Beneficial Effect of a Selective Adenosine A2A Receptor Antagonist in the APPswe/PS1dE9 Mouse Model of Alzheimer's Disease

Abstract

Consumption of caffeine, a non-selective adenosine A_2A receptor (A_2AR) antagonist, reduces the risk of developing Alzheimer's disease (AD) and mitigates both amyloid and Tau lesions in transgenic mouse models of the disease. While short-term treatment with A_2AR antagonists have been shown to alleviate cognitive deficits in mouse models of amyloidogenesis, impact of a chronic and long-term treatment on the development of amyloid burden, associated neuroinflammation and memory deficits has never been assessed. In the present study, we have evaluated the effect of a 6-month treatment of APPsw/PS1dE9 mice with the potent and selective A_2AR antagonist MSX-3 from 3 to 9-10 months of age. At completion of the treatment, we found that the MSX-3 treatment prevented the development of memory deficits in APP/PS1dE9 mice, without significantly altering hippocampal and cortical gene expressions. Interestingly, MSX-3 treatment led to a significant decrease of Aβ1-42 levels in the cortex of APP/PS1dE9 animals, while Aβ1-40 increased, thereby strongly affecting the Aβ1-42/Aβ1-40 ratio. Together, these data support the idea that A_2AR blockade is of therapeutic value for AD.

Keywords: A2A; Alzheimer’s disease; adenosine receptor; amyloid; memory.

Figure 1

Astrocytic upsurge of A_2ARs in APP/PS1 mice. Representative photomicrographs of hippocampal immunostaining for the A_2A receptor (A_2AR; red) (A–C,G–I) and merged with the astrocyte marker GFAP (green) (D–F,J–L) from WT mice (A–F) and the APP/PS1 mice (G–L), at different ages: 3 months (A,D,G,J), 6 months (B,E,H,K), and 9–10 months (C,F,I,L). Representative photomicrographs of A_2AR expression (red) (M) and the merged with GFAP marker (green; N) in the cortex of 9-month-old APP/PS1 mice. Representative photomicrograph of A_2AR expression (red) GFAP (yellow) and 6E10-positive amyloid plaque marker (green) in the hippocampus of 9 month-old APP/PS1 mice (O). Cell nuclei were labeled with DAPI (blue). Scale bar = 20 μm.

Figure 2

A_2AR blockade prevents spatial memory impairments in APP/PS1 transgenic mice. Effect of MSX-3 treatment on spatial learning and memory using the Morris water-maze task. (A) Learning, as indicated by the equivalent path length needed to find the hidden platform (PF). At day 2, APP/PS1 mice exhibited a slight but significant higher path length as compared to WT animals (***p < 0.001 vs. WT H₂O using One-way analysis of variance (ANOVA) followed by LSD Fisher post hoc test). (B) All genotypes exhibited a comparable velocity in the maze, suggesting no motor deficits. (C) Spatial memory was assessed 72 h after the last day of learning. Results represent the percentage of time spent in the target (T) vs. non-target (O) quadrants. WT mice (both treated with water or MSX-3) spent significantly more time in the T quadrant, indicative of a preserved spatial memory. While APP/PS1 mice exhibited spatial memory deficits as underlined by their lack of preference for the T quadrant, APP/PS1-MSX-3 treated mice behaved as WT mice, suggesting a rescue of memory impairment. *p < 0.05 ***p < 0.001 T vs. O; ^$p < 0.05 WT vs. APP/PS1; ^#p < 0.05 APP/PS1-H₂O vs. APP/PS1 MSX-3 using One-way ANOVA followed by LSD Fisher post hoc test; N = 12–16 per group; Results are expressed as mean ± SEM.

Figure 3

Impact of MSX-3 treatment on amyloid load, Aβ levels and phagocytic capacity of microglia in APP/PS1 mice. Representative images of 6E10 staining in the brains of 10 months old APP/PS1 treated with water (A) or MSX-3 (B). Scale bar = 500 μm. Distribution of amyloid plaques size were examined in the hippocampus (C) and cortex (D) of APP/PS1 mice. We found that the treatment with MSX-3 significantly reduced, in the cortex (D) but not in the hippocampus (C), the density of plaques of lower size (between 50–150 μm²) as compared with APP/PS1 H₂O animals (50–150 μm², p < 0.001; 100–150 μm², p < 0.05 vs. APP/PS1 H₂O using Two-Way ANOVA followed by LSD Fisher post hoc test; N = 7–9/group). (E) Effect of acute MSX-3 treatment on microglial phagocytic index determined in an in situ live cerebral slice assay of 12-month-old APP/PS1-Csf1r-EGFP mice and Csf1r-EGFP wildtype littermates after 60 min pre-incubation with the indicated concentration of MSX-3. Datasets were tested for significance with the One-way ANOVA and represent data from three independent experiments. ***p < 0.001 vs. WT (F) As measured by ELISA, MSX-3 treatment decreased Aβ1–42 levels in the cortex of APP/PS1 mice while Aβ1–40 levels was found increased. Overall, the Aβ1–42/Aβ1–40 ratio was found significantly reduced by the A_2A antagonist treatment (*p < 0.05, **p < 0.01 vs. APP/PS1 H₂O using Student’s t-test; N = 7–11/group). (G) Western blot analysis performed in cortex of water and MSX-3 treated APP/PS1dE9 mice did not revealed any change in APP and Carboxyterminal fragments (CTFs) expression (N = 6/group). Results are expressed as mean ± SEM.