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. 2014 Sep;1842(9):1806-15.
doi: 10.1016/j.bbadis.2014.06.029. Epub 2014 Jul 2.

Mixed oligomers and monomeric amyloid-β disrupts endothelial cells integrity and reduces monomeric amyloid-β transport across hCMEC/D3 cell line as an in vitro blood-brain barrier model

Hisham Qosa  1 , Harry LeVine 3rd  2 , Jeffrey N Keller  3 , Amal Kaddoumi  4
Affiliations

Mixed oligomers and monomeric amyloid-β disrupts endothelial cells integrity and reduces monomeric amyloid-β transport across hCMEC/D3 cell line as an in vitro blood-brain barrier model

Hisham Qosa et al. Biochim Biophys Acta. 2014 Sep.

Abstract

Senile amyloid plaques are one of the diagnostic hallmarks of Alzheimer's disease (AD). However, the severity of clinical symptoms of AD is weakly correlated with the plaque load. AD symptoms severity is reported to be more strongly correlated with the level of soluble amyloid-β (Aβ) assemblies. Formation of soluble Aβ assemblies is stimulated by monomeric Aβ accumulation in the brain, which has been related to its faulty cerebral clearance. Studies tend to focus on the neurotoxicity of specific Aβ species. There are relatively few studies investigating toxic effects of Aβ on the endothelial cells of the blood-brain barrier (BBB). We hypothesized that a soluble Aβ pool more closely resembling the in vivo situation composed of a mixture of Aβ40 monomer and Aβ42 oligomer would exert higher toxicity against hCMEC/D3 cells as an in vitro BBB model than either component alone. We observed that, in addition to a disruptive effect on the endothelial cells integrity due to enhancement of the paracellular permeability of the hCMEC/D3 monolayer, the Aβ mixture significantly decreased monomeric Aβ transport across the cell culture model. Consistent with its effect on Aβ transport, Aβ mixture treatment for 24h resulted in LRP1 down-regulation and RAGE up-regulation in hCMEC/D3 cells. The individual Aβ species separately failed to alter Aβ clearance or the cell-based BBB model integrity. Our study offers, for the first time, evidence that a mixture of soluble Aβ species, at nanomolar concentrations, disrupts endothelial cells integrity and its own transport across an in vitro model of the BBB.

Keywords: Alzheimer's disease; Amyloid-β; Blood–brain barrier; Clearance.

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Conflict of interest statement

Conflict of interest

The authors declare no competing financial interest.

Figures

Figure 1
Figure 1
Preparation of Aβ mixtures. Starting from synthetic Aβ42 monomer, a stable oligomer was prepared and fractionated by SEC. (A) A representative blot of crude Aβ42 oligomers. (B) SEC profile of Aβ42 oligomers as measured by an oligomer-specific ELISA. Fractions 4–8 containing oligomers were collected for further analysis. (C) A representative western blot analysis for purified Aβ42 oligomer, Aβ mixture and a calibration curve of monomeric synthetic Aβ40 standards to adjust the concentration of each species for cell treatment. (D) Densitometry versus concentration calibration curve that was used to quantify Aβ concentrations.
Figure 2
Figure 2
Cell toxicity assays after treatment with Aβ preparations. (A) Concentration dependent MTT cytotoxcity assay of 0, 50, 100 and 250 nM of monomeric Aβ40 with or without 50 nM Aβ42 oligomer. (B) Concentration dependent MTT cytotoxcity assay of 0, 25, 50 and 100 nM of Aβ42 oligomer with or without 100 nM Aβ40 monomer. Significant reductions in cell viability as measured by MTT reduction to formazan were observed only after 24 h treatment with 250 nM monomeric Aβ40, 100 nM Aβ42 oligomer and their corresponding mixtures. (C) Microscopic images of cells after exposure to control media or Aβ mixture for 24 h. The data are expressed as mean ± SEM of n = 3 independent experiments.
Figure 3
Figure 3
Effect of Aβ preparations on the integrity of an hCMEC/D3 monolayer model of the BBB endothelium. Apical to basolateral 14C-inulin permeation across the hCMEC/D3 cell monolayer was monitored for 2 h after 24 h treatment with different Aβ preparations at sub-toxic nanomolar concentrations. (A) Schematic presentation of hCMEC/D3 monolayer shows the direction of inulin permeation when added to the apical side. (B) Concentration dependent studies on the effect of Aβ40 monomer with or without 50 nM Aβ42 oligomer on inulin permeation, and (C) Concentration dependent studies on the effect of Aβ42 oligomer with or without 100 nM Aβ40 monomer on inulin permeation. 14C-inulin permeaion in the apical to basolateral direction was significantly enhanced after treatment with Aβ mixture of 100 nM Aβ40 monomer and 50 nM Aβ40 oligomer indicating the disruptive effect of this Aβ mixture on the integrity of the hCMEC/D3 cells. Data represent mean ± SEM from three independent experiments, * P<0.05.
Figure 4
Figure 4
Transport of 125I-Aβ40 across hCMEC/D3 cell monolayer after exposure to Aβ preparations. (A) Schematic presentation of hCMEC/D3 monolayer shows the direction of transport of 125I-Aβ40 added to the basolateral side. (B) Transport quotient CQB→A of 125I-Aβ40 across hCMEC/D3 monolayer treated for 24 h with increasing concentrations of Aβ40 monomer with or without 50 nM Aβ42 oligomers, and (C) Transport quotient CQB→A of 125I-Aβ40 across hCMEC/D3 monolayer treated for 24 h with increasing concentrations of Aβ42 oligomers with or without 100 nM Aβ40 monomer. While Aβ mixture of 100 nM Aβ40 monomer and 50 nM Aβ42 oligomer significantly reduced CQB→A of 125I-Aβ40, other Aβ preparations did not affect 125I-Aβ40 transport. Data represent mean ± SEM from three independent experiments; * P<0.05.
Figure 5
Figure 5
Degradation of 125I-Aβ40 by hCMEC/D3 cell monolayer after exposure to Aβ preparations. (A) % degradation of 125I-Aβ40 by hCMEC/D3 monolayer treated for 24 h with increasing concentrations of Aβ40 monomer with or without 50 nM Aβ42 oligomers, and (B) % degradation of 125I-Aβ40 by hCMEC/D3 monolayer treated for 24 h with increasing concentrations of Aβ42 with or without 100 nM Aβ40 monomer. None of Aβ preparations altered % degradation of 125I-Aβ40 after 24 h treatment. Data represent mean ± SEM from three independent experiments; * P<0.05.
Figure 6
Figure 6
Expression of P-gp, LRP1 and RAGE in hCMEC/D3 cells. (A) Western blot analysis of P-gp, LRP1, and RAGE protein expressions in hCMEC/D3 cells after 24 h exposure to control media, 100 nM monomeric Aβ40, 50 nM Aβ42 oligomers, or Aβ mixture of both. (B) Densitometry analyses showed similar expression level of P-gp in all treatment groups, significantly higher RAGE expression and lower LRP1 expression in hCMEC/D3 cells treated with Aβ mixture. Data represent mean ± SEM from three independent experiments; ** P<0.01.
Figure 7
Figure 7
(A) Western blot analysis of IDE and NEP protein expression in hCMEC/D3 cells after 24 h exposure to control media, 100 nM monomeric Aβ40, 50 nM Aβ42 oligomers or Aβ mixture of both. (B) Corresponding densitometry analysis showed that none of the Aβ preparations altered the expression of IDE or NEP. Data represent mean ± SEM from three independent experiments.
Figure 8
Figure 8
Schematic presentation for a model describing toxic effect of soluble Aβ pool against BBB endothelial cells. Faulty clearance of monomeric Aβ results in its brain accumulation. Accumulated Aβ initiates a cascade of Aβ aggregation to form soluble aggregates of different sizes and types. In addition to their intrinsic neurotoxicity, soluble Aβ aggregates act synergistically with monomeric Aβ40 in the form of Aβ mixture to disrupt BBB endothelial cells and enhance more Aβ accumulation by halting its clearance across BBB. This accelerates the formation of a wide range of soluble and insoluble Aβ assemblies enhancing the development of CAA and AD.
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