Marie-Christine Boucau

Apical-to-Basolateral Transport of Amyloid-β Peptides through Blood-Brain Barrier Cells is Mediated by the Receptor for Advanced Glycation End-Products and is Restricted by P-Glycoprotein

Several studies have highlighted the close relationship between Alzheimers disease (AD) and alterations in the bidirectional transport of amyloid-β (Aβ) peptides across the blood-brain barrier (BBB). The brain capillary endothelial cells (BCECs) that compose the BBB express the receptors and transporters that enable this transport process. There is significant in vivo evidence to suggest that P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) restrict Aβ peptides entry into the brain, whereas the receptor for advanced glycation end-products (RAGE) seems to mediate apical-to-basolateral passage across the BBB. However, deciphering the molecular mechanisms underlying these in vivo processes requires further in vitro characterization. Using an in vitro BBB model and specific competition experiments against RAGE, we have observed a significant decrease in apical-to-basolateral (but not basolateral-to-apical) transport of Aβ1-40 and Aβ1-42 peptides through BCECs. This transport is a caveolae-dependent process and fits with the apical location of RAGE observed in confocal microscopy experiments. Inhibition of P-gp and BCRP using different inhibitors increases transport of Aβ peptides suggesting that these efflux pumps are involved in Aβ peptide transport at the BCECs level. Taken as a whole, these results demonstrate the involvement of the caveolae-dependent transcytosis of Aβ peptides through the BBB in a RAGE-mediated transport process, reinforcing the hypothesis whereby this receptor is a potential drug target in AD.

Brain pericytes ABCA1 expression mediates cholesterol efflux but not cellular amyloid-β peptide accumulation.

In brain, excess cholesterol is metabolized into 24S-hydroxycholesterol (24S-OH-chol) and eliminated into the circulation across the blood-brain barrier. 24S-OH-chol is a natural agonist of the nuclear liver X receptors (LXRs) involved in peripheral cholesterol homeostasis. The effects of this oxysterol on the pericytes embedded in the basal lamina of this barrier (close to the brain compartment) have not been previously studied. We used primary cultures of brain pericytes to demonstrate that the latter express LXR nuclear receptors and their target gene ATP-binding cassette, sub-family A, member 1 (ABCA1), known to be one of the major transporters involved in peripheral lipid homeostasis. Treatment with 24S-OH-chol caused an increase in ABCA1 expression that correlated with a reverse cholesterol transfer to apolipoprotein E, apolipoprotein A-I, and high density lipoprotein particles. Inhibition of ABCA1 decreased this efflux. As pericytes are able to internalize the amyloid-β peptides which accumulate in brain of Alzheimers disease patients, we then investigated the effects of 24S-OH-chol on this process. We found that the cellular accumulation process was not modified by 24S-OH-chol treatment. Overall, our results highlight the importance of the LXR/ABCA1 system in brain pericytes and suggest a new role for these cells in brain cholesterol homeostasis.

In vitro discrimination of the role of LRP1 at the BBB cellular level: Focus on brain capillary endothelial cells and brain pericytes

Several studies have demonstrated that the blood-brain barrier (BBB) (dynamic cellular complex composed by brain capillary endothelial cells (BCECs) and surrounded by astrocytic end feet and pericytes) regulates the exchanges of amyloid β (Aβ) peptide between the blood and the brain. Deregulation of these exchanges seems to be a key trigger for the brain accumulation of Aβ peptide observed in Alzheimers disease (AD). Whereas the involvement of receptor for advanced glycation end-products in Aβ peptide transcytosis has been demonstrated in our laboratory, low-density lipoprotein receptors role at the cellular level needs to be clarified. For this, we used an in vitro BBB model that consists of a co-culture of bovine BCECs and rat glial cells. This model has already been used to characterize low-density lipoprotein receptor-related peptide (LRP)s involvement in the transcytosis of molecules such as tPA and angiopep-2. Our results suggest that Aβ peptide efflux across the BCEC monolayer involves a transcellular transport. However, the experiments with RAP discard an involvement of LRP family members at BCECs level. In contrast, our results show a strong transcriptional expression of LRP1 in pericytes and suggest its implication in Aβ endocytosis. Moreover, the observations of pericytes contraction and local downregulation of LRP1 in response to Aβ treatment opens up perspectives for studying this cell type with respect to Aβ peptide metabolism and AD.

Oxysterols decrease apical-to-basolateral transport of Aß peptides via an ABCB1-mediated process in an in vitro Blood-brain barrier model constituted of bovine brain capillary endothelial cells

It is known that activation of the liver X receptors (LXRs) by natural or synthetic agonists decreases the amyloid burden and enhances cognitive function in transgenic murine models of Alzheimers disease (AD). Recent evidence suggests that LXR activation may affect the transport of amyloid ß (Aß) peptides across the blood-brain barrier (the BBB, which isolates the brain from the peripheral circulation). By using a well-characterized in vitro BBB model, we demonstrated that LXR agonists (24S-hydroxycholesterol, 27-hydroxycholesterol and T0901317) modulated the expression of target genes involved in cholesterol homeostasis (such as ATP-binding cassette sub-family A member 1 (ABCA1)) and promoted cellular cholesterol efflux to apolipoprotein A-I and high density lipoproteins. Interestingly, we also observed a decrease in Aß peptide influx across brain capillary endothelial cells, although ABCA1 did not appear to be directly involved in this process. By focusing on others receptors and transporters that are thought to have major roles in Aß peptide entry into the brain, we then demonstrated that LXR stimulation provoked an increase in expression of the ABCB1 transporter (also named P-glycoprotein (P-gp)). Further investigations confirmed ABCB1s involvement in the restriction of Aß peptide influx. Taken as a whole, our results not only reinforce the BBBs key role in cerebral cholesterol homeostasis but also demonstrate the importance of the LXR/ABCB1 axis in Aß peptide influx-highlighting an attractive new therapeutic approach whereby the brain could be protected from peripheral Aß peptide entry.

Bexarotene Promotes Cholesterol Efflux and Restricts Apical-to-Basolateral Transport of Amyloid-β Peptides in an In Vitro Model of the Human Blood-Brain Barrier

One of the prime features of Alzheimers disease (AD) is the excessive accumulation of amyloid-β (Aβ) peptides in the brain. Several recent studies suggest that this phenomenon results from the dysregulation of cholesterol homeostasis in the brain and impaired bidirectional Aβ exchange between blood and brain. These mechanisms appear to be closely related and are controlled by the blood-brain barrier (BBB) at the brain microvessel level. In animal models of AD, the anticancer drug bexarotene (a retinoid X receptor agonist) has been found to restore cognitive functions and decrease the brain amyloid burden by regulating cholesterol homeostasis. However, the drugs therapeutic effect is subject to debate and the exact mechanism of action has not been characterized. Therefore, the objective of this present study was to determine bexarotenes effects on the BBB. Using an in vitro model of the human BBB, we investigated the drugs effects on cholesterol exchange between abluminal and luminal compartments and the apical-to-basolateral transport of Aβ peptides across the BBB. Our results demonstrated that bexarotene induces the expression of ABCA1 but not ApoE. This upregulation correlates with an increase in ApoE2-, ApoE4-, ApoA-I-, and HDL-mediated cholesterol efflux. Regarding the transport of Aβ peptides, bexarotene increases the expression of ABCB1, which in turn decreases Aβ apical-to-basolateral transport. Our results showed that bexarotene not only promotes the cholesterol exchange between the brain and the blood but also decreases the influx of Aβ peptides across BBB, suggesting that bexarotene is a promising drug candidate for the treatment of AD.

Transient oxygen-glucose deprivation sensitizes brain capillary endothelial cells to rtPA at 4h of reoxygenation.

Thrombolysis treatment of acute ischemic stroke is limited by the pro-edematous and hemorrhagic effects exerted by reperfusion, which disrupts the blood-brain barrier (BBB) capillary endothelium in the infarct core. Most studies of the ischemic BBB overlook the complexity of the penumbral area, where the affected brain cells are still viable following deprivation. Our present objective was to examine in vitro the kinetic impact of reoxygenation on the integrity of ischemic BBB cells after oxygen-glucose deprivation. Through the use of a co-culture of brain capillary endothelial cells and glial cells, we first showed that the transendothelial permeability increase induced by deprivation can occur with both preserved cell viability and interendothelial tight junction network. The subtle and heterogeneous alteration of the tight junctions was observable only through electron microscopy. A complete permeability recovery was then found after reoxygenation, when Vimentin and Actin networks were reordered. However, still sparse ultrastructural alterations of tight junctions suggested an acquired vulnerability. Endothelial cells were then exposed to recombinant tissue-type plasminogen activator (rtPA) to define a temporal profile for the toxic effect of this thrombolytic on transendothelial permeability. Interestingly, the reoxygenated BBB broke down with aggravated tight junction disruption when exposed to rtPA only at 4h after reoxygenation. Moreover, this breakdown was enhanced by 50% when ischemic glial cells were present during the first hours of reoxygenation. Our results suggest that post-stroke reoxygenation enables retrieval of the barrier function of brain capillary endothelium when in a non-necrotic environment, but may sensitize it to rtPA at the 4-hour time point, when both endothelial breakdown mechanisms and glial secretions could be identified and targeted in a therapeutical perspective.

β-Cyclodextrins Decrease Cholesterol Release and ABC-Associated Transporter Expression in Smooth Muscle Cells and Aortic Endothelial Cells

Atherosclerosis is an inflammatory disease that leads to an aberrant accumulation of cholesterol in vessel walls forming atherosclerotic plaques. During this process, the mechanism regulating complex cellular cholesterol pools defined as the reverse cholesterol transport (RCT) is altered as well as expression and functionality of transporters involved in this process, namely ABCA1, ABCG1, and SR-BI. Macrophages, arterial endothelial and smooth muscle cells (SMCs) have been involved in the atherosclerotic plaque formation. As macrophages are widely described as the major cell type forming the foam cells by accumulating intracellular cholesterol, RCT alterations have been poorly studied at the arterial endothelial cell and SMC levels. Amongst the therapeutics tested to actively counteract cellular cholesterol accumulation, the methylated β-cyclodextrin, KLEPTOSE® CRYSMEβ, has recently shown promising effects on decreasing the atherosclerotic plaque size in atherosclerotic mouse models. Therefore we investigated in vitro the RCT process occurring in SMCs and in arterial endothelial cells (ABAE) as well as the ability of some modified β-CDs with different methylation degree to modify RCT in these cells. To this aim, cells were incubated in the presence of different methylated β-CDs, including KLEPTOSE® CRYSMEβ. Both cell types were shown to express basal levels of ABCA1 and SR-BI whereas ABCG1 was solely found in ABAE. Upon CD treatments, the percentage of membrane-extracted cholesterol correlated to the methylation degree of the CDs independently of the lipid composition of the cell membranes. Decreasing the cellular cholesterol content with CDs led to reduce the expression levels of ABCA1 and ABCG1. In addition, the cholesterol efflux to ApoA-I and HDL particles was significantly decreased suggesting that cells forming the blood vessel wall are able to counteract the CD-induced loss of cholesterol. Taken together, our observations suggest that methylated β-CDs can significantly reduce the cellular cholesterol content of cells forming atherosclerotic lesions and can subsequently modulate the expression of ABC transporters involved in RCT. The use of methylated β-CDs would represent a valuable and efficient tool to interfere with atherosclerosis pathogenesis in patients, nonetheless their mode of action still needs further investigations to be fully understood and finely controlled at the cellular level.

Disease-Induced Alterations in Brain Drug Transporters in Animal Models of Alzheimer’s Disease

PurposeAlzheimer’s disease (AD) may disturb functions of the blood-brain barrier and change the disposition of drugs to the brain. This study assessed the disease-induced changes in drug transporters in the brain capillaries of transgenic AD mice.MethodsEighteen drug transporters and four tight junction-associated proteins were analyzed by RT-qPCR in cortex, hippocampus and cerebellum tissue samples of 12–16-month-old APdE9, Tg2576 and APP/PS1 transgenic mice and their healthy age-matched controls. In addition, microvessel fractions enriched from 1-3-month-old APdE9 mice were analyzed using RT-qPCR and Western blotting. Brain transport of methotrexate in APdE9 mice was assessed by in vivo microdialysis.ResultsThe expression profiles of studied genes were similar in brain tissues of AD and control mice. Instead, in the microvessel fraction in APdE9 mice, >2-fold alterations were detected in the expressions of 11 genes but none at the protein level. In control mice strains, >5-fold changes between different brain regions were identified for Slc15a2, Slc22a3 and occludin. Methotrexate distribution into hippocampus of APdE9 mice was faster than in controls.ConclusionsThe expression profile of mice carrying presenilin and amyloid precursor protein mutations is comparable to controls, but clear regional differences exist in the expression of drug transporters in brain.

ABCA7 Downregulation Modifies Cellular Cholesterol Homeostasis and Decreases Amyloid-β Peptide Efflux in an in vitro Model of the Blood-Brain Barrier

The role of ABCA7 in brain homeostasis and Alzheimers disease (AD) is currently under intense scrutiny, since it has been reported that polymorphisms in the Abca7 gene and a loss of function of the protein are closely linked to excessive accumulation of amyloid peptides and disturbed cholesterol homeostasis. The blood-brain barrier (BBB), which isolates the brain from the blood compartment, is involved in both of these processes. We therefore hypothesized that ABCA7 downregulation might affect cholesterol and amyloid exchanges at the BBB. Using siRNA and primary cultures of mouse endothelial cells purified from brain microvessels and seeded on Transwell ® inserts, we investigated the role of ABCA7 in cholesterol and amyloid exchanges across the BBB. Our results showed that a decrease in ABCA7 expression at the BBB provokes in vitro a reduction in ABCA1 expression and a decrease in APOE secretion. In vitro, these decreases reduce cholesterol exchange across the BBB, particularly for high-density lipoproteins and ApoA-I particles. When ABCA7 was absent, we observed a reduction in Aβ peptide basolateral-to-apical transport in the presence of ApoA-I, with non-significant changes in the expression levels of Rage, Lrp1, Abcb1, Abcc1, and Abcg2. Our study in murine BBB model highlighted a putative new role for ABCA7 in AD via the proteins involvement in cholesterol metabolism and amyloid clearance at the BBB.