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Comparative Study
. 2020 Apr;57(4):2131-2141.
doi: 10.1007/s12035-020-01873-x. Epub 2020 Jan 17.

Reduced Influence of apoE on Aβ43 Aggregation and Reduced Vascular Aβ43 Toxicity as Compared with Aβ40 and Aβ42

Lieke Jäkel  1   2 , Elisanne A L M Biemans  1   2 , Catharina J M Klijn  1 , H Bea Kuiperij  1   2 , Marcel M Verbeek  3   4   5
Affiliations
Comparative Study

Reduced Influence of apoE on Aβ43 Aggregation and Reduced Vascular Aβ43 Toxicity as Compared with Aβ40 and Aβ42

Lieke Jäkel et al. Mol Neurobiol. 2020 Apr.

Abstract

The amyloid-β 43 (Aβ43) peptide has been shown to be abundantly expressed in Alzheimer's disease plaques, whereas only relatively low levels have been demonstrated in cerebral amyloid angiopathy (CAA). To better understand this discrepant distribution, we studied various biochemical properties of Aβ43, in comparison with Aβ40 and Aβ42. We assessed the interaction of Aβ43 with the three apoE isoforms (apoE2, apoE3, and apoE4) using SDS-PAGE/Western blotting and ELISA, aggregation propensity using thioflavin T assays, and cytotoxicity towards cerebrovascular cells using MTT assays. We found that Aβ43 did not differ from Aβ42 in its interaction with apoE, whereas Aβ40 had a significantly lower degree of interaction with apoE. At a molar ratio of 1:100 (apoE:Aβ), all apoE isoforms were comparably capable of inhibiting aggregation of Aβ40 and Aβ42, but not Aβ43. All Aβ variants had a concentration-dependent negative effect on metabolic activity of cerebrovascular cells. However, the degree of this effect differed for the three Aβ isoforms (Aβ40 > Aβ42 > Aβ43), with Aβ43 being the least cytotoxic peptide towards cerebrovascular cells. We conclude that Aβ43 has different biochemical characteristics compared with Aβ40 and Aβ42. Aggregation of Aβ43 is not inhibited by apoE, in contrast to the aggregation of Aβ40 and Aβ42. Furthermore, cerebrovascular cells are less sensitive towards Aβ43, compared with Aβ40 and Aβ42. In contrast, Aβ43 neither differed from Aβ42 in its aggregation propensity (in the absence of apoE) nor in its apoE-binding capacity. Altogether, our findings may provide an explanation for the lower levels of Aβ43 accumulation in cerebral vessel walls.

Keywords: Aggregation; Alzheimer’s disease; Amyloid-β (1–43); Apolipoprotein E; Cerebral amyloid angiopathy; Cytotoxicity.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Fig. 1
Fig. 1
Aβ-apoE complex formation. The complex formation between different isoforms of Aβ and apoE was assessed semi-quantitatively using ELISA and SDS-PAGE/Western blotting. (a) Under the non-reducing conditions of the ELISA, apoE isoforms bound to the Aβ peptides in a comparable way. Aβ40 bound significantly less efficient to all apoE isoforms as compared with Aβ42 and Aβ43. (b) Under the reducing conditions of SDS-PAGE/Western blot analysis, Aβ40 bound less efficient to apoE2 and apoeE3 compared with Aβ42 and Aβ43. In addition, no interaction between apoE4 and any of the Aβ isoforms was observed using SDS-PAGE/Western blot analysis. The upper panel shows the quantification of the apoE-Aβ complex band of the blot that is shown in the lower panel. For ELISA experiments, samples were assessed in duplicates. Data represent mean (sd) of n = 4 (ELISA) and n = 2 (SDS-PAGE/Western blot) experiments. *p < 0.05; **p < 0.01; and ***p < 0.001 as analyzed by two-way ANOVA including apoE and Aβ isoform as variables
Fig. 2
Fig. 2
Aggregation kinetics of Aβ. Compared with Aβ42 and Aβ43, Aβ40 aggregated slower (a) and had a significantly higher t50 value, at which ThT fluorescence reached 50% of the maximum amplitude (b). ApoE3 inhibited Aβ40 aggregation in a concentration-dependent manner (c). Aggregation of Aβ40 (d) and Aβ42 (e) was inhibited by the addition of 0.1 μM apoE2, apoE3, or apoE4. Aβ43 aggregation was not affected by the addition of 0.1 μM of any apoE isoform (f). Aβ concentrations in all experiments were 10 μM. a, b Data represent mean (sd) of n = 4 experiments performed. c–f Representative data of n = 3 experiments. The t50 times were analyzed by ANOVA, followed by Bonferroni’s post hoc testing. *p < 0.05 and **p < 0.01
Fig. 3
Fig. 3
Effect of Aβ on metabolic activity of cerebrovascular cells. Aβ40, Aβ42, and Aβ43 reduced metabolic activity of HBPs (a), SMCs (b), and hBMECs (c) in a concentration-dependent manner. This effect was most pronounced for Aβ40, and least pronounced for Aβ43. HBMECs were less sensitive to Aβ compared with SMCs and HBPs, as only relatively high concentrations of 1 and 10 μM reduced metabolic activity. Data represent mean (sd) of 3 experiments. Data were analyzed by ANOVA, followed by Dunnet’s post hoc testing *p < 0.05; **p < 0.01; and ***p < 0.001 for Aβ-treated cells versus vehicle-treated cellsz
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