Restoring Blood-Brain Barrier P-Glycoprotein Reduces Brain
Amyloid-? in a Mouse Model of Alzheimer’s Disease□
Anika M. S. Hartz, David S. Miller, and Bjo ¨rn Bauer
Department of Biochemistry and Molecular Biology, University of Minnesota Medical School, Duluth, Minnesota (A.M.S.H.);
Laboratory of Toxicology and Pharmacology, National Institute of Environmental Health Sciences, National Institutes of Health,
Research Triangle Park, North Carolina (D.S.M.); and Department of Pharmaceutical Sciences, College of Pharmacy, University
of Minnesota, Duluth, Minnesota (B.B.)
Received October 13, 2009; accepted January 25, 2010
Reduced clearance of amyloid-? (A?) from brain partly under-
lies increased A? brain accumulation in Alzheimer’s disease
(AD). The mechanistic basis for this pathology is unknown, but
recent evidence suggests a neurovascular component in AD
etiology. We show here that the ATP-driven pump, P-glyco-
protein, specifically mediates efflux transport of A? from mouse
brain capillaries into the vascular space, thus identifying a
critical component of the A? brain efflux mechanism. We dem-
onstrate in a transgenic mouse model of AD [human amyloid
precursor protein (hAPP)-overexpressing mice; Tg2576 strain]
that brain capillary P-glycoprotein expression and transport
activity are substantially reduced compared with wild-type con-
trol mice, suggesting a mechanism by which A? accumulates in
the brain in AD. It is noteworthy that dosing 12-week-old,
asymptomatic hAPP mice over 7 days with pregnenolone-16?-
carbonitrile to activate the nuclear receptor pregnane X recep-
tor restores P-glycoprotein expression and transport activity in
brain capillaries and significantly reduces brain A? levels com-
pared with untreated control mice. Thus, targeting intracellular
signals that up-regulate blood-brain barrier P-glycoprotein in
the early stages of AD has the potential to increase A? clear-
ance from the brain and reduce A? brain accumulation. This
mechanism suggests a new therapeutic strategy in AD.
A hallmark of Alzheimer’s disease (AD) is the accumu-
lation of neurotoxic amyloid-? (A?) peptide within the
brain. The A? transport-clearance hypothesis of AD pro-
posed by Zlokovic and coworkers (Zlokovic and Frangione,
2003; Deane et al., 2004b; Zlokovic, 2005) states that re-
duced A? clearance (reduced A? efflux transport) from the
brain underlies A? brain accumulation (see also Moora-
dian et al., 1997). This hypothesis suggests that the mech-
anism responsible for A? brain clearance itself could be a
therapeutic target in AD.
A? clearance from brain to blood has to be a two-step
process. A? must first pass through the abluminal (brain
side) and then the luminal (blood side) plasma membranes of
the brain capillary endothelial cells that comprise the blood-
brain barrier. Given that A? is a peptide, both steps must be
facilitated, involving receptors or transporters. At the ablu-
minal membrane, the receptor low-density lipoprotein recep-
tor-related protein 1 (LRP1) seems to be the major protein
responsible for A? uptake from brain into capillary endothe-
lial cells (Shibata et al., 2000; Deane et al., 2004a,b). How-
ever, the luminal membrane protein mediating the critical
second step, A? efflux from the endothelial cells into the
blood, has not been identified.
One candidate is P-glycoprotein, an ATP-driven efflux
transporter that under normal physiological conditions is
highly expressed at the luminal membrane of the brain cap-
illary endothelium. This transporter handles a wide spec-
trum of nonpolar, therapeutic drugs, some of which are small
polypeptide derivatives (Miller et al., 2008). Limited data
This research was supported in part by the Intramural Research Pro-
gram of the National Institutes of Health National Institute of Environ-
mental Health Sciences [Grant Z01-ES080048]; a Duluth Medical Research
Institute Grant; and University of Minnesota College of Pharmacy startup
Article, publication date, and citation information can be found at
The online version of this article (available at http://molpharm.
aspetjournals.org) contains supplemental material.
ABBREVIATIONS: A?, amyloid-?; hA?, human A?; AD, Alzheimer’s disease; BCRP, breast cancer resistance protein; FTC, fumitremorgin C;
hAPP, human amyloid precursor protein; LTC4, leukotriene C4; LRP1, low-density lipoprotein receptor-related protein 1; MRP, multidrug
resistance-associated protein; NBD-CSA, [N-? (4-nitrobenzofurazan-7-yl)-D-Lys8]-cyclosporine A; PCN, pregnenolone-16?-carbonitrile; PSC833,
valspodar; PXR, pregnane X receptor; RAGE, receptor for advanced glycation end products; RAP, receptor-associated protein; PBS, phosphate-
buffered saline; BSA, bovine serum albumin; IACUC, Institutional Animal Care and Use Committee; ELISA, enzyme-linked immunosorbent assay;
GLUT-1, glucose transporter 1.
U.S. Government work not protected by U.S. copyright
Mol Pharmacol 77:715–723, 2010
Vol. 77, No. 5
Printed in U.S.A.
with artificial model systems such as transporter-overex-
pressing cell lines of nonbrain endothelial origin and mem-
brane vesicles from these cell lines suggest that P-glycopro-
tein can transport A? (Lam et al., 2001; Cirrito et al., 2005;
Kuhnke et al., 2007), and one report suggests reduced A?
efflux from the brain in P-glycoprotein-null mice (Cirrito et
al., 2005). However, those mice also exhibit substantially
reduced expression of LPR1 in cerebral vessels, so the in-
volvement of blood-brain barrier P-glycoprotein in A? efflux
in AD remains unclear.
Here, we establish a role for blood-brain barrier P-glycopro-
tein in the efflux transport of A? from the brain into capillary
lumen. We use a transgenic mouse model of AD [human amy-
loid precursor protein (hAPP)-overexpressing mice; Tg2576
strain] to test the hypotheses that defective transport mediated
by P-glycoprotein contributes to A? accumulation within the
brain and that restoring such transport can reduce brain A?
levels. We demonstrate here for the first time that P-glycopro-
tein mediates efflux transport of A? in intact brain capillaries.
We show that P-glycoprotein expression and transport activity
are substantially reduced in brain capillaries from hAPP mice.
These experiments were done with 12-week-old transgenic
mice, at a time when brain hAPP and human A? (hA?) levels
are substantial, but when there is not yet evidence of cognitive
impairment (Hsiao et al., 1996; Kawarabayashi et al., 2001).
Finally, and most importantly, using this AD model we show
that in vivo activation of the nuclear receptor pregnane X re-
ceptor (PXR) over 7 days both restores P-glycoprotein expres-
sion and activity at the blood-brain barrier and significantly
reduces A? brain levels.
These results suggest that up-regulating blood-brain barrier
that could lower A? brain levels and delay onset and progres-
sion of AD.
Materials and Methods
Chemicals. Antibodies against RAGE, human APP, human
A?40, human A?42, and ?-actin were purchased from Abcam, Inc.
(Cambridge, MA). Note that in Western blots using the antibodies to
detect human APP, human A?40, and human A?42, we observed a
single band at the right molecular weight for each of these proteins,
indicating that the antibodies used did not cross-react with other
amyloid species and are thus specific for the corresponding amyloid
isoform. C219 antibody against P-glycoprotein was from Signet Lab-
oratories (Dedham, MA), LRP1 antibody was obtained from Calbio-
chem-Novabiochem (San Diego, CA), and PXR antibody was from
BioLegend (San Diego, CA). Fluorescein-hA?42 [fluorescein-?A(1–
42)] was purchased from rPeptide (Bogart, GA), fumitremorgin C
(FTC) was from Alexis Axxora (San Diego, CA), receptor-associated
protein (RAP) was from Calbiochem-Novabiochem, and safflower oil
was from MP Biomedicals (Solon, OH). [N-? (4-nitrobenzofurazan-7-
yl)-D-Lys8]-cyclosporine A (NBD-CSA) was custom-synthesized by R.
Wenger (Basel, Switzerland) (Wenger, 1986). PSC833 (valspodar)
was a kind gift from Novartis (Basel, Switzerland). Sulforhodamine
101 free acid, NaCN, mannitol, leukotriene C4(LTC4), pregnenolone-
16?-carbonitrile (PCN), and all other chemicals were of the highest
grade and purchased from Sigma-Aldrich (St. Louis, MO).
Animals. Male transgenic human APP-overexpressing mice
[Tg2576 strain; 129S6.Cg-Tg(APPSWE)2576Kha] and corresponding
male wild-type mice were purchased from Taconic Farms (German-
town, NY). Mice were 12 weeks old with an average body weight of
29 g for wild-type mice and 31 g for transgenic hAPP mice. Animals
were kept under controlled environmental conditions (23°C, 35%
relative humidity, 12-h dark-light cycle) with free access to tap water
and standard rodent feed. After shipping, animals were allowed
to adapt to the new environment for at least 1 week before experi-
ments. Animal protocols were approved by the Institutional Animal
Care and Use Committees (IACUC) of the University of Minnesota
(IACUC protocol 0801A25321; principal investigator Bjoern Bauer)
and National Institutes of Health/National Institute on Environmen-
tal Health Sciences (IACUC protocol LPC99-14; principal investiga-
tor David Miller) and were in accordance with the Guides to Animal
Use of the University of Minnesota, National Institutes of Health
guidelines, Association for Assessment and Accreditation of Labora-
tory Animal Care regulations, and the U.S. Department of Agricul-
ture Animal Welfare Act.
PCN Dosing and Facial Vein Bleeding. Twenty hAPP trans-
genic mice received daily one injection of 25 mg/kg i.p. PCN for 7
days. Twenty hAPP transgenic controls and 20 wild-type mice re-
ceived vehicle (safflower oil). Blood samples were taken by facial vein
bleeding on days 0, 1, 3, and 5 from seven to eight animals per group.
Twenty-four hours after the last injection, mice were euthanized by
CO2inhalation and decapitated, and trunk blood was collected.
Brains from each animal were collected for A? determination and
isolation of brain capillaries.
Brain Capillary Isolation. Brain capillaries were isolated as
described previously (Hartz et al., 2008). In brief, mice were eutha-
nized by CO2inhalation and then decapitated. Brains were removed,
dissected, and homogenized in ice-cold phosphate-buffered saline
(PBS) 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 MgCl2supplemented
with 5 mM D-glucose, 1 mM sodium pyruvate, pH 7.4). Ficoll was
added to a final concentration of 15%, and the homogenate was
centrifuged at 5,800g for 20 min at 4°C. After resuspending the pellet
in 1% bovine serum albumin (BSA)/PBS, the capillary suspension
was passed over a glass bead column. Capillaries adhering to the
glass beads were collected by gentle agitation in 1% BSA/PBS. Cap-
illaries were washed with BSA-free PBS and used for experiments.
P-Glycoprotein Transport Assay. To determine P-glycoprotein
transport activity, freshly isolated brain capillaries were incubated
for 1 h at room temperature with the fluorescent P-glycoprotein-
specific substrate NBD-CSA (2 ?M in PBS buffer) (Hartz et al.,
2008). For A?-transport studies, capillaries were incubated for 1 h at
room temperature with 5 ?M fluorescein-hA?42 in PBS buffer. For
each treatment, images of 10 capillaries were acquired by confocal
microscopy [Nikon C1 LSC microscope unit, Nikon TE2000 inverted
microscope, 40? oil immersion objective, numerical aperture 1.3,
488-nm line of an argon laser, Nikon Instruments Inc. (Melville, NY),
or Zeiss LSM 510 META inverted confocal microscope, 40? water
immersion objective, NA 1.2, 488-nm line of argon laser, Carl Zeiss
Inc. (Thornwood, NY)]. Images were analyzed by quantitating lumi-
nal NBD-CSA fluorescence with Zeiss Image Examiner and Image J
1.41 software (Research Services Branch, National Institute of Mental
Health/National Institutes of Health, Bethesda, MD). As before, spe-
cific, luminal NBD-CSA fluorescence was taken as the difference be-
tween total luminal fluorescence and fluorescence in the presence of the
P-glycoprotein-specific inhibitor PSC833 (5 ?M) (Hartz et al., 2008).
Sulforhodamine 101 Accumulation Assay. Freshly isolated
mouse brain capillaries were incubated with 2 ?M sulforhodamine
101 for 1 h alone or transporter inhibitors as indicated. For each
treatment group, confocal images (Nikon C1 LSC microscope unit,
Nikon TE2000 inverted microscope, 40? oil immersion objective,
numerical aperture 1.3, 488-nm line of an argon laser; Nikon Instru-
ments Inc.) were obtained from 10 capillaries. Luminal sulforhodam-
ine 101 fluorescence intensity was measured with Image J software
(ver. 1.41; http://rsbweb.nih.gov/ij/index.html) as described previ-
ously (Bauer et al., 2008).
A? Immunostaining of Mouse Brain Capillaries. Isolated
mouse brain capillaries were fixed with 3% paraformaldehyde/0.25%
glutaraldehyde for 15 min at room temperature. After washing with
PBS, capillaries were permeabilized with 0.5% Triton X-100 for 30
Hartz et al.
min and washed with PBS. Capillaries were blocked with 1% BSA for
60 min and incubated overnight at 4°C with a 1:500 dilution of
monoclonal antibody against hA?40 or polyclonal antibody against
hA?42. Capillaries were washed and incubated with Alexa-Fluor
488-conjugated secondary IgG (1:750; Invitrogen, Carlsbad, CA) for
1 h at 37°C. Nuclei were counterstained with 2 ?g/ml of propidium
iodide. Negative controls for each treatment that were processed
without primary antibody showed negligible background fluores-
cence (data not shown). A? immunofluorescence was visualized by
confocal microscopy (Nikon C1 LSC microscope unit, Nikon TE2000
inverted microscope, 40? oil immersion objective, NA 1.3, 488 nm
line of an argon laser, 543 nm line of a HeNe laser; Nikon Instru-
ments Inc.). For each treatment, confocal images of 10 capillaries
were acquired. A? membrane immunofluorescence for each capillary
was quantitated with Image J software. A 10 ? 10 grid was super-
imposed on each image, and fluorescence measurements of capillary
membranes were taken between intersecting grid lines. Fluores-
cence intensity for each capillary was the mean of six measurements
Western Blotting. Protein expression levels from different tissues
were analyzed by Western blotting as described previously (Hartz et al.,
2008). Isolated capillaries were homogenized in lysis buffer (Sigma-
Aldrich) containing Complete protease inhibitor (Roche Diagnostics,
Mannheim, Germany). Homogenized samples were centrifuged at
10,000g for 15 min, and denucleated supernatants were centrifuged at
100,000g for 90 min to obtain brain capillary membranes. Brain capil-
lary membranes were resuspended and stored at ?80°C.
Western blots were performed by using the Invitrogen NuPage Bis-
Tris electrophoresis and blotting system. After protein electrophoresis
and transfer, blotting membranes were blocked and incubated over-
night with the primary antibody as indicated. Membranes were washed
and incubated with horseradish peroxidase-conjugated ImmunoPure
secondary IgG (1:15,000; Thermo Fisher Scientific, Waltham, MA) for
1 h. Proteins were detected by using SuperSignal West Pico Chemolu-
minescent Substrate (Thermo Fisher Scientific). Protein bands were
system (Bio-Rad Laboratories, Hercules, CA). QuantityOne 1-D soft-
ware (vers. 4.6.5; Bio-Rad Laboratories) was used for densitometric
analyses of band intensities and digital molecular weight analyses;
molecular weight markers used were RPN800 (GE Healthcare, Chal-
font St. Giles, Buckinghamshire, UK) and NOVEX Sharp PS (Invitro-
gen). Linear adjustments of contrast and brightness were applied to
entire Western blot images. None of the Western blots shown were
modified by nonlinear adjustments.
ELISA for hA?40 and hA?42. hA?40 and hA?42 was quanti-
tated in plasma and brain samples by enzyme-linked immunosor-
bent assay (ELISA) (Invitrogen) according to the manufacturer’s
protocol. Plasma was obtained from blood samples by centrifugation
at 5,000g for 5 min at 4°C. To determine hA?40 plasma levels,
samples were diluted 1:20 before analysis; to determine hA?42 lev-
els, samples were diluted 1:4 before analysis. hA?40 and hA?42 were
extracted from brain tissue from APP and APP-PCN mice by homog-
enization with Tris-HCl buffer containing 5 M guanidine-HCl. Sam-
ples were diluted 1:20 in Dulbecco’s PBS buffer containing 5% BSA
and 0.03% Tween 20 and centrifuged at 16,000g for 20 min at 4°C.
Supernatant was collected and diluted 1:1 with buffer before ELISA
Statistical Analysis. Data are presented as mean ? S.E.M. Two-
tailed unpaired Student’s t test was used to evaluate differences
between controls and treated groups; differences were considered to
be statistically significant at P ? 0.05.
Blood-Brain Barrier P-Glycoprotein Transports hA?42.
To investigate the role of P-glycoprotein in A? efflux at the
blood-brain barrier, we used freshly isolated brain capillaries
from normal wild-type mice. Our previous studies have
shown that isolated brain capillaries from both mouse and
rat are morphologically intact and remain metabolically ac-
tive and functionally capable of ATP-driven transport for up
to 12 h (Bauer et al., 2006; Hartz et al., 2008; data not
shown). Thus, isolated brain capillaries closely mimic the
blood-brain barrier in vivo and are well suited to study trans-
port processes across the brain capillary endothelium.
In the present study, we incubated isolated mouse brain
capillaries with fluorescein-hA?42 and measured its accumu-
lation in capillary lumens by using confocal microscopy and
quantitative imaging. Fluorescein-hA?42 accumulated to
high levels in the lumens of control capillaries, indicating
active transport from bath to vascular space (Fig. 1, A and B;
Supplemental Fig. 1). Luminal fluorescence was significantly
(P ? 0.001) reduced by the P-glycoprotein-specific inhibitors
PSC833 (valspodar), XR9576 (tariquidar), ivermectin, cyclo-
sporin A, and verapamil (Mayer et al., 1997; Fellner et al.,
2002), the metabolic inhibitor NaCN, and the LRP1-specific
inhibitor RAP. FTC, Ko143, LTC4, and probenecid, inhibitors
of the ATP-driven efflux transporters, breast cancer resis-
tance protein (BCRP), and multidrug resistance-associated
proteins (MRPs) were without effect. These results are con-
sistent with blood-brain barrier A? efflux being an active and
ATP-dependent two-step process, involving LRP1-mediated
A? uptake from brain into capillary endothelial cells followed
by P-glycoprotein-mediated A? efflux from endothelium into
blood (Fig. 1C).
P-Glycoprotein Is Compromised at the Blood-Brain
Barrier of hAPP Transgenic Mice. To test the hypothesis
that P-glycoprotein is compromised at the blood-brain barrier
in AD, we measured P-glycoprotein expression and transport
activity in brain capillaries isolated from 12-week-old, male
wild-type and hAPP transgenic mice (Tg2576; overexpress-
ing hAPP with the Swedish mutation), a well established
animal model of AD (Hsiao et al., 1996). At 12 weeks of age,
hAPP transgenic mice exhibit accumulation of human A? in
brain, but show no evidence of cognitive impairment, which
does not start until approximately 6 months of age and then
progressively worsens (Hsiao et al., 1996; Kawarabayashi et
al., 2001). Figure 2A shows representative images of isolated
brain capillaries from wild-type and hAPP mice that were
incubated to steady state with the fluorescent dye NBD-CSA,
a P-glycoprotein substrate. Using isolated brain capillaries
from rats and mice we previously demonstrated that
PSC833-sensitive luminal accumulation of this dye is a spe-
cific measure of P-glycoprotein transport activity (Hartz et
al., 2008). In capillaries from wild-type mice, luminal NBD-
CSA fluorescence was high and sensitive to P-glycoprotein
inhibition with PSC833. Comparison of luminal NBD-CSA
fluorescence in capillaries from wild-type and hAPP mice
indicated a 70% decrease in P-glycoprotein transport activity
for the latter (Fig. 2B). Consistent with A? being a P-glyco-
protein substrate, isolated brain capillaries from hAPP mice
exhibited substantially reduced fluorescein-hA?42 transport
Reduced luminal accumulation of NBD-CSA and fluores-
cein-hA?42 in brain capillary lumens from hAPP mice could
reflect reduced P-glycoprotein expression or increased per-
meability of the tight junctions that separate endothelial
cells within the endothelium (Hartz et al., 2004). If tight
junctional permeability were altered, one would expect to see
P-Glycoprotein in Alzheimer’s Disease
Fig. 1. P-glycoprotein mediates hA?42
transport in mouse brain capillaries. A, rep-
resentative confocal images of brain capillar-
ies isolated from wild-type mice. Capillaries
for 1 h alone (control) or with 5 ?M fluo-
rescein-hA?42 plus PSC833 (P-glycopro-
tein inhibitor), NaCN (metabolic inhibitor),
fluorescein-hA?42 fluorescence after image
analysis. Residual fluorescence is caused by
nonspecific binding (Hartz et al., 2008). Data
represent mean ? S.E.M. for 10 capillaries
from one preparation (pooled tissue from 10
wild-type mice). Shown are arbitrary fluo-
rescence units (scale 0–255). ???, signifi-
cantly lower than control, P ? 0.001. C,
proposed two-step mechanism of blood-
brain barrier A? efflux involving the A?
receptor LRP1, on the abluminal membrane
and the efflux transporter, P-glycoprotein, on
the luminal membrane of brain capillaries.
Fig. 2. P-glycoprotein expression and trans-
port activity are reduced at the blood-brain
barrier of hAPP mice. A, representative im-
ages of brain capillaries isolated from 12-
week-old wild-type and hAPP mice. Capillar-
ies were incubated with 2 ?M NBD-CSA, a
fluorescent P-glycoprotein-specific substrate,
for 1 h alone or with PSC833. B, specific
(PSC833-sensitive) luminal NBD-CSA fluo-
rescence after image analysis of brain capil-
laries. C, luminal fluorescein-hA?42 fluores-
cence in brain capillaries from wild-type and
hAPP mice. D, luminal fluorescence of the
damine 101, in brain capillaries alone (con-
trol) or with mannitol (osmotic tight junction
disruptor), LTC4(MRP inhibitor), NaCN
inhibitor), RAP (LRP1 inhibitor), or FTC
(BCRP inhibitor). Data in B–D are mean ?
(pooled tissue from 10–20 mice per group).
Shown are arbitrary fluorescence units (scale
0–255). ???, significantly lower than control,
P ? 0.001. E and F, Western blots for P-
GLUT-1 (F) of brain capillary membranes
from wild-type and hAPP mice. ?-Actin was
used as protein loading control (pooled tissue
from 20 mice per group).
Hartz et al.
similar effects for all transporters that are capable of driving
concentrative luminal substrate accumulation. In this re-
gard, we recently demonstrated that transport of a fluores-
cent organic anion, sulforhodamine 101, into brain capillary
lumens is mediated by another ATP-driven efflux pump,
Mrp2 (multidrug resistance-associated protein isoform 2)
(Bauer et al., 2008). When we compared the ability of brain
capillaries from wild-type and hAPP mice with transport
sulforhodamine 101, we found no difference (Fig. 2D, Supple-
mental Fig. 2). Unaltered luminal accumulation of sulforho-
damine 101 indicates intact tight junctions in brain capillar-
ies from hAPP mice. Thus, reduced luminal NBD-CSA and
fluorescein-hA?42 accumulation found in brain capillaries
from hAPP mice is caused by reduced P-glycoprotein trans-
port activity at the blood-brain barrier of these mice.
Consistent with reduced transport activity, we found a
marked decrease of P-glycoprotein protein in brain capillary
plasma membranes isolated from hAPP mice compared with
membranes from wild-type controls (Fig. 2E). Western blot
density measurements showed that P-glycoprotein levels
were significantly decreased by approximately 60% [P-glyco-
protein in hAPP mice: 39 ? 6% (S.E.M., P ? 0.01) of wild-type
control, P-glycoprotein levels normalized to ?-actin]. Thus,
despite the absence of cognitive impairment, 12-week-old
hAPP mice display early biochemical and physiological
changes in expression and transport activity of P-glycopro-
tein at the blood-brain barrier. In agreement with previous
reports (Mooradian et al., 1997; Hooijmans et al., 2007), we
also detected reduced protein expression of glucose trans-
porter 1 (GLUT-1) in brain capillaries from hAPP mice. In
contrast, in capillaries from hAPP mice, expression of the
abluminal A?-uptake receptor LRP1 was slightly increased
and that of the luminal A?-uptake receptor RAGE was un-
changed (Fig. 2F). This indicates that decreased A? efflux
transport in brain capillaries from hAPP mice was not caused
by altered LRP1 or RAGE expression. Thus, these data sug-
gest that the critical limiting step in A? brain clearance in
AD seems to be A? transport across the luminal membrane
into the vascular space.
PXR Activation Restores Blood-Brain Barrier P-Gly-
coprotein in hAPP Mice. We previously demonstrated that
blood-brain barrier P-glycoprotein can be up-regulated by in
vivo exposure to ligands that activate PXR, a nuclear receptor
that controls expression of drug metabolizing enzymes and ef-
flux transporters (Kliewer et al., 1998; Geick et al., 2001; Bauer
et al., 2004, 2006). In preliminary experiments we found that
PXR protein was expressed in brain capillaries from wild-type
and hAPP mice, but expression was slightly lower in capillaries
from hAPP mice (Supplemental Fig. 3). To determine whether
PXR activation in vivo could restore P-glycoprotein expression
and transport activity at the blood-brain barrier, we dosed
hAPP mice with the prototypical rodent PXR ligand, PCN, at 25
mg/kg i.p. once a day for 7 days (Kliewer et al., 1998). PCN
dosing of hAPP mice increased P-glycoprotein expression in
type mice [Fig. 3A; hAPP: 56 ? 8% (S.E.M.; P ? 0.03) of control;
hAPP?PCN: 92 ? 9% (S.E.M.; not statistically significant, P ?
0.49) of control as determined by optical density measurements;
P-glycoprotein levels normalized to ?-actin]. Consistent with
increased protein expression, specific (PSC833-sensitive) P-gly-
coprotein transport activity in brain capillaries from PCN-
treated hAPP mice was restored to levels in wild-type mice for
both NBD-CSA and fluorescein-hA?42 (Figs. 3, B and C and 4).
PXR protein expression in brain capillaries was slightly in-
creased by PCN dosing as reported previously (Pascussi et al.,
2000); LRP1, RAGE, and GLUT-1 protein levels were unaf-
fected (Fig. 5). Thus, PCN dosing of hAPP mice selectively
up-regulated P-glycoprotein at the blood-brain barrier and the
newly synthesized transporter protein was functionally active.
Fig. 3. PXRactivationinhAPPmicerestores
P-glycoprotein expression and transport of
NBD-CSA. A, P-glycoprotein (P-gp) Western
blot of brain capillary membranes isolated
from vehicle-treated wild-type and hAPP
mice and hAPP mice dosed with 25 mg/kg
PCN once a day for 7 days. ?-Actin was
used as protein loading control (pooled
tissue from 20 mice per group). B, accu-
mulation of NBD-CSA in brain capillaries
from vehicle-treated wild-type and hAPP
mice and PCN-treated hAPP mice. Capil-
laries were incubated with 2 ?M NBD-
CSA for 1 h alone or with PSC833. C, data
after capillary image analysis. Data are
mean ? S.E.M. for 10 capillaries from one
preparation (pooled tissue from 20 mice
per group). Shown are arbitrary fluores-
cence units (scale 0–255). ???, signifi-
cantly lower than control, P ? 0.001.
P-Glycoprotein in Alzheimer’s Disease
Restoring Blood-Brain Barrier P-Glycoprotein Re-
duces A? Brain Levels. We next determined the conse-
quences of restoring blood-brain barrier P-glycoprotein on
hA? levels in hAPP mice. We initially measured hA?40 and
hA?42 in plasma by ELISA but found no difference between
untreated hAPP mice and PCN-treated hAPP mice over the
7-day period of PCN dosing (Supplemental Fig. 4, A and B).
This finding is consistent with rapid A? clearance from blood
by filtration at the kidney.
An early sign of AD is cerebral amyloid angiopathy, char-
acterized by A? deposition in brain capillary membranes
(Greenberg et al., 2004; Iadecola, 2004; Zlokovic, 2008). To
measure capillary-associated A?, we isolated brain capillar-
ies from hAPP control and PCN-dosed mice and immuno-
stained for hA?40 and hA?42. Capillaries from untreated
hAPP mice stained positive for both A? peptides (Fig. 6,
A–D). Seven days of PCN treatment significantly decreased
membrane immunofluorescence of both peptides in brain
capillaries from hAPP mice by 28% for hA?40 (P ? 0.05) and
31% for hA?42 (P ? 0.01) compared with capillaries from
untreated hAPP mice (Fig. 6, A–D). Western blotting of brain
capillary membranes also showed reduced levels of hA?40
and hA?42 in hAPP mice treated with PCN compared with
Fig. 4. PXR activation in hAPP mice restores P-glycoprotein-mediated
hA?42 transport. A, representative images of brain capillaries isolated from
wild-type, hAPP, and PCN-treated hAPP mice. Capillaries were incubated
with 5 ?M fluorescein-hA?42 for 1 h. B, data after capillary digital image
analysis. Data are mean ? S.E.M. for 10 capillaries from one preparation
(pooled tissue from 20 mice per group). Shown are arbitrary fluorescence
units (scale 0–255). ???, significantly lower than control, P ? 0.001.
Fig. 5. Western blots for indicated proteins of capillary membranes from
vehicle- and PCN-treated hAPP mice (pooled tissue from 20 mice per group).
Fig. 6. Restoring P-glycoprotein in hAPP mice reduces A? levels in brain
capillaries. A and B, representative images of hA?40-immunostained (A)
and hA?42-immunostained (B) brain capillaries from vehicle- and PCN-
treated hAPP mice. C and D, data from membrane hA?-immunofluores-
cence analysis. Data are mean ? S.E.M. for 10 capillaries (pooled tissue
from 20 mice per group). Shown are arbitrary fluorescence units (scale
0–255). ?, significantly lower than control, P ? 0.05; ??, significantly
lower than control, P ? 0.01. E, hA?40 and hA?42 Western blots of brain
Hartz et al.
untreated hAPP mice [Fig. 6E; hA?40: 82 ? 1.4% (S.E.M.;
P ? 0.05) of control, hA?42: 45 ? 4.4% (S.E.M.; P ? 0.001) of
control as determined by optical density measurements;
hA? levels normalized to ?-actin]. Thus, PCN dosing
clearly reduced capillary-associated A?, suggesting re-
Finally, Western blots and ELISA analysis showed signif-
icantly (P ? 0.001) reduced hA?40 and hA?42 levels in brain
tissue of hAPP mice dosed with PCN compared with un-
treated hAPP control mice (Fig. 7). Optical density measure-
ments of Western blots revealed that PCN dosing reduced
brain levels of hA?40 and hA?42 by approximately 50 and
60%, respectively [Fig. 7A; hA?40: 50 ? 4.5% (S.E.M., P ?
0.001) of control; hA?42: 36 ? 1.0% (S.E.M., P ? 0.001) of
control; hA? levels normalized to ?-actin]. This was con-
firmed by ELISA measurements that demonstrated that
brain levels of hA?40 and hA?42 were reduced by approxi-
mately 35 and 60%, respectively [Fig. 7, B and C; hA?40:
66 ? 3% (S.E.M., P ? 0.001) of control; hA?42: 38 ? 2%
(S.E.M., P ? 0.001) of control]. It is noteworthy that hAPP
brain levels were not affected (Fig. 7A), indicating that PCN-
induced PXR activation did not change A? precursor levels.
Thus, activation of PXR increased blood-brain barrier P-
glycoprotein expression and transport activity, which acutely
reduced hA?40 and hA?42 brain levels in hAPP mice.
The present study reconciles three disparate observations,
each of which suggests that P-glycoprotein is a critical com-
ponent of the A? brain clearance mechanism: 1) cell lines
that overexpress human P-glycoprotein transport A? (Lam et
al., 2001; Kuhnke et al., 2007; Tai et al., 2009); 2) injecting a
P-glycoprotein inhibitor into hAPP transgenic mice increases
A? brain levels (Cirrito et al., 2005); and 3) brain deposition
of A? in elderly, nondemented patients is inversely correlated
with brain capillary expression of P-glycoprotein (Vogelgesang
et al., 2002, 2004). Indeed, we provide here compelling evidence
for both P-glycoprotein contributing significantly to A? efflux
across the luminal membrane of the brain capillary endothe-
lium and defective P-glycoprotein-mediated A? efflux transport
in a mouse model of AD.
Recent studies and our current work argue for an impor-
tant neurovascular component in AD etiology. They show
that at least three plasma membrane proteins facilitate A?
movement across the brain capillary endothelium: RAGE,
LRP1, and P-glycoprotein (Fig. 1C) (present study; Deane et
al., 2003, 2004b). Note that a recent publication suggests that
the blood-brain barrier ATP-driven efflux pump BCRP could
affect brain levels of A? (Xiong et al., 2009). This work
indicates that BCRP transports A? in BCRP-overexpressing
cell lines, A? accumulates in the brain of BCRP null mice,
and BCRP expression is increased in brain samples from
patients with AD and AD brains from two AD mouse models.
However, the latter results are contrary to what one would
expect if BCRP contributed to A? efflux from the brain and
reduced BCRP-mediated efflux contributed to A? accumula-
tion. In addition, the findings by Xiong et al. (2009) in pa-
tients were made after AD diagnosis, which is based on
cognitive symptoms. Likewise, based on the age of the AD
mice used in the study by Xiong et al. (2009) (3XTg and
Tg-SwDI models), cognitive symptoms were already evident
in those animals. In the present study, we used hAPP mice at
12 weeks of age, a time well before cognitive symptoms and
presumably complex neuropathology set in (Hsiao et al.,
1996; Kawarabayashi et al., 2001). Because we found no
evidence for BCRP-mediated A? efflux in intact mouse brain
capillaries, increased BCRP expression may appear at later
stages of the disease, be model-dependent, or both. Thus, at
this time the role of BCRP in AD etiology remains unclear.
On the other hand, the A? receptors RAGE and LRP1 have
been shown to contribute to A? trafficking across the brain
capillary endothelium, but not at early stages of the disease
(Deane et al., 2003, 2004b). In both AD mouse models and
patients with AD in whom cognitive symptoms are evident,
RAGE has been shown to be increased and LRP1 to be
decreased (Deane et al., 2003, 2004a; Donahue et al., 2006).
In the present study with 12-week-old asymptomatic hAPP
mice, unchanged protein expression of the luminal A? uptake
receptor RAGE in brain capillaries suggests that A? uptake
from blood into the endothelium may not be increased before
the onset of cognitive impairment in AD. Slightly increased
expression of the abluminal A? uptake receptor LRP1 sug-
gests increased A? uptake from brain into the endothelium,
again in very early stages of the disease, which may contrib-
Fig. 7. Restoring P-glycoprotein in hAPP
mice reduces A? brain levels. A, hA?40,
hA?42, and hAPP Western blots of total
brain from vehicle- and PCN-treated
hAPP mice. B and C, hA?40 (B) and
hA?42 (C) ELISA analysis of brain tissue
from vehicle- and PCN-treated hAPP
mice (pooled tissue from 19 vehicle-
treated hAPP mice and 20 PCN-treated
hAPP mice). ???, significantly lower than
control, P ? 0.001.
P-Glycoprotein in Alzheimer’s Disease
ute to A? accumulation within the capillary endothelium in
cerebral amyloid angiopathy. However, such changes in ex-
pression of these receptors cannot underlie reduced brain
efflux of A? in the early-stage AD mouse model used in the
present study. Thus, our results point to P-glycoprotein as
the final step in clearing A? from the brain. They indicate
that efflux mediated by P-glycoprotein may well be the lim-
iting factor in A? brain clearance and the critical step that is
defective in AD. Thus, reduced blood-brain barrier P-glyco-
protein expression in AD would be a major contributor to A?
brain accumulation. These results have two important impli-
cations for how we view the disease.
First, the present findings indicate that reduced P-glyco-
protein expression at the blood-brain barrier is an early
biochemical manifestation of AD pathology that occurs before
cognitive symptoms are evident. The mechanism by which
the disease signals the loss of P-glycoprotein is unknown. In
the early stages of the disease, accumulation of A?42 levels
in the brain capillary plasma membrane could directly im-
pair P-glycoprotein function, lead to A? accumulation, and
reduce P-glycoprotein expression. In this regard, our unpub-
lished experiments show that exposure of rat or mouse brain
capillaries to low levels of A?40 selectively removes P-glyco-
protein from the plasma membrane and sends it to the pro-
teasome, which implies that a particularly pernicious posi-
tive feedback loop drives the loss of efflux transporter
expression, leading to a further increase in A? brain levels.
We posit that this loop contributes to the progressive nature
of AD, at least in the early stages. Second, P-glycoprotein is
one transporter for which few endogenous substrates have
been identified (King et al., 2001). Our results indicate that
A? is one such substrate and that P-glycoprotein is respon-
sible for the final critical step in A? efflux across the blood-
brain barrier (Fig. 1C). P-glycoprotein is also a major protec-
tive element of the blood-brain barrier, limiting a large
number of xenobiotics, including many therapeutic drugs,
from entering into the central nervous system (Schinkel et
al., 1996; Fellner et al., 2002). Thus, substantially reduced
protein expression and efflux transport activity of blood-
brain barrier P-glycoprotein in AD could have significant
consequences for patients with AD. These include increased
brain uptake of xenobiotics, altered dose response relation-
ships for therapeutic drugs, and increased neurotoxicity for
those drugs that exhibit a narrow therapeutic index and
neurotoxicants that are P-glycoprotein substrates. All of
these could aggravate the progression of AD.
It is clear from the present experiments with hAPP trans-
genic mice that targeting PXR, a ligand-activated nuclear
receptor that modulates blood-brain barrier P-glycoprotein
(Bauer et al., 2004, 2006), restores transporter expression
and function, which in turn reduces brain A? burden. PXR is
activated by a number of drugs and dietary constituents, and
potent ligands for human PXR include the antibiotic, rifam-
picin, and the St. John’s Wort constituent hyperforin (Jones
et al., 2000; Watkins et al., 2001). We previously showed that
rifampicin dosing of transgenic mice expressing human PXR
increased expression and transport activity of P-glycoprotein
at the blood-brain barrier (Bauer et al., 2006). In these mice,
the rifampicin dose was adjusted so that free plasma levels of
the drug were comparable with those in patients receiving
rifampicin treatment. In this regard, a recent clinical trial
showed that rifampicin dosing lessened cognitive decline in
patients with AD over the 12-month treatment period (Loeb
et al., 2004). The mechanistic basis for this observation is not
known, but rifampicin activation of PXR leading to induction
of blood-brain barrier P-glycoprotein is a likely possibility.
These findings and the present results indicate that PXR
activation to increase blood-brain barrier P-glycoprotein
could be used in the clinic to increase A? brain efflux and
transport and lower A? brain burden. This therapeutic strat-
egy implies PXR protein expression in human brain capillar-
ies. Although PXR mRNA has previously been detected in
human whole brain and brain capillaries (Lamba et al., 2004;
Dauchy et al., 2009), PXR protein expression has not yet been
demonstrated. However, our study provides proof of princi-
ple, and we anticipate that up-regulation of P-glycoprotein
expression through PXR or other signaling pathways has the
potential to increase A? brain efflux transport and lower A?
Finally, although brain accumulation of A? is not the only
major contributor to cognitive impairment in AD (Iadecola,
2004; Blennow et al., 2006), reducing A? accumulation in the
transgenic hAPP (Tg2576) mouse model does delay pathology
(Karlnoski et al., 2009). It remains to be seen to what extent
a general, long-term strategy of targeting signals that up-
regulate blood-brain barrier P-glycoprotein will reduce brain
A? burden over the long term and thus prove to be a useful
therapeutic strategy for delaying the onset of AD and slowing
the progression of the disease.
We thank Destiny Sykes, Jonathan Lucking, Sylvia Notenboom,
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P-Glycoprotein in Alzheimer’s Disease