Short-term treatment with dabigatran alters protein expression patterns in a late-stage tau-based Alzheimer's disease mouse model

Abstract

Proteins that regulate the coagulation cascade, including thrombin, are elevated in the brains of Alzheimer's disease (AD) patients. While studies using amyloid-based AD transgenic mouse models have implicated thrombin as a protein of interest, the role of thrombin in tau-based animal models has not been explored. The current study aims to determine how inhibiting thrombin could alter oxidative stress, inflammation, and AD-related proteins in a tau-based mouse model, the Tg4510. Aged Tg4510 mice were treated with the direct thrombin inhibitor dabigatran or vehicle for 7 days, brains collected, and western blot and data-independent proteomics using mass spectrometry with SWATH-MS acquisition performed to evaluate proteins related to oxidative stress, intracellular signaling, inflammation, and AD pathology. Dabigatran reduced iNOS, NOX4, and phosphorylation of tau (S396, S416). Additionally, dabigatran treatment increased expression of several signaling proteins related to cell survival and synaptic function. Increasing evidence supports a chronic procoagulant state in AD, highlighting a possible pathogenic role for thrombin. Our data demonstrate that inhibiting thrombin produces alterations in the expression of proteins involved in oxidative stress, inflammation, and AD-related pathology, suggesting that thrombin-mediated signaling affects multiple AD-related pathways providing a potential future therapeutic target.

Keywords: Alzheimer's disease; Cell signaling; Oxidative stress; Thrombin.

Fig. 1

Dabigatran reduces expression of oxidative stress-related proteins in Tg4510 mouse brains. Brain homogenates from wild type mice, Tg4510 mice (Tg-Vehicle), and Tg4510 mice treated with dabigatran (Tg-Dabigatran) were analyzed by western blot (A) for iNOS (B) and NOX4 (C). Mean ± SEM (n = 4), significance determined by one-way ANOVA with post-hoc Bonferroni, **p ≤ 0.01 vs. Wild Type, ^#p < 0.05 vs. Tg-Vehicle, ^##p < 0.01 vs. Tg-Vehicle.

Fig. 2

Dabigatran diminishes levels of phosphorylated tau species in Tg4510 mice. Brain homogenates from wild type mice, Tg4510 mice (Tg-Vehicle), and Tg4510 mice treated with dabigatran (Tg-Dabigatran) were analyzed by western blot for total tau (A) tau S396 (B) and tau S416 (C). Mean ± SEM (WT n = 5, Tg-Vehicle n = 6, Tg-Dabigatran n = 4), significance determined one-way ANOVA with post-hoc Bonferroni, ***p < 0.001 vs. Wild Type, ^#p ≤ 0.05 vs. Tg-Vehicle.

Fig. 3

Proteomic expression differences between wild type mice, Tg-Vehicle, and Tg-Dabigatran mice are detected by LC-MS/MS SWATH analysis. (A) Volcano plot of data sets from brain homogenates of wild type mice and Tg4510 mice (Tg-Vehicle). (B) Volcano plot of data sets from brain homogenates of Tg4510 mice (Tg-Vehicle) and Tg4510 mice treated with dabigatran (Tg-Dabigatran).

Fig. 4

Treatment of Tg4510 mice with dabigatran causes widespread differences in protein expression. Proteomic expression differences between wild type mice, Tg4510 mice (Tg-Vehicle), and Tg4510 mice treated with dabigatran (Tg-Dabigatran) are detected by LC-MS/MS SWATH analysis. Functional categories of proteins that show significant expression differences are shown as follows: (A) coagulation cascade, (B) cell signaling, and (C) inflammation-related. Mean ± SEM (n = 5), statistical differences determined by multiple t-tests, *p ≤ 0.05 vs. Wild Type, ^#p ≤ 0.05 vs. Tg-Vehicle.

Fig. 5

Treatment of Tg4510 mice with dabigatran alters expression of proteins related to Alzheimer's disease pathology. Proteomic expression differences between wild type mice, Tg4510 mice (Tg-Vehicle), and Tg4510 mice treated with dabigatran (Tg-Dabigatran) are detected by LC-MS/MS SWATH analysis. (A) classic AD-associated proteins and (B) proteins determined by literature search to be altered in sporadic AD or tau pathology that show significant expression differences are shown. Mean ± SEM (n = 5), statistical differences determined by multiple t-tests, *p ≤ 0.05 vs. Wild Type, ^#p ≤ 0.05 vs. Tg-Vehicle.