(M0930-03-17) Effect of Vasculotropic Dutch Amyloid Beta 40 on Blood-Brain Barrier (BBB) Endothelial Trafficking and Signaling

Purpose: Alzheimer's disease (AD) is a multifactorial disease. The AD is clinically characterized by the presence of amyloid beta (Aβ) and intraneuronal tangles of hyperphosphorylated tau protein. Vascular dysfunction, genetic mutations, and metabolic disorders increase the AD risk. The accumulation of Aβ peptides in the media and adventitia of cerebral arteries and arterioles 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 generally accumulates in the brain parenchyma as amyloid plaques, Aβ40 predominantly accumulates in the cerebral vasculature as amyloid deposits. Various mutant forms of Aβ40, such as Dutch (E22Q) Aβ40, tend to accumulate in cerebral vessels more readily than native Aβ40. The molecular mechanisms underlying the preferential accumulation of Dutch Aβ40 (D-Aβ40) in the cerebral vasculature are poorly understood. We hypothesize that both Aβ40 and D-Aβ40 differentially affect the cellular and molecular pathways governing their trafficking at the BBB endothelium and their accumulation in the cerebral vasculature. To test this hypothesis, Wild-type (B6SJLF1) mice were infused with Aβ40 or D-Aβ40 peptides. Additionally, human cerebral microvascular endothelial cells (hCMEC/D3) were treated with Aβ40 and D-Aβ40 to study the underlying molecular mechanisms.

Methods: In vivo: The B6SJLF1/J  female mice were purchased from the Jackson Laboratory (Bar Harbor, ME) and were housed in the Mayo Clinic animal care facility, and experiments were conducted when they were 8-12 months old. Both Aβ40 and D-Aβ40 peptides were radiolabeled with ¹²⁵I using the chloramine-T procedure (Kandimalla 2005). A single intravenous injection (100 μCi) of ¹²⁵I-Aβ was administered in the femoral vein, and blood (20 μL) was collected at various time points. TCA precipitation of plasma was conducted to separate intact and degraded peptides. Total radioactivity counts in supernatant and precipitate (intact peptide) were analyzed using a gamma counter (Cobra II; Amersham Biosciences, Piscataway, NJ). A two-compartment model was fitted to plasma concentration-time data of intact peptides using SAAM II (version 2.3; SAAM Institute, University of Washington, Seattle, WA). The statistics were conducted using GraphPad Prism 9.1 (GraphPad Software, La Jolla, CA). In vitro: Molecular mechanisms involved in the differential accumulation of Aβ40 and D-Aβ40 peptides in the BBB endothelial cells were investigated. The effects of Aꞵ peptides on the RAGE-TRX-p-STAT3 axis were examined in human cerebral microvascular endothelial cell lines (hCMEC/D3 cells). These cells were pretreated with Aβ40 (12.5 μg/ml) and D-Aβ40 (12.5 μg/ml) for 1 h. Cell lysates were collected, and western blot analysis was performed to quantify protein expression. The blots were probed for proteins that are involved with Aꞵ transport at the BBB (RAGE & P-gp), antioxidant marker (TRX), and inflammatory marker p-STAT3. Immunoblots were quantified using LICOR imaging. Further, the impact of RAGE inhibition by FPS-ZM1 on FITC-tagged Aꞵ₄₀ and D-Aꞵ₄₀ accumulation in the BBB endothelial cells was determined by flow cytometry. Data was quantified with FlowJo and analyzed using GraphPad Prism.

Results: Plasma PK analysis was conducted; significant changes in the brain influx rate constants were observed between Aβ40 and D-Aβ40 (Table 1). Further, a reverse-phase protein array (RPPA) assay was conducted, which demonstrated an increase in the expression of Aβ influx receptor RAGE and decreased expression of P-gp that drives Aβ efflux from the brain. The RPPA results were further validated by Western blot studies (Figure 2). Moreover, the expression of TRX and p-STAT3, downstream regulators of RAGE, was assessed. Both Aβ40 and D-Aβ40 significantly decreased TRX expression, whereas D-Aβ40 increased the expression of p-STAT3 compared to Aβ40 (Figure 2) (p < 0.05, one-way ANOVA followed by Bonferroni multiple comparison test). Band intensities of western blots were quantified by densitometry using Image Studio™ Lite software. Further, inhibitory studies were conducted to assess if the RAGE-TRX-p-STAT3 axis is involved in this differential accumulation of both peptides. The hCMEC/D3 cells were pretreated with FPS-ZM1, a RAGE inhibitor, for 2 h, followed by peptide treatment for 1 h. The FPS-ZM1 increased the accumulation of both peptides; however, the extent of accumulation differs for Aꞵ₄₀ and D-Aꞵ₄₀ compared to the respective controls (Figure 3).

Conclusion: We observed differential accumulation of Aβ40 and D-Aβ40 in BBB endothelial cells The RAGE-TRX-p-STAT3 pathway could govern this differential accumulation pattern. Increased uptake of D-Aβ40 through this pathway disrupts the cerebral vessels' antioxidant and inflammatory potential, which could result in CAA, which trigger massive lobar hemorrhages.


Figure 1: Plasma PK concentration changes in Aβ40 and D-Aβ40


Figure 2: Comparative effect of Aβ40 and D-Aβ40 on RAGE, P-gp, TRX & p-STAT3 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 in low serum medium at 37°C. A: Western blots showed differential expression of RAGE, P-gp, & p-STAT3 with Aβ40 compared to D-Aβ40 pre-treatment 1 h B, C, D, E: Quantification of RAGE, P-gp, TRX & p-STAT3 blots, respectively. *p-value <0.05, **p-value <0.01, One-way ANOVA followed by Bonferroni post-test.


Figure 3: Studies with FPS-ZM1, a RAGE inhibitor in hCMEC/D3 cell lysates: Cells were treated with FPS-ZM1 for 2 h in 6-well plates and in low serum medium at 37°C. A: Western blots showed differential expression of RAGE and TRX. B, C: Quantification of RAGE and TRX with and without FPS-ZM1, respectively. D, E: Fold change differences in mean fluorescence intensity for Aβ40 and D-Aβ40 peptides treated with and without FPS-ZM1 and analyzed with flow cytometry. *p-value <0.05, **p-value <0.01, One-way ANOVA followed by Bonferroni post-test.