Oleocanthal-Rich Extra-Virgin Olive Oil Restores the Blood−BrainBarrier Function through NLRP3 Inflammasome InhibitionSimultaneously with Autophagy Induction in TgSwDI MiceSweilem B. Al Rihani,†,‡ Lucy I. Darakjian,† and Amal Kaddoumi*,†,‡

†Department of Drug Discovery and Development, Harrison School of Pharmacy, Pharmacy Research Building, Auburn University,Auburn, Alabama 36849, United States‡Center for Neuroscience Initiative, Auburn University, Auburn, Alabama 36849, United States

*S Supporting Information

ABSTRACT: Alzheimer’s disease (AD) is a complex neurodegenerative disordercharacterized 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 pathwayswould be essential to prevent, halt, or treat the disease. A growing body of evidenceincluding our studies supports a protective effect of oleocanthal (OC) and extra-virginolive oil (EVOO) at early AD stages before the onset of pathology. In addition, wereported previously that OC and EVOO exhibited such effect by restoring the BBBfunction; however, the mechanism(s) by which OC and EVOO exert such an effect andwhether this effect extends to a later stage of AD remain unknown. In this work, wesought first to test the effect of OC-rich EVOO consumption at an advanced stage of thedisease in TgSwDI mice, an AD mouse model, starting at the age of 6 months for 3months treatment, and then to elucidate the mechanism(s) by which OC-rich EVOOexerts the observed beneficial effect. Overall findings demonstrated that OC-rich EVOOrestored the BBB function and reduced AD-associated pathology by reducing neuroinflammation through inhibition ofNACHT, LRR, and PYD domain-containing protein 3 (NLRP3) inflammasome and inducing autophagy through activation ofAMP-activated protein kinase (AMPK)/Unc-51-like autophagy activating kinase 1 (ULK1) pathway. Thus, dietsupplementation 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

■ INTRODUCTION

Alzheimer’s disease (AD) is the most common neuro-degenerative disease and form of dementia and accounts for60−80% of all cases worldwide.1 Unfortunately, currently thereare no disease modifying treatments for AD. The pathology ofthis devastating neurodegenerative disease is complex. Whilethe cause of AD is not clear, the cardinal features commonlyobserved in the brains of AD patients are the deposition ofamyloid-β (Aβ) plaques, aggregation of hyperphosphorylatedtau protein, abnormal neurites, neuropil threads, synapsis andneuronal loss, astrogliosis, microglial activation, cerebralamyloid angiopathy (CAA), and a disrupted blood−brainbarrier (BBB).2−7

Current evidence suggests that aging, cerebrovasculardisruption, or Aβ or tau accumulation can induce BBBdysfunction by altering the neurovascular unit.8,9 Disruption ofthe BBB results in neuronal damage and reduced Aβ clearance,which would further accumulate Aβ and BBB dysfunctionleading to AD progression. Therefore, to maintain healthy BBBfunction or restore BBB function provides a novel strategy toprevent or treat AD.

A primary risk factor for AD is advanced age.10 While agingitself is not a disease, it is usually accompanied by a low degreeof chronic inflammation.11 Therefore, the term inflamm-agingwas introduced, which involves a number of factors andprocesses characterized by complex interactions of severalmolecular mediators such as inflammatory markers.11,12

Examples of such markers include C-reactive protein andcytokines.11,12 In AD, cerebral neuroinflammation contributesto the disease pathogenesis. Growing evidence has demon-strated increased interleukin-1β (IL-1β) levels, activation ofNACHT, LRR, and PYD domain-containing protein 3(NLRP3) inflammasome in microglia, and subsequentinflammatory events as downstream consequences of Aβdeposition.13,14 The continuous presence of stimulus in the ADbrain, such as Aβ, has been recently reported to activateNLRP3 and promote formation of the inflammasome complex,which results in the activation of caspase-1 and production ofthe pro-inflammatory cytokines IL-1β and IL-18.15,16 These

Received: March 23, 2019Accepted: June 12, 2019

Research Article

pubs.acs.org/chemneuroCite This: ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

© XXXX American Chemical Society A DOI: 10.1021/acschemneuro.9b00175ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

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findings suggest that the NLRP3 inflammasome is animportant contributor to cerebral neuroinflammation in theAD brain, and its inhibition could provide a therapeuticstrategy to prevent, halt or treat AD. In addition, considerableevidence suggests that the dysregulation of autophagy, anessential homeostatic pathway, occurs in the brains of ADanimal models and AD patients.17−19 Autophagy plays animportant role in the clearance of Aβ,20 and the clearance ofAPP and tau.21−23 Thus, autophagy dysregulation could play akey role in the pathology of AD by enhancing theaccumulation of Aβ, APP, and tau. Collectively, there is asubstantial interest in the discovery of therapeutic inflamma-some and autophagy modulators.Currently, there is sufficient scientific evidence supporting

that chronic exposure to the Mediterranean diet (MedD) islinked to low risk of cognitive impairment and AD.24 Amongthe key elements of the MedD is the daily consumption ofextra-virgin olive oil (EVOO), which provides anti-inflamma-tory and antioxidant effects,25 in addition to other healthbenefits.26 Data from our group have shown that EVOO addedto the diet of AD model mice at early age and before thepathology onset was able to protect mice from Aβ-relatedpathology by restoring the BBB function.27,28 EVOOconsumption for 6 months starting at one month of agesignificantly enhanced BBB function, reduced parenchymal Aβload, reduced Aβ production, enhanced Aβ clearance, anddecreased vascular deposits of Aβ and total tau and itsphosphorylation in the brains of the AD model TgSwDImice.29 In addition, available studies have shown that EVOOadministration to triple transgenic mice (3xTg) as model ofAD, improved memory function, reduced synaptic toxicity, andsignificantly reduced Aβ deposition by autophagy activation.30

In addition, EVOO increased antioxidants levels, such asglutathione, in mouse brains.25 EVOO contains severalphenolic compounds, among which is oleocanthal (OC).When tested in mice, OC restored the BBB function, reducedAβ brain levels, and reduced inflammation as evident byreduced astrogliosis and brain levels of cytokines.27,28,31,32

These results suggest that OC could largely contributed toEVOO observed and reported benefits. Besides OC, EVOOcontains hydroxyoleocanthal (also known as oleacein) andoleuropein aglycone; while studies reporting hydroxyoleocan-thal activity are lacking, several studies demonstrated thebeneficial effect of oleuropein aglycone against Aβ relatedpathology when added to mouse diet at 50 mg/kg.33

The potential beneficial effect of OC and EVOO on AD indisease models, especially at the very early stages, have beenstudied previously by us and others.29,30,32 However, themechanism(s) by which OC and EVOO rectified the BBBfunction, and exerted the reported beneficial effect remainsunclear; also, whether EVOO effectiveness against AD extendsto late stages is unknown. Thus, the purpose of this study wasto evaluate the beneficial effect and associated mechanism(s)of OC-rich EVOO consumption at an advanced stage of thedisease in TgSwDI mice starting at the age of 6 months for 3months treatment.

■ RESULTSOC-Rich EVOO Improved BBB Tightness in TgSwDI

Mouse Brains. Effect of OC-rich EVOO (hereafter EVOO)on BBB leakiness was evaluated by immunostaining ofimmunoglobulin-G (IgG) extravasation, which was used asan endogenous BBB permeability marker. As shown in Figure

1, EVOO consumption significantly reduced IgG extravasationby 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 previousresults.27,29

EVOO Reduces Total Aβ Load and Plaques in TgSwDIMouse Brains. As shown in Figure 2, compared to controlgroup, EVOO significantly reduced total 6E10-detected Aβ by61% and 73% in brain cortex and hippocampus, respectively(Figure 2A). In addition, thioflavin-S positive Aβ plaquesstaining showed similar pattern with an overall 47% and 79%reduction in cortex and hippocampus regions, respectively(Figure 2B).

Figure 1. Representative brain sections stained with anti-mouse IgGantibody to detect IgG extravasation (green) and anti-collagenantibody (red) in (A) brain cortex and (B) hippocampus. (C) IgGoptical density in mouse cortexes and hippocampi were quantified forIgG extravasation. Data are presented as box-and-whisker plotsrepresenting median and interquartile range (IQR), with minimumand maximum values. Statistical analysis was determined by Student’st test for n = 7 mice/group. **P < 0.01, ***P < 0.001 versus controlgroup. Scale bar, 100 μm.

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EVOO Modulates APP Processing, Total Tau, and ItsPhosphorylation 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

Figure 2. (A) Representative cortex and hippocampus sections stained with 6E10 (green) antibody against Aβ with corresponding quantificationfor 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 areacovered with Aβ plaques; anti-collagen IV (red) was used to stain microvessels. Data are presented as box-and-whisker plots representing medianand IQR, with minimum and maximum values. Statistical analysis was determined by Student’s t test for n = 7 mice/group. ***P < 0.001 versuscontrol group. Scale bar, 100 μm.

Figure 3. EVOO consumption effect on APP processing, tau, and tau phosphorylation at amino acid residue threonine 231 in TgSwDI mousebrains. 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 asbox-and-whisker plots representing median and IQR, with minimum and maximum values. Statistical analysis was determined by Student’s t test ofn = 7 mice/group. **P < 0.01 versus control group.

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EVOO effect on the expression of tau protein and itsphosphorylation in TgSwDI mice with advanced diseasepathology at 9 months by Western blot. A significant reductionin total tau by 48% and tau phosphorylation at threonineposition 231 (p-Tau-Thr231) by 38% was observed.EVOO Attenuates Brain Neuroinflammation. Astrocyte

inflammatory activation is recognized by increased levels ofglial fibrillary acidic protein (GFAP) with elongated shape andthick branches.34 As shown in Figure 4A, in comparison to thecontrol group, EVOO significantly reduced astrocyte activationand ameliorated the astrocyte shape in mouse cortexes andhippocampi. Moreover, EVOO significantly reduced brainlevels of the microglial marker ionized calcium binding adaptormolecule 1 (Iba1) as analyzed by immunostaining (Figure4C), which was further confirmed by immunoblottingdemonstrating a 43% reduction in Iba1 (Figure 4D). Reducedmicroglial activation was also associated with 40% reduction inFEPPA levels, a specific marker for activated microglia, as

determined by HPLC analysis, in the brains of mice consumingEVOO when compared to the control group (Figure 4B). Inaddition, Western blot analysis of metalloproteinase-9(MMP9) enzyme showed EVOO to significantly reduceMMP9 by 57% compared to control group (Figure 4D).Transient receptor potential ankyrin 1 (TRPA1) is a

membrane-associated cation channel that is widely expressedin neuronal and non-neuronal cells.35−37 TRPA1 activation hasbeen linked with inflammation.35−37 Here and as shown in(Figure 4D), TRPA-1 expression was significantly reduced byEVOO 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 reducedIL-1β brain levels by 75% and increased IL-10 levels by 31%.

EVOO Reduces Oxidative Stress Markers in TgSwDIMouse Brains. Carbonyl formation is an important detectablemarker of protein oxidation, and protein carbonyls aresignificantly increased in AD;38 therefore we evaluated the

Figure 4. (A) Representative brain sections from 9-month-old TgSwDI mice were colabeled with GFAP antibody (red) to detect activatedastrocytes and thioflavin-S (green) to detect Aβ plaques in control and EVOO groups. Hippocampus is seen at higher magnification. Activatedastrocytes in cortex are shown with long and thick branches in control group that were significantly reduced by EVOO as demonstrated at highermagnification in the closed inserts. Scale bar, 100 μm. (B) Results from HPLC analysis of FEPPA levels, a specific marker for microglial activationin 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 6E10antibody (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 onIL-10 levels in mouse brain homogenates. For panels D−F, data are presented as box-and-whisker plots representing median and IQR, withminimum and maximum values (n = 7 mice/group). For panel B, data is presented as mean + SEM for n = 3 mice/group. Statistical analysis wasdetermined by Student’s t test. **P < 0.01, and ***P < 0.001 versus control group.

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effect of EVOO on total protein carbonyl formation. EVOOconsumption significantly reduced protein carbonyl levels by64% (Figure 5A). In addition, the effect of EVOOconsumption on superoxide dismutase (SOD) brain levelswas evaluated. SOD is an enzyme that acts as an antioxidantthat catalyzes the dismutation of superoxide anion free radical

into molecular oxygen and hydrogen peroxide, which isdecreased in AD suggesting oxidative stress.39 EVOOsignificantly increased SOD formation by 79% compared tocontrol group (Figure 5B).

EVOO Reduces IL-1β Production through the NLRP3Pathway in TgSwDI Mouse Brains. The IL-1β upstream

Figure 5. (A) Effect of EVOO on protein carbonyl levels in mouse brain homogenates. (B) Effect of EVOO on SOD levels in mouse brainhomogenates. Data are presented as box-and-whisker plots representing median and IQR, with minimum and maximum values of n = 7 mice ineach group. Statistical analysis was determined by Student’s t test. *P < 0.05 and ***P < 0.001 versus control group.

Figure 6. EVOO reduced inflammasome formation by modulating NLRP-3/caspase-1 and -8 pathway. Representative blots and densitometryanalysis 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 arepresented as box-and-whisker plots representing median and IQR, with minimum and maximum values of n = 7 mice in each group. Statisticalanalysis was determined by Student’s t test. **P < 0.01, and ***P < 0.001 versus control group.

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 IIin brain homogenates of mice that consumed EVOO compared to control group. Data are presented as box-and-whisker plots representing medianand IQR, with minimum and maximum values of n = 7 mice in each group. Statistical analysis was determined by Student’s t test. ns = notsignificant, *P < 0.05, **P < 0.01, and ***P < 0.001 versus control group.

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inflammasome activation and production pathway wasevaluated. Findings revealed that EVOO significantly reducedNLRP3 protein expression by 41%, which was also associatedwith a significant reduction in the expression levels ofprocaspase-1 and cleaved caspase-1 p20 and p10 by 31%,45%, and 28%, respectively. In addition, this effect was alsoassociated with a significant reduction in procaspase-8 by 56%and cleaved caspase-8 p18 by 60% when compared to controldiet (Figure 6).EVOO Induces Autophagy in TgSwDI Mouse Brains.

Recent studies established that autophagy induction plays akey role in the regulation of BACE1 turnover and APPprocessing and that the autophagy process is downregulatedwith aging.40 Several autophagy markers affecting either theinitiation or the maturation of autophagosomes and autophagicflux were analyzed in this study. As shown in Figure 7, EVOOsignificantly induced major autophagosome initiation proteinsAMP-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 theincrease in beclin-1 levels did not reach a significant level, asignificant increase in ATG7 and ATG3 protein levels by 2.4-

and 1.7-fold, respectively, was observed without significantalteration of ATG-12/5 level (Figure 7). In addition, themicrotubule-associated protein light chain 3 conversion (LC3-I) and the ubiquitin-binding protein p62 were significantlyreduced by 22% and 37%, respectively, which was associatedwith 1.6-fold increase in the level of LC3-II (Figure 7). On theother hand, EVOO consumption has no significant effect onthe mTOR pathway or its downstream proteins P70S6K and4E-BP1 and their phosphorylation (Supporting Information,Figure S1).

Effect of EVOO on Neurosynaptic Markers and MorrisWater Maze Test Performance. As shown in Figure 8,Western blot findings demonstrated EVOO significantlyincreased 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 effecton learning and memory on the fourth day of training. Thefollowing parameters were analyzed: the time a mouse takes tofind the platform (latency, s), swimming distance (cm), andswimming speed (cm/s). As shown in Figure 8B−E, EVOOfed mice found the platform faster than vehicle-treated mice

Figure 8. (A) Representative blots and densitometry analysis of the neurosynaptic markers SNAP-25, synapsin-1, and PSD-95 in brainhomogenates 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 distancecompared 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.

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(15.5 ± 3.1 vs 43.9 ± 6.0 s, respectively, P < 0.01), and EVOOdiet significantly reduced mouse swimming distance to 476.4 ±97.8 cm from 1124 ± 135.7 cm for the control group. Theseresults suggest that EVOO improved MWM performance.There was no difference between the two groups in swimmingspeed, thus excluding motor changes as a cause of the observedimprovement.

■ DISCUSSIONRecently, we have reported that OC and EVOO improvedBBB integrity and function, increased endothelial cell tightness,upregulated Aβ major transport proteins localized at theendothelial cells of the BBB, and enhanced clearance of Aβacross the BBB.27−29,32 The in vivo studies were performed intwo mouse models, namely, TgSwDI and 5xFAD mice, used atthe age of one month and before the disease starts as aprevention mode.27−29,32 In the current study, we evaluatedthe effect of 3 months consumption of OC-rich EVOO addedto the mouse diet, on the BBB function, Aβ-related pathology,and MWM performance in TgSwDI mice at an older age withadvanced pathology and investigated potential mechanisms bywhich OC-rich EVOO rectified BBB function and reduced Aβrelated pathology. To the best of our knowledge, findings ofthis study demonstrated for the first time that the long-termdietary supplementation with OC-rich EVOO significantlyreduced inflammasome activation through NLRP3 inhibitionand increased autophagy through AMPK−ULK1 pathwayactivation when compared to mice consuming a refined oliveoil-enriched diet. Reduced neuroinflammation and inducedautophagy by EVOO could explain, at least in part, theimproved BBB function as determined by the significantreduction in IgG extravasation in mouse cortexes andhippocampi. This effect was concomitant to reduced Aβ-loadand associated synaptotoxicity, reduced tau hyperphosphor-ylation, and reduced oxidative stress biomarkers in TgSwDImice with extensive disease pathology, which collectivelyenhanced learning.During aging and in AD, the BBB function has been

reported to deteriorate.8,41,42 Neuroinflammation and autoph-agy dysregulation in aging and AD,19,40,43 have also been linkedto BBB disruption.44,45 Thus, their simultaneous restorationcould provide an effective approach for functional BBB, healthyaging, and reduced risk of AD or halt in AD progression.NLRP3 inflammasome activation in microglia and astrocytes

has been associated with numerous chronic inflammatorydiseases. NLRP3 can detect inflammatory and Aβ aggre-gates.13,46 The activation of NLRP3 inflammasome by Aβenhances AD progression by facilitating chronic inflammatoryresponses,13 which are partly involved in restricting glialfunction and mediating synaptic dysfunction and cognitiveimpairment.13 In addition, available studies reported thatNLRP3 inflammasome activation associated with IL-1β releasecontributed to BBB damage and increased BBB permeabilityand that the knockout of NLRP3 in mice with ischemic strokereduced BBB damage.47 Therefore, blocking NLRP3 inflam-masome activity could effectively interfere with the progressionof AD.13,46 NLRP3 inflammasome processes pro-IL-1 tomature and functional IL-1β. In TgSwDI mice, EVOOconsumption significantly reduced IL-1β levels in mousebrains compared to that in mice treated with refined oliveoil. This effect was accompanied by a significant reduction inthe expression of two major caspases responsible for theincreased formation of IL-1β, namely, caspase-148 and caspase-

8.49 In addition, consequent to NLRP3-inflammasomeinhibition by EVOO, a significant reduction in astrogliosis,measured by GFAP, and microglial activation, measured byIba1 and FEPPA, was also observed. FEPPA is a specific ligandfor the translocator protein tryptophan-rich sensory protein(TSPO), which is used as a biomarker to assess glial activationand neuroinflammation by positron emission tomography.50

TSPO is located in the outer mitochondrial membrane ofmicroglia; its expression increases in response to microglialactivation, and thus it is considered a valid biomarker forneuroinflammation.51 Our results from FEPPA study demon-strated that EVOO significantly reduced TSPO, which suggestsreduced inflammation. A recent study reported a link betweenNLRP3 inflammasome activation and TSPO upregulation,which is implicated in mitochondrial dysfunction,52 suggestingan additional therapeutic target for EVOO.Moreover, calcium signaling has been identified as a critical

mediator pathway in NLRP3 inflammasome activation whereincreased levels of intracellular calcium were associated withNLRP3 inflammasome activation.53 In the brain, TRPA1receptor is expressed in primary sensory neurons and in non-neuronal cells mainly in astrocytes.35,37 TRPA1 plays a key rolein pain and inflammation and is recognized as a gatekeeper ofinflammation.36 It acts as a sensor for detecting several toxicsignals such as reactive oxygen species, inflammatory cytokines,and Aβ, upon detection of which, TRPA1 is activated. In AD, ithas been shown that in the brains of APP/PS1 mice, Aβ-mediated chronic inflammation caused TRPA1 activation,which triggered calcium influx and inflammation in astro-cytes.54 Our results demonstrated that EVOO significantlyreduced TRPA1 expression, which is the molecular target ofOC,55 suggesting another mechanism by which EVOO wasable to reduce neuroinflammation. While additional studies arerequired to understand the link between TRPA1 and NLRP3inflammasome activation, a very recent study reported thatcigarette smoke-induced TRPA1 was associated with increasedNLRP3 inflammasome activation and ROS production in thealveolar epithelial cells A549, and this effect was calciummediated.56 These results suggest that OC-rich EVOO reducedinflammation directly by inhibition of NLRP3 inflammasomeactivation or indirectly by TRPA1 down-regulation thattriggered NLRP3 inflammasome inhibition and reduced IL-1β production. Collectively, our findings support TRPA1 andNLRP3−IL-1β axis as targets for the overall beneficial effect ofEVOO.Chronic inflammasome activation and neuroinflammation

associated with prolonged accumulation of Aβ and tauhyperphosphorylation could damage autophagy.57 Autophagydysregulation impairs effective elimination of aggregates anddamaged mitochondria leading to their accumulation asso-ciated with toxicity and oxidative stress.57 In this study, the 3-months consumption of EVOO significantly increased severalautophagy markers that regulate autophagy induction andmaturation in the brains of TgSwDI mice. Our findingsconfirm an earlier observation for EVOO effect on inducingautophagy where its chronic administration significantlyincreased levels of the autophagy activation biomarkersATG5 and ATG7 in the brains of 3xTg mice.30 In an effortto expand on the mechanism by which EVOO inducedautophagy, AMPK and mTOR pathways were evaluated. Basedon the results, EVOO induced autophagy through AMPKpathway activation but not through mTOR pathway inhibition.Besides acting as a positive regulator of autophagy, through

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ULK1 phosphorylation, AMPK has multiple functionsincluding general regulation of cellular metabolism, NLRP3inflammasome regulation, and mitochonrdrial function.58 Inaddition, several recent studies have shown that autophagyinduction by AMPK can reduce the levels of Aβ load in AD59

and α-synuclein in Parkinson’s disease60 and amelioratesHuntington’s disease pathology and prevents neurodegenera-tion.61 A recent study examined post-mortem brains of ADpatients and showed the possible relation between autophagyand the upregulation of NLRP3 in response to Aβaggregation.62 The autophagy markers SQSTM1/p62 andLC3 were abnormally accumulated and colocalized with Aβdeposits and hyperphosphorylated tau in the brains of ADpatients.62 Autophagy induction, thus, has been proposed as astrategy to rectify the pathology of numerous diseases bypromoting clearance of aggregated and hyperphosphorylatedproteins in the cytoplasm.57 Our immunostaining analysisdemonstrated fewer Aβ plaques and lower Aβ levels in thebrains of TgSwDI mice consuming EVOO compared to micefed with refined olive oil. This significant reduction in Aβ loadcould be related to autophagy induction through AMPKpathway.Besides autophagy, another significant outcome that could

be caused by neuroinflammation inhibition,63 is reduced levelsof BACE1 in EVOO mouse brains, which could also contributeto sAPPβ reduced levels and thus brain Aβ load. The levels of

sAPPα, on the other hand, were significantly increased; sAPPαpossesses neurotrophic and neuroprotective activities,64 whichcould contribute to the enhanced learning and memoryobserved in TgSwDI mice consuming EVOO. In addition,decreased brain Aβ deposits contributed to the increasedproduction of IL-10, which is consistent with previousreports,55 and reduced MMP9 and oxidative stress. Withinthis scenario, interestingly, the 3-months consumption ofEVOO significantly reduced full-APP and tau levels in mousebrain homogenates suggesting the re-establishment of a properprocessing of both proteins. While additional studies arerequired to explain this observation, available studies in mousemodels have already shown that autophagy−lysosomaldegradation of APP and Aβ21 and tau and hyperphosphory-lated tau22 reduce AD. Furthermore, a recent study reportedcorrelation between autophagy induction and reduced tau-protein expression in a mouse model of tauopathy.65 TgSwDImouse model does not overexpress tau, and studies evaluatingthe development of tau pathology in this model with aging arenot available. However, evidence exists that suggest Aβ and taupathology to reciprocally drive each other.66 In addition,Roberson et al.67 reported tau depletion in hAPP mouse modelthat was associated with revoked learning and memoryimpairment. Also, Yoshikawa et al., recently reported that thedepletion of tau protein ameliorated the hyper-locomotorbehavior in J20 transgenic mice, an APP model, which signifies

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 AbcamAlexa-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 signalingphospho-4E-BP1(Ser65) (D9G1Q)(cat. no. 2855)

WB Cell signaling

AMPKα (D63G4) (cat. no. 5832) WB Cell signalingphospho-AMPKα (Thr172) (cat.no. 2535)

WB Cell signaling

ULK1 (cat. no. 8054) WB Cell signalingphospho-ULK1 (Ser555) (cat. no.5869)

WB Cell signaling

beclin-1 (cat. no. 3495) WB Cell signalingATG-7 (cat. no. 8558) WB Cell signalingATG-5/12 (cat. no. 2011) WB Cell signalingATG-3 (cat. no. 3415) WB Cell signalingSQSTM1/p62 (cat. no. 88588) WB Cell signalingLC3-I (cat. no. 3868) WB Cell signalingLC3-II (cat. no. 12741) WB Cell signalingsynapsin-1 (cat. no. 5297) WB Cell signalingIKB-α (cat. no. 4814) WB Cell signalinganti-collagen-IV (cat. no. AB756P) IHC EDM-Millipore

(Burlington, MA)APP-TOTAL (cat. no. MAB348) WB EDM-MilliporeTRPA-1 (cat. no. ABN1009) WB EDM-MilliporeSNAP-25 (cat. no. GTX113839) WB GeneTex (Irvin, CA)

antibody application source

PSD-95 (cat. no. GTX42033) WB GeneTexsAPPα (cat. no. 11088) WB Immuno-Biological

Laboratories(Minneapolis, MN)

sAPPβ (cat. no. 18957) WB Immuno-BiologicalLaboratories

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 CruzMMP9 (E-11) (cat. no. sc-393859) WB Santa Cruzβ-tubulin (5F131) (cat. no. sc-55529)

WB Santa Cruz

caspase-1 (cat. no. sc-56036) WB Santa Cruzcleaved caspase-1 (P20)cleaved caspase-1 (P10)caspase-8 (cat. no. sc-56070) WB Santa Cruzcleaved 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) secondaryantibody, HRP-labeled (cat. no.PI31460)

WB ThermoFisher

anti-mouse IgG (H+L) secondaryantibody, HRP-labeled (cat. no.PI31430)

WB ThermoFisher

aCFL594, CruzFluor 594; HRP, horseradish peroxidase; IHC,immunohistochemistry; WB, Western blot.

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the functional outcome of Aβ/tau interaction.68 Similarly, inour study, EVOO significantly reduced tau, which is consistentwith our previous findings with EVOO;29 however, it could benecessary to evaluate the effect of EVOO consumption onhAPP and tau mRNA levels to explain the observed reductionand confirm this effect is mediated by induced autophagy.Several studies linked Aβ toxicity,69 tau aggregation,70 and

memory impairment to neuroinflammation and impairedautophagy.17,19,69,70 Thus, reduced neuroinflammation andinduced autophagy associated with reduced Aβ-relatedpathology and oxidative stress by EVOO could explain, atleast in part, the enhanced BBB function. Functional BBB isvital to limit brain access of unwanted molecules, asdemonstrated 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 effectsof OC-rich EVOO to protect, halt, or stop the progression ofAD even when given at an advanced stage of the disease. Inaddition, our results suggest that OC-rich EVOO exerts sucheffects by reducing NLRP3 inflammasome activation andinducing autophagy through the AMPK−ULK1 pathway.Collectively, our findings support OC-rich EVOO supplemen-tation as a medical food to ameliorate AD pathology.

■ METHODSMaterials. Bovine serum albumin (BSA) and thioflavin-S (Thio-S)

were purchased from Sigma-Aldrich (St. Louis, MO). Total proteinmeasurement reagents with the bicinchoninic acid (BCA) methodwere obtained from Pierce (Rockford, IL). Mouse IL-1β ELISA kitwas purchased from R&D Systems (Minneapolis, MN, USA). MouseIL-10 ELISA kit was obtained from Mabtech (Nacka Strand,Sweden). Protein carbonyl colorimetric assay kit and SODcolorimetric assay kit were purchased from Cayman ChemicalCompany (Ann Arbor, MI, USA). All other chemicals and reagentswere of analytical grade and were readily available from commercialsources.Antibodies. The antibodies used for Western blot and

immunostaining are summarized in Table 1.Animals. TgSwDI mice were purchased from Jackson Laboratories

(Bar Harbor, ME). TgSwDI mice express human APP under controlof Thy 1.2 neuronal promoter harboring double Swedish mutationsand the Dutch and Iowa vasculotropic Aβ mutations leading to earlyand aggressive Aβ accumulation associated with neuroinflammation,astrocyte activation, and memory decline.71 In the brains of TgSwDImice, Aβ begins to accumulate at 2−3 months of age and depositsextensively at age of 12 months.71 In addition, learning and memorydeficits have been detected in TgSwDI mice at 3, 9, and 12 months inthe Barnes maze task.72 All mice were housed in plastic cages understandard conditions, 12-h light/dark cycle, 22 °C, 35% relativehumidity, and ad libitum access to water and food. All animalexperiments and procedures were approved by the InstitutionalAnimal Care and Use Committee of the University of Louisiana atMonroe and according to the National Institutes of Health guidelinesPrinciples 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 regularpowdered 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 mostcommon used types of olive oil, and unlike EVOO, it loses beneficialphenolic compounds during the refining process.31 The treatmentgroup was fed with powdered diet enriched with oleocanthal-richEVOO, namely, “The Governor” brand from Corfu Greece (http://

thegovernor.gr/index.php/our-products/the-governor-premium). Ac-cording to the producer’s Web site, this oil contains 680 mg/kgoleocanthal as determined by a validated NMR method.73 Weconfirmed the reported amount by our HPLC-DAD methoddeveloped based on previous reports.74 Besides oleocanthal andbased on the producer’s Web site, the EVOO contains 315 mg/kgoleacein, 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 monthsending the experiment at the age of 9 months. Diets enriched withrefined olive oil and EVOO were prepared using similar protocols tothose we used previously by mixing oil with powdered diet to producea dose of 0.714 (g/kg)/day, which contains 476 (μg/kg)/dayoleocanthal in the EVOO group.27,29 This dose was selected toresemble the dose of EVOO in Mediterranean diet followers andbased 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 wererecorded weekly, and health status and normal behavior were checkeddaily. Because the diet was enriched with similar volumes of refinedolive oil and EVOO, it is expected that both diets would providecomparable calories, which could also be concluded by theinsignificant difference between the two groups in mouse bodyweights that ranged between 23.0 ± 2.9 and 25.2 ± 3.1 g. Five daysbefore the end of the treatment period, all mice underwent Morriswater maze task as described below. In addition, at the end oftreatment and before euthanizing the mice to collect tissues, threemice from each group received an intravenous injection of 10 mg/kgFEPPA (N-acetyl-N-(2-[18F]fluoroethoxybenzyl)-2-phenoxy-5-pyri-dinamine, kindly provided by Dr. Pradeep Garg, Center for MolecularImaging and Therapy, Biomedical Research Foundation, Shreveport,LA), a specific marker for activated microglia.50 One hour later, allmice were anesthetized with xylazine and ketamine (ip, 20 and 125mg/kg, respectively) followed by decapitation. Mouse brains wereextracted, and the two hemispheres of each brain were separated andused for analysis; blood samples were also collected for HPLCanalysis of plasma FEPPA levels. All samples were stored at −80 °Cuntil analysis.

Western Blot Analysis. Protein samples (25 μg) of total brainhomogenate were loaded and resolved using 10% SDS−polyacryla-mide gel at 200 V for 1 h and transferred electrophoretically to PVDFmembrane (Millipore) at 300 mA for 1.5 h at 4 °C. Nonspecificbinding was blocked by preincubation of the PVDF membrane in PBSsolution containing 3% BSA with rocking at room temperature for 1 h.Membranes were then incubated with primary antibodies overnight at4 °C. Primary and secondary antibodies used for brain homogenatesamples are listed in Table 1. Protein blots were developed using achemiluminescence detection kit (SuperSignal West Femto substrate;ThermoFisher). Bands were visualized using ChemiDoc MP ImagingSystem (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 thickwere prepared using Leica CM3050S Research Cryostat (BuffaloGrove, IL, USA). Sections were fixed by incubation in methanol for10 min at −20 °C. Sections were then washed 5 times in PBS andblocked with PBS containing 10% donkey serum for 1 h at roomtemperature. Immunostaining was performed for brain hippocampiand cortices of TgSwDI mice. The entire cortex and hippocampusregions were included in the analysis spanning the dentate gyrus andCA1−CA3 regions. To assess IgG extravasation, brain sections wereprobed by dual immunohistochemical staining for collagen IV andmouse IgG using rabbit anti-collagen-IV and fluorescein-conjugateddonkey anti-IgG (both at 1:200 dilution), respectively. The secondaryantibody used for collagen-IV was CFL594-conjugated donkey anti-rabbit IgG. For total Aβ detection, TgSwDI brain sections wereimmunostained with Alexa-fluor 488 labeled 6E10 human-specificanti-Aβ antibody at 1:200 dilution. For detection of Aβ-plaque load inhippocampus, the brain tissue sections were stained with a freshlyprepared and filtered 0.02% Thio-S solution in 70% ethanol for 30min followed by incubation in 70% ethanol. For reactive astrocytes,

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TgSwDI sections were probed with rabbit anti-GFAP polyclonal IgGat 1:100 dilution followed by anti-rabbit IgG−CFL594 secondaryantibody. For reactive microglia, TgSwDI sections were probed withgoat anti-Iba1 polyclonal IgG at 1:100 dilution followed by anti-goatIgG−CFL594 secondary antibody for each treatment; imageacquisition was performed in 4 tissue sections spanning thehippocampus, each separated by 150 μm (total of approximately 40sections per mouse). Total Aβ load and Thio-S were captured andquantified at a total magnification of 4×; GFAP, Iba1, and IgGextravasation images were captured at a total magnification of 20×.Quantification of fluorescence intensity was performed using ImageJversion 1.6.0 software (Research Services Branch, National Instituteof Mental Health, National Institutes of Health, Bethesda, MD) afteradjusting for threshold. All images were visualized using NikonEclipse Ti−S inverted fluorescence microscope (Melville, NY).Determination of Oxidative Stress and Neuroinflammation

Markers. Brain homogenates were centrifuged for 15 min at 14000rpm, and the supernatants were used to assay protein carbonyl, SOD,IL-1β, and IL-10 levels following the manufacturers’ instructions. Allsamples were run at least in duplicate and corrected to the totalprotein amount in each sample using BCA assay. In addition, thequantification of FEPPA in brains and plasma samples collected fromthe three mice receiving the injections was conducted using isocraticShimadzu LC-20AB HPLC equipped with a Shimadzu SIL-20A HTautosampler and LC-20AB pump connected to a DGU-20A3 degasser(Shimadzu, OR) using the following separation method. Briefly, anacetonitrile/water (45:50 v/v) mobile phase was used for theseparation of brain and blood samples and was delivered at 1.0mL/min flow rate. The separation was performed at roomtemperature using an Agilent eclipse XDB-C18 column (5 μm, 4.6× 150 mm2 ID; Agilent Technologies Inc., CA, USA). Thewavelength was set at 210 nm, and the injection volume was 20 μL.Each chromatographic run was completed in 10 min with FEPPAeluted at a retention time of 6.8 min. One hour later, FEPPA wasdetected in the brains but not in the in the plasma (SupportingInformation, Figure S2).Morris Water Maze Testing. The Morris water maze (MWM)

test was performed for TgSwDI mice to assess learning and memoryat the end of the treatment using protocols similar to those describedpreviously with modification.76,77 All mice underwent training 3 timesa day for 4 consecutive days. The platform was kept in the samequadrant during the entire course of the experiment. Mice wererequired to find the hidden platform utilizing the distal spatial cuesavailable in the room. Conditions were kept the same during all theexperiments. An overhead camera connected to a computerizedtracking system (SMART 3.0 Platform, Panlab Harvard apparatus;Holliston, MA) was used to record the movements of the mice. Theparameters latency and swimming distance were calculated andanalyzed for treatment effect on the fourth day of training to compareperformance using statistical analysis.Statistical Analysis. Data are presented as box-and-whisker plots

representing median and interquartile range (IQR) with minimumand maximum values of n = 7 mice/group. FEPPA data was presentedas mean + SEM for n = 3 mice/group. The experimental results werestatistically analyzed for significant difference using Student’s t test fortwo groups. Values of P < 0.05 were considered statistically significant.Data analyses were performed using GraphPad Prism, version 6.0. Inthe figures, the fold change for control groups was calculated bydividing each control value on the average of all control mice valuesfor a specific protein. Treatment values were then normalized tocontrol (1.0).

■ ASSOCIATED CONTENT*S Supporting InformationThe Supporting Information is available free of charge on theACS Publications website at DOI: 10.1021/acschemneur-o.9b00175.

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

■ AUTHOR INFORMATIONCorresponding Author*A.K. E-mail: kaddoumi@auburn.edu. Mailing address:Department of Drug Discovery & Development, HarrisonSchool of Pharmacy, Auburn University, 720 S. Donahue Dr.,Auburn, AL 36849. Phone: +1-334-844-7239.ORCIDAmal Kaddoumi: 0000-0001-9792-7766Author ContributionsS.B.R. designed and performed the experiments, analyzed andinterpreted data, and wrote the manuscript; L.I.D. performedexperiments; A.K. designed the study and experiments,analyzed and interpreted data, and wrote the manuscript.NotesThe authors declare the following competing financialinterest(s): The corresponding author, Amal Kaddoumi, is aChief Scientific Officer without compensation in theShreveport, Louisiana, based Oleolive, LLC.

■ ACKNOWLEDGMENTSThis research work was funded by the National Institute ofNeurological Disorders and Stroke (NIH/NINDS) underGrant Number R15NS091934 (A.K.) and Center forMolecular Imaging and Therapy (CMIT). We thank PradeepGarg and Sudha Garg from CMIT for providing FEPPAcompound and Olive Fabrica Spyros and George Dafnis(Corfu, Greece) for providing us with “The Governor” extra-virgin olive oil to perform the studies.

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ACS Chemical Neuroscience Research Article

DOI: 10.1021/acschemneuro.9b00175ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

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