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Amyloid-beta-induced occludin down-regulation and increased permeability in human brain endothelial cells is mediated by MAPK activation.

Abstract Vascular dysfunction is emerging as a key pathological hallmark in Alzheimer’s disease (AD). A leaky blood–brain barrier (BBB) has been described in AD patient tissue and in vivo AD mouse models. Brain endothelial cells (BECs) are linked together by tight junctional (TJ) proteins, which are a key determinant in restricting the permeability of the BBB. The amyloid β (Aβ) peptides of 1–40 and 1–42 amino acids are believed to be pivotal in AD pathogenesis. We therefore decided to investigate the effect of Aβ 1–40, the Aβ variant found at the highest concentration in human plasma, on the permeability of an immortalized human BEC line, hCMEC/D3. Aβ 1–40 induced a marked increase in hCMEC/D3 cell permeability to the paracellular tracer 70 kD FITC-dextran when compared with cells incubated with the scrambled Aβ 1–40 peptide. Increased permeability was associated with a specific decrease, both at the protein and mRNA level, in the TJ protein occludin, whereas claudin-5 and ZO-1 were unaffected. JNK and p38MAPK inhibition prevented both Aβ 1–40-mediated down-regulation of occludin and the increase in paracellular permeability in hCMEC/D3 cells. Our findings suggest that the JNK and p38MAPK pathways might represent attractive therapeutic targets for preventing BBB dysfunction in AD.

environment, and has been demonstrated in AD patients by the abnormal presence of plasma proteins in the CSF or brain parenchyma, although some contrasting results exist [15][16][17]. Most recent studies have demonstrated a correlation between BBB leakage and advanced-stage AD [14] as well as medial temporal atrophy [13]. Animals receiving a peripheral A␤ injection [18] and transgenic AD mouse models [19] display increased BBB permeability. A␤ peptides have also been demonstrated to increase the permeability of non-human endothelial cell cultures [20,21]. Changes in TJ organization may in part be responsible for the increased BBB permeability found in AD. Indeed, morphological alterations of BEC TJs, suggestive of a leaky BBB, have been observed in AD patients' brain biopsies [22], and occludin downregulation is found in APP/PS1 transgenic mice [23]. A␤ 1-42 has also been noted to transiently decrease occludin expression and cause cldn-5 translocation from TJs in rat primary BECs [24].
In this study, we have investigated the effects of A␤ on the permeability of human BECs in vitro. To do so, we used a wellestablished immortalized human BEC line, hCMEC/D3 cells [25], to determine whether A␤ peptides could induce increased BEC permeability and to explore the signalling pathways that could mediate such changes.

A␤ 1-40 increases hCMEC/D3 cell paracellular permeability
Increased BBB permeability has been demonstrated in AD patient brain tissue and in vivo AD models [13,14,19]. We therefore investigated the effect of A␤ peptides on the paracellular permeability of hCMEC/D3 cells to 70 kD FITC-dextran. As shown in Fig. 2 [26] and in cerebrovascular deposits [27].

A␤ 1-40 specifically decreases levels of occludin at the protein and mRNA levels
The mechanisms behind A␤ 1-40-induced increases in hCMEC/D3 cell paracellular permeability were investigated next. TJ proteins mediate BEC cell-cell contacts at the BBB, and changes in their expression are associated with increased BEC permeability. We therefore investigated the expression of occludin, cldn-5 and ZO-1, the most extensively investigated TJ proteins expressed by BECs, following A␤ 1-40 or sA␤ 1-40 treatment. As shown in Fig. 3A (Fig. 4F), staining was reduced both at the cell borders and intracellularly and in some instances, a complete loss of occludin at the cell junctions was observed. In contrast, there were no changes in either overall expression levels or in the sub-cellular localization of ZO-1 (Fig. 4C and D) or cldn-5 ( Fig. 4A and B) in hCMEC/D3 cells after A␤ 1-40 incubation, compared with sA␤ 1-40 incubation. These data suggest that the A␤ 1-40-induced increase in hCMEC/D3 cell paracellular permeability may be a consequence of reduced occludin levels at cell junctions.

Occludin down-regulation is mediated by the JNK and p38MAPK signalling pathways
We next sought to identify the contribution of various signalling pathways in A␤-mediated occludin down-regulation, using flow cytometry as a quantitative measure of protein expression analysis. We found that inhibitors for pathways that have been previously shown to regulate TJ expression (ROCK, Src, PKC, PI3K) [28] and those known to be activated by A␤ in neurons (NF-␤,  GSK-3␤) [29] did not prevent occludin down-regulation in hCMEC/D3 cells mediated by A␤ 1-40 (Suppl. Fig. 2).
MAPK family members ERK [30], JNK [31] and p38MAPK [32] have been reported to be up-regulated in neurons from AD patients. A␤ 1-40 treatment induced a time-dependent activation of JNK (46-kD form), ERK (42-kD form) and p38MAPK (42-kD form) in hCMEC/D3 cells (Fig. 5A and B). JNK activation was detected as early as 6 hrs post-incubation with A␤ 1-40 and increased with time up to 48 hrs, whereas ERK2 and p38MAPK were also activated by A␤ 1-40 but from 24 hrs onwards.
Inhibitors for each MAPK family member were used in order to investigate the signalling pathways mediating A␤ 1-40-induced occludin down-regulation (Fig. 6A). In this set of experiments, hCMEC/D3 occludin levels were decreased to 71% in A␤ 1-40-treated cells compared with those treated with sA␤ 1-40, and pre-incubation with 50 M of the MEK inhibitor PD98059, which inhibits ERK, did not prevent this down-regulation.

Fig. 4 The expression of TJ proteins by hCMEC/D3 cells after 48 hrs of A␤ 1-40 incubation as assessed by immunocytochemistry. hCMEC/D3 cells were incubated with 5 M (A, C, E) sA␤ 1-40 or (B, D, F) A␤ 1-40 for 48 hrs, fixed in methanol and the expression of cldn-5 (A, B), ZO-1, (C, D) or occludin (E, F) visualized by fluorescence microscopy. Image shown is of one experiment representative of three. Scale bar ϭ 50 M.
brain endothelial occludin down-regulation in a p38MAPK-and JNK-dependent manner.

Discussion
In this study, we initially determined the cytotoxicity of A␤ peptides to hCMEC/D3 cells. We then selected non-cytotoxic concentrations to further investigate specific A␤ effects on BBB permeability, rather than A␤-induced gross permeability changes due to BEC death. The lowest dose of A␤ 1-40 (Fig. S1). Although A␤ 1-40 aggregates and/or oligomers may still induce EC death at high concentrations, as previously shown in neurons [4], the soluble monomeric form of A␤ 1-40 may be responsible for subtler changes in BBB function that do not involve cell death.
Our in vitro findings are consistent with observations in AD patients, where a leaky BBB could potentially disrupt the CNS homeostatic environment and lead to neuronal degeneration.  Increased CSF/plasma protein ratio of albumin and IgG has been noted in AD patients, suggestive of a leaky BBB [13,14,[33][34][35][36]. However, other studies have not confirmed this finding [15][16][17]. The most compelling evidence for increased BBB permeability in AD patients was provided by Zipser et al. [14], who found that prothrombin leakage from the blood into the CNS was significant in advanced-stage AD. In addition, increased cerebrospinal fluid levels of albumin are associated with medial temporal atrophy as assessed by MRI measurements in AD patients [13]. A␤ could play a central role in mediating increased BBB permeability. Indeed, Tg2576 mice, which harbour the human Swedish mutation of APP, display increased BBB permeability after 4 months of age, prior to plaque deposition and disease onset, and these effects can be reversed via A␤ peptide immunization [19,37]. Further evidence comes from studies showing that intravascular jugular vein injections of A␤ 1-40 can induce a leaky BBB in rats [18]. In vitro endothelial cells of non-human origin also display increased permeability after A␤ treatment [20,21]. In the present study, A␤ 1-40 was added to the luminal compartment, which corresponds to the blood side in vivo. Strazielle [21]. They observed that although both luminal and abluminal A␤ increased BEC permeability, the rise in PEG efflux was not observed until much later when A␤ 25-35 was applied abluminally. These authors speculated that the lag period might be explained as the time required for the peptide to diffuse from the lower compartment through the BEC monolayer and subsequently reach high enough levels on the luminal interface to induce barrier changes. Because of the polarized nature of BECs, it is possible that the receptor(s) activated by A␤ (e.g. RAGE, megalin, P-gp or an unknown receptor) mediating permeability effects might be differentially expressed between the luminal and abluminal membranes. Whether A␤ acts on the luminal or abluminal membranes or on both to increase BEC permeability remains to be determined.

et al. compared the effects of luminal and abluminal application of 5 M A␤ 25-35 on the permeability of bovine BECs to the paracellular tracer PEG
Measurements of plasma A␤ levels have been inconclusive as to whether A␤ levels are increased or unaltered in AD [26], although Van Oijen et al. [38] found that high A␤ 1-40 levels in plasma in early life corresponded with an increased risk of developing dementia in later life. Increased plasma levels of A␤, acting at the luminal surface, might induce subtle changes in BBB permeability and lead to the leakage of plasma proteins observed in AD. On the abluminal side of the BBB, increased A␤ production by neurons, stiffening of arteries during aging and/or increased plasma A␤ leakage (as a result of occludin down-regulation in BECs) in AD could potentially reduce the clearance of A␤ from the brain via drainage of interstitial fluid and lead to cerebral amyloid angiopathy (CAA), a frequent occurrence in AD patients [39,40]. In CAA, cerebrovascular deposits of A␤ (of both 1-40 and 1-42 amino acids in length) are found aggregated with other proteins, including apoE and ␣-2 macroglobulin. CAA occurs in both capillaries and small arteries and, to a lesser extent, veins. With respect to capillaries, A␤ deposition in CAA occurs on the BEC basement membrane and protrudes into the neuropil, and can result in capillary occlusion. Whether luminal or abluminal, soluble A␤ may induce occludin down-regulation in BECs prior to or at early stages of CAA, thereby inducing an increased BBB permeability. Plasma proteins that cross the endothelium could in turn bind to and exacerbate the deposition of A␤ around cerebrovascular vessels.
Occludin is an important TJ protein and its down-regulation is associated with increased permeability in BECs [2]. Indeed, the importance of occludin in conferring restrictive TJs has been demonstrated in vitro and in vivo. Transfection of truncated occludin or siRNA knockdown in MDCK cells [41] or Xenopus embryo cells [42] results in increased paracellular permeability. In addition, occludin down-regulation and increased permeability has been observed in models of inflammatory pain [43] and oxidative stress [44], and CNS disorders associated with BBB breakdown such as multiple sclerosis [45]. Here, we found that occludin down-regulation was the most likely cause of the A␤-induced increase in hCMEC/D3 cell permeability. Marco and Skaper [24] have observed that 20 M A␤ 1-42 can induce a decrease in occludin protein levels after 24 hrs, but not 48 hrs, of incubation in rat BECs. In agreement with our study, the authors did not detect any alterations in overall cldn-5 or ZO-1 levels, although cldn-5 appeared to translocate to the cytoplasm. Cerebral extravasation of IgG in APP/PS1 mice fed on a high saturated-fat diet has also been associated with decreased occludin expression at the BBB [23]. A disruption in TJ organization has also been noted in AD patient's biopsy samples [22]. Intriguingly, neuronal occludin over-expression has been detected in brains of AD individuals [46], although the significance of this observation is unclear.
Changes in the levels of occludin protein at 48 hrs were preceded by a decrease in occludin mRNA at 24 hrs. At earlier timepoints, A␤ may still induce changes in BBB permeability by directly acting on TJ organization, either by post-translational modifications of TJ proteins such as phosphorylation or by inducing TJ protein degradation and/or sub-cellular reorganization. Whether A␤ induces post-translational modifications of the occludin protein requires further investigation. Our observation that inhibition of JNK restores occludin mRNA levels to those of controls would indicate that A␤ acts on occludin expression either by suppression of occludin gene transcriptional activity or by decreasing occludin mRNA stability. A␤-mediated occludin downregulation in BECs could therefore represent an important mechanism of increased BBB permeability in AD, as suggested by the present study.
The observation that p38MAPK or JNK inhibition could prevent the A␤-induced increase in BEC permeability suggests that these signalling pathways are potential targets for therapeutic treatments in AD. p38MAPK activation has been associated with occludin down-regulation in response to different stimuli, including combined tumour necrosis factor-␣ (TNF-␣) and interferon-␥ (IFN-␥) incubation [47], transforming growth factor-␤ (TGF-␤) [48] and alcohol [49]. Indeed,Lui et al. [48] have demonstrated that CdCl2-induced TGF-␤3 production, and occludin down-regulation, can be prevented by p38MAPK inhibition at the rat blood-testis barrier. Crucially, in a study by Munoz and co-workers [50], a novel p38MAPK inhibitor was observed to improve synaptic dysfunction and behavioural deficits after A␤ 1-42 intracerebroventricular injections into mice. Although they did not investigate the permeability of the BBB, the authors did find that p38MAPK inhibition suppressed an A␤ 1-42-induced increase in TNF-␣ and IL-1␤ levels in the hippocampus of these mice. A␤mediated activation of astrocytes and microglia can induce cytokine production in vitro [7,[51][52][53] and astrocytic end feet swelling [54] and microglia activation [51] have been observed in AD tissue sections. IL-1␤, TNF-␣ and IL-6 levels are found increased in the CSF or serum of AD patients [55] and cytokines can induce a leaky TJ in vitro [56][57][58][59]. p38MAPK inhibition could therefore act as a dual target in AD, preventing cytokine production and A␤-induced increased BBB permeability.
The JNK pathway also represents a potential novel potential therapeutic target for AD, with its inhibition not only preventing A␤-induced increase in BBB permeability, as demonstrated here, but also neuronal atrophy [60]. There are three genes that encode JNK in mammalian systems, jnk1, jnk2 and jnk3, and each gene is alternatively spliced to create 46 and 55-kD forms of the proteins [61,62]. The significance of each variant is not fully understood. However, JNK1 and -2 are expressed ubiquitously, whereas JNK3 is largely restricted to the brain (reviewed in [63]). Morishima et al. [64] have demonstrated that A␤ 25-35 toxicity is significantly reduced in cortical neurons derived from JNK3 knock-out mice compared with the controls; however, they also state that JNK1-deficient neurons are also resistant to apoptosis. Using antisense technology, JNK1-depleted rat cortical neurons are found to be resistant to A␤ 1-40-induced apoptosis [65]. A␤ in this study was found to activate the 46-kD JNK isoform, which in some studies was found to correspond to JNK1 [65]. Regardless of the isoform, JNK inhibition protects SH-SY5Y neuroblastoma, rat cortical neurons and PC12 arrhenoblastoma cells from A␤-induced apoptosis in vitro [66][67][68]. A further interesting study has demonstrated that JNK inhibition can prevent H2O2-induced ␥-secretase-mediated APP cleavage and A␤ production in SH-SY5Y cells [69]. The JNK pathway might therefore represent a very attractive therapeutic target, to prevent A␤ production, neuronal atrophy and BBB dysfunction.
In summary, we have found that A␤ 1-40 can increase the paracellular permeability of human BECs and this correlates with occludin down-regulation. p38MAPK and JNK inhibition prevented both occludin down-regulation and increased paracellular permeability of BECs, as induced by A␤. The p38MAPK and JNK path-way therefore potentially represent interesting therapeutic targets to ameliorate cerebrovascular dysfunction in AD.

Fig. S1. A␤ 1-40 after 48 hr incubation in the presence (right) or absence (left) of hCMEC/D3 cells. (A)
Soluble A␤ 1-40 monomer was added to culture medium and then incubated either in a collagencoated tissue culture plate or with confluent monolayers of hCMEC/D3 cells grown on a collagen-coated tissue culture plate for 1, 3, 6, 24 or 48 hr. The supernatant was collected and the A␤ peptide levels (6E10, mouse monoclonal IgG1) was investigated using western blotting techniques and a 15% acrylamide gel. (B) A␤ peptide levels were measured as above in whole cell lysates or in scraped collagen-coated dishes at each time point on 15% acrylamide gels.