Age-Dependent Regulation of the Blood-Brain Barrier Influx/Efflux Equilibrium of Amyloid-β Peptide in a Mouse Model of Alzheimer’s Disease (3xTg-AD)

Abstract

The involvement of transporters located at the blood-brain barrier (BBB) has been suggested in the control of cerebral Aβ levels, and thereby in Alzheimer’s disease (AD). However, little is known about the regulation of these transporters at the BBB in animal models of AD. In this study, we investigated the BBB expression of Aβ influx (Rage) and efflux (Abcb1-Abcg2-Abcg4-Lrp-1) transporters and cholesterol transporter (Abca1) in 3–18-month-old 3xTg-AD and control mice. The age-dependent effect of BBB transporters regulation on the brain uptake clearance (Cl_up) of [³H]cholesterol and [³H]Aβ_1 - 40 was then evaluated in these mice, using the in situ brain perfusion technique. Our data suggest that transgenes expression led to the BBB increase in Aβ influx receptor (Rage) and decrease in efflux receptor (Lrp-1). Our data also indicate that mice have mechanisms counteracting this increased net influx. Indeed, Abcg4 and Abca1 are up regulated in 3- and 3/6-month-old 3xTg-AD mice, respectively. Our data show that the balance between the BBB influx and efflux of Aβ is maintained in 3 and 6-month-old 3xTg-AD mice, suggesting that Abcg4 and Abca1 control the efflux of Aβ through the BBB by a direct (Abcg4) or indirect (Abca1) mechanism. At 18 months, the BBB Aβ efflux is significantly increased in 3xTg-AD mice compared to controls. This could result from the significant up-regulation of both Abcg2 and Abcb1 in 3xTg-AD mice compared to control mice. Thus, age-dependent regulation of several Aβ and cholesterol transporters at the BBB could ultimately limit the brain accumulation of Aβ.

Keywords

ABCA1 ABCB1 ABCG2 ABCG4 Alzheimer’s disease amyloid-β blood-brain barrier cholesterol Oatp1a4 RAGE 3xTg-AD

INTRODUCTION

The accumulation of amyloid-β peptide (Aβ) in the brain is an important histological feature of Alzheimer’s disease (AD), but recent work indicates that brain Aβ concentrations are dynamically regulated by several mechanisms. As the clearance of Aβ by AD brains is 30% less efficient than the clearance by normal brains [1], the blood-brain barrier (BBB) may be important for regulating brain Aβ concentrations [1 –3].

The BBB is formed by brain capillary endothelial cells (BCECs) which are connected together by tight junctions. The barrier effect is reinforced by influx and efflux transporters at the BCECs, such as solute carriers (SLC) and ATP-binding cassette (ABC) transporters [4]. Unlike the endothelium of systemic capillaries, which have a very permeable barrier allowing transport of solutes and bigger molecules into the interstitial fluid [5], the BBB generally restricts entry of polar molecules into the brain. However, essential nutrients for the brain such as glucose, amino acids, and vitamins can cross the BBB using specific transporters expressed in the brain endothelium [6, 7]. The blood-brain exchanges of larger molecules, such as several regulatory neuroactive peptides and proteins, have also been described [8 –11].

Recently, the transport of Aβ across the BCECs has been demonstrated as a two-directional process mediated by several transporters [12, 13]. The influx of Aβ from blood to brain across the BBB is mediated by at least two mechanisms. One is a receptor-mediated mechanism involving receptor for advanced glycation end products (Rage) [14]. This mechanism has been validated in vivo (in mice) [15, 16] and in vitro (in a bovine model of BBB) [17]. A second mechanism, involving the SLC transporter Oatp1a4, has recently been demonstrated by our group [12]. The several mechanisms involved in the efflux of Aβ from the BBB are not yet fully understood. One involves the low density lipoprotein-related receptor 1 (Lrp-1) [18, 19]. There is also evidence that three ABC transporters (Abcg2, Abcg4 and Abcb1) restrict the passage of Aβ across the BBB both in vivo (in mice) and in vitro (transfected cells or in vitro models of BBB) [20 –22]. Lastly, unidentified insulin-sensitive or vitamin E-sensitive transporters/receptors may take part in the clearance of Aβ across the BBB [23 –25]. All these data show that the amount of Aβ in the brain is strictly controlled by a complex of coordinated transporters at the BBB. They work together to maintain the brain influx/efflux of Aβ in equilibrium in normal situations. Any change in the synthesis and/or function of these BBB transporters can influence the amount of Aβ in the brain and lead to disorders such as AD [26]. Both aging and AD are associated with altered regulation of several transporters in the brain. The amounts of Abcb1 and Lrp-1 in normal aging brains are sub-normal [27, 28], as are their amounts in the BBB of AD patients [29]. The amount of Rage in the BBB also increases with normal aging [30] and in AD [31]. On the other hand, the amount of Abcg2 is elevated in the BBB of AD patients who are also suffering from a cerebral amyloid angiopathy disorder [20], as is that of Abcg4 in the microglial cells of AD patients [32]. Finally, the amount of the cholesterol efflux transporter Abca1 in the brains of AD sufferers is above normal [33, 34]. The amount of Abca1 in the brains of transgenic mouse models of AD varies inversely with the brain Aβ concentration[35, 36, 35, 36].

We therefore postulated that the transporters involved in the BBB exchanges of Aβ and/or cholesterol are regulated in AD [29, 31]. But most studies on these regulations have been done on whole brain homogenates or using brain cells other than brain endothelial cells where Aβ influx and efflux are controlled [32, 34]. It is therefore important to work with these particular cells to better understand the role of the BBB in AD. Another question is whether these regulations have a direct impact on the BBB transport of Aβ and when regulation occurs. To answer these questions, we have measured the amounts of several transporters involved in the Aβ (Abcb1, Abcg2, Abcg4,Lrp-1, Rage and Oatp1a4) and cholesterol (Abca1) exchanges through the BBB in brain capillaries at different stages of AD development (3 to 18 months), in a mouse model of AD (3xTg-AD) that harbors three mutant genes (AβPP_swe/PS1_M146_V/Tau_P301_L). These mice are one of the few transgenic models that mimic both major pathological lesions of AD: amyloid and tau, in the same time-dependent manner as AD patients [37]. We also investigated the impact of these modifications on the BBB transport of Aβ and cholesterol, two major markers in AD.

MATERIALS AND METHODS

Animals

Triple-transgenic mice (3xTg-AD) were produced in our laboratories and used as models of AD [37, 38]. As previously described, 3xTg-AD mice were studied at 3, 6, and 18 months of age to coincide with early, middle, and late plaque and tangle pathology, respectively [39 –41].

They were genotyped using the polymerase chain reaction (PCR) and DNA extracted from tail biopsies as described previously [37]. The mice had free access to standard laboratory food and water and were kept on a 12 h light-dark cycle at 22±1°C. Studies involving animals and their care were performed according to the National Research Council guidelines for the care and use of laboratory animals. The experimental protocols were approved by the Faculty Committee for Animal Care and Use.

Reagents

[³H]Aβ_1 - 40 (15 Ci/mmol) was purchased from Ambios Labs (Newington, CT, USA). [³H]cholesterol (44.5 Ci/mmol), [¹⁴C]sucrose (588.0 mCi/mmol), liquid scintillation cocktails Ultima Gold and Soluene were purchased from PerkinElmer Life Sciences (Courtaboeuf, France). Triton X-100 and bicinchoninic protein assay kits were obtained from Sigma-Aldrich (Saint-Quentin-Fallavier, France). Dulbecco’s Modified Eagles Medium (DMEM), fetal calf serum (FCS) and phosphate-buffered saline (PBS) were purchased from Gibco Invitrogen (Cergy-Pontoise, France). Bovine serum albumin (BSA) was purchased from EuroMedex (Souffelweyersheim, France). Rat anti-human/mouse Abcg2 antibody (BXP-53), mouse anti-mouse Abcb1 antibody (C219), mouse anti-mouse β-actin antibody (AC-74) and peroxidase-conjugated secondary antibodies were purchased from Dako (Glostrup, Denmark). Rat anti-mouse Abca1 antibody (NB400-164) and goat anti-mouse Oatp1a4 antibody (M-13) were from Novus Biological (Littleton, USA). Rabbit anti-mouse Abcg4 and rabbit anti-mouse Rage antibodies were purchased from Alpha Diagnostic International (San Antonio, TX, USA) and Sigma-Aldrich (Saint-Quentin-Fallavier, France), respectively. Rabbit anti-mouse Lrp-1 antibody was from Abcam (Toronto, ON, Canada).

All other chemicals were reagent grade commercial products.

In situ brain perfusion

This method was developed to study drug transport through the BBB [42]. It can be used to measure several transport parameters, including the distribution volume and the brain uptake clearance (Cl_up) of a target molecule over very short initial periods (15–120 s) [43, 44, 43, 44].

Surgery and perfusion

Mice were anesthetized by intra-peritoneal injection of a xylazine and ketamine mixture (8/140 mg/kg). 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. The right common carotid artery was then catheterized with polyethylene tubing. A syringe containing the perfusion fluid was placed in an infusion pump (PHD 2000; Harvard Apparatus, Les Ulis, France) and connected to the catheter. The thorax of the animal was opened, the heart was cut and 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 in a water bath.

Each mouse was perfused with [³H]Aβ_1 - 40 (0.1μCi/ml) for 30 s or with [³H]cholesterol (0.2μCi/ml) for 60 s. The molar concentration of Aβ_1 - 40 was 6.6 nM which is equivalent to the plasma concentrations of Aβ_1 - 40 found in several transgenic mouse models of AD [45, 46]. For each animal, [¹⁴C]sucrose (0.1-0.2μCi/ml) was perfused with the tritiated substrate to check the physical integrity of the BBB and to correct for vascular space contamination. Perfusion was terminated by decapitating the mouse. The right cerebral hemisphere and aliquots of the perfusion fluid were sampled. Tissue samples were digested in 1 ml Soluene at 50°C and mixed with 10 ml Ultima Gold scintillation cocktail. The two labels were counted simultaneously in a scintillation counter (LS 6500, Beckman, Ireland).

Calculation of BBB transport parameters

All the calculations were performed as described previously [42].

The brain vascular volume of each animal (V_vasc, μl/g) was estimated from the tissue distribution of [¹⁴C]sucrose, which does not measurably cross the BBB during short perfusions, by dividing the amount of [¹⁴C]sucrose (dpm/g) in the right brain hemisphere by the concentration of [¹⁴C]sucrose in the perfusion fluid (dpm/μl). All the values were below 20μl/g, indicating that the BBB was not altered during the experiments.

The apparent tissue distribution volume (V_brain, μl/g) of [³H]Aβ_1 - 40 or [³H]cholesterol wascalculated from the amount of radioactivity in the right brain hemisphere using the following equation: $V_{brain} = X_{brain} / C_{perf}$ (1) where C_perf (dpm/μl) is the concentration of tritiated tracer in the perfusion fluid, and X_brain (dpm/g) is the calculated amount of tritiated tracer in the right cerebral parenchyma, corrected for vascular contamination with the following equation: X_brain = X_tot – (V_vasc×C_perf), where X_tot (dpm/g) is the total amount of tritiated tracer measured in the brain tissue sample (vascular + extravascular).

Brain uptake clearance, expressed as Cl_up (μl/g/s), was calculated by dividing V_brain by the perfusiontime (s).

The resulting Cl_up integrates the enhancing or limiting factors introduced by the transporters located at the luminal side of BCECs. Thus, this technique enabled us to evaluate a given transporter’s function at the BBB level. For example, up-regulation of an efflux transporter may result in a more efficient export of the transporter’s substrate from BCECs to the circulation and therefore a decrease in the substrate’s Cl_up. On the contrary, up-regulation of an influx transporter could enhance the substrate’s Cl_up as more substrate is transferred from blood to BCECs.

Capillary isolation

Mouse brain microvessels were isolated by density-gradient centrifugation, essentially as described previously [21]. Briefly, a mouse brain was immersed in ice-cold phosphate-buffered saline (PBS), and the cerebellum, meninges, brainstem, and large superficial blood vessels removed. The cortex was gently homogenized in ice-cold DMEM containing 10% FCS in a Teflon Potter homogenizer. The resulting homogenate was centrifuged at 500 g for 10 min at 4°C and the supernatant discarded. The pellet was homogenized in 5 ml of DMEM, 25% bovine serum albumin, and centrifuged at 1500 g for 20 min at 4°C. This pellet was homogenized in DMEM containing 10% FCS and the homogenate passed through a 140-μm mesh nylon filter. This filtrate was passed through a 40-μm mesh nylon filter. The fraction retained on the 40-μm filter was collected, suspended in ice-cold DMEM - 10% FCS and centrifuged at 12000 g for 45 min at 4°C. The pellet containing the microvessels was washed in ice-cold PBS and centrifuged again at 12000 g for 20 min at 4°C. The supernatant was discarded and the pellets were stored at –80°C.

Western blotting

Total proteins were extracted from the pellets containing microvessels by homogenizing them in TENTS (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 for 1 h at 4°C and then centrifuged at 12000 g at 4°C for 20 min. The protein content of the supernatant was determined with the bicinchoninic acid assay.

Proteins (10 or 25μg per lane) were separated by electrophoresis on 8% SDS-polyacrylamide gels and transferred electrophoretically to nitrocellulose membranes. Free binding sites on the membranes were blocked by incubation with Tris-buffered saline containing 0.1% Tween-20 (TTBS) and 10% non-fat dried milk for 1 h at 20–25°C. The membranes were washed with TTBS and incubated with primary antibody for 2 h at 20–25°C. The dilutions of primary antibodies were: 1:1500 for Abca1, 1:100 for Abcb1, 1:80 for Abcg2, 1:1000 for Abcg4, 1:200 for Oatp1a4, 1:1000 for Rage, 1:10000 for Lrp-1, and 1:2000 for β-actin. The membranes were then washed with TTBS (5 times for 10 min each) and then incubated with secondary antibodies diluted 1:5000 for 1 h at 20–25°C. The membranes were again washed (5 times for 10 min) with TTBS and probed with the Western Lightning Chemiluminescence Reagent (Perkin Elmer Life Sciences, Courtaboeuf, France). The intensities of the bands were quantified with the Scion Image program (NIH, Scion Corporation, Bethesda, MD, USA).

Statistical analysis

The results are expressed as means±S.D. Samples were compared by Student’s t test when appropriate. Differences were considered to be statistically significant when p < 0.05.

RESULTS

The Aβ influx transporters Rage and Oatp1a4 in mouse BCECs

The amount of Rage in the brain capillary fractions of 3xTg-AD mice were greater than in their age-matched controls: 1.4-times at 3 months and 1.5- and 1.8-times at 6 and 18 months, respectively (Fig. 1A, B). The 18-month-old 3xTg-AD mice also had slightly less Oatp1a4, statistically non-significant (17% , p = 0.07), than age-matched control mice (Fig. 1C, D). The amount of Rage at the BBB tended to be positively (r² = 0.9796) correlated with the age of the 3xTg-AD mice, while that of Oatp1a4 tended to be negatively correlated (r² = 0.9357) (Fig. 1E).

The Aβ efflux transporters Abcb1, Abcg2, Abcg4 and Lrp-1 in mouse BCECs

The amounts of Abcb1, Abcg2 and Abcg4 in the brain capillary fractions of wild-type and 3xTg-AD mice aged 3 to 18 months were measured by western blotting. The amount of Abcg4 protein in the BCECs of 3-month-old 3xTg-AD mice was 1.4-times higher than in their age-matched wild-type controls, but that of Abcb1 and Abcg2 proteins was not different from controls. In contrast, the capillary fractions from 18-month-old 3xTg-AD mice contained 1.7-times more of both Abcb1 and Abcg2 proteins than did age-matched controls, while those of Abcg4 in the 3xTg-AD and control mice were the same (Fig. 2A-F). Finally, the amounts of Abcb1, Abcg2 and Abcg4 in the capillary fractions of 6-month-old control and 3xTg-AD mice were not significantly different (Fig. 2A-F). Figure 2G also indicates that the amounts of Aβ efflux transporters tended to vary inversely: the BBB amounts of Abcb1 and Abcg2 increased with age in 3xTg-AD mice while that of Abcg4 decreased.

We also measured the amount of Lrp-1 in the brain capillary fractions of 18-month-old 3xTg-AD and wild-type control mice. Our results showed that the amount Lrp-1 was significantly decreased in the BCECs of 3xTg-AD mice as compared to age-matched control mice (Fig. 3A, B).

The cholesterol efflux transporter Abca1 in mouse BCECs

The BCECs of 3-month-old and 18-month-old 3xTg-AD mice contained 1.3-times more Abca1 than their controls; the greatest increase in Abca1 (3.2-times control) was detected in 3xTg-AD mice aged 6 months (Fig. 4A, B).

Brain uptake of [³H]Aβ_1 - 40

In AD, Aβ mainly exists as 40 or 42 amino-acids forms, which are thought to be transported by the same set of transporters at the BBB level [17 , 48]. Although the Aβ_1 - 42 peptide has been shown to be more amyloidogenic, in this study, we used the Aβ_1 - 40 peptide for in vivo BBB transport studies as it is the most abundant Aβ peptide found in the cerebral vasculature and it is more soluble and stable in solution than Aβ_1 - 42 peptide [49 –51]. The brain uptake clearance (Cl_up) of [³H]Aβ_1 - 40 was measured by in situ brain perfusion. It was the same in 3xTg-AD and age-matched controls aged from 3 to 6 months (Fig. 5). But [³H]Aβ_1 - 40 Cl_up was significantly lower (17% ) in 3xTg-AD mice aged 18 months than in their age-matched controls (Fig. 5).

Brain uptake of [³H]cholesterol

The brain uptake clearance (Cl_up) of [³H]cholesterol, measured by in situ brain perfusion, was significantly lower (15% ) in 3 and 6-month-old 3xTg-AD mice than in their age-matched controls (Fig. 6). But the [³H]cholesterol Cl_up in 3xTg-AD and control mice aged 18 months were not significantly different (Fig. 6).

DISCUSSION

We first studied the mechanisms involved in the BBB influx of Aβ. Our results demonstrate that the amount of Rage in brain capillaries increased linearly with the severity of the disorder (Fig. 1A, B, and E). This agrees well with data from AD patients; the Rage amount in endothelial cells increases linearly from early stage AD as does the severity of the AD [52]. These mouse-age data confirm that the mouse model of AD we used reproduces events observed in AD. Conversely, we find that the amount of Oatp1a4 in brain capillaries tends to decrease linearly with the severity of the disorder (Fig. 1C-E), although the decrease was not statistically significant (p = 0.07 in 18-month-old mice). These opposing changes in the amounts of BBB Rage and Oatp1a4 with the severity of AD suggest that the late decrease tendency in Oatp1a4 might partially compensate for the increase in Rage in the BBB.

Secondly, we studied the mechanisms involved in the BBB efflux of Aβ/cholesterol. Our data show that the regulation of the Aβ/cholesterol efflux mechanisms in the BBB begins early (3 months) in the development of AD-like neuropathology in 3xTg-AD mice. Abcg4 and Abca1 are the first efflux transporters to become over-abundant in brain capillary extracts from 3-month-old 3xTg-AD mice (Fig. 2A, B and Fig. 4A, B). This shows that the BBB regulation begins before the formation of senile plaques or neurofibrillary tangles which appear in 3xTg-AD mice at about 9-10 months [37]. The increase in Abcg4 was transient since it did not occur in later stages of AD (in mice aged 6 and 18 months). Our results agree with those of studies showing that Abcg4 and Abca1 amounts are above-normal in human AD brains (microglial cells for Abcg4 and hippocampus for Abca1) and in hippocampal neurons of AβPP/PS1 mice (Abca1) [32 –34].

At early (3 months) stage we saw no change in the brain uptake clearance of [³H]Aβ_1 - 40 (Fig. 5). We suggest that the efflux transporters Abcg4 and Abca1 are regulated to offset the increase in Rage. If so, these early changes in BBB transporters could be an initial defense against the accumulation of Aβ in the brain. There is evidence that Abcg4 is important for clearing Aβ from mouse cerebral tissue via the BBB [21]. The role of Abca1 is, however, not very clear since Abca1 itself does not directly transport Aβ across the mouse BBB [21, 53].

The amount of Abca1 also increased in older 3xTg-AD mice BBB and was maximal (x 3.2) in 6-month-old 3xTg-AD mice. Besides, the BBB efflux of [³H]cholesterol was enhanced in 3 and 6 months but not in 18-month-old 3xTg-AD mice (Fig. 6). In 3-month-old 3xTg-AD mice BBB, the 1.3-fold increase of Abca1 amount was associated with a 1.4-fold increase of Abcg4 amount, which was not the case in 6- and 18-month-old 3xTg-AD mice. This suggests that the increase of [³H]cholesterol efflux occurring in 3-month-old 3xTg-AD mice might be mediated by Abca1 and/or Abcg4. On the contrary, the increase of the BBB [³H]cholesterol efflux occurring in 6-month-old 3xTg-AD mice might only be mediated by Abca1, since the BBB Abcg4 amount was unchanged in 6-month-old 3xTg-AD mice. But despite a 1.3-fold increase of the Abca1 amount, the [³H]cholesterol efflux was unchanged in 18-month-old 3xTg-AD mice BBB. This suggests that a 3.2-fold enhance in Abca1 protein is needed to observe an Abca1-mediated decrease of [³H]cholesterol efflux at the BBB level. The increased of Abca1 production and function could provide an indirect control of brain Aβ by regulating brain cholesterol. We suggest that this is a second mechanism of preventing the accumulation Aβ in the brain. There have been several reports of a reciprocal relationship between the amount of Abca1 in the brain and the cerebral Aβ level [35 , 55]. But exactly how Abca1 affects the amount of Aβ in the brain is not fully understood. A recent in vitro study suggests that Abca1 mediates the transport of an Aβ-cholesterol complex from the brain to the peripheral compartment [56]. However, studies in our laboratory found no difference in the Cl_up of [³H]Aβ_1 - 40 of Abca1(+/+) and Abca1(-/-) mice perfused with [³H]Aβ_1 - 40 and unlabeled cholesterol (data not shown). Other studies suggest that Abca1 could indirectly regulate the amount of Aβ in the brain via an ApoE-dependent pathway. Indeed, Abca1 was found to mediate the lipidation of ApoE to form PC-rich ApoE-HDL [57 –59]. Interestingly, it has been shown that substrates of Abcg4 like cholesterol were effluxed from cells to ApoE-HDL [21, 60]. We then hypothesized that an increase of Abca1 amount and function at the mouse BBB level could lead to an increase of ApoE-HDL which in turns stimulates the Abcg4-mediated efflux of Aβ. This hypothesis is supported by recent studies showing that the stimulation of the retinoid X receptor (RXR), an Abca1 and Abcg4 transcription factor [61], resulted in an increase of the BBB clearance of Aβ in ApoE4 competent mice but not in ApoE4 knock-out mice [57, 62]. Further studies are needed to determine the exact role of Abca1 in the regulation of brain Aβ and the part it plays in AD. Nevertheless, there appears to be a close relationship between cholesterol/Aβ transporters like Abca1 and Abcg4 in the regulation of brain Aβ.

Our data also evidenced a late regulation of the BBB Aβ transporters. Indeed, while the amount of Rage (influx of Aβ) is still increased (1.8-times) and the amount of Lrp-1 (efflux of Aβ) is decreased in 18-month-old 3xTg-AD mice (Fig. 1A, B and Fig. 3A, B), the amounts of two ABC efflux transporters, Abcb1 and Abcg2, are significantly increased (1.7-fold) in 18-month-old 3xTg-AD mice (Fig. 2C-F). Our results agree with previous studies showing a late increase of Abcg2 and a decrease of Lrp-1 in the BBB oftransgenic AD mice and in human AD brains [20 , 63]. On the contrary, some other studies have detected a downregulation of Abcb1 in transgenic mice or AD patients [29 , 64]. The discrepancy between our study and previous animal studies could be due to the differences in the AD animal models used and/or the time at which the BBB amounts Abcb1 were measured. For example, a decrease in the BBB amount of Abcb1 has been detected in 3-month-old Tg2576 transgenic mice, as compared to wild-type controls [63]. However, the Tg2576 mice are different from 3xTg-AD mice in term of genetic modifications. While 3xTg-AD mice express three human mutant genes, the Tg2576 mice have only a mutant form of AβPP. Consequently, the 3xTg-AD mice develop both plaques and tangles while the latter is absent in Tg2576 mice. This difference might have an impact on the type of Aβ-transporters regulated and the moments at which these regulations begin. Thus, while the increase in BBB amount of Abcb1 was observed in old 3xTg-AD mice aged 18 months, the downregulation of Abcb1 amount was found in young 3-month-old Tg2576 mice. In addition, no difference in the BBB amount of Rage has been observed in young Tg2576 mice while the up-regulation of this receptor has been observed early in 3-month-old 3xTg-AD mice.

In humans, however, the BBB amounts of Abcb1 are dependent on multiple factors other than AD, such as age, genetic polymorphisms, and coexisting diseases. Thus, the number of patients included in these studies must be large enough to confirm the expression pattern of Abcb1 in AD.

Abcb1 and Abcg2 have been recently suggested to function as gatekeepers at the BBB to prevent the circulated Aβ peptides from entering the brain [65, 66]. Up-regulation of Abcb1 or Abcg2 in response to cerebral amyloid accumulation have also been observed in old rats’ choroid plexus or in late-stage AD brains, respectively [20, 67]. In the current study, the down regulation of Lrp-1, another Aβ efflux transporter, and the up-regulation of Rage in 18-month-old 3xTg-AD mice could induce an increase in the blood-to-brain flux of Aβ. However, this increase in the Aβ influx was counterbalanced by an increase in the amount of both Abcb1 and Abcg2 in combination with a slight decrease of Oatp1a4 in 3xTg-AD mice. Consequently, the Cl_up of [³H]Aβ_1 - 40 was significantly decreased in these mice (Fig. 5). This suggests that there is a third defense mechanism operating in 18-month-old 3xTg-AD mice to reduce the cerebral amyloid burden in these mice. Our results agree with recent studies showing that the plasma concentrations of Aβ tend to increase in 15- and 22-month-old 3xTg-AD mice compared to younger mice aged 9 and 17 months, respectively, maybe as the result of an enhanced Aβ efflux from the brain [68 –70]. However, this late-onset system may not be sufficient to reverse the accumulation of Aβ in the brains of AD patients or those of 3xTg-AD mice [37, 71].

As the regulation of the transporters involved in the mouse BBB Aβ efflux occurs at different stage of the pathology (i.e., 3 months for Abcg4 and 18 months for Abcb1/Abcg2), we hypothesized that the mechanisms involved in the regulation of Abcg4 and Abcb1/Abcg2 are different. The mechanisms underlying the early increase in the amount of Abcg4 at the BBB of 3xTg-AD mice remain unknown. There is little published information on the regulation of this transporter in the brain. Although the sequence of Abcg4 is very similar to that of Abcg1, another well-known cholesterol transporter, it seems to be regulated differently. Abcg1 synthesis is increased in response to both RXR and LXR (liver X receptor) agonists, while that of Abcg4 is only regulated by RXR agonists [61, 72]. In contrast, Abcb1 and Abcg2 share the same transcription factors (e.g., pregnane X receptor) and constitutive androstane receptor) [73, 74]. It has also been suggested that inflammatory conditions can modify the synthesis and/or function of both Abcb1 and Abcg2 at the BBB [75 –77]. Elevated concentrations of cytokines such as TNF-alpha were detected recently in 3xTg-AD mice aged 16 months [77]. These inflammatory conditions may favor the late increases in Abcb1/Abcg2 in these mice. Further mechanistic studies are thus necessary to confirm and establish the factors regulating the synthesis of Aβ efflux and/or influx transporters in the brain capillaries. A thorough understanding of these regulatory mechanisms could lead to the development of therapeutic strategies eliminating Aβ from the brain.

In this study, we did not use transporter inhibitors to evaluate the extent to which each transporter exerts an effect on the brain uptake clearance of Aβ because of the non-specificity of these compounds. Moreover, at each stage of the disease, there were often more than one Aβ transporter that were regulated at the BBB of 3xTg-AD mice. The results obtained with non-specific inhibitors could be very difficult to interpret. However, it would be interesting to study the BBB transport of Aβ in 3xTg-AD mice that are knocked-out or knocked-in for an Aβ transporter. The use of transporter inhibitors in these cases would be more valuable to confirm the effect of the studied transporter on the BBB transport of Aβ in the AD transgenic mice.

In conclusion, the increase in Rage is associated with compensatory regulation of Aβ and cholesterol efflux transporters in the BBB so as to reduce the amount of Aβ in the brain. We have identified three lines of defense (early, mid, and late) at the BBB.Figure 7 summarizes the changes at the BBB during the AD-like aging of our murine model of AD. The early increase in brain Aβ uptake mediated by the receptor Rage is offset by increased elimination of Aβ from the brain mediated by the efflux transporter Abcg4. This seems to be the initial defense system limiting the accumulation of Aβ in the brain; it keeps the flux of Aβ across the BBB in equilibrium. The increase in the Rage-mediated influx of Aβ into the brain is no longer offset by an increase in Aβ efflux transporter Abcg4 from the brain in older mice (6 months). The influx and efflux of Aβ should be therefore no longer in equilibrium. However, Aβ Cl_up is still maintained. This could be due to an increase in the synthesis of the cholesterol transporter Abca1 whose level is maximal in 6-month-old mice and may indirectly control the transport of Aβ across the BBB. This is our postulated indirect second mechanism of limiting the accumulation of Aβ in the brain. Lastly, 18-month-old mice with late stage AD have a third mechanism for clearing Aβ from the brain. This involves the increased synthesis of two Aβ efflux transporters (Abcg2/Abcb1) and a slight decrease in an influx transporter (Oatp1a4) which results in a significant decrease in the uptake of Aβ by the brain. Our results provide further evidence that the BBB is a dynamic barrier, adapting its transporter profile in response to pathological conditions. The most efficient therapeutic strategy for limiting the accumulation of Aβ in the brain as early as possible in disease development could be to block Aβ influx by inhibiting Rage and/or Oatp1a4, or to stimulate the synthesis and/or function of Abca1 and/or Abcg4 at the BBB.

Footnotes

ACKNOWLEDGMENTS

We thank Dr. Owen Parkes for editing the English text and Valérie Domergue-Dupont, head of the Faculty of Pharmacy central animal facility, for taking care of the mice. We are grateful for the assistance of Claudine Deloménie (Trans-Prot, IFR141-IPSIT) in technical support for this work, particularly relating to mouse genotyping.

This work was supported by Paris Sud University (France), Paris Descartes University (France), Laval University (Quebec, Qc, Canada), the Canadian Institutes of Health Research (MOP 102532) and the “Fonds de la recherche en santé du Québec” (Canada).

Authors’ disclosures available online ().

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Age-Dependent Regulation of the Blood-Brain Barrier Influx/Efflux Equilibrium of Amyloid-β Peptide in a Mouse Model of Alzheimer’s Disease (3xTg-AD)

Abstract

Keywords

INTRODUCTION

MATERIALS AND METHODS

Animals

Reagents

Surgery and perfusion

Calculation of BBB transport parameters

Capillary isolation

Western blotting

Statistical analysis

RESULTS

The Aβ influx transporters Rage and Oatp1a4 in mouse BCECs

The Aβ efflux transporters Abcb1, Abcg2, Abcg4 and Lrp-1 in mouse BCECs

The cholesterol efflux transporter Abca1 in mouse BCECs

Brain uptake of [3H]Aβ1 - 40

Brain uptake of [3H]cholesterol

DISCUSSION

Footnotes

ACKNOWLEDGMENTS

References

Brain uptake of [³H]Aβ_1 - 40

Brain uptake of [³H]cholesterol