Direct evidence of abca1-mediated efflux of cholesterolat the mouse blood–brain barrier

Tuan Minh Do • Melissa Ouellet • Frederic Calon •

Giovanna Chimini • Helene Chacun •

Robert Farinotti • Fanchon Bourasset

Received: 9 March 2011 / Accepted: 28 May 2011 / Published online: 10 June 2011

� Springer Science+Business Media, LLC. 2011

Abstract We investigated the expression and function

of Abca1 in wild-type C57BL/6, abca1(?/?), and

abca1(-/-) mice brain capillaries forming the blood–brain

barrier (BBB). We first demonstrated by quantitative RT-

PCR and Western immunoblot that Abca1 was expressed

and enriched in the wild-type mouse brain capillaries. In

abca1(-/-) mice, we reported that the lack of Abca1

resulted in an 1.6-fold increase of the Abcg4 expression

level compared to abca1(?/?) mice. Next, using the in situ

brain perfusion technique, we showed that the [3H]cho-

lesterol brain uptake clearance (Clup, ll/s/g brain), was

significantly increased (107%) in abca1(-/-) mice com-

pared to abca1(?/?) mice, meaning that the deficiency of

Abca1 conducted to a significant decrease of the choles-

terol efflux at the BBB level. In addition, the co-perfusion

of probucol (Abca1 inhibitor) with [3H]cholesterol resulted

in an increase of [3H]cholesterol Clup (115%) in abca1

(?/?) but not in abca1(-/-) mice, meaning that probucol

inhibited selectively the efflux function of Abca1. In

conclusion, our results demonstrated that Abca1 was

expressed in the mouse brain capillaries and that Abca1

functions as an efflux transporter through the mouse BBB.

Keywords Blood–brain barrier � Cholesterol � Abca1 �Abcg4 � Abcb1 � Abcg2 � Mouse � In situ brain perfusion

Abbreviations

ABC ATP-binding cassette

AD Alzheimer’s disease

BBB Blood–brain barrier

BCECs Brain capillary endothelial cells

Clup Brain uptake clearance

PBS Phosphate-buffered saline

Introduction

ABCA1, which belongs to the superfamily of the ATP-

binding cassette (ABC) proteins, is known for its efflux

function of cholesterol from intracellular compartment to

systemic and brain apolipoproteins [1]. ABCA1 is

expressed in several tissues, including brain, placenta, and

liver [2]. In the brain, ABCA1 is particularly expressed in

neurons and glial cells [3, 4]. ABCA1 mRNA has also been

detected in cultured human and rat brain capillaries endo-

thelial cells (BCECs) [5]. In addition, the Abca1 protein

has been identified in the brain capillaries of transgenic

mice lacking Abca1 selectively in neurons and glial cells

[4]. Associated with the basement membrane, pericytes and

astrocytes end-foot, the brain capillary endothelial cells

(BCECs) constitute the blood–brain barrier (BBB), which

represents the main interface separating the blood circu-

lation from the brain. The barrier effect is due to tight

T. M. Do � R. Farinotti � F. Bourasset (&)

Laboratory of Clinical Pharmacy, EA4123, Faculty of Pharmacy,

University of Paris Sud 11, 5 rue Jean-Baptiste Clement,

92296 Chatenay-Malabry, France

e-mail: fanchon.bourasset@wanadoo.fr

M. Ouellet � F. Calon

Faculty of Pharmacy, Laval University, Quebec, QC, Canada

G. Chimini

Centre d’Immunologie de Marseille-Luminy, INSERM-CNRS-

Universite de La Mediterranee, Marseille, France

H. Chacun

Laboratory of Biopharmacy and Pharmaceutical Technology,

CNRS UMR 8612, Faculty of Pharmacy, University of Paris

Sud 11, Chatenay-Malabry, France

123

Mol Cell Biochem (2011) 357:397–404

DOI 10.1007/s11010-011-0910-6

junctions between BCECs as well as to the presence of

efflux transporters that limit the entry of their substrates

into the brain. Among these efflux transporters, ABC

proteins such as ABCB1 (P-glycoprotein, P-gp) and

ABCG2 (Breast Cancer Resistance Protein, BCRP) have

been shown to play prominent roles in brain homeostasis.

In addition to limiting the brain entrance of several drugs,

these proteins are also involved in a wide range of

pathologies, such as inflammatory or neurodegenerative

diseases [6–9].

Like other ABC proteins, ABCA1 has been suggested to

play a key role in Alzheimer’s disease (AD) [10, 11]. The

direct link between ABCA1 and AD has not been evi-

denced, but several studies showed relationships between

the brain level of ABCA1 and amyloid-beta peptide (Ab)

(see review [11]). Recently, Kim et al. [12] showed that

Abca1 was over-expressed in hippocampal neurons of

APP/PS1 mice. In addition, the link between cholesterol

metabolism and AD has been established by series of in

vivo and in vitro studies [13, 14].

So far, the exact mechanisms by which brain cholesterol

homeostasis, ABCA1 and AD are linked are unknown. A

hypothesis is that cholesterol could interfere with the BBB

efflux of Ab, by an ABCA1-mediated pathway. However,

the efflux function of ABCA1 in the BBB has not been

evidenced. Since the most commonly used animal models

of AD are developed in the mouse, it appeared necessary to

quantify the expression and efflux function of Abca1 in the

mouse BBB.

For this purpose, we performed quantitative RT-PCR

(qRT-PCR) and Western immunoblot to measure the

mRNA and protein expression of Abca1 in the C57BL/6

mouse BBB. We then performed in situ brain perfusion to

evaluate the Abca1 function at the mouse BBB by mea-

suring the brain uptake of [3H]cholesterol (used as a probe)

in the presence or absence of probucol, a known Abca1

inhibitor [15], in abca1(?/?), abca1(±), or abca1(-/-)

mice.

Materials and methods

Animals

C57BL/6 mice (6–8 weeks old) were purchased from

Janvier (Le Genest-St-Ile, France). The heterozygote

Abca1-deficient mice are on a C57BL/6 genetic back-

ground. These mice were bred to yield abca1(-/-),

abca1(?/-), and abca1(?/?) littermate mice [16]. The

genotype of offspring was checked by PCR analysis using

genomic DNA isolated from tail biopsies, with the fol-

lowing pairs of primers: forward, 50 TGG GAA CTC CTG

CTA AAA T 30, and reverse, 50 CCA TGT GGT GTG TAG

ACA 30 for the wild-type allele; forward, 50 TTT CTC

ATA GGG TTG GTC A 30, and reverse, 50 TGC AAT

CCA TCT TGT TCA AT 30 for the mutant allele. The wild-

type allele generated a 750 bp product and the mutant

allele a 500 bp product. Male and female mice, 6- to

8-week-old, had free access to standard laboratory food and

water and kept on a 12 h light–dark cycle at 22 ± 1�C.

They were housed under these conditions for at least

5 days before being used. Studies involving animals and

their care were performed according to the guidelines

issued by the European Economic Community for the care

and use of laboratory animals (Official Journal of the

European Community, 18/2/86-authorization L3600).

Reagents and antibodies

[3H]cholesterol (44.5 Ci/mmol), [14C]sucrose (588.0 mCi/

mmol), Soluene, and Ultima Gold scintillation cocktails

were purchased from PerkinElmer Life and Analytical

Sciences (Courtaboeuf, France). Probucol was obtained

from Sigma-Aldrich (St. Quentin Fallavier, France). Rat

monoclonal anti-mouse Abca1 antibody (clone NB400-

164) was purchased from Novus Biological (Littleton,

USA). Rat monoclonal anti-mouse Abcg2 antibody (clone

BXP-53) and mouse monoclonal anti-mouse Abcb1 anti-

body (clone C219) were obtained from Dako (Glostrup,

Denmark). Rabbit polyclonal anti-mouse Abcg4 antibody

was purchased from Alpha Diagnostic International (San

Antonio, TX, USA). Mouse monoclonal anti-mouse b-actin

antibody (clone AC74) was from Sigma-Aldrich (St Louis,

MO, USA). All the other chemicals were commercial

products of reagent grade.

Transporter expression studies

mRNA extraction and quantitative RT-PCR

The mRNA was extracted from cortex homogenate or from

mouse brain capillaries. The capillary depletion method of

Triguero et al. [17] was used with some modifications [18]

to obtain capillary-enriched fractions of mouse brains. To

remove the blood from brain capillaries, seven mice were

perfused with saline by in situ brain perfusion for 30 s.

After decapitation, the brain was removed and meninges

were discarded on ice. Brains were homogenized in 7 ml of

buffer (10 mM HEPES, 141 mM NaCl, 4 mM KCl, 1 mM

NaH2PO4, 2.8 mM CaCl2, 1 mM MgSO4, and 10 mM

D-Glucose (pH 7.4) at 4�C). Chilled 37% dextran solution

was then added to obtain a final dextran concentration of

18.5%. The homogenates were centrifuged at 54009g for

15 min at 4�C in a swinging-bucket rotor. In these condi-

tions, the supernatant corresponds to the brain parenchyma,

and the pellet constitutes the enriched-capillary fraction

398 Mol Cell Biochem (2011) 357:397–404

123

[17]. All these fractions were carefully separated. The

purity of the fractions has been controlled by measuring the

gamma-glutamyl transferase activity which is maximal in

the capillary fraction [19]. Total RNA was extracted from

the isolated mouse brain capillaries and brain cortex

homogenate by using the RNeasy Mini (Qiagen) extraction

kit, according to the manufacturer’s instructions. Random

Hexamers (pDN6) were used to synthesize cDNA from

total RNA (1 lg) using a SuperScript II cDNA synthesis

kit (Invitrogen, Carlsbarg, CA) according to the manufac-

turer’s instructions. The primers for the target gene abca1

were purchased from Applied Biosystems (Mm0044

2646_m1 and Mm00440736_m1, respectively). Mouse b-

actin (Applied Biosystems) was used as normalization

control as it was shown that b-actin was stably expressed in

the brain [20, 21]. The conditions were 95�C for 10 min,

followed by 42 cycles of 15 s at 95�C, and 1 min at 60�C,

as recommended by the primer’s provider. The method

used for the mRNA quantification was the comparative CT

method with the formula 2-DDCt, as described previously

[22, 23].

Protein extraction and Western immunoblot analysis

Total proteins were extracted from C57BL/6, abca1(?/?),

abca1(?/-), and abca1(-/-) mice brain capillaries and

brain cortex. To extract total protein from the capillary

fraction, mice brain microvessels were first isolated using a

density-gradient procedure [24]. The mouse brain cortex

and the pellets containing microvessels were homogenized

in a mixture of TENTS (containing 10 mM Tris–HCl at pH

7.4, 5 mM EDTA at pH 8, 126 mM NaCl, 1% (v/v) Triton

X-100, and 0.1% (v/v) SDS) supplemented with leupeptin,

aprotinin, pepstatin, and phenyl methane sulfonyl fluoride

(Sigma-Aldrich, France). The suspensions were agitated

gently during 1 h at 4�C and then centrifuged at

120009g at 4�C for 20 min. Protein content in the super-

natant was determined by using the bicinchoninic acid

protein assay kit (Sigma-Aldrich, France).

Samples containing 10–25 lg of total proteins were

subjected to electrophoresis on an 8% SDS-polyacrylamide

gel and transferred electrophoretically on nitrocellulose

membranes (Amersham Biosciences, UK). The blots were

blocked with nonfat dry milk/Tween Tris base solution

(TTBS) 10% for 1 h at 20-25�C. After washing with TTBS,

the blots were incubated for 2 h at 20–25�C with primary

antibody. The dilutions of primary antibodies used were:

1:1500 for Abca1, 1:100 for Abcb1, 1:80 for Abcg2,

1:1000 for Abcg4 and 1:5000 for b-actin. After five washes

in TTBS (10 min each), they were further incubated for 1 h

at 20–25�C with horseradish peroxidase-conjugated anti-

bodies diluted at 1:10000 (Dako, Glostrup, Danemark).

The membranes were washed five times for 10 min each in

TTBS and then probed with the Western lightning chemi-

luminescence reagent (PerkinElmer, Courtaboeuf, France).

The intensity of the bands was quantified by using Scion

Image (NIH, Scion Corporation, Bethesda, MD, USA).

In vivo transport studies

Surgical procedure and perfusion technique

The mouse brain transport of [3H]cholesterol was measured

using the in situ brain perfusion method [25]. Mice were

anesthetized by intra-peritoneal injection of a mixture of

xylazine (Merial, Lyon, France) and ketamine (Bayer,

Puteaux, France) at 8/140 mg/kg. Briefly, the right com-

mon carotid artery was catheterized with polyethylene

tubing (0.30 mm 9 0.70 mm; Folioplast, Sarcelles, France)

filled with heparin (25 IU/ml). Before inserting the cathe-

ter, the common carotid artery was ligated on the heart side

and the right external carotid artery was ligated rostral to

the occipital artery at the level of the bifurcation of the

common carotid. Before perfusion, the heart was cut and

the perfusion was started immediately at a flow rate of

2.5 ml/min. The perfusion fluid consisted of bicarbonate-

buffered physiological saline containing 128 mM NaCl,

24 mM NaHCO3, 4.2 mM KCl, 2.4 mM NaH2PO4,

1.5 mM CaCl2, 0.9 mM MgCl2, and 9 mM D-glucose. The

solution was gassed with 95% O2–5% CO2 for pH control

(7.4) and warmed to 37�C. The syringe containing the

perfusate was placed in an infusion pump (Harvard

Apparatus, Les Ulis, France) and connected to the catheter.

For the time course study, each mouse was perfused

with [3H]cholesterol (0.2 lCi/ml) and [14C]sucrose

(0.3 lCi/ml) for 15, 30, or 60 s. For all other single-time

point studies, each mouse was perfused for 60 s with

[3H]cholesterol and [14C]sucrose to check the physical

integrity of the blood–brain barrier. The brain uptake of

[3H]cholesterol was also measured in the presence of

unlabeled inhibitor, probucol (10 lM). The stock solution

of probucol was prepared in dimethyl sulfoxide (DMSO;

\0.25% in final perfusate) and then added to the perfusate.

Perfusion was terminated by decapitating the animal. The

right cerebral hemisphere and aliquots of the perfusate

were weighed. Samples were digested in 1 ml of Soluene at

50�C and mixed with 9 ml of Ultima Gold scintillation

cocktail. Both labels were counted simultaneously in a

scintillation counter (LS6000LL, Beckman, Galway,

Ireland).

Calculation of BBB transport parameters [25, 26]

The brain vascular volume of each animal (Vvasc, ll/g

brain) was estimated from the tissue distribution of

Mol Cell Biochem (2011) 357:397–404 399

123

[14C]sucrose which does not measurably cross the BBB

[27], using the following equation:

Vvasc ¼X�

C�perf

where X* (dpm/g) is the amount of [14C]sucrose in the right

brain hemisphere and C�perf (dpm/ll) is the concentration of

[14C]sucrose in the perfusate.

The apparent tissue distribution volume (Vbrain, ll/g

brain) was calculated from the amount of radioactivity in

the right brain hemisphere using the following equation:

Vbrain ¼Xbrain

Cperf

where Cperf (dpm/ll) is the concentration of [3H]cholesterol

in the perfusate, and Xbrain (dpm/g) is the amount of

[3H]cholesterol in the right cerebral parenchyma, corrected

for vascular contamination:

Xbrain ¼ Xtot � VvascCperf

where Xtot (dpm/g) is the total amount of [3H]cholesterol

measured in the brain tissue sample (vascular and

extravascular).

The brain uptake clearance, expressed as Clup (ll/s/g

brain), was calculated from:

Clup ¼Vbrain

Tperf

where Tperf is the perfusion time (s).

Efflux ratios were calculated as:

Clup(-/-)/Clup

(?/?) = Clup obtained in abca1(-/-) mice/Clup

obtained in abca1(?/?) mice,

Clup(prob?)/Clup

(prob-) = Clup obtained with probucol/Clup

obtained without probucol, in Abca1-competent or -defi-

cient mice.

Data and statistical analysis

Data were shown as means ± S.D. Student’s unpaired

t test was used to identify significant differences between

groups when appropriate. All the tests were two-tailed and

statistical significance was set at * P \ 0.05, ** P \ 0.01,

*** P \ 0.001.

Results

Mouse BBB expression of Abca1

Abca1 mRNA was detected in the brain cortex homogenate

as well as in the brain capillary fraction of C57BL/6 mice.

The mRNA level of abca1 was significantly 4.2-fold

enriched in the brain capillary fraction compared to the

brain cortex homogenate (Fig. 1a).

The protein expression of Abca1 in C57BL/6 mice brain

capillary fraction was shown in Fig. 1b and c. The level of

Abca1 protein expression is 2.5-fold increased in the brain

capillary fraction compared to the brain cortex homogenate

(Fig. 1c).

To validate the model of Abca1-deficient mice, we

confirmed that Abca1 was expressed in the brain capillary

fraction of abca1(?/?) but not in that of abca1(-/-) mice

(Fig. 1d). We also observed a slight, but not significant

decrease of Abca1 expression in the brain capillary fraction

of abca1(?/-) mice, compared to abca1(?/?) mice

(Fig. 1d, Table 1).

Linearity of the cholesterol brain distribution over time

A first set of perfusion experiments was performed to study

the linearity of the brain volume of distribution (Vbrain, ll/g

brain) of [3H]cholesterol over time in wild-type C57BL/6

mice (Fig. 2a). The [3H]cholesterol Vbrain increased linearly

with the perfusion time suggesting that the transport rate

remained unchanged over time. A perfusion time of 60 s

was used for subsequent single-time point experiments.

Physical blood–brain barrier integrity

The brain vascular volume (Vvasc, ll/g brain) was measured

in each mouse by co-perfusing [14C]sucrose, a vascular

space marker, during the in situ brain perfusion experi-

ments. All the values were under 20 ll/g brain, indicating

that the BBB was intact during each experiment [25].

Abca1 function at the mouse BBB

The function of Abca1 at the BBB level was evaluated by

measuring the transport of [3H]cholesterol in abca1(?/?),

abca1(?/-), and abca1(-/-) mice, using the in situ brain

perfusion technique, with or without probucol. As shown in

Fig. 2b, the deficiency of Abca1 resulted in an increase of

107% in [3H]cholesterol Clup (P \ 0.01) in abca1(-/-)

mice (Clup = 21.93 ± 4.43 ll/s/g brain) as compared to

abca1(?/?) mice (Clup = 10.6 ± 2.98 ll/s/g brain). Thus,

the [3H]cholesterol efflux ratio (Clup(-/-)/Clup

(?/?)) was equal

to 2.1. The slight decrease of Abca1 expression in

abca1(?/-) mice resulted only in a nonsignificant increase

of the [3H]cholesterol Clup (Clup = 12.5 ± 1.2 ll/s/g

brain), compared to the abca1(?/?) mice. The addition of

probucol significantly increased the Clup of [3H]cholesterol

in abca1(?/?) mice by 115% (Clup = 22.74 ± 5.48 ll/s/g

brain) (P \ 0.01), but not in abca1(-/-) mice (Clup =

19.75 ± 2.90 ll/s/g brain) (Fig. 2b). The [3H]cholesterol

400 Mol Cell Biochem (2011) 357:397–404

123

efflux ratio (Clup(prob?)/Clup

(prob-)) was then equal to 2.1 in

abca1(?/?) mice, whereas this ratio was equal to the unity

in abca1(-/-) mice.

Expression of Abcb1, Abcg2, and Abcg4 in brain

capillary fraction of abca1(?/?), abca1(?/-),

and abca1(-/-) mice

We measured the levels of expression of Abcb1, Abcg2,

and Abcg4 in the brain capillary fraction of abca1(?/?),

(?/-), and (-/-) mice. These three transporters are

known as possible efflux transporters of cholesterol [28–

30]. Our results showed no changes in the expression of

Abcb1 and Abcg2 in both abca1(-/-) and abca1(?/-)

mice compared to abca1(?/?) mice (Table 1). On the

contrary, we evidenced a significant 1.6-fold increase of

the Abcg4 expression in abca1(-/-) mice compared to

abca1(?/?) mice, whereas the increase of Abcg4 expres-

sion observed in abca1(?/-) mice compared to abca1

(?/?) mice was nonsignificant (P = 0.06) (Table 1).

Discussion

The present work was undertaken to study the expression

and the function of Abca1 in the mouse BBB. We first

Fig. 1 Abca1 expression in the mouse brain capillary fraction and

brain cortex homogenate. a Abca1 mRNA expression in C57BL/6

mouse brain cortex homogenate (BC open square; n = 3 mouse

brains) and brain capillary fraction (Capillary filled square; n = 7

mouse brains). mRNA quantifications were determined by qRT-PCR

and normalized to b-actin. Data are means ± S.D. b Abca1

immunoblots from brain cortex homogenate (BC; n = 5 mouse

brains) and brain capillary fraction (Capillary; n = 5 mouse brains) of

C57BL/6 mice, b-actin was used as loading control. c Densitometric

analysis of Abca1 immunoblots, normalized by b-actin. BC opensquare: brain cortex homogenate. Capillary filled square: brain

capillary fraction. Values shown are in relative optical density values

as means ± S.D. of 5 mice. d Abca1 immunoblots from the brain

capillary fractions of abca1(?/?), abca1(?/-) and abca1(-/-) mice

Table 1 Protein expression of transporters in the mouse brain capillary fraction

Transporter/b-actin Ratio abca1(-/-)/abca1(?/?)

abca1(?/?) abca1(?/-) abca1(-/-)

Abca1 0.51 ± 0.23 0.31 ± 0.03

Abcb1 1.15 ± 0.24 1.12 ± 0.11 1.03 ± 0.15 0.97

Abcg2 1.21 ± 0.4 1.24 ± 0.16 1.36 ± 0.14 1.02

Abcg4 0.56 ± 0.09 0.86 ± 0.29 0.89 ± 0.28* 1.60

Protein levels were determined by Western blot and normalized to b-actin. Data are means ± S.D. (n = 3–5 mice per group). * P \ 0.05 for

abca1(-/-) mice versus abca1(?/?) mice

Mol Cell Biochem (2011) 357:397–404 401

123

demonstrated that abca1 mRNA and protein were expres-

sed in the brain capillary fraction of C57BL/6 mice

(Fig. 1). Our results are consistent with earlier in vitro data

from cultured human, rat, bovine, and porcine BCECs [5,

31, 32]. In addition, our results demonstrated that mRNA

and protein expression of Abca1 was higher in the mouse

brain capillary fraction compared to the total brain cortex

homogenate (Fig. 1a–c). This demonstrates that the BBB

expression of Abca1 has the same pattern as that of other

known efflux ABC proteins expressed at the mouse BBB,

like Abcb1 and Abcg2, which are also over-expressed in

brain capillary fraction compared to total brain cortex

homogenate [33, 34].

To study the function of Abca1 at the mouse BBB, we

measured the ability of Abca1 to efflux its known substrate,

cholesterol, using the in situ brain perfusion technique. In

our study, we did not focus on the BBB uptake of

cholesterol, but we rather used it as a probe of Abca1 to

investigate its efflux function at the mouse BBB. However,

it is important to note that, in healthy mammals, brain

cholesterol is produced in situ because blood-borne cho-

lesterol does not cross the BBB, mainly due to its binding

to circulating lipoproteins [35, 36]. But a specific choles-

terol brain clearance mechanism is needed to compensate

for the cholesterol synthesis under steady-state conditions.

One of these specific brain clearance mechanisms is the

BBB OATP2-mediated efflux of 24(S)-hydroxycholesterol,

a cholesterol metabolite resulting from the action of the

cholesterol 24-hydroxylase which is specifically expressed

in the brain [37, 38]. But the balance studies in rodents

revealed that the rate of excretion of sterol from the CNS

equals to 1.4 mg/day/kg among which only 0.9 mg/day/kg

was 24(S)-hydroxycholesterol. The remainder has to be

excreted across the BBB, maybe as free cholesterol, by

unidentified pathways [39]. It has been hypothesized that

several ABCA, ABCB, or ABCG proteins, could be

involved in the brain cholesterol efflux [28–31]. Our results

indeed suggest that Abca1 might play a role in the efflux of

cholesterol produced in the brain. Thus, the brain choles-

terol level should be higher in abca1(-/-) mice than in

abca1(?/?) mice. But Burns et al. reported no changes in

the brain cholesterol levels in abca1(-/-) compared to

abca1(?/?) mice [10]. This result suggested that another

cholesterol transporter, like Abcg1, Abcg4, Abcg2, or

Abcb1, could be over-expressed to compensate for the lack

of Abca1. In their report, Burns et al. showed that Abcg1

expression was unaffected by the lack of Abca1. Therefore,

we measured the BBB expression level of Abcg2, Abcb1,

and Abcg4 and found that Abcg4, but not Abcb1 or Abcg2,

was significantly over-expressed in abca1(-/-) mice

compared to abca1(?/?) mice. This result suggests that

Abcg4 could partially compensate for the lack of Abca1 in

abca1(-/-) mice.

The in situ brain perfusion technique was developed in

the rat and mouse, to study drug transport through the BBB

[25, 27]. It allows for the measuring of several transport

parameters like the volume of distribution (Vbrain) and the

brain uptake clearance (Clup) of the studied molecule, in

very short times (from 15 to 120 s), in order to avoid

metabolism and transporter expression regulation pro-

cesses. The resulting Clup integrates the enhancing or

limiting factors introduced by the transporters located at

the luminal side of BCECs [40]. Thus, using transporter

inhibitors and/or transporter deficient mice, this technique

enabled us to determine whether or not a given transporter

was involved. In other words, if the Clup increases when a

transporter inhibitor is added or when a transporter is

missing, we can conclude that the transporter concerned is

an efflux carrier involved in the BBB transport of the

molecule studied. Thus, the efflux ratio of a molecule can

Fig. 2 Abca1-mediated efflux of [3H]cholesterol through the mouse

BBB. a Time course of [3H]cholesterol uptake by the right hemisphere

(solid line) of C57BL/6 mice, expressed as apparent volume of

distribution (Vbrain, ll/g brain), determined by in situ brain perfusion

technique. Regression analysis of individual data gives r2 = 0.985.

Data are means ± S.D. of 4–6 animals per data point. b Brain uptake

clearance (Clup, ll/s/g brain) of [3H]cholesterol measured in abca1(?/?) (black filled square), abca1(?/-) (filled square) and

abca1(-/-) mice (grey open square) by in situ brain perfusion

technique. [3H]cholesterol was perfused alone or with probucol

(10 lM). Data are means ± S.D. of 4–6 mice. NS nonsignificant

402 Mol Cell Biochem (2011) 357:397–404

123

be calculated [26]. In our case, we calculated the

[3H]cholesterol efflux ratio by comparing abca1(-/-) and

abca1(?/?) mice or by comparing mice perfused with or

without probucol.

In the current study, we demonstrated that the [3H]cho-

lesterol Clup was enhanced when probucol was co-perfused

with [3H]cholesterol in abca1(?/?) mice (Fig. 2b). The

[3H]cholesterol efflux ratio (Clup(prob?)/Clup

(prob-)) was calcu-

lated at 2.1, suggesting that probucol inhibited an efflux

transporter, likely to be Abca1. This result agrees with a

preliminary study, in which we showed that the brain cho-

lesterol Clup was enhanced when probucol was added [41].

To demonstrate that the probucol-sensitive transporter

evidenced here was Abca1, we measured the [3H]choles-

terol Clup in abca1(-/-) mice with and without probucol.

Without probucol, the [3H]cholesterol Clup was signifi-

cantly increased in abca1(-/-) mice compared to

abca1(?/?) mice (Fig. 2b), meaning that Abca1 was

actually involved in the BBB cholesterol efflux. The value

of the [3H]cholesterol efflux ratio (Clup(-/-)/Clup

(?/?)) was

equal to (Clup(prob?)/Clup

(prob-)) ratio obtained in abca1(?/?)

mice, suggesting that probucol inhibited specifically Abca1.

This is in agreement with the literature, since, to our

knowledge, probucol has been shown to inhibit solely

Abca1 [15, 42]. To confirm this point, we co-perfused

probucol with [3H]cholesterol in abca1(-/-) mice

(Fig. 2b). The lack of Abca1 led to the lack of the

probucol effect as illustrated by the value of the efflux ratio

(Clup(prob?)/Clup

(prob-)) obtained in abca1(-/-) mice, which

failed to unity. This result confirmed the specific effect of

probucol on Abca1-mediated transport of cholesterol at the

mouse BBB level. In addition, the higher [3H]cholesterol

Clup obtained in abca1(-/-) mice compared to abca1(?/

?) mice perfused without probucol suggests that the BBB

over-expression of Abcg4 is not sufficient to fully com-

pensate for the lack of Abca1.

Altogether, our results strongly support the hypothesis

that Abca1 is a cholesterol efflux transporter at the mouse

BBB. Our results are in agreement with two in vitro studies

in which Abca1 was shown to transport cholesterol through

cultures of bovine and porcine BCECs [31, 32]. But to our

knowledge, we have been the first to demonstrate, in vivo,

the efflux function of Abca1 at the mouse BBB level.

Conclusion

In summary, our results evidenced that Abca1 is enriched

in mouse brain capillaries compared to total brain cortex,

reminiscent to the expression pattern of other ABC trans-

porters ABCB1 and ABCG2. In addition, our results

underscore the role of Abca1 as an efflux transporter at the

mouse BBB. Finally, our data demonstrate that the lack of

Abca1 resulted in an over-expression of Abcg4, to partially

compensate for the lack of Abca1-mediated efflux of

cholesterol. Bearing in mind that several relationships were

established between cerebral cholesterol homeostasis and

AD, our results encourage further investigations to eluci-

date if Abca1- and/or Abcg4-mediated cholesterol efflux at

the mouse BBB could be one of the key segments of the

AD-cholesterol link.

Acknowledgments We thank Chantal Guillemette for helping us

with the qRT-PCR technique. We thank Valerie Domergue-Dupont,

the head of the central animal house of the faculty of Pharmacy, for

taking care of animals. The authors thank the Banting Research

Foundation for their financial supports.

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