Research Article

Olive-Oil-Derived Oleocanthal Enhances β‑Amyloid Clearance as a
Potential Neuroprotective Mechanism against Alzheimer’s Disease:
In Vitro and in Vivo Studies
Alaa H. Abuznait, Hisham Qosa, Belnaser A. Busnena, Khalid A. El Sayed, and Amal Kaddoumi*
Department of Basic Pharmaceutical Science, College of Pharmacy, University of Louisiana at Monroe, 1800 Bienville Drive, Monroe,
Louisiana 71201, United States

ABSTRACT: Oleocanthal, a phenolic component of extra-
virgin olive oil, has been recently linked to reduced risk of
Alzheimer’s disease (AD), a neurodegenerative disease that is
characterized by accumulation of β-amyloid (Aβ) and tau
proteins in the brain. However, the mechanism by which
oleocanthal exerts its neuroprotective effect is still incom-
pletely understood. Here, we provide in vitro and in vivo
evidence for the potential of oleocanthal to enhance Aβ
clearance from the brain via up-regulation of P-glycoprotein
(P-gp) and LDL lipoprotein receptor related protein-1
(LRP1), major Aβ transport proteins, at the blood-brain
barrier (BBB). Results from in vitro and in vivo studies demonstrated similar and consistent pattern of oleocanthal in controlling
Aβ levels. In cultured mice brain endothelial cells, oleocanthal treatment increased P-gp and LRP1 expression and activity. Brain
efflux index (BEI%) studies of 125I-Aβ40 showed that administration of oleocanthal extracted from extra-virgin olive oil to
C57BL/6 wild-type mice enhanced 125I-Aβ40 clearance from the brain and increased the BEI% from 62.0 ± 3.0% for control mice
to 79.9 ± 1.6% for oleocanthal treated mice. Increased P-gp and LRP1 expression in the brain microvessels and inhibition studies
confirmed the role of up-regulation of these proteins in enhancing 125I-Aβ40 clearance after oleocanthal treatment. Furthermore,
our results demonstrated significant increase in 125I-Aβ40 degradation as a result of the up-regulation of Aβ degrading enzymes
following oleocanthal treatment. In conclusion, these findings provide experimental support that potential reduced risk of AD
associated with extra-virgin olive oil could be mediated by enhancement of Aβ clearance from the brain.
KEYWORDS: Oleocanthal, β-amyloid clearance, β-amyloid degradation, P-glycoprotein, LRP1, BBB

T he Mediterranean diet is associated with beneficial health
properties against Alzheimer’s disease (AD), a neuro-
degenerative disease that affects about 30 million people
vitro and in vivo studies have demonstrated that olive oil
phenolics have positive effects on certain physiological
parameters such as plasma lipoproteins, oxidative damage,
worldwide.1 Epidemiological studies indicate that the preva- inflammatory markers, platelet and cellular function, antimicro-
lence of AD and cognitive decline is low among the bial activity, and bone health.10
Mediterranean area populations compared to those of other Among the phenolic olive oil constituents, (−)-oleocanthal, a
geographical regions of the world.2−4 One integral component naturally occurring phenolic secoiridoid isolated from EVOO,
of the Mediterranean dietary pattern is the consumption of has shown an anti-inflammatory and antioxidant properties
extra-virgin olive oil (EVOO).5 Typically, the intake of EVOO similar to the nonsteroidal anti-inflammatory drug ibuprofen.11
ranges from 25 to 50 mL per day in the Mediterranean diet.6 (−)-Oleocanthal is the dialdehydic form of (−)-deacetoxylig-
Therefore, the apparent health benefits have been partially stroside glycoside responsible for the bitter taste of EVOO, and
attributed to the dietary consumption of EVOO by its chemical structure is related to the secoiridoid glycosides
Mediterranean populations. ligstroside and oleuropein, which are also common in EVOO.
Historically, the health promoting properties of EVOO were Chemical structure of oleocanthal is shown in Figure 1.
attributed to the high concentration of monounsaturated fatty Recently, oleocanthal has been demonstrated to have
acids, in particular oleic acid, contained in EVOO. However, potential neuroprotective properties and contribute to
other seed oils (i.e., sunflower, soybean, and rapeseed), which preventing cognitive decline due to neurodegenerative
also contain high concentrations of oleic acid, do not exhibit diseases.12−14 This has been supported by population-based
the same health benefits as EVOO.7−9 In addition to oleic acid, studies indicating that Mediterranean diet, rich in olive oil and
EVOO contains a minor, yet significant phenolic fraction that
other seed oils lack and this fraction has generated much Received: January 21, 2013
interest regarding its health promoting properties. Currently, 36 Accepted: February 15, 2013
phenolic compounds have been identified in EVOO and in Published: February 15, 2013

© 2013 American Chemical Society 973 | ACS Chem. Neurosci. 2013, 4, 973−982

the role of oleocanthal in enhanced bEnd3 cells treated with oleocanthal.13 Additional potential mechanism suggests that oleocanthal reduce the formation of β-amyloid (Aβ) senile plaques. mediates and regulates Aβ influx to the brain.30 Thus. 0. degrading enzymes.25 On the other hand. including enzymatic degradation by a variety of proteases such as neprilysin (NEP) and insulin degrading enzyme (IDE). 4. and then the consequences of such treatment on the clearance of Aβ across the BBB.35-fold increase. by acting on microtubule associated proteins known as tau proteins. ■ increase. treatment of bEnd3 cells populations consuming Mediterranean style diet. respectively. a multiligand receptor in the immunoglobulin superfamily.2− new insight on the protective effect of oleocanthal against AD 1.05).8. P < 0.15. Chemical structure of (−)-oleocanthal.1021/cn400024q | ACS Chem. Representative Western blots for P-gp (A) and LRP1 (B) in and in vivo studies. perivascular lymphatic drainage.17.26. Pitt et al. in the brain. 2013. Neurosci.24. Cells were treated for 72 h with clearance of Aβ from the brain as an additional possible increasing concentrations of the indicated compounds in the range of mechanism for its neuroprotective effect via its potential to up. another pathological hallmark of AD.4 Different mechanisms have been proposed to describe the role that oleocanthal plays in the reduced incidence of AD.12 This study was followed by Monti et al. Li and co-workers showed that oleocanthal inhibits the formation of neurofibrillary tangles.2-fold increase in P-gp and LRP1 Available experimental data strongly suggest impaired clearance expressions.. The ability of oleocanthal at different concentrations to monounsaturated fats. the greater fold increase in P-gp and LRP1 expressions 974 dx.05) and LRP1 levels (1. when compared to control cells of Aβ across the BBB might largely contribute to the formation (Figure 3). Also. which are involved in the promotion of microtubule assembly and stability in the neurons.doi. 973−982 .org/10.5−50 μM.14 Several mechanisms have been proposed to account for the clearance of Aβ. the receptor for advanced glycation end products (RAGE).29 In the present study. removal through the brain ISF bulk flow into the bloodstream.25−1.27 Numerous attempts have been made to decrease Aβ accumulation. regulate P-gp and LRP1 at the BBB. we aimed to demonstrate.18 Increasing evidence from the literature show that AD mainly develop due to the excessive accumulation of Aβ in the brain as a result of its faulty clearance across the BBB. and transport across the BBB.16 One cohort study performed on 1880 elders in the Western blotting using bEnd3 cells as a representative model of United States showed a 40% decrease in the incidence of AD in mouse BBB. demonstrated that oleocanthal can interact with Aβ and alter the oligomerization state of Aβ oligomers and protect the neurons from the synaptopathological effects associated with Aβ aggregation and plaque formation. in the current study we aimed to investigate the effect of oleocanthal treatment on the levels of P-gp. a key hallmark in the pathogenesis of AD. protects against age-related cognitive induce P-gp and LRP1 expressions at the BBB was assessed by decline. using in vitro Figure 2. While both methods provide semiquantitative of Aβ brain deposits and AD progression.19 It has been reported that Aβ removal from the brain across the BBB is mediated by two major transport proteins. LRP1 and Aβ Figure 1. recent evidence indicated a progressive decline in the levels of P-gp and LRP1 at the BBB during normal aging. where oleocanthal treatment RESULTS AND DISCUSSION resulted in 2. and its ability to enhance Aβ degradation. P < 0. Based on the findings. it has been results.ACS Chemical Neuroscience Research Article demonstrated that P-gp and LRP1 play substantial role in the elimination of Aβ from the brain across the BBB.30-fold by enhancing Aβ clearance from the brain. who investigated the mechanism by which oleocanthal inhibits tau fibrillization and aggregation in vitro via covalent chemical interaction with the fibrillogenic fragment of tau proteins.20−23 Moreover. we hope to provide a with oleocanthal resulted in significant increase in P-gp (1. and the enhanced Aβ clearance across BBB was one potential therapeutic approach that has been thoroughly reviewed. P-glycoprotein (P-gp) and LDL lipoprotein receptor related protein-1 (LRP1). As shown in Figure 2.and 2. Similar pattern has been observed in immunofluorescence studies.20−23.28. and this decline was positively correlated with accumulation of Aβ in AD.

Representative fluorescent micrographs of (A) P-gp (green) and (B) LRP1 (red) for control and bEnd3 cells treated with 25 μM of oleocanthal.05 compared to control untreated cells. or RAGE (N- cellular uptake. cellular uptake studies were conducted. The data are expressed as mean ± SD (n = 4). 973−982 . have been P-gp limits 125I-Aβ40 cellular entry due to its efflux function.44.33 The results showed conclusive treatment in the presence and absence of 100 μM verapamil evidence for the specific roles of P-gp and LRP1 in 125I-Aβ40 (P-gp inhibitor).org/10. observed by the immunofluorescence method compared to level. in uptake studies P-gp and LRP1 are localized accumulation of Aβ.ACS Chemical Neuroscience Research Article Figure 3. *P < 0. from abluminal To determine the effect of oleocanthal treatment on the to luminal side).e. Quantitative folds change in P-gp and LRP1 expression were measured using ImageJ version 1. 4. Aβ40 and Aβ42. Unlike in transport studies where cells are polarized and Western blotting could be related to methods sensitivity. and their up-regulation on 125I-Aβ40 cellular 16) antibody (as RAGE inhibitor) at a dilution ratio 1:100.doi.31 P-gp and LRP1 work in the same direction (i. at the cell membrane and function in opposite directions where While both Aβ peptide species.1021/cn400024q | ACS Chem.32 Aβ40 was used in this while LRP1 enhances cellular uptake of 125I-Aβ40. The 975 dx. Neurosci. 1 μM RAP (LRP1 inhibitor). implicated in the pathogenesis of AD. 2013.. Scale bar = 50 μm. The cellular work because it is practically feasible as it has much faster uptake of 125I-Aβ40 was evaluated following oleocanthal clearance rate than Aβ42.

BEI% analysis demonstrated about 18% oleocanthal has no significant effect on its expression or increase in the clearance of 125I-Aβ40 in oleocanthal treated activity (Figure 5).05). In oleocanthal treated cells. In contrast to the function of P-gp. cpm/mg protein. The role of P-gp in 125I-Aβ40 clearance across the studies was intraperitoneal administration at 10 mg/kg/day BBB of C57BL/6 mice was evaluated by preinjection of twice daily for 2 weeks to wild-type mice. indicating enhanced P-gp function Aβ is cleared from the brain by nonsaturable (passive) and by oleocanthal. Cells were treated for 72 h with The data are progress to investigate oleocanthal metabolism. With regard to RAGE. we aimed to investigate the effect of oleocanthal < 0. In line with the expressed as mean ± SEM (n = 3 independent experiments). Effect of treatment of bEnd3 cells with vehicle (CTRL) or unstable suggesting formation of active metabolites that may oleocanthal (OLC) in presence or absence of inhibitors on the exert the up-regulation effect. 25.01). CSF turnover). metabolically it could be Aβ40/14C-inulin solution.01) compared expression of RAGE has been observed in control or treated to 28% in vehicle treated cells (from 837 ± 36 to 1068 ± 102 animals (data not shown). At this dose.9 ± 1. To study whether the enhanced brain clearance of 125I- treatment on the clearance of 125I-Aβ40 from mouse brain. respectively.05. both mechanisms were control suggesting that oleocanthal possibly induced an investigated. mice (79. 4. Quantitative analysis for RAGE in bEnd3 cells treated with oleocanthal. unknown uptake mechanism that is not inhibited by RAP. Western blot analysis (Figure 6) revealed # Significantly different from control treated cells with inhibitors (P < significant increase in the expression of P-gp and LRP1 in the 0. brain microvessels of C57BL/6 mice by oleocanthal treatment.02). P < 0. In concept.38 into the brain of C57BL/6 animals were healthy and no weight loss was observed. However.05). Clearance BEI% experiments were performed at 24 h after This finding is further supported by the in vivo data discussed the last injection of oleocanthal or vehicle (normal saline). of 125I-Aβ40 by 34% (from 837 ± 36 to 555 ± 8 cpm/mg Saturable pathways involve degradation and BBB efflux protein) for vehicle treated cells. The brain efflux index (BEI) method was used 812 ± 12 to 671 ± 30 cpm/mg protein) for oleocanthal treated to study brain 125I-Aβ40 clearance with 14C-inulin as a reference cells.e. LRP1 saturable pathways (transport and metabolism). in vitro results. 2013.ACS Chemical Neuroscience Research Article results showed significant alteration in the activities of P-gp and LRP1 but not RAGE caused by oleocanthal treatment (Figure 4). Figure 4).0 ± 3. P-gp inhibition caused Figure 5.35 Nonsaturable functions by cellular internalization. P < 0.doi. shown in Figure 7A.25-fold significant increase in 125I-Aβ40 cellular uptake by 69% (from compared to control. a specific P-gp inhibitor. Densitometric analysis of the bands showed that oleocanthal increased P-gp and LRP1 expression by 1.0%. Neurosci. To Aβ40 is specifically due to increased expression of P-gp and our knowledge. Consequently.36 The BEI% method is commonly used to treated cells was significantly lower than that of vehicle treated investigate molecules’ brain clearance via degradation/metab- cells (P < 0. and 50 μM concentrations of oleocanthal. 973−982 .and 1. Valspodar was administered first to 976 dx. The dosage regimen selected to conduct these performed. changes in BEI% of 125 induction by oleocanthal. Given mice 5 min before the intracerebral microinjection of 125I- the aldehyde structure of oleocanthal. this study is the first to in vivo investigate LRP1 at the BBB. As below. RAP inhibition should reduce 125I. compared to only 17% (from components.6%) compared to control mice (62. *Significantly different from no inhibitor treated cells (P < 0.30.37 Thus. P Next. unlike P-gp and LRP1. The percent reduction in 125I-Aβ40 uptake in oleocanthal compound. I-Aβ40 following oleocanthal treatment compared to control Aβ40 uptake equally in both oleocanthal and control treated demonstrates alteration in 125I-Aβ40 removal from the brain by cells..34 Further studies are currently in intracellular accumulation of radiolabeled 125I-Aβ40. all valspodar. thus its inhibition by RAP pathway involves passive removal of soluble Aβ through bulk caused a significant reduction (P < 0. No significant change in the 812 ± 12 to 1376 ± 132 cpm/mg protein. assuming lack of LRP1 olism and/or transport across BBB. Figure 4. the reduction in 125I-Aβ40 cellular level in enhanced enzymatic degradation and/or clearance across BBB oleocanthal treated cells was less than that observed with via the transport system.1021/cn400024q | ACS Chem.02) in the cellular uptake flow of cerebrospinal fluid (CSF) (i. inhibitory studies of each protein were oleocanthal.

23 obtained from the in vitro inhibition studies. (data not shown).6-fold) in control and oleocanthal-treated mice with valspodar resulted in the expression of IDE and small but insignificant increase in about 13% (from 62. The reduction in BEI% following P-gp bEnd3 cells resulted in significant increase in the expression of inhibition by valspodar in oleocanthal treated group compared IDE (1. Both valspodar and allow its brain distribution and interaction with P-gp prior to RAP caused a significant reduction in the BEI% in oleocanthal treated 125 I-Aβ40 microinjection which has rapid brain clearance. the reduction in BEI% values of 125I-Aβ40. in vitro treatment of % values. Figure 7B) in oleocanthal-treated mice. Consistent with in vivo findings. 2013.5%. P < 0. P < 0. (A) Brain efflux index (BEI) of 125I-Aβ40 in control (CTRL).3%) compared to control or enhanced degradation of 125I-Aβ40 as a result of the (40. the pretreatment of with oleocanthal resulted in a significant increase (1.5 ng/0. P < 0.doi.05). (B) Quantitative fold increase in injection of oleocanthal or normal saline.05) enzymes to control group was statistically insignificant (P > 0. (A) Representative Western blot lanes for P-gp. which is consistent with the data P-gp and LRP1 in the clearance of Aβ across the BBB. (B) Effect of P-gp and LRP1 inhibition by valspodar (24 ng/0.9 ± 1. inhibition significantly lowered BEI% value of 125I-Aβ40 in ***P < 0. the above results corroborate the assumption resulted in about 33% (from 62.0% to 29. Our results confirm both. 973−982 . The percent of degraded Aβ clearance of 125I-Aβ40. mostly via induction of IDE Aβ40 BEI% (P < 0. and oleocanthal (OLC) treated groups measured 24 h after the last and protein loading control (β-actin).3-fold.8%.1-fold) in mice brain microvessels.001).5 μL injection). These inhibition of LRP1 by RAP was more apparent in control mice results are in line with similar studies demonstrating the role of than oleocanthal-treated mice.3 ± 4. Further.1 ± 1. oleocanthal treatment enhanced the Similarly. Thus.6% to 59. treatment of mice treatment. enzyme and possibly NEP.2%.001) reduction in BEI 8B. The data are expressed as mean ± SEM of n = 4−6 (*P < 0.9 ± 1. P < 0. P < 0. ± 3. group (CTRL).ACS Chemical Neuroscience Research Article Figure 6.4 ± 2. degradation of Aβ40 in mice brain. Aβ clearance To characterize the effect of oleocanthal treatment on the from the brain of oleocanthal-treated mice was greater than degradation of 125I-Aβ40 in C57BL/6 mice brain. Thirty minutes post microinjection of 125I-Aβ40.6-fold. respectively. Significantly higher expression levels of P-gp and LRP1 were detected in oleocanthal (OLC) treated mice compared to control Figure 7.6% to 39. control and oleocanthal-treated group (Figure 7B).0 ± 3.5 μL injection) and RAP (19. Pretreatment of control and oleocanthal-treated mice with RAP Collectively.1021/cn400024q | ACS Chem.05).22. P < that oleocanthal improves 125I-Aβ40 clearance by enhancing P- 0. respectively (Figure 40% (79. Yet. n = 3 independent experiments (*P < 0. and/ oleocanthal treated group (60.05.1 ± 1. suggesting the TCA degradation assay and measured the expression of Aβ induction of another mechanism(s) contributing to the degrading enzymes NEP and IDE. Western blot analysis of P-gp and LRP1 in mice brain microvessels.33 P-gp group.05) and NEP (1.05) gp and LRP1 expression/activity at the BBB. P < 0. Significantly higher BEI% P-gp and LRP1 expressions.05) and NEP (1.6%. 4. LRP1 inhibition by RAP significantly reduced 125I. Neurosci. respectively.001) and 21% (79.0% to 49. which could be an unknown trans- peptide (cpm in supernatant) were significantly higher in porter(s)/receptor(s).23 most likely at the abluminal side. C).2 ± 3.05. Figure 8A). However.05. The data are expressed as mean ± SEM of was observed in oleocanthal treated mice compared to control group.20. we performed control mice even in the presence of RAP. induction of an unknown 977 dx.0 ± 2. on BEI% of 125I-Aβ40 in CTRL and OLC treated groups.5%. respectively.

Beside its role in the up-regulation of P-gp and cognitive decline. oleocanthal enhanced the clearance of 125I-Aβ40 by up. two peptidases that more recently been demonstrated to possess therapeutic have been reported to cleave and degrade Aβ implicated in AD. and protein loading control (β-actin) in mice brain microvessels. 4. Significantly higher expression levels of IDE but not NEP were detected in OLC treated mice compared to control. and lation.ACS Chemical Neuroscience Research Article Figure 8.40−42 LRP1.12. supported by the in vitro data. NEP.4 Our findings described in this study provide further potential means of protecting the brain against Aβ accumu. (C) Quantitative fold increase in IDE and NEP expressions. (A) Effect of oleocanthal (OLC) treatment on Aβ degradation in mice brain homogenate compared to control (CTRL) measured using TCA 2013. The data are expressed as mean ± SEM of n = 4−6 (*P < 0. prevention of amyloid plaque formation. transport mechanism. The data are expressed as mean ± SEM of n = 3 independent experiments (*P < 0.05). Neurosci.05).14 These studies Several studies reported overexpression of NEP to protect support research surveys showing a 40% decrease in AD in hippocampal neurons from Aβ-mediated toxicity in vitro.1021/cn400024q | ACS Chem. (B) Representative Western blots lanes for IDE. Significantly higher Aβ degradation% was observed in OLC treated mice compared to control. and consequent degradation. In support of its putative health benefits. 973−982 . evidence on the role of oleocanthal as a neuroprotective agent 978 dx. oleocanthal has regulating NEP (in vitro studies) and IDE.39 and populations consuming a Mediterranean style diet comprising the up-regulation of NEP or IDE has been proposed as a olive oil. activities in the treatment of AD disease.

3. 1% olive oil-derived oleocanthal associated with the consumption NP-40. which substantiate the in vitro findings reported cells were allowed to grow up to 50% confluence and then treated with by us and other research laboratories. The cells were then washed with PBS and blocked for 30 min a humidified atmosphere (5%CO2/95% air) at 37 °C. At the end of treatment period with oleocanthal or control. An uptake study using light chain LRP1 was obtained from Epitomics (Burlingame. Louis. 14C-Inulin (2. evidence for the role of oleocanthal on Aβ degradation as Western Blot Analysis of P-gp. 4. LRP1. Blotting membranes were blocked with 2% BSA and incubated METHODS overnight with antibodies for LRP1 (light chain). (Cambridge. from Covance Research Products (Dedham. CA). 1:3000. protease inhibitor for 15 min at 4 °C. IDE. TX) and band intensity was measured by densitometric blotting were purchased from Bio-Rad (Hercules. the cells mL). For immunohistochemistry. or NEP at dilutions 1:1500. or RAGE (N-16) Immunochemicals Inc. Following 72 h of treatment with 25 Biotechnology Inc. mouse monoclonal In Vitro Uptake Study of 125I-Aβ40 in bEnd3 Cells. CA).2%. in complete Flour 594 was purchased from Invitrogen (Carlsbad. Cells were seeded in 10 mm cell of primary antibody against P-gp or LRP1 in solution composed of 1% 979 dx. MI). 1H NMR spectroscopy. Inc. Rockford. Donkey anti-rabbit Alexa antibody (RAGE inhibitor) at a dilution ratio 1:100. NY) at a density of 1 × 106 cells per dish. Ketamine and xylazine for mice CO2/95% air) at 37 °C. The bands were MA). Mallinckrodt Baker. PA). MA). RAGE Reagents and Antibodies. this study provides conclusive harvested and total protein was extracted for Western blot analysis.07 nM of 125I-Aβ40 in complete culture media with or Santa Cruz Biotechnology (Santa Cruz. addition to control medium were added to the respective treatment In conclusion. The human receptor substrate detection kit (Thermo Scientific. The activity experiment was then terminated anesthetization were purchased from Henry Schein Inc. 1:200. Waltham. Rockford. 125 I-Aβ40 was conducted as described previously. 2200 Ci/mmol) was purchased from PerkinElmer (Boston. β-actin (C-11).1. our results show that extra-virgin with lysis buffer (RIPA buffer. The Aβ40 clearance. total protein was extracted from bEnd3 cells possibly NEP. At the end of treatment period. CA). MO). (Melville.3% Triton X-100/PBS. After incubation. and/or LRP1 in bEnd3 Cells. 0. A purity of >90% was established for Immunofluorescence Staining and Imaging of P-gp and oleocanthal as assessed by TLC. Additional research is oleocanthal or control (vehicle) in a humidified atmosphere (5%CO2/ required to determine the therapeutic benefits of oleocanthal 95% air) at 37 °C. The data were EVOO (Member’s Mark. To confirm effect of drugs treatment on the HPLC analysis. CA). P-gp. expression and up-regulation of P-gp and LRP1. Inc. 1:200.6). (Gilbertsville.3-Hexafluoro-2-propanol (N-16). 25 mM Tris. IDE. RAGE (N-16). bEnd3 cells (5 × 104) were Manassas. VF1_US102808.1021/cn400024q | ACS Chem. CA). 150 mM NaCl. NJ). 1 μM RAP (LRP1 inhibitor).5 medium supplemented with 10% fetal bovine serum (FBS). 2013. Furthermore. were cultured in DMEM growth seeded on 35 mm poly-D-lysine coated glass bottom plates no. Valspodar was obtained from XenoTech luminescent image analyzer (Scientific Resources Southwest Inc. Media containing oleocanthal at different concentrations in been investigated. RAGE.. anti-CD10) were obtained from were washed three times with phosphate buffer-saline (PBS) solution. our findings demonstrated that the beneficial cells in duplicate at a maximum methanol concentration of 0. For Western blot of Mediterranean diet has the potential to reduce the risk of AD analysis. Methanolic stock solution of oleocanthal was diluted to a final concentration of 0. For protein detection. For Western analysis. 1% w/v nonessential amino acids. passage 25−30. and cultured onto 48 well plates at a density of 5 × 104 cells/well and were HRP-labeled secondary antibodies were purchased from Santa Cruz grown to 50−60% confluence. Quantitative associated protein (RAP) was purchased from Oxford Biomedical analysis of the immunoreactive bands was performed using GeneSnap Research (Oxford. Ultrapure EM grade) was purchased from RAGE (N-16) antibody for 15 min in a humidified atmosphere (5% Polysciences. P-gp (C-219). The reagents and supplements required for Western Stafford. rabbit polyclonal antibody μM of oleocanthal or vehicle. the Louis. (Lenexa. which are responsible for Aβ CO2/95% air) at 37 °C. cells antibody against neprilysin (NEP.ACS Chemical Neuroscience Research Article against AD in vivo in wild-type mice brains by increasing 125I. IL). NEP (antimouse). The activity experiments were started by anti-mouse IgG-FITC labeled secondary antibody was obtained from the addition of 0. β-actin (antigoat) at 1:5000 dilution. IL). by washing the cells three times with ice-cold PBS. Three independent Western blotting experiments were blot. kit (Thermo Scientific. IL) according to the manufacturer’s phase Bakerbond octadecyl (40 μm. (Warrington. accumulated inside the cells was measured using Wallac 1470 Wizard Oleocanthal was extracted as reported by Elnagar et al. rabbit monoclonal antibody against on the accumulation of 125I-Aβ40 in bEnd3 cells. KS). and NEP shown by the up-regulation of Aβ degrading enzymes IDE and Proteins in bEnd3 Cells. The radioactivity of 125I-Aβ40 Extraction of Oleocanthal from Extra-Virgin Olive Oil. IDE (antirabbit) and (human. 1 μM RAP. the rabbit The cells were then preincubated with or without 100 μM verapamil polyclonal antibody against P-gp was purchased from Rockland (P-gp inhibitor). ■ polyacrylamide gels and transferred onto nitrocellulose membrane. and goat culture medium for 30 min. Also.5% SDS- or related neurodegenerative dementias. the medium was aspirated and the cells against insulin degrading enzyme (IDE) and mouse monoclonal were incubated in fresh growth medium for 4 1:100. The mouse brain endothelial cells (bEnd3. PA). bead size 25−100 μm) determined using the Pierce bicinchoninic acid (BCA) protein assay using n-hexane-CH2Cl2 (1:9) and finally purified on C-18 reversed. visualized using confocal microscopy. Formaldehyde 16% without 100 μM verapamil. and its bioavailability which has not before use. PA). RAGE. The cells were washed three times with PBS and fixed with 4% formaldehyde for were grown to confluence in 75 cm2 cell culture flasks for 1−2 days in 10 min. the mouse monoclonal antibody C-219 against P-gp was obtained carried out for each treatment group. ATCC. and 1:100. isocratic CH3CN−H2O (40:60). The protein concentrations were lipophilic Sephadex LH20 (Sigma Aldrich. MA) and treated with 25 μM of antibiotics penicillin G (100 units/mL) and streptomycin (100 μg/ oleocanthal or vehicle as described above.2 mCi/g) was purchased from American visualized using SuperSignal West Femto Maximum Sensitivity Radiolabeled Chemicals (St. (Santa Cruz. (HFP) and Tween 20 were purchased from Sigma-Aldrich (St.. 25 μg of cellular protein was resolved on 7.3. 1. In Vitro Induction of P-gp and LRP1 Expression by The cells were then incubated overnight at 4 °C with a 1:200 dilution Oleocanthal in bEnd3 Cells. Italy) on normalized for the protein content.43 from Gamma Counter (PerkinElmer Inc. cells were clearance across the BBB. Synthetic monoiodinated and nonoxidized 125I-Aβ40 IgG antibody for LRP1. 1. culture dishes (Corning. Neurosci. both proteins were Cell Culture. bEnd3 cells goat polyclonal antibodies against. VA).1. Ashland. After treatment. RIPA buffer was purchased from Thermo Scientific membranes were washed and incubated with HRP-labeled secondary (Rockford. and the (MatTek Corporation. 1% sodium deoxylate.) using instruction with bovine serum albumin (BSA) as a standard. pH 7. All other reagents and supplies were purchased from VWR disrupted with the lysis buffer containing complete mammalian (West Chester.5−50 μM in growth medium using AD animal model. MA). batch no.1% SDS. The effect of oleocanthal against AD could be extended to its ability cells were then incubated for 72 h in a humidified atmosphere (5% to induce P-gp and LRP1.HCl. β-actin (C-11). The aim of antibody against light chain LRP1 (5A6) was obtained from this experiment was to investigate the effect of oleocanthal treatment Calbiochem (Gibbstown. glutamine 2 mM.doi.31 Briefly. MA). MO). respectively. or 1:100 dilution ratio of (Methanol Free. Abcam Inc. 973−982 . with 10% of normal donkey and goat sera in 0. The cells were then NY).

47 were intracerebrally preadministered 5 min prior to 125I-Aβ40 injection at concentrations of 1 μM (19.).9 mm as described previously by us. The in vivo Aβ clearance The percent degradation for each sample was calculated by dividing experiments (BEI%) were performed using the intracerebral micro. To characterize the role of LRP1 and P-gp standard error of mean (SEM). 1. a well-established P-gp inhibitor23.5 μL) containing 125I-Aβ40 (30 nM) and Western Blot Analysis of P-gp. 0.4) was administered into the caudate nucleus over a period of 3 min. A p-value less than 0. 1. one volume of TCA (Research Services Branch. 10 min and supernatant was used for further Western blot analysis as dried. About half objective lens with numerical aperture = 1. All animal experiments Wallac 1414 WinSpectral Liquid Scintillation Counter (PerkinElmer were approved by the Institutional Animal Care and Use Committee Inc. Mice were kept under standard 125 I-Aβ40 (intact peptide) and TCA supernatant (degraded peptide) environmental conditions (22 °C. 125I-Aβ40 was initially solubilized in HFP. intactness and quality of 125I-Aβ40 was initially confirmed by Statistical Analysis.5 μL Co.4 (Carl Zeiss lateral to bregma.23 Brain tissues were excised were incubated for 30 min with antirabbit Alexa flour 594 conjugated and homogenized by Dounce tissue grinder with seven strokes in two secondary IgG for the detection of LRP1 and antimouse FITC for the volumes of DPBS buffer (2. Following centrifugation.doi. MD). inhibitor.23 Briefly. Fax: 318-342-1737. 25 mM NaHCO3. mice were precipitate and the supernatant cpm) and the resulting percent were anesthetized with intraperitoneal xylazine and ketamine (20 and 125 subtracted from the percent of free 125I which determined from mg/kg. 0. 30 min post 125I-Aβ40 treated groups. 12 h dark/ were measured using Wallac 1470 Wizard Gamma mice were allowed to adapt to the new environment for cocktail and the beta radioactivity of 14C-inulin was measured using one week before initiating the experiments. The BEI% was defined by eq 1 and the percentage of substrate of the University of Louisiana at Monroe and all surgical and treatment remaining in the brain (100 − BEI%) was determined using eq 2. both at 1:250 dilution in PBS. and 10 mM HEPES.0 μL used for Western blotting and immunohistochemistry studies of LRP1 gastight Hamilton microsyringe was inserted into the guide cannula.9 mm below the surface of the brain as brain was immediately put in ice-cold normal saline. and 2. the microsyringe was left in place for 3 min to minimize unpaired Student’s t-test to evaluate differences between controls and any backflow.05 was considered to be injection.02 μCi) prepared in extracellular fluid buffer (ECF. At the end of treatment period.22. supernatant and precipitate were then mixed with 5 mL scintillation After shipping. and resolubilized in ECF buffer prior to the experiment. mM NaCl. 122 microvessels were analyzed by Western blotting as described above. Neurosci. Thornwood. gamma radioactivity of precipitated with an average body weight of 20 g. 125I-Aβ40 is characterized by *Telephone: 318-342-1460. the supernatant cpm by the total cpm (sum of the cpm in the injection technique reported previously. 980 dx. Homogenized samples were centrifuged at 13 000 rpm for To prevent aggregation. TX). Drug administration was started at 7−8 weeks of age and continued for 2 weeks.20 After the micro- were analyzed for statistically significant difference using Two-tailed injection. Bethesda. Trichloroacetic acid (TCA) precipitation ground fluorescence. Mice of oleocanthal group received amount of 14C‐inulin in the brain intraperitoneal oleocanthal at dose of 10 mg/kg twice daily. 100 − BEI% = × 100 amount of intact 125I‐Aβ injected Brain Efflux Index Study. NIMH/NIH. and 0. The mice were 6−7 weeks old 30 min.23 To measure intact 125I-Aβ40. mice 14 amount of C‐inulin injected (2) were prepared for brain efflux index study (BEI) to assess the clearance of Aβ 24 h after the last dose of oleocanthal. that coincide with the right caudate nucleus.46 or valspodar.23. the cells postinjection to determine Aβ BEI%. LRP1.5 μL injection volume).75 mm Michel suture clips. A stainless steel guide Isolation of Brain Microvessels.20. and centrifuged at 14 000 rpm (4 °C) for Harlan Laboratories (Houston. Brain microvessels were isolated cannula was implanted stereotaxically at 0. homogenized and previously reported.20. 0. 10 mM D-glucose.9 mM CaCl2.5 ng/0. Mice of control group received intraperitoneal amount of intact 125I‐Aβ in the brain vehicle (normal saline) twice daily. 2013. Microvessels adhering to the glass beads were collected due to insertion of guide cannula in order to restore BBB integrity.5 mM MgCl2 LRP1 were captured using Zeiss LSM 5 Pascal confocal microscope supplemented with 5 mM D-glucose and 1 mM sodium pyruvate. 136.23.5 μL injection volume) and 40 μM (24 ng/0. and P-gp.5 μL of ECF containing RAP.44 software in the brain.45 by gentle agitation in 1% BSA in DPBS. Wood Dale.44. 4 °C). equipped with 543 nm line of HeNe Laser and 63X oil immersion pH 7. and then samples were vortexed. 3 m M KCl.23 plasma and brain tissues were rapidly collected for statistically significant. P-gp and LRP1 membrane immunofluorescence method was used to calculate the amount of intact 125I-Aβ40 remained for each sample was quantified using ImageJ version 1.4 mM K2HPO4.. the experimental results trichloacetic acid (TCA) precipitation assay.1021/cn400024q | ACS Chem. Each group contained at least four I‐Aβ injected into the brain (1) animals.2 mM Total protein was extracted from isolated brain microvessels by MgSO4. Protein expression levels in isolated brain 14 C-inulin (0. containing 1% bovine serum albumin (BSA) and passed over glass Animals were then allowed for 12 h recovery from acute brain injury bead column. Negative controls for each treatment that quantification and the second half for microvessels isolation and were processed without primary antibody showed negligible back.22.7 mM KCl.9 mm anterior. One volume Ficoll (30%) was skull with binary dental cement. (20%) was added to the sample.20.1 mM Na2HPO4. and RAGE in Isolated Brain Microvessels.9 mM detection of LRP1. the animal was removed and the mixture was mixed. Wherever possible. Isolated microvessels were Animals were reanesthetized with intraperitoneal xylazine/ketamine and an injector cannula connecting via Teflon tubing to a 1. The resulting pellets were suspended in ice-cold DPBS posterior to the guide assembly using 1. an LRP1 inhibitor20. 1.45 In brief. At the designated time. After washing with PBS.33 thus brain was collected 30 min kaddoumi@ulm. Animals. C57BL/6 wild-type male mice were purchased from incubated in ice for 30 min. and a stylet was introduced into the added to the half of brain homogenate to a final concentration of 15% guide cannula. 973−982 .36. and the wound was closed anterior and min. The described above. function. after decapitation of mouse. All results were expressed as means and Aβ measurements.ACS Chemical Neuroscience Research Article normal donkey and goat sera in PBS.4 mM CaCl2. The light cycle) with free access to tap water and standard rodent food. In Vivo Induction of P-gp and LRP1 Expression by 125 I‐Aβ brain clearance Oleocanthal. of the brain homogenate was used for 125I-Aβ40 and 14C-inulin LLC. protein expression study. IL) to determine the coordinates of the mice brain injectate solution and then processed as sample homogenate.4) containing mammalian protease inhibitor cocktail. The applied tracer fluid (0. respectively) and placed in a stereotaxic apparatus (Stoelting preinjected sample by spiking mice brain homogenate with 0. and then centrifuged (5000 rpm for 10 from the stereotaxic device.46 procedures were consistent with the IACUC policies and procedures. E-mail: its rapid clearance across the BBB. pH homogenization with lysis buffer (RIPA buffer) containing protease 7. 8. Animals were divided into two treatment groups: BEI% = 125 × 100 control and oleocanthal groups. 4. ■ AUTHOR INFORMATION Corresponding Author Calculation of 125I-Aβ40 Clearance. Once the cement was firm. 1. Images for P-gp and NaCl. respectively. NY).46 mM KH2PO4. 35% relative humidity.20 The guide cannula and screw were fixed to the divided into halves as described above.

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