(T1530-06-35) Effect of Vasculotropic Dutch Amyloid Beta 40 on Blood-Brain Barrier (BBB) Endothelial Trafficking and Signaling

Purpose: Cerebrovascular dysfunction, which culminates in the Blood Brain Barrier (BBB) disruption is one of the major pathological changes in Alzheimer’s disease (AD) brain. The BBB disruption in AD is likely caused by the accumulation of amyloid beta (Aβ) peptides in the media and adventitia of cerebral arteries and arterioles, which results in a condition commonly referred to as cerebral amyloid angiopathy (CAA). The two most common isoforms of Aβ often implicated in AD are Aβ40 and Aβ42. While Aβ42 primarily accumulates in the brain parenchyma as amyloid plaques, Aβ40 accumulates in the cerebral vasculature as amyloid deposits. Other mutant forms of Aβ40 such as Dutch (E22Q) Aβ40 (D-Aβ40) tend to accumulate in cerebral vessels more predominantly than native Aβ40. However, molecular mechanisms driving the preferential accumulation of D-Aβ40 in the cerebral vasculature compared to Aβ40 or Aβ42. We hypothesize that both Aβ40 and D-Aβ40 will differentially affect cellular and molecular pathways governing their accumulation in the BBB endothelium. We further hypothesize that these vasculotropic peptides have distinct effects on BBB endothelial signaling. These hypotheses were tested in polarized human cerebral microvascular endothelial cell monolayers (hCMEC/D3) in vitro.

Methods: Western blot analysis was performed to assess the effect of Aβ40, Aβ42, and D-Aβ40 on protein expression of receptors in the BBB endothelium that are involved in ECM turnover. The hCMEC/D3 cells were pretreated with Aβ40, Aβ42, or D-Aβ40 for 1 h along with co-incubation of insulin for 5 min. Cell lysates were collected and western blot analysis was performed to identify and quantify proteins. The blots were probed for phosphorylated extracellular signal-regulated kinase (p-ERK), protein kinase-B (p-AKT), and tissue inhibitors of metalloproteinases 1, 2 (TIMP-1, TIMP-2). For confocal analysis, hCMEC/D3 cells were grown on μ-slide (chamber slide; 8-well) and treated as described in the western blot study to analyze the expression of matrix metalloproteinase-2 (MMP-2) antibody labeled with Alexa fluor 647 (AF647). After washing and fixation, cells were stained for nuclei with DAPI (4′,6-diamidino-2-phenylindole). Images are taken using Nikon A1Rsi HD Confocal with SIM super-resolution microscopy. Images were analyzed by FIJI software.

Results: Western blot analysis showed decreased p-ERK expression when hCMEC/D3 cell monolayers are treated with Aβ40 and insulin, whereas the p-ERK expression  significantly increased with D-Aβ40 and Aβ42 co-incubated with insulin treatment (Figure 1-A, C), however, p-AKT levels were decreased with all the Aβ treatments co incubated with insulin (Figure 1-B, D). Further hCMEC/D3 cell monolayers were treated with various Aβ peptides to identify the underlying molecular markers involved in ECM turnover, which include: TIMP-1, TIMP-2, and MMP-2. Western blot analysis on TIMPs has shown that Aβ40 significantly (p < 0.05) elevated TIMP-1 (Figure 2-A, B) and TIMP-2 (Figure 2-A, C) levels compared to D-Aβ40 and Aβ42. Band intensities of western blots were quantified by densitometry using Image studio^TM lite software. Confocal micrographs demonstrated differential MMP-2 expression in hCMEC/D3 cells upon treatment with Aβ peptides with Aβ40 showing decreased MMP-2 expression (figure 3-E) compared to D-Aβ40 (figure 3-D). Overall, these results suggest that Aβ40 differentially affected p-ERK, TIMP-1, TIMP-2, and MMP-2 but not p-AKT expression compared to D-Aβ40 and Aβ42. (*p-value < 0.05, **p-value < 0.01, ***p-value < 0.001, ****p-value < 0.0001, One-way ANOVA followed by Bonferroni multiple comparison test).

Conclusion: In the present investigation, we observed differential effects of Aβ peptides on various proteins regulating insulin signaling and ECM turnover. Collectively, the results suggest that both vasculotropic peptides, Aβ40 and D-Aβ40, differentially activate insulin signaling pathway (ERK) and the proteins responsible for ECM remodeling and degradation (MMP-2, TIMP-1, TIMP-2). Differential effects exhibited by Aβ40 and D-Aβ40 are associated with higher accumulation of D-Aβ40 than native Aβ40, and may contribute to extracellular matrix disruption in the cerebral vessels that may eventually lead to cerebral hemorrhages that are commonly observed in patients with CAA. 

Figure 1: Comparative effects of various amyloid variants on phosphorylation of AKT, ERK in hCMEC/D3 cell lysates: Cells were treated with 12.5 μg/ml of Aβ40, Aβ42, and D-Aβ40 in 6-well plates for 1 h with 50 nM insulin stimulation for 5 min in low serum medium at 37°C. A: Western blots showed increased ERK phosphorylation with 1 h of Aβ42, and D-Aβ40 and decreased ERK phosphorylation with pre-treatment followed by 5 min insulin treatment compared to the group that received insulin treatment only. B: Western blots showed decreased AKT phosphorylation with 1 h of Aβ40, Aβ42, and D-Aβ40 pre-treatment followed by 5 min insulin treatment compared to the group that received insulin treatment only. C & D: Quantification of ERK and AKT blots.*p-value <0.05, ***p-value <0.001, ****p-value <0.0001 One-way ANOVA followed by Bonferroni post-test.


Figure 2: Comparative effect of Aβ40, Aβ42, and D-Aβ40 on TIMP-1 and TIMP-2 in hCMEC/D3 cell lysates: Cells were treated with 12.5 μg/ml of Aβ40, Aβ42, and D-Aβ40 in 6-well plates for 1 h with 50 nM insulin stimulation for 5 min in low serum medium at 37°C.A: Western blots showed differential expression of TIMP-1 and TIMP-2 with Aβ40 compared to Aβ42, and D-Aβ40 pre-treatment 1 h followed by 5 min insulin treatment. B, C: Quantification of TIMP-1 and TIMP-2 blots respectively. *p-value <0.05, **p-value <0.01, One-way ANOVA followed by Bonferroni post-test.

Figure 3: Comparative effect of Aβ40, and D-Aβ40 on MMP-2 in hCMEC/D3 cell lysates: Cells were grown on μ-slide (chamber slide; 8-well) and treated with 12.5 μg/ml of Aβ40, Aβ42, and D-Aβ40 for 1 h with 50 nM insulin stimulation for 5 min in low serum medium at 37°C. A-C Confocal microscopy images in various treatment groups. D Fluorescence intensity quantification of MMP-2 (*p-value <0.05, **p-value <0.01, ***p-value <0.001, ****p-value <0.0001, One-way ANOVA followed by Bonferroni multiple comparison test).