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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 Aβtransport proteins, at the blood-brain
barrier (BBB). Results from in vitro and in vivo studies demonstrated similar and consistent pattern of oleocanthal in controlling
Aβlevels. 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 Aβdegrading 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 Aβclearance from the brain.
KEYWORDS: Oleocanthal, β-amyloid clearance, β-amyloid degradation, P-glycoprotein, LRP1, BBB
The Mediterranean diet is associated with beneficial health
properties against Alzheimer’s disease (AD), a neuro-
degenerative disease that affects about 30 million people
worldwide.
1
Epidemiological studies indicate that the preva-
lence of AD and cognitive decline is low among the
Mediterranean area populations compared to those of other
geographical regions of the world.
2−4
One integral component
of the Mediterranean dietary pattern is the consumption of
extra-virgin olive oil (EVOO).
5
Typically, the intake of EVOO
ranges from 25 to 50 mL per day in the Mediterranean diet.
6
Therefore, the apparent health benefits have been partially
attributed to the dietary consumption of EVOO by
Mediterranean populations.
Historically, the health promoting properties of EVOO were
attributed to the high concentration of monounsaturated fatty
acids, in particular oleic acid, contained in EVOO. However,
other seed oils (i.e., sunflower, soybean, and rapeseed), which
also contain high concentrations of oleic acid, do not exhibit
the same health benefits as EVOO.
7−9
In addition to oleic acid,
EVOO contains a minor, yet significant phenolic fraction that
other seed oils lack and this fraction has generated much
interest regarding its health promoting properties. Currently, 36
phenolic compounds have been identified in EVOO and in
vitro and in vivo studies have demonstrated that olive oil
phenolics have positive effects on certain physiological
parameters such as plasma lipoproteins, oxidative damage,
inflammatory markers, platelet and cellular function, antimicro-
bial activity, and bone health.
10
Among the phenolic olive oil constituents, (−)-oleocanthal, a
naturally occurring phenolic secoiridoid isolated from EVOO,
has shown an anti-inflammatory and antioxidant properties
similar to the nonsteroidal anti-inflammatory drug ibuprofen.
11
(−)-Oleocanthal is the dialdehydic form of (−)-deacetoxylig-
stroside glycoside responsible for the bitter taste of EVOO, and
its chemical structure is related to the secoiridoid glycosides
ligstroside and oleuropein, which are also common in EVOO.
Chemical structure of oleocanthal is shown in Figure 1.
Recently, oleocanthal has been demonstrated to have
potential neuroprotective properties and contribute to
preventing cognitive decline due to neurodegenerative
diseases.
12−14
This has been supported by population-based
studies indicating that Mediterranean diet, rich in olive oil and
Received: January 21, 2013
Accepted: February 15, 2013
Research Article
pubs.acs.org/chemneuro
© XXXX American Chemical Society Adx.doi.org/10.1021/cn400024q |ACS Chem. Neurosci. XXXX, XXX, XXX−XXX
monounsaturated fats, protects against age-related cognitive
decline.
15,16
One cohort study performed on 1880 elders in the
United States showed a 40% decrease in the incidence of AD in
populations consuming Mediterranean style diet.
4
Different mechanisms have been proposed to describe the
role that oleocanthal plays in the reduced incidence of AD; Li
and co-workers showed that oleocanthal inhibits the formation
of neurofibrillary tangles, a key hallmark in the pathogenesis of
AD, by acting on microtubule associated proteins known as tau
proteins, which are involved in the promotion of microtubule
assembly and stability in the neurons.
12
This study was
followed by Monti et al., 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.
13
Additional potential mechanism
suggests that oleocanthal reduce the formation of β-amyloid
(Aβ) senile plaques, another pathological hallmark of AD, in
the brain. Pitt et al. demonstrated that oleocanthal can interact
with Aβand alter the oligomerization state of Aβoligomers and
protect the neurons from the synaptopathological effects
associated with Aβaggregation and plaque formation.
14
Several mechanisms have been proposed to account for the
clearance of Aβ, including enzymatic degradation by a variety of
proteases such as neprilysin (NEP) and insulin degrading
enzyme (IDE), removal through the brain ISF bulk flow into
the bloodstream, perivascular lymphatic drainage, and transport
across the BBB.
17,18
Increasing evidence from the literature
show that AD mainly develop due to the excessive
accumulation of Aβin the brain as a result of its faulty
clearance across the BBB.
19
It has been reported that Aβ
removal from the brain across the BBB is mediated by two
major transport proteins; P-glycoprotein (P-gp) and LDL
lipoprotein receptor related protein-1 (LRP1).
20−23
Moreover,
recent evidence indicated a progressive decline in the levels of
P-gp and LRP1 at the BBB during normal aging, and this
decline was positively correlated with accumulation of Aβin
AD.
24,25
On the other hand, the receptor for advanced glycation
end products (RAGE), a multiligand receptor in the
immunoglobulin superfamily, mediates and regulates Aβinflux
to the brain.
26,27
Numerous attempts have been made to
decrease Aβaccumulation, and the enhanced Aβclearance
across BBB was one potential therapeutic approach that has
been thoroughly reviewed.
28,29
In the present study, we aimed to demonstrate, using in vitro
and in vivo studies, the role of oleocanthal in enhanced
clearance of Aβfrom the brain as an additional possible
mechanism for its neuroprotective effect via its potential to up-
regulate P-gp and LRP1 at the BBB, and its ability to enhance
Aβdegradation. Based on the findings, we hope to provide a
new insight on the protective effect of oleocanthal against AD
by enhancing Aβclearance from the brain.
■RESULTS AND DISCUSSION
Available experimental data strongly suggest impaired clearance
of Aβacross the BBB might largely contribute to the formation
of Aβbrain deposits and AD progression. Also, it has been
demonstrated that P-gp and LRP1 play substantial role in the
elimination of Aβfrom the brain across the BBB.
20−23,30
Thus,
in the current study we aimed to investigate the effect of
oleocanthal treatment on the levels of P-gp, LRP1 and Aβ
degrading enzymes, and then the consequences of such
treatment on the clearance of Aβacross the BBB.
The ability of oleocanthal at different concentrations to
induce P-gp and LRP1 expressions at the BBB was assessed by
Western blotting using bEnd3 cells as a representative model of
mouse BBB. As shown in Figure 2, treatment of bEnd3 cells
with oleocanthal resulted in significant increase in P-gp (1.2−
1.35-fold increase, P< 0.05) and LRP1 levels (1.25−1.30-fold
increase, P< 0.05). Similar pattern has been observed in
immunofluorescence studies, where oleocanthal treatment
resulted in 2.8- and 2.2-fold increase in P-gp and LRP1
expressions, respectively, when compared to control cells
(Figure 3). While both methods provide semiquantitative
results, the greater fold increase in P-gp and LRP1 expressions
Figure 1. Chemical structure of (−)-oleocanthal.
Figure 2. Representative Western blots for P-gp (A) and LRP1 (B) in
bEnd3 cells treated with oleocanthal. Cells were treated for 72 h with
increasing concentrations of the indicated compounds in the range of
0.5−50 μM.
ACS Chemical Neuroscience Research Article
dx.doi.org/10.1021/cn400024q |ACS Chem. Neurosci. XXXX, XXX, XXX−XXXB
observed by the immunofluorescence method compared to
Western blotting could be related to methods sensitivity.
31
To determine the effect of oleocanthal treatment on the
accumulation of Aβ, cellular uptake studies were conducted.
While both Aβpeptide species, Aβ40 and Aβ42, have been
implicated in the pathogenesis of AD,
32
Aβ40 was used in this
work because it is practically feasible as it has much faster
clearance rate than Aβ42.
33
The results showed conclusive
evidence for the specific roles of P-gp and LRP1 in 125I-Aβ40
cellular uptake, and their up-regulation on 125I-Aβ40 cellular
level. Unlike in transport studies where cells are polarized and
P-gp and LRP1 work in the same direction (i.e., from abluminal
to luminal side), in uptake studies P-gp and LRP1 are localized
at the cell membrane and function in opposite directions where
P-gp limits 125I-Aβ40 cellular entry due to its efflux function,
while LRP1 enhances cellular uptake of 125I-Aβ40. The cellular
uptake of 125I-Aβ40 was evaluated following oleocanthal
treatment in the presence and absence of 100 μM verapamil
(P-gp inhibitor), 1 μM RAP (LRP1 inhibitor), or RAGE (N-
16) antibody (as RAGE inhibitor) at a dilution ratio 1:100. The
Figure 3. Representative fluorescent micrographs of (A) P-gp (green) and (B) LRP1 (red) for control and bEnd3 cells treated with 25 μMof
oleocanthal. Quantitative folds change in P-gp and LRP1 expression were measured using ImageJ version 1.44. The data are expressed as mean ±SD
(n= 4). *P< 0.05 compared to control untreated cells. Scale bar = 50 μm.
ACS Chemical Neuroscience Research Article
dx.doi.org/10.1021/cn400024q |ACS Chem. Neurosci. XXXX, XXX, XXX−XXXC
results showed significant alteration in the activities of P-gp and
LRP1 but not RAGE caused by oleocanthal treatment (Figure
4). In oleocanthal treated cells, P-gp inhibition caused
significant increase in 125I-Aβ40 cellular uptake by 69% (from
812 ±12 to 1376 ±132 cpm/mg protein, P< 0.01) compared
to 28% in vehicle treated cells (from 837 ±36 to 1068 ±102
cpm/mg protein, P< 0.01), indicating enhanced P-gp function
by oleocanthal. In contrast to the function of P-gp, LRP1
functions by cellular internalization, thus its inhibition by RAP
caused a significant reduction (P< 0.02) in the cellular uptake
of 125I-Aβ40 by 34% (from 837 ±36 to 555 ±8 cpm/mg
protein) for vehicle treated cells, compared to only 17% (from
812 ±12 to 671 ±30 cpm/mg protein) for oleocanthal treated
cells. The percent reduction in 125I-Aβ40 uptake in oleocanthal
treated cells was significantly lower than that of vehicle treated
cells (P< 0.05; Figure 4). In concept, assuming lack of LRP1
induction by oleocanthal, RAP inhibition should reduce 125I-
Aβ40 uptake equally in both oleocanthal and control treated
cells. However, the reduction in 125I-Aβ40 cellular level in
oleocanthal treated cells was less than that observed with
control suggesting that oleocanthal possibly induced an
unknown uptake mechanism that is not inhibited by RAP.
This finding is further supported by the in vivo data discussed
below. With regard to RAGE, unlike P-gp and LRP1,
oleocanthal has no significant effect on its expression or
activity (Figure 5).
Next, we aimed to investigate the effect of oleocanthal
treatment on the clearance of 125I-Aβ40 from mouse brain. To
our knowledge, this study is the first to in vivo investigate
oleocanthal. The dosage regimen selected to conduct these
studies was intraperitoneal administration at 10 mg/kg/day
twice daily for 2 weeks to wild-type mice. At this dose, all
animals were healthy and no weight loss was observed. Given
the aldehyde structure of oleocanthal, metabolically it could be
unstable suggesting formation of active metabolites that may
exert the up-regulation effect.
34
Further studies are currently in
progress to investigate oleocanthal metabolism. In line with the
in vitro results, Western blot analysis (Figure 6) revealed
significant increase in the expression of P-gp and LRP1 in the
brain microvessels of C57BL/6 mice by oleocanthal treatment.
Densitometric analysis of the bands showed that oleocanthal
increased P-gp and LRP1 expression by 1.30- and 1.25-fold
compared to control, respectively. No significant change in the
expression of RAGE has been observed in control or treated
animals (data not shown).
Aβis cleared from the brain by nonsaturable (passive) and
saturable pathways (transport and metabolism).
35
Nonsaturable
pathway involves passive removal of soluble Aβthrough bulk
flow of cerebrospinal fluid (CSF) (i.e., CSF turnover).
Saturable pathways involve degradation and BBB efflux
components. The brain efflux index (BEI) method was used
to study brain 125I-Aβ40 clearance with 14C-inulin as a reference
compound.
36
The BEI% method is commonly used to
investigate molecules’brain clearance via degradation/metab-
olism and/or transport across BBB.
37
Thus, changes in BEI% of
125I-Aβ40 following oleocanthal treatment compared to control
demonstrates alteration in 125I-Aβ40 removal from the brain by
enhanced enzymatic degradation and/or clearance across BBB
via the transport system. Consequently, both mechanisms were
investigated.
Clearance BEI% experiments were performed at 24 h after
the last injection of oleocanthal or vehicle (normal saline). As
shown in Figure 7A, BEI% analysis demonstrated about 18%
increase in the clearance of 125I-Aβ40 in oleocanthal treated
mice (79.9 ±1.6%) compared to control mice (62.0 ±3.0%; P
< 0.05). To study whether the enhanced brain clearance of 125I-
Aβ40 is specifically due to increased expression of P-gp and
LRP1 at the BBB, inhibitory studies of each protein were
performed. The role of P-gp in 125I-Aβ40 clearance across the
BBB of C57BL/6 mice was evaluated by preinjection of
valspodar, a specific P-gp inhibitor,
38
into the brain of C57BL/6
mice 5 min before the intracerebral microinjection of 125I-
Aβ40/14C-inulin solution. Valspodar was administered first to
Figure 4. Effect of treatment of bEnd3 cells with vehicle (CTRL) or
oleocanthal (OLC) in presence or absence of inhibitors on the
intracellular accumulation of radiolabeled 125I-Aβ40. The data are
expressed as mean ±SEM (n= 3 independent experiments).
*Significantly different from no inhibitor treated cells (P< 0.02).
#Significantly different from control treated cells with inhibitors (P<
0.05).
Figure 5. Quantitative analysis for RAGE in bEnd3 cells treated with
oleocanthal. Cells were treated for 72 h with 0, 25, and 50 μM
concentrations of oleocanthal.
ACS Chemical Neuroscience Research Article
dx.doi.org/10.1021/cn400024q |ACS Chem. Neurosci. XXXX, XXX, XXX−XXXD
allow its brain distribution and interaction with P-gp prior to
125I-Aβ40 microinjection which has rapid brain clearance.
33
P-gp
inhibition significantly lowered BEI% value of 125I-Aβ40 in
control and oleocanthal-treated group (Figure 7B). Thirty
minutes post microinjection of 125I-Aβ40, the pretreatment of
control and oleocanthal-treated mice with valspodar resulted in
about 13% (from 62.0 ±3.0% to 49.3 ±4.6%; P< 0.05) and
40% (79.9 ±1.6% to 39.4 ±2.8%; P< 0.001) reduction in BEI
% values, respectively. The reduction in BEI% following P-gp
inhibition by valspodar in oleocanthal treated group compared
to control group was statistically insignificant (P> 0.05).
Similarly, LRP1 inhibition by RAP significantly reduced 125I-
Aβ40 BEI% (P< 0.05, Figure 7B) in oleocanthal-treated mice.
Pretreatment of control and oleocanthal-treated mice with RAP
resulted in about 33% (from 62.0 ±3.0% to 29.1 ±1.5%; P<
0.001) and 21% (79.9 ±1.6% to 59.2 ±3.5%; P< 0.05)
reduction in BEI% values of 125I-Aβ40, respectively. These
results are in line with similar studies demonstrating the role of
P-gp and LRP1 in the clearance of Aβacross the BBB.
20,22,23
To characterize the effect of oleocanthal treatment on the
degradation of 125I-Aβ40 in C57BL/6 mice brain, we performed
TCA degradation assay and measured the expression of Aβ
degrading enzymes NEP and IDE. The percent of degraded Aβ
peptide (cpm in supernatant) were significantly higher in
oleocanthal treated group (60.0 ±2.3%) compared to control
(40.1 ±1.2%, P< 0.05, Figure 8A). Further, treatment of mice
with oleocanthal resulted in a significant increase (1.6-fold) in
the expression of IDE and small but insignificant increase in
NEP (1.1-fold) in mice brain microvessels, respectively (Figure
8B, C). Consistent with in vivo findings, in vitro treatment of
bEnd3 cells resulted in significant increase in the expression of
IDE (1.6-fold, P< 0.05) and NEP (1.3-fold, P< 0.05) enzymes
(data not shown). Thus, oleocanthal treatment enhanced the
degradation of Aβ40 in mice brain, mostly via induction of IDE
enzyme and possibly NEP.
Collectively, the above results corroborate the assumption
that oleocanthal improves 125I-Aβ40 clearance by enhancing P-
gp and LRP1 expression/activity at the BBB. However, the
inhibition of LRP1 by RAP was more apparent in control mice
than oleocanthal-treated mice, which is consistent with the data
obtained from the in vitro inhibition studies. Yet, Aβclearance
from the brain of oleocanthal-treated mice was greater than
control mice even in the presence of RAP, suggesting the
induction of another mechanism(s) contributing to the
clearance of 125I-Aβ40, which could be an unknown trans-
porter(s)/receptor(s),
23
most likely at the abluminal side, and/
or enhanced degradation of 125I-Aβ40 as a result of the
treatment. Our results confirm both, induction of an unknown
Figure 6. Western blot analysis of P-gp and LRP1 in mice brain
microvessels. Significantly higher expression levels of P-gp and LRP1
were detected in oleocanthal (OLC) treated mice compared to control
group (CTRL). (A) Representative Western blot lanes for P-gp, LRP1,
and protein loading control (β-actin). (B) Quantitative fold increase in
P-gp and LRP1 expressions. The data are expressed as mean ±SEM of
n= 3 independent experiments (*P< 0.05).
Figure 7. (A) Brain efflux index (BEI) of 125I-Aβ40 in control (CTRL),
and oleocanthal (OLC) treated groups measured 24 h after the last
injection of oleocanthal or normal saline. Significantly higher BEI%
was observed in oleocanthal treated mice compared to control group.
(B) Effect of P-gp and LRP1 inhibition by valspodar (24 ng/0.5 μL
injection) and RAP (19.5 ng/0.5 μL injection), respectively, on BEI%
of 125I-Aβ40 in CTRL and OLC treated groups. Both valspodar and
RAP caused a significant reduction in the BEI% in oleocanthal treated
group. The data are expressed as mean ±SEM of n=4−6(*P< 0.05,
***P< 0.001).
ACS Chemical Neuroscience Research Article
dx.doi.org/10.1021/cn400024q |ACS Chem. Neurosci. XXXX, XXX, XXX−XXXE
transport mechanism, supported by the in vitro data, and
degradation. Beside its role in the up-regulation of P-gp and
LRP1, oleocanthal enhanced the clearance of 125I-Aβ40 by up-
regulating NEP (in vitro studies) and IDE, two peptidases that
have been reported to cleave and degrade Aβimplicated in AD.
Several studies reported overexpression of NEP to protect
hippocampal neurons from Aβ-mediated toxicity in vitro,
39
and
the up-regulation of NEP or IDE has been proposed as a
potential means of protecting the brain against Aβaccumu-
lation, prevention of amyloid plaque formation, and consequent
cognitive decline.
40−42
In support of its putative health benefits, oleocanthal has
more recently been demonstrated to possess therapeutic
activities in the treatment of AD disease.
12,14
These studies
support research surveys showing a 40% decrease in AD in
populations consuming a Mediterranean style diet comprising
olive oil.
4
Our findings described in this study provide further
evidence on the role of oleocanthal as a neuroprotective agent
Figure 8. (A) Effect of oleocanthal (OLC) treatment on Aβdegradation in mice brain homogenate compared to control (CTRL) measured using
TCA assay. Significantly higher Aβdegradation% was observed in OLC treated mice compared to control. The data are expressed as mean ±SEM of
n=4−6(*P< 0.05). (B) Representative Western blots lanes for IDE, NEP, and protein loading control (β-actin) in mice brain microvessels.
Significantly higher expression levels of IDE but not NEP were detected in OLC treated mice compared to control. (C) Quantitative fold increase in
IDE and NEP expressions. The data are expressed as mean ±SEM of n= 3 independent experiments (*P< 0.05).
ACS Chemical Neuroscience Research Article
dx.doi.org/10.1021/cn400024q |ACS Chem. Neurosci. XXXX, XXX, XXX−XXXF
against AD in vivo in wild-type mice brains by increasing 125I-
Aβ40 clearance, which substantiate the in vitro findings reported
by us and other research laboratories. Additional research is
required to determine the therapeutic benefits of oleocanthal
using AD animal model, and its bioavailability which has not
been investigated.
In conclusion, our findings demonstrated that the beneficial
effect of oleocanthal against AD could be extended to its ability
to induce P-gp and LRP1, which are responsible for Aβ
clearance across the BBB. Also, this study provides conclusive
evidence for the role of oleocanthal on Aβdegradation as
shown by the up-regulation of Aβdegrading enzymes IDE and
possibly NEP. Furthermore, our results show that extra-virgin
olive oil-derived oleocanthal associated with the consumption
of Mediterranean diet has the potential to reduce the risk of AD
or related neurodegenerative dementias.
■METHODS
Reagents and Antibodies. 1,1,1,3,3,3-Hexafluoro-2-propanol
(HFP) and Tween 20 were purchased from Sigma-Aldrich (St.
Louis, MO). RIPA buffer was purchased from Thermo Scientific
(Rockford, IL). Synthetic monoiodinated and nonoxidized 125I-Aβ40
(human, 2200 Ci/mmol) was purchased from PerkinElmer (Boston,
MA). 14C-Inulin (2.2 mCi/g) was purchased from American
Radiolabeled Chemicals (St. Louis, MO). The human receptor
associated protein (RAP) was purchased from Oxford Biomedical
Research (Oxford, MI). Valspodar was obtained from XenoTech
(Lenexa, KS). The reagents and supplements required for Western
blotting were purchased from Bio-Rad (Hercules, CA). For Western
blot, the mouse monoclonal antibody C-219 against P-gp was obtained
from Covance Research Products (Dedham, MA); mouse monoclonal
antibody against light chain LRP1 (5A6) was obtained from
Calbiochem (Gibbstown, NJ); rabbit monoclonal antibody against
light chain LRP1 was obtained from Epitomics (Burlingame, CA);
goat polyclonal antibodies against, RAGE (N-16), β-actin (C-11), and
HRP-labeled secondary antibodies were purchased from Santa Cruz
Biotechnology Inc. (Santa Cruz, CA); rabbit polyclonal antibody
against insulin degrading enzyme (IDE) and mouse monoclonal
antibody against neprilysin (NEP, anti-CD10) were obtained from
Abcam Inc. (Cambridge, MA). For immunohistochemistry, the rabbit
polyclonal antibody against P-gp was purchased from Rockland
Immunochemicals Inc. (Gilbertsville, PA). Donkey anti-rabbit Alexa
Flour 594 was purchased from Invitrogen (Carlsbad, CA), and goat
anti-mouse IgG-FITC labeled secondary antibody was obtained from
Santa Cruz Biotechnology (Santa Cruz, CA). Formaldehyde 16%
(Methanol Free, Ultrapure EM grade) was purchased from
Polysciences, Inc. (Warrington, PA). Ketamine and xylazine for mice
anesthetization were purchased from Henry Schein Inc. (Melville,
NY). All other reagents and supplies were purchased from VWR
(West Chester, PA).
Extraction of Oleocanthal from Extra-Virgin Olive Oil.
Oleocanthal was extracted as reported by Elnagar et al.
43
from
EVOO (Member’s Mark, batch no. VF1_US102808, Italy) on
lipophilic Sephadex LH20 (Sigma Aldrich, bead size 25−100 μm)
using n-hexane-CH2Cl2(1:9) and finally purified on C-18 reversed-
phase Bakerbond octadecyl (40 μm; Mallinckrodt Baker, Inc.) using
isocratic CH3CN−H2O (40:60). A purity of >90% was established for
oleocanthal as assessed by TLC, 1H NMR spectroscopy, and/or
HPLC analysis.
Cell Culture. The mouse brain endothelial cells (bEnd3; ATCC,
Manassas, VA), passage 25−30, were cultured in DMEM growth
medium supplemented with 10% fetal bovine serum (FBS), and the
antibiotics penicillin G (100 units/mL) and streptomycin (100 μg/
mL), 1% w/v nonessential amino acids, glutamine 2 mM. The cells
were grown to confluence in 75 cm2cell culture flasks for 1−2 days in
a humidified atmosphere (5%CO2/95% air) at 37 °C.
In Vitro Induction of P-gp and LRP1 Expression by
Oleocanthal in bEnd3 Cells. Cells were seeded in 10 mm cell
culture dishes (Corning, NY) at a density of 1 ×106cells per dish. The
cells were allowed to grow up to 50% confluence and then treated with
oleocanthal or control (vehicle) in a humidified atmosphere (5%CO2/
95% air) at 37 °C. Methanolic stock solution of oleocanthal was
diluted to a final concentration of 0.5−50 μM in growth medium
before use. Media containing oleocanthal at different concentrations in
addition to control medium were added to the respective treatment
cells in duplicate at a maximum methanol concentration of 0.2%. The
cells were then incubated for 72 h in a humidified atmosphere (5%
CO2/95% air) at 37 °C. At the end of treatment period, cells were
harvested and total protein was extracted for Western blot analysis.
Western Blot Analysis of P-gp, LRP1, RAGE, IDE, and NEP
Proteins in bEnd3 Cells. At the end of treatment period with
oleocanthal or control, total protein was extracted from bEnd3 cells
with lysis buffer (RIPA buffer; 25 mM Tris.HCl, 150 mM NaCl, 1%
NP-40, 1% sodium deoxylate, 0.1% SDS, pH 7.6). For Western blot
analysis, 25 μg of cellular protein was resolved on 7.5% SDS-
polyacrylamide gels and transferred onto nitrocellulose membrane.
Blotting membranes were blocked with 2% BSA and incubated
overnight with antibodies for LRP1 (light chain), P-gp (C-219), RAGE
(N-16), β-actin (C-11), IDE, or NEP at dilutions 1:1500, 1:200, 1:200,
1:3000, 1:100, and 1:100, respectively. For protein detection, the
membranes were washed and incubated with HRP-labeled secondary
IgG antibody for LRP1, P-gp, NEP (antimouse), IDE (antirabbit) and
RAGE, β-actin (antigoat) at 1:5000 dilution. The bands were
visualized using SuperSignal West Femto Maximum Sensitivity
substrate detection kit (Thermo Scientific; Rockford, IL). Quantitative
analysis of the immunoreactive bands was performed using GeneSnap
luminescent image analyzer (Scientific Resources Southwest Inc.,
Stafford, TX) and band intensity was measured by densitometric
analysis. Three independent Western blotting experiments were
carried out for each treatment group.
In Vitro Uptake Study of 125I-Aβ40 in bEnd3 Cells. The aim of
this experiment was to investigate the effect of oleocanthal treatment
on the accumulation of 125I-Aβ40 in bEnd3 cells. An uptake study using
125I-Aβ40 was conducted as described previously.
31
Briefly, bEnd3 cells
cultured onto 48 well plates at a density of 5 ×104cells/well and were
grown to 50−60% confluence. Following 72 h of treatment with 25
μM of oleocanthal or vehicle, the medium was aspirated and the cells
were incubated in fresh growth medium for 4 h. After treatment, cells
were washed three times with phosphate buffer-saline (PBS) solution.
The cells were then preincubated with or without 100 μM verapamil
(P-gp inhibitor), 1 μM RAP (LRP1 inhibitor), or RAGE (N-16)
antibody (RAGE inhibitor) at a dilution ratio 1:100, in complete
culture medium for 30 min. The activity experiments were started by
the addition of 0.07 nM of 125I-Aβ40 in complete culture media with or
without 100 μM verapamil, 1 μM RAP, or 1:100 dilution ratio of
RAGE (N-16) antibody for 15 min in a humidified atmosphere (5%
CO2/95% air) at 37 °C. The activity experiment was then terminated
by washing the cells three times with ice-cold PBS. The cells were then
disrupted with the lysis buffer containing complete mammalian
protease inhibitor for 15 min at 4 °C. The radioactivity of 125I-Aβ40
accumulated inside the cells was measured using Wallac 1470 Wizard
Gamma Counter (PerkinElmer Inc., Waltham, MA). The data were
normalized for the protein content. The protein concentrations were
determined using the Pierce bicinchoninic acid (BCA) protein assay
kit (Thermo Scientific, Rockford, IL) according to the manufacturer’s
instruction with bovine serum albumin (BSA) as a standard.
Immunofluorescence Staining and Imaging of P-gp and
LRP1 in bEnd3 Cells. To confirm effect of drugs treatment on the
expression and up-regulation of P-gp and LRP1, both proteins were
visualized using confocal microscopy. bEnd3 cells (5 ×104) were
seeded on 35 mm poly-D-lysine coated glass bottom plates no. 1.5
(MatTek Corporation, Ashland, MA) and treated with 25 μMof
oleocanthal or vehicle as described above. After incubation, the cells
were washed three times with PBS and fixed with 4% formaldehyde for
10 min. The cells were then washed with PBS and blocked for 30 min
with 10% of normal donkey and goat sera in 0.3% Triton X-100/PBS.
The cells were then incubated overnight at 4 °C with a 1:200 dilution
of primary antibody against P-gp or LRP1 in solution composed of 1%
ACS Chemical Neuroscience Research Article
dx.doi.org/10.1021/cn400024q |ACS Chem. Neurosci. XXXX, XXX, XXX−XXXG
normal donkey and goat sera in PBS. After washing with PBS, the cells
were incubated for 30 min with antirabbit Alexa flour 594 conjugated
secondary IgG for the detection of LRP1 and antimouse FITC for the
detection of LRP1, both at 1:250 dilution in PBS. Images for P-gp and
LRP1 were captured using Zeiss LSM 5 Pascal confocal microscope
equipped with 543 nm line of HeNe Laser and 63X oil immersion
objective lens with numerical aperture = 1.4 (Carl Zeiss MicroImaging,
LLC, Thornwood, NY). Negative controls for each treatment that
were processed without primary antibody showed negligible back-
ground fluorescence. P-gp and LRP1 membrane immunofluorescence
for each sample was quantified using ImageJ version 1.44 software
(Research Services Branch, NIMH/NIH, Bethesda, MD).
Animals. C57BL/6 wild-type male mice were purchased from
Harlan Laboratories (Houston, TX). The mice were 6−7 weeks old
with an average body weight of 20 g. Mice were kept under standard
environmental conditions (22 °C, 35% relative humidity, 12 h dark/
light cycle) with free access to tap water and standard rodent food.
After shipping, mice were allowed to adapt to the new environment for
one week before initiating the experiments. All animal experiments
were approved by the Institutional Animal Care and Use Committee
of the University of Louisiana at Monroe and all surgical and treatment
procedures were consistent with the IACUC policies and procedures.
In Vivo Induction of P-gp and LRP1 Expression by
Oleocanthal. Animals were divided into two treatment groups:
control and oleocanthal groups. Each group contained at least four
animals. Drug administration was started at 7−8 weeks of age and
continued for 2 weeks. Mice of control group received intraperitoneal
vehicle (normal saline) twice daily. Mice of oleocanthal group received
intraperitoneal oleocanthal at dose of 10 mg/kg twice daily.
Brain Efflux Index Study. At the end of treatment period, mice
were prepared for brain efflux index study (BEI) to assess the clearance
of Aβ24 h after the last dose of oleocanthal. The in vivo Aβclearance
experiments (BEI%) were performed using the intracerebral micro-
injection technique reported previously.
23,36,44,45
In brief, mice were
anesthetized with intraperitoneal xylazine and ketamine (20 and 125
mg/kg, respectively) and placed in a stereotaxic apparatus (Stoelting
Co., Wood Dale, IL) to determine the coordinates of the mice brain
that coincide with the right caudate nucleus. A stainless steel guide
cannula was implanted stereotaxically at 0.9 mm anterior, 1.9 mm
lateral to bregma, and 2.9 mm below the surface of the brain as
previously reported.
20
The guide cannula and screw were fixed to the
skull with binary dental cement, and a stylet was introduced into the
guide cannula. Once the cement was firm, the animal was removed
from the stereotaxic device, and the wound was closed anterior and
posterior to the guide assembly using 1.75 mm Michel suture clips.
Animals were then allowed for 12 h recovery from acute brain injury
due to insertion of guide cannula in order to restore BBB integrity.
45
Animals were reanesthetized with intraperitoneal xylazine/ketamine
and an injector cannula connecting via Teflon tubing to a 1.0 μL
gastight Hamilton microsyringe was inserted into the guide cannula.
The applied tracer fluid (0.5 μL) containing 125I-Aβ40 (30 nM) and
14C-inulin (0.02 μCi) prepared in extracellular fluid buffer (ECF; 122
mM NaCl, 25 mM NaHCO3, 3 m M KCl, 1.4 mM CaCl2, 1.2 mM
MgSO4, 0.4 mM K2HPO4,10mMD-glucose, and 10 mM HEPES, pH
7.4) was administered into the caudate nucleus over a period of 3 min.
To prevent aggregation, 125I-Aβ40 was initially solubilized in HFP,
dried, and resolubilized in ECF buffer prior to the experiment. The
intactness and quality of 125I-Aβ40 was initially confirmed by
trichloacetic acid (TCA) precipitation assay.
20
After the micro-
injection, the microsyringe was left in place for 3 min to minimize
any backflow. At the designated time, 30 min post 125I-Aβ40
injection,
20,22,23
plasma and brain tissues were rapidly collected for
Aβmeasurements. To characterize the role of LRP1 and P-gp
function, 0.5 μL of ECF containing RAP, an LRP1 inhibitor
20,23,46
or
valspodar, a well-established P-gp inhibitor
23,47
were intracerebrally
preadministered 5 min prior to 125I-Aβ40 injection at concentrations of
1μM (19.5 ng/0.5 μL injection volume) and 40 μM (24 ng/0.5 μL
injection volume), respectively.
Calculation of 125I-Aβ40 Clearance. 125I-Aβ40 is characterized by
its rapid clearance across the BBB,
33
thus brain was collected 30 min
postinjection to determine AβBEI%.
20,22,23
Brain tissues were excised
and homogenized by Dounce tissue grinder with seven strokes in two
volumes of DPBS buffer (2.7 mM KCl, 1.46 mM KH2PO4, 136.9 mM
NaCl, 8.1 mM Na2HPO4, 0.9 mM CaCl2, and 0.5 mM MgCl2
supplemented with 5 mM D-glucose and 1 mM sodium pyruvate,
pH 7.4) containing mammalian protease inhibitor cocktail. About half
of the brain homogenate was used for 125I-Aβ40 and 14C-inulin
quantification and the second half for microvessels isolation and
protein expression study. Trichloroacetic acid (TCA) precipitation
method was used to calculate the amount of intact 125I-Aβ40 remained
in the brain.
20,23
To measure intact 125I-Aβ40, one volume of TCA
(20%) was added to the sample, and then samples were vortexed,
incubated in ice for 30 min, and centrifuged at 14 000 rpm (4 °C) for
30 min. Following centrifugation, gamma radioactivity of precipitated
125I-Aβ40 (intact peptide) and TCA supernatant (degraded peptide)
were measured using Wallac 1470 Wizard Gamma Counter. The
supernatant and precipitate were then mixed with 5 mL scintillation
cocktail and the beta radioactivity of 14C-inulin was measured using
Wallac 1414 WinSpectral Liquid Scintillation Counter (PerkinElmer
Inc.). The BEI% was defined by eq 1 and the percentage of substrate
remaining in the brain (100 −BEI%) was determined using eq 2.
46
β
β
=
‐
‐
×BEI% I A brain clearance
I A injected into the brain 100
125
125 (1)
−= ×
β
β
‐
‐
‐
‐
100 BEI% 100
amount of intact I A in the brain
amount of C inulin in the brain
amount of intact I A injected
amount of C inulin injected
125
14
125
14 (2)
The percent degradation for each sample was calculated by dividing
the supernatant cpm by the total cpm (sum of the cpm in the
precipitate and the supernatant cpm) and the resulting percent were
subtracted from the percent of free 125I which determined from
preinjected sample by spiking mice brain homogenate with 0.5 μL
injectate solution and then processed as sample homogenate.
Isolation of Brain Microvessels. Brain microvessels were isolated
as described previously by us.
23
Briefly, after decapitation of mouse,
brain was immediately put in ice-cold normal saline, homogenized and
divided into halves as described above. One volume Ficoll (30%) was
added to the half of brain homogenate to a final concentration of 15%
and the mixture was mixed, and then centrifuged (5000 rpm for 10
min, 4 °C). The resulting pellets were suspended in ice-cold DPBS
containing 1% bovine serum albumin (BSA) and passed over glass
bead column. Microvessels adhering to the glass beads were collected
by gentle agitation in 1% BSA in DPBS. Isolated microvessels were
used for Western blotting and immunohistochemistry studies of LRP1
and P-gp.
Western Blot Analysis of P-gp, LRP1, and RAGE in Isolated
Brain Microvessels. Protein expression levels in isolated brain
microvessels were analyzed by Western blotting as described above.
Total protein was extracted from isolated brain microvessels by
homogenization with lysis buffer (RIPA buffer) containing protease
inhibitor. Homogenized samples were centrifuged at 13 000 rpm for
10 min and supernatant was used for further Western blot analysis as
described above.
Statistical Analysis. Wherever possible, the experimental results
were analyzed for statistically significant difference using Two-tailed
unpaired Student’st-test to evaluate differences between controls and
treated groups. A p-value less than 0.05 was considered to be
statistically significant. All results were expressed as means and
standard error of mean (SEM).
■AUTHOR INFORMATION
Corresponding Author
*Telephone: 318-342-1460. Fax: 318-342-1737. E-mail:
kaddoumi@ulm.edu.
ACS Chemical Neuroscience Research Article
dx.doi.org/10.1021/cn400024q |ACS Chem. Neurosci. XXXX, XXX, XXX−XXXH
Author Contributions
All in vitro and in vivo experiments and data analysis were
performed by A.H.A. and H.Q. Oleocanthal extraction from
EVOO and characterization were performed by B.A.B. and
supervised by K.A.E. All experiments were designed and
supervised by A.K.
Funding
This research work was funded by an Institutional Develop-
ment Award (IDeA) from the National Institute of General
Medical Sciences of the National Institutes of Health under
Grant Number P20GM103424.
Notes
The authors declare no competing financial interest.
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