Effect of the ROCK inhibitor fasudil on the brain proteomic profile in the tau transgenic mouse model of Alzheimer's disease

Abstract

Introduction: The goal of this study is to explore the pharmacological potential of the amyloid-reducing vasodilator fasudil, a selective Ras homolog (Rho)-associated kinases (ROCK) inhibitor, in the P301S tau transgenic mouse model (Line PS19) of neurodegenerative tauopathy and Alzheimer's disease (AD).

Methods: We used LC-MS/MS, ELISA and bioinformatic approaches to investigate the effect of treatment with fasudil on the brain proteomic profile in PS19 tau transgenic mice. We also explored the efficacy of fasudil in reducing tau phosphorylation, and the potential beneficial and/or toxic effects of its administration in mice.

Results: Proteomic profiling of mice brains exposed to fasudil revealed the activation of the mitochondrial tricarboxylic acid (TCA) cycle and blood-brain barrier (BBB) gap junction metabolic pathways. We also observed a significant negative correlation between the brain levels of phosphorylated tau (pTau) at residue 396 and both fasudil and its metabolite hydroxyfasudil.

Conclusions: Our results provide evidence on the activation of proteins and pathways related to mitochondria and BBB functions by fasudil treatment and support its further development and therapeutic potential for AD.

Keywords: Alzheimer's disease; P301S; PS19; fasudil; proteomic; tau.

Figure 1

PS19 transgenic mice brain proteomic profile. (A) Graphical representation of the experimental timeline of this study including groups, drug treatment, biochemical and statistical analysis performed. (B) Levels of total tau (Tau) and phosphorylated tau (pTau)-181, −202, −231, and −396 in the brain of PS19 transgenic mice as compared to non-carrier (NC) mice measured with MSD ELISA. (C) Gene ontology enrichment analysis of differentially expressed proteins in the brain of PS19 as compared to NC mice. Significantly enriched biological process, cellular components, and molecular functions are represented and were obtained with DAVID. (D) KEGG and Reactome pathway enrichment analysis of differentially expressed proteins in the brain of PS19 as compared to NC mice. Bubble plot represents the top significant pathways.

Figure 2

Network of differentially expressed proteins upon fasudil treatment. A total of 252 proteins are shown, with 182/253 proteins in F100 group with p > 0.1 for F30 vs. Veh and 70/149 proteins from F30 group with p > 0.1 for F100 vs. Veh. Each node represents one protein, and each node face was divided into two halves. The sector on the left was colored based on -log10 transformation of p-value from Tukey's test for F100 vs. Veh, while the sector on the right was colored according to F30 vs. Veh. P-values corresponding to up-regulation after treatment were red, while blue represented down-regulation after treatment (p ≥ 0.05 was represented in white). Each edge represents one PPI record with combined confidence score ≥ 0.7 from STRING database.

Figure 3

Analysis of the differentially expressed proteins responding to fasudil. Gene ontology and pathway enrichment analysis of differentially expressed proteins in the brain of PS19 transgenic mice dosed with fasudil 30 mg/kg/day [(A, C) respectively] or 100 mg/kg/day [(B, D) respectively] as compared to the control group (Veh, saline). The most significantly enriched biological process, cellular components and molecular functions are represented by bar graphs, while the top significantly affected pathways are represented by bubble plots.

Figure 4

Fasudil affects mitochondrial proteins expression. Proteins showing a significant differential expression in the brain of PS19 mice compared to NC mice (A), PS19 mice dosed with 30 mg/kg/day fasudil compared to vehicle-dosed mice (B), and PS19 mice dosed with 100 mg/kg/day fasudil compared to vehicle-dosed mice (C). The significant proteins were obtained from the Reactome pathway for TCA cycle (R-MMU-148517, the citric acid cycle and respiratory electron transport) ranked among the top significantly enriched pathways in PS19 transgenic mice and after treatment with fasudil.

Figure 5

Gap junction proteins are affected by fasudil treatment. Differentially expressed proteins in the brain of PS19 mice compared to NC mice (A), and PS19 mice dosed with 100 mg/kg/day fasudil compared to vehicle-dosed mice (B) obtained from the KEGG pathway for Gap junction (MMU04540) ranked among the top significantly enriched pathways in PS19 transgenic mice and after treatment with fasudil.

Figure 6

Effect of fasudil on the brain tau pathology in PS19 mice. PS19 transgenic mice received daily intraperitoneal injections with vehicle (Veh), fasudil 30 mg/kg (F30), and fasudil 100 mg/kg (F100) for 2 weeks. At the end of the treatment whole brains were collected and processed to quantify levels of fasudil and its metabolite hydroxyfasudil, as well as brain concentrations of total tau (Tau) and phosphorylated tau (pTau) isoforms. Brain levels of fasudil (A) and hydroxyfasudil (B) increased in a dose dependent way in PS19 mice administered 30 and 100 mg/kg/day. Brain levels of pTau-396 showed a significant negative correlation with fasudil (C) and hydroxyfasudil (D). Data are presented as mean ± standard error of means (SEM; ***p < 0.001).