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

Tai LM, Holloway KA, Male DK, Loughlin AJ, Romero IA - J. Cell. Mol. Med. (2009)

Bottom Line: We therefore decided to investigate the effect of Abeta 1-40, the Abeta variant found at the highest concentration in human plasma, on the permeability of an immortalized human BEC line, hCMEC/D3.Abeta 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 Abeta 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Sciences, The Open University, Milton Keynes, UK.

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 beta (Abeta) 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 Abeta 1-40, the Abeta variant found at the highest concentration in human plasma, on the permeability of an immortalized human BEC line, hCMEC/D3. Abeta 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 Abeta 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 Abeta 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.

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Related in: MedlinePlus

Cell viability of hCMEC/D3 cells incubated with Aβ 1–40 and Aβ 1–42 peptides for 48 hrs. hCMEC/D3 cells were incubated with (A) Aβ 1–40 or sAβ 1–40 and (B) Aβ 1–42 or sAβ 1–42 for 48 hrs at the concentrations indicated. hCMEC/D3 cell viability was measured using an MTT assay and expressed as a percentage of untreated cells. Data represent mean ± S.E.M., n= 3 experiments with quintuplet samples. *P < 0.05 compared with control.
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fig01: Cell viability of hCMEC/D3 cells incubated with Aβ 1–40 and Aβ 1–42 peptides for 48 hrs. hCMEC/D3 cells were incubated with (A) Aβ 1–40 or sAβ 1–40 and (B) Aβ 1–42 or sAβ 1–42 for 48 hrs at the concentrations indicated. hCMEC/D3 cell viability was measured using an MTT assay and expressed as a percentage of untreated cells. Data represent mean ± S.E.M., n= 3 experiments with quintuplet samples. *P < 0.05 compared with control.

Mentions: An aim of this study was to investigate the effect of non-cytotoxic concentrations of Aβ peptides on hCMEC/D3 cell permeability. We therefore measured hCMEC/D3 cell viability in the presence of Aβ 1–42 or Aβ 1–40 or their scrambled counterparts using an MTT assay. As shown in Fig. 1, hCMEC/D3 cell viability was unaffected by 48 hrs of incubation with Aβ 1–42 or Aβ 1–40 at concentrations up to 5 μM. However, at 10 μM, both Aβ 1–40 and Aβ 1–42, but not sAβ peptides, reduced cell viability compared with vehicle-treated cells (70 and 74%, respectively). Therefore, for subsequent investigations, to avoid cytotoxic effects, treatments with Aβ peptides were at 5 μM for 48 hrs. It should be noted that the 4 kD form of Aβ was added initially to the culture medium, and no soluble oligomers were detected by Western blotting over 48 hrs of incubation (Suppl. Fig. 1).


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

Tai LM, Holloway KA, Male DK, Loughlin AJ, Romero IA - J. Cell. Mol. Med. (2009)

Cell viability of hCMEC/D3 cells incubated with Aβ 1–40 and Aβ 1–42 peptides for 48 hrs. hCMEC/D3 cells were incubated with (A) Aβ 1–40 or sAβ 1–40 and (B) Aβ 1–42 or sAβ 1–42 for 48 hrs at the concentrations indicated. hCMEC/D3 cell viability was measured using an MTT assay and expressed as a percentage of untreated cells. Data represent mean ± S.E.M., n= 3 experiments with quintuplet samples. *P < 0.05 compared with control.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3822747&req=5

fig01: Cell viability of hCMEC/D3 cells incubated with Aβ 1–40 and Aβ 1–42 peptides for 48 hrs. hCMEC/D3 cells were incubated with (A) Aβ 1–40 or sAβ 1–40 and (B) Aβ 1–42 or sAβ 1–42 for 48 hrs at the concentrations indicated. hCMEC/D3 cell viability was measured using an MTT assay and expressed as a percentage of untreated cells. Data represent mean ± S.E.M., n= 3 experiments with quintuplet samples. *P < 0.05 compared with control.
Mentions: An aim of this study was to investigate the effect of non-cytotoxic concentrations of Aβ peptides on hCMEC/D3 cell permeability. We therefore measured hCMEC/D3 cell viability in the presence of Aβ 1–42 or Aβ 1–40 or their scrambled counterparts using an MTT assay. As shown in Fig. 1, hCMEC/D3 cell viability was unaffected by 48 hrs of incubation with Aβ 1–42 or Aβ 1–40 at concentrations up to 5 μM. However, at 10 μM, both Aβ 1–40 and Aβ 1–42, but not sAβ peptides, reduced cell viability compared with vehicle-treated cells (70 and 74%, respectively). Therefore, for subsequent investigations, to avoid cytotoxic effects, treatments with Aβ peptides were at 5 μM for 48 hrs. It should be noted that the 4 kD form of Aβ was added initially to the culture medium, and no soluble oligomers were detected by Western blotting over 48 hrs of incubation (Suppl. Fig. 1).

Bottom Line: We therefore decided to investigate the effect of Abeta 1-40, the Abeta variant found at the highest concentration in human plasma, on the permeability of an immortalized human BEC line, hCMEC/D3.Abeta 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 Abeta 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Sciences, The Open University, Milton Keynes, UK.

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 beta (Abeta) 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 Abeta 1-40, the Abeta variant found at the highest concentration in human plasma, on the permeability of an immortalized human BEC line, hCMEC/D3. Abeta 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 Abeta 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 Abeta 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.

Show MeSH
Related in: MedlinePlus