a key role in pain and inflammation and is recognized as a gatekeeper of inflammation.36 It acts as a sensor for detecting several toxic signals such as reactive oxygen species, inflammatory cytokines, and Aβ, upon detection of which, TRPA1 is activated. In AD, it has been shown that in the brains of APP/PS1 mice, Aβ-mediated chronic inflammation caused TRPA1 activation, which triggered calcium influx and inflammation in astrocytes.54 Our results demonstrated that EVOO significantly reduced TRPA1 expression, which is the molecular target of OC,55 suggesting another mechanism by which EVOO was able to reduce neuroinflammation. While additional studies are required to understand the link between TRPA1 and NLRP3 inflammasome activation, a very recent study reported that cigarette smoke-induced TRPA1 was associated with increased NLRP3 inflammasome activation and ROS production in the alveolar epithelial cells A549, and this effect was calcium mediated.56 These results suggest that OC-rich EVOO reduced inflammation directly by inhibition of NLRP3 inflammasome activation or indirectly by TRPA1 down-regulation that triggered NLRP3 inflammasome inhibition and reduced IL-1β production. Collectively, our findings support TRPA1 and NLRP3−IL-1β axis as targets for the overall beneficial effect of EVOO.

Chronic inflammasome activation and neuroinflammation associated with prolonged accumulation of Aβ and tau hyperphosphorylation could damage autophagy.57 Autophagy dysregulation impairs effective elimination of aggregates and damaged mitochondria leading to their accumulation associated with toxicity and oxidative stress.57 In this study, the 3-months consumption of EVOO significantly increased several autophagy markers that regulate autophagy induction and maturation in the brains of TgSwDI mice. Our findings confirm an earlier observation for EVOO effect on inducing autophagy where its chronic administration significantly increased levels of the autophagy activation biomarkers ATG5 and ATG7 in the brains of 3xTg mice.30 In an effort to expand on the mechanism by which EVOO induced autophagy, AMPK and mTOR pathways were evaluated. Based on the results, EVOO induced autophagy through AMPK pathway activation but not through mTOR pathway inhibition. Besides acting as a positive regulator of autophagy, through ULK1 phosphorylation, AMPK has multiple functions including general regulation of cellular metabolism, NLRP3 inflammasome regulation, and mitochonrdrial function.58 In addition, several recent studies have shown that autophagy induction by AMPK can reduce the levels of Aβ load in AD59 and α-synuclein in Parkinson’s disease60 and ameliorates Huntington’s disease pathology and prevents neurodegeneration.61 A recent study examined post-mortem brains of AD patients and showed the possible relation between autophagy and the upregulation of NLRP3 in response to Aβ aggregation.62 The autophagy markers SQSTM1/p62 and LC3 were abnormally accumulated and colocalized with Aβ deposits and hyperphosphorylated tau in the brains of AD patients.62 Autophagy induction, thus, has been proposed as a strategy to rectify the pathology of numerous diseases by promoting clearance of aggregated and hyperphosphorylated proteins in the cytoplasm.57 Our immunostaining analysis demonstrated fewer Aβ plaques and lower Aβ levels in the brains of TgSwDI mice consuming EVOO compared to mice fed with refined olive oil. This significant reduction in Aβ load could be related to autophagy induction through AMPK pathway.

Besides autophagy, another significant outcome that could be caused by neuroinflammation inhibition,63 is reduced levels of BACE1 in EVOO mouse brains, which could also contribute to sAPPβ reduced levels and thus brain Aβ load. The levels of sAPPα, on the other hand, were significantly increased; sAPPα possesses neurotrophic and neuroprotective activities,64 which could contribute to the enhanced learning and memory observed in TgSwDI mice consuming EVOO. In addition, decreased brain Aβ deposits contributed to the increased production of IL-10, which is consistent with previous reports,55 and reduced MMP9 and oxidative stress. Within this scenario, interestingly, the 3-months consumption of EVOO significantly reduced full-APP and tau levels in mouse brain homogenates suggesting the re-establishment of a proper processing of both proteins. While additional studies are required to explain this observation, available studies in mouse models have already shown that autophagy−lysosomal degradation of APP and Aβ21 and tau and hyperphosphorylated tau22 reduce AD. Furthermore, a recent study reported correlation between autophagy induction and reduced tau-protein expression in a mouse model of tauopathy.65 TgSwDI mouse model does not overexpress tau, and studies evaluating the development of tau pathology in this model with aging are not available. However, evidence exists that suggest Aβ and tau pathology to reciprocally drive each other.66 In addition, Roberson et al.67 reported tau depletion in hAPP mouse model that was associated with revoked learning and memory impairment. Also, Yoshikawa et al., recently reported that the depletion of tau protein ameliorated the hyper-locomotor behavior in J20 transgenic mice, an APP model, which signifies the functional outcome of Aβ/tau interaction.68 Similarly, in our study, EVOO significantly reduced tau, which is consistent with our previous findings with EVOO;29 however, it could be necessary to evaluate the effect of EVOO consumption on hAPP and tau mRNA levels to explain the observed reduction and confirm this effect is mediated by induced autophagy.

Several studies linked Aβ toxicity,69 tau aggregation,70 and memory impairment to neuroinflammation and impaired autophagy.17,19,69,70 Thus, reduced neuroinflammation and induced autophagy associated with reduced Aβ-related pathology and oxidative stress by EVOO could explain, at least in part, the enhanced BBB function. Functional BBB is vital to limit brain access of unwanted molecules, as demonstrated by reduced IgG extravasation, and to clear Aβ, findings that are consistent with our previous reports.27

CONCLUSION

In conclusion, our findings demonstrated the beneficial effects of OC-rich EVOO to protect, halt, or stop the progression of AD even when given at an advanced stage of the disease. In addition, our results suggest that OC-rich EVOO exerts such effects by reducing NLRP3 inflammasome activation and inducing autophagy through the AMPK–ULK1 pathway. Collectively, our findings support OC-rich EVOO supplementation as a medical food to ameliorate AD pathology.

METHODS

Materials.

Bovine serum albumin (BSA) and thioflavin-S (Thio-S) were purchased from Sigma-Aldrich (St. Louis, MO). Total protein measurement reagents with the bicinchoninic acid (BCA) method were obtained from Pierce (Rockford, IL). Mouse IL-1β ELISA kit was purchased from R&D Systems (Minneapolis, MN, USA). Mouse IL-10 ELISA kit was obtained from Mabtech (Nacka Strand, Sweden). Protein carbonyl colorimetric assay kit and SOD colorimetric assay kit were purchased from Cayman Chemical Company (Ann Arbor, MI, USA). All other chemicals and reagents were of analytical grade and were readily available from commercial sources.

Antibodies.

The antibodies used for Western blot and immunostaining are summarized in Table 1.

Table 1.

List of Antibodies Used in the Studya

antibody application source
BACE-1 (cat. no. ab108394) WB Abcam (Cambridge, MA)
Iba1 (cat. no. ab5076) WB, IHC Abcam
Alexa-fluor 488-labeled 6E10 (cat. no. 803013) IHC BioLegend (San Diego, CA)
mTOR (7C10) (cat. no. 2983) WB Cell signaling (Danvers, MA)
phospho-Mtor (Ser2448) (D9C2) (cat. no. 5536) WB Cell signaling
p70 S6 kinase (49D7) (cat. no. 2708) WB Cell signaling
phospho-p70 S6 kinase (Ser371) (cat. no. 9205) WB Cell signaling
4E-BP1 (53H11) (cat. no. 9644) WB Cell signaling
phospho-4E-BP1(Ser65) (D9G1Q) (cat. no. 2855) WB Cell signaling
AMPKα (D63G4) (cat. no. 5832) WB Cell signaling
phospho-AMPKα (Thr172) (cat. no. 2535) WB Cell signaling
ULK1 (cat. no. 8054) WB Cell signaling
phospho-ULK1 (Ser555) (cat. no. 5869) WB Cell signaling
beclin-1 (cat. no. 3495) WB Cell signaling
ATG-7 (cat. no. 8558) WB Cell signaling
ATG-5/12 (cat. no. 2011) WB Cell signaling
ATG-3 (cat. no. 3415) WB Cell signaling
SQSTM1/p62 (cat. no. 88588) WB Cell signaling
LC3-I (cat. no. 3868) WB Cell signaling
LC3-II (cat. no. 12741) WB Cell signaling
synapsin-1 (cat. no. 5297) WB Cell signaling
IKB-α (cat. no. 4814) WB Cell signaling
anti-collagen-IV (cat. no. AB756P) IHC EDM-Millipore (Burlington, MA)
APP-TOTAL (cat. no. MAB348) WB EDM-Millipore
TRPA-1 (cat. no. ABN1009) WB EDM-Millipore
SNAP-25 (cat. no. GTX113839) WB GeneTex (Irvin, CA)
PSD-95 (cat. no. GTX42033) WB GeneTex
sAPPα (cat. no. 11088) WB Immuno-Biological Laboratories (Minneapolis, MN)
sAPPβ (cat. no. 18957) WB Immuno-Biological
Laboratories
NLRP3 (cat. no. NBP212446SS) WB Novus Biologicals (Centennial, CO)
anti-goat HRP-labeled secondary (cat. no. HAF109) WB R&D systems (Minneapolis, MN)
β-actin (C4) (cat. no. sc-47778) WB Santa Cruz (Santa Cruz, CA)
GFAP (N18) (cat. no. sc-6171) IHC Santa Cruz
MMP9 (E-11) (cat. no. sc-393859) WB Santa Cruz
β-tubulin (5F131) (cat. no. sc-55529) WB Santa Cruz
caspase-1 (cat. no. sc-56036) WB Santa Cruz
cleaved caspase-1 (P20)
cleaved caspase-1 (P10)
caspase-8 (cat. no. sc-56070) WB Santa Cruz
cleaved caspase-8 (P18)
anti-rabbit IgG-CFL594 (cat. no. sc-516250) IHC Santa Cruz
anti-goat IgG-CFL594 (cat. no. sc-362275) IHC Santa Cruz
phospho-Tau (Thr231) (cat. no. 11110–1) WB Signalway antibody (College Park, MD)
Tau-Total (TAU-5) (cat. no. MS247P0) WB ThermoFisher (Waltham, MA)
anti-rabbit IgG (H+L) secondary antibody, HRP-labeled (cat. no. PI31460) WB ThermoFisher
anti-mouse IgG (H+L) secondary antibody, HRP-labeled (cat. no. PI31430) WB ThermoFisher
a

CFL594, CruzFluor 594; HRP, horseradish peroxidase; IHC, immunohistochemistry; WB, Western blot.

Animals.

TgSwDI mice were purchased from Jackson Laboratories (Bar Harbor, ME). TgSwDI mice express human APP under control of Thy 1.2 neuronal promoter harboring double Swedish mutations and the Dutch and Iowa vasculotropic Aβ mutations leading to early and aggressive Aβ accumulation associated with neuroinflammation, astrocyte activation, and memory decline.71 In the brains of TgSwDI mice, Aβ begins to accumulate at 2–3 months of age and deposits extensively at age of 12 months.71 In addition, learning and memory deficits have been detected in TgSwDI mice at 3, 9, and 12 months in the Barnes maze task.72 All mice were housed in plastic cages under standard conditions, 12-h light/dark cycle, 22 °C, 35% relative humidity, and ad libitum access to water and food. All animal experiments and procedures were approved by the Institutional Animal Care and Use Committee of the University of Louisiana at Monroe and according to the National Institutes of Health guidelines Principles of laboratory animal care (NIH publication No. 86–23, revised 1985).

Animal Treatment.

Mice, all females, were divided into two groups (n = 7 mice/group). The control group was fed with regular powdered diet (Teklad Laboratory diets, Harlan Laboratories, Madison, WI) enriched with Great-Value classic Olive Oil diet (Walmart, LA, USA). This oil was selected as one of the most common used types of olive oil, and unlike EVOO, it loses beneficial phenolic compounds during the refining process.31 The treatment group was fed with powdered diet enriched with oleocanthal-rich EVOO, namely, “The Governor” brand from Corfu Greece (http://thegovernor.gr/index.php/our-products/the-governor-premium). According to the producer’s Web site, this oil contains 680 mg/kg oleocanthal as determined by a validated NMR method.73 We confirmed the reported amount by our HPLC-DAD method developed based on previous reports.74 Besides oleocanthal and based on the producer’s Web site, the EVOO contains 315 mg/kg oleacein, 389 mg/kg hydroxytyrosol, 994 mg/kg tyrosol, and 72 mg/kg oleuropein aglycon. Olive oil (control) and OC-rich EVOO (EVOO) treatments were started at the age of 6 months for 3 months ending the experiment at the age of 9 months. Diets enriched with refined olive oil and EVOO were prepared using similar protocols to those we used previously by mixing oil with powdered diet to produce a dose of 0.714 (g/kg)/day, which contains 476 (μg/kg)/day oleocanthal in the EVOO group.27,29 This dose was selected to resemble the dose of EVOO in Mediterranean diet followers and based on the dietary intake of olive oil in the Greek population (50 g/day).75 Diet was changed every other day to maintain freshness. During the 3 months of treatment, mouse body weights were recorded weekly, and health status and normal behavior were checked daily. Because the diet was enriched with similar volumes of refined olive oil and EVOO, it is expected that both diets would provide comparable calories, which could also be concluded by the insignificant difference between the two groups in mouse body weights that ranged between 23.0±2.9 and 25.2±3.1 g. Five days before the end of the treatment period, all mice underwent Morris water maze task as described below. In addition, at the end of treatment and before euthanizing the mice to collect tissues, three mice from each group received an intravenous injection of 10 mg/kg FEPPA (N-acetyl-N-(2-[18F]fluoroethoxybenzyl)-2-phenoxy-5-pyridinamine, kindly provided by Dr. Pradeep Garg, Center for Molecular Imaging and Therapy, Biomedical Research Foundation, Shreveport, LA), a specific marker for activated microglia.50 One hour later, all mice were anesthetized with xylazine and ketamine (ip, 20 and 125 mg/kg, respectively) followed by decapitation. Mouse brains were extracted, and the two hemispheres of each brain were separated and used for analysis; blood samples were also collected for HPLC analysis of plasma FEPPA levels. All samples were stored at −80 °C until analysis.

Western Blot Analysis.

Protein samples (25 μg) of total brain homogenate were loaded and resolved using 10% SDS−polyacrylamide gel at 200 V for 1 h and transferred electrophoretically to PVDF membrane (Millipore) at 300 mA for 1.5 h at 4 °C. Nonspecific binding was blocked by preincubation of the PVDF membrane in PBS solution containing 3% BSA with rocking at room temperature for 1 h. Membranes were then incubated with primary antibodies overnight at 4 °C. Primary and secondary antibodies used for brain homogenate samples are listed in Table 1. Protein blots were developed using a chemiluminescence detection kit (SuperSignal West Femto substrate; ThermoFisher). Bands were visualized using ChemiDoc MP Imaging System (Bio-Rad Hercules, CA, USA) and quantified by densito-metric analysis using Image Lab Software V.6.0 (Bio-Rad).

Immunohistochemical Analysis.

Brain sections 16 μm thick were prepared using Leica CM3050S Research Cryostat (Buffalo Grove, IL, USA). Sections were fixed by incubation in methanol for 10 min at −20 °C. Sections were then washed 5 times in PBS and blocked with PBS containing 10% donkey serum for 1 h at room temperature. Immunostaining was performed for brain hippocampi and cortices of TgSwDI mice. The entire cortex and hippocampus regions were included in the analysis spanning the dentate gyrus and CA1–CA3 regions. To assess IgG extravasation, brain sections were probed by dual immunohistochemical staining for collagen IV and mouse IgG using rabbit anti-collagen-IV and fluorescein-conjugated donkey anti-IgG (both at 1:200 dilution), respectively. The secondary antibody used for collagen-IV was CFL594-conjugated donkey anti-rabbit IgG. For total Aβ detection, TgSwDI brain sections were immunostained with Alexa-fluor 488 labeled 6E10 human-specific anti-Aβ antibody at 1:200 dilution. For detection of Aβ-plaque load in hippocampus, the brain tissue sections were stained with a freshly prepared and filtered 0.02% Thio-S solution in 70% ethanol for 30 min followed by incubation in 70% ethanol. For reactive astrocytes, TgSwDI sections were probed with rabbit anti-GFAP polyclonal IgG at 1:100 dilution followed by anti-rabbit IgG−CFL594 secondary antibody. For reactive microglia, TgSwDI sections were probed with goat anti-Iba1 polyclonal IgG at 1:100 dilution followed by anti-goat IgG−CFL594 secondary antibody for each treatment; image acquisition was performed in 4 tissue sections spanning the hippocampus, each separated by 150 μm (total of approximately 40 sections per mouse). Total Aβ load and Thio-S were captured and quantified at a total magnification of 4×; GFAP, Iba1, and IgG extravasation images were captured at a total magnification of 20×. Quantification of fluorescence intensity was performed using ImageJ version 1.6.0 software (Research Services Branch, National Institute of Mental Health, National Institutes of Health, Bethesda, MD) after adjusting for threshold. All images were visualized using Nikon Eclipse Ti−S inverted fluorescence microscope (Melville, NY).

Determination of Oxidative Stress and Neuroinflammation Markers.

Brain homogenates were centrifuged for 15 min at 14000 rpm, and the supernatants were used to assay protein carbonyl, SOD, IL-1β, and IL-10 levels following the manufacturers’ instructions. All samples were run at least in duplicate and corrected to the total protein amount in each sample using BCA assay. In addition, the quantification of FEPPA in brains and plasma samples collected from the three mice receiving the injections was conducted using isocratic Shimadzu LC-20AB HPLC equipped with a Shimadzu SIL-20A HT autosampler and LC-20AB pump connected to a DGU-20A3 degasser (Shimadzu, OR) using the following separation method. Briefly, an acetonitrile/water (45:50 v/v) mobile phase was used for the separation of brain and blood samples and was delivered at 1.0 mL/min flow rate. The separation was performed at room temperature using an Agilent eclipse XDB-C18 column (5 μm, 4.6 × 150 mm2 ID; Agilent Technologies Inc., CA, USA). The wavelength was set at 210 nm, and the injection volume was 20 μL. Each chromatographic run was completed in 10 min with FEPPA eluted at a retention time of 6.8 min. One hour later, FEPPA was detected in the brains but not in the in the plasma (Supporting Information, Figure S2).

Morris Water Maze Testing.

The Morris water maze (MWM) test was performed for TgSwDI mice to assess learning and memory at the end of the treatment using protocols similar to those described previously with modification.76,77 All mice underwent training 3 times a day for 4 consecutive days. The platform was kept in the same quadrant during the entire course of the experiment. Mice were required to find the hidden platform utilizing the distal spatial cues available in the room. Conditions were kept the same during all the experiments. An overhead camera connected to a computerized tracking system (SMART 3.0 Platform, Panlab Harvard apparatus; Holliston, MA) was used to record the movements of the mice. The parameters latency and swimming distance were calculated and analyzed for treatment effect on the fourth day of training to compare performance using statistical analysis.

Statistical Analysis.

Data are presented as box-and-whisker plots representing median and interquartile range (IQR) with minimum and maximum values of n = 7 mice/group. FEPPA data was presented as mean + SEM for n = 3 mice/group. The experimental results were statistically analyzed for significant difference using Student’s t test for two groups. Values of P < 0.05 were considered statistically significant. Data analyses were performed using GraphPad Prism, version 6.0. In the figures, the fold change for control groups was calculated by dividing each control value on the average of all control mice values for a specific protein. Treatment values were then normalized to control (1.0).

Supplementary Material

Supp M

ACKNOWLEDGMENTS

This research work was funded by the National Institute of Neurological Disorders and Stroke (NIH/NINDS) under Grant Number R15NS091934 (A.K.) and Center for Molecular Imaging and Therapy (CMIT). We thank Pradeep Garg and Sudha Garg from CMIT for providing FEPPA compound and Olive Fabrica Spyros and George Dafnis (Corfu, Greece) for providing us with “The Governor” extra-virgin olive oil to perform the studies.

Footnotes

Supporting Information

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acschemneuro.9b00175.

Effect of EVOO on mTOR pathway and FEPPA analysis by HPLC (PDF)

The authors declare the following competing financial interest(s): The corresponding author, Amal Kaddoumi, is a Chief Scientific Officer without compensation in the Shreveport, Louisiana, based Oleolive, LLC.

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