Microglial P2X₇ receptor expression is accompanied by neuronal damage in the cerebral cortex of the APPswe/PS1dE9 mouse model of Alzheimer's disease

Abstract

The possibility that P2X₇ receptor (P2X₇R) expression in microglia would mediate neuronal damage via reactive oxygen species (ROS) production was examined in the APPswe/PS1dE9 mouse model of Alzheimer's disease (AD). P2X7R was predominantly expressed in CD11b-immunopositive microglia from 3 months of age before Abeta plaque formation. In addition, gp91phox, a catalytic subunit of NADPH oxidase, and ethidium fluorescence were detected in P2X₇R-positive microglial cells of animals at 6 months of age, indicating that P2X₇R-positive microglia could produce ROS. Postsynaptic density 95-positive dendrites showed significant damage in regions positive for P2X₇R in the cerebral cortex of 6 month-old mice. Taken together, up-regulation of P2X₇R activation and ROS production in microglia are parallel with Aβ increase and correlate with synaptotoxicity in AD.

Figure 1

Aβ accumulation and P2X₇R expression in APP/PS1 mice. (A) Western blot analysis of P2X₇R and actin in the cerebral cortex from 1, 3, 6, and 12 month-old Tg mice and non-Tg control. (B) P2X₇R levels were measured at indicated points of time, normalized to those of β-actin and presented as means ± SEM (n = 3 animals for each condition). ^*P < 0.01 compared with age-matched non-Tg control, using ANOVA and Student-Newman-Keuls analyses. (C) Fluorescent photomicrographs of cerebral cortex section from 3, 6, 10, and 14 month-old Tg mouse labeled with the antibody against P2X₇R and thioflavin-S. P2X₇R (red) expression increased in parallel with accumulation of thioflavin-S-positive fibrillar Aβ (green). Abbreviations: N, non-Tg; T, Tg. Scale bar: 100 µm.

Figure 2

P2X₇R expression in CD11b-positive microglia. Fluorescent photomicrographs of the cerebral cortex from 3, 6, 10, and 14 month-old Tg mice labeled with antibodies against CD11b (green), P2X₇R (red) and Aβ_17-24 (4G8) (blue). Double labeling with CD11b and P2X₇R showed the P2X₇R expression in CD11b-positive microglia (yellow). P2X₇R immunostaining co-localized with microdeposits of Aβ (arrowheads). Triple labeling with CD11b, P2X₇R, and 4G8 showed a P2X₇R-negative microglial cell in contact with a plaque in the brain of a 10 month-old mouse (arrow). Scale bar: 20 µm.

Figure 3

Gp91^phox expression and ROS production in P2X₇R-positive microglia. (A) Western blot analysis of gp91^phox and actin in the cerebral cortex from 1, 3, 6, and 12 month-old Tg mice and non-Tg control. (B) gp91^phox levels were measured at indicated points of time, normalized to those of β-actin and presented as means ± SEM (n = 3 animals for each condition). ^*P < 0.01 compared with age-matched non-Tg control, using ANOVA and Student-Newman-Keuls analyses. (C) Fluorescent photomicrographs of cerebral cortex sections of 6 month-old Tg mice immunolabeled with either antibodies against CD11b (green), P2X₇R (red), and gp91^phox (blue) or antibodies against CD11b (green) and P2X₇R (blue), and the chemical hydroethidine (HE) (red). Abbreviations: N, non-Tg; T, Tg. Scale bar: 20 µm.

Figure 4

Microglial P2X₇R expression and their association with PSD loss in APP/PS1 mice. (A) Western blot analysis of PSD95 and actin in the cerebral cortex from 1, 3, 6, and 12 month-old Tg mice and non-Tg control. (B) PSD95 levels were measured at indicated points of time, normalized to those of β-actin and presented as means ± SEM (n = 3 animals for each condition). ^*P < 0.01 compared with age-matched non-Tg control, using ANOVA and Student-Newman-Keuls analyses. (C) Fluorescent photomicrographs of cerebral cortex sections of 6- and 12-month-old APP/PS1 (b, c) and 6-month-old non-Tg mice (a) immunolabeled with antibodies against PSD95 (green), P2X₇R (red), and Aβ_17-24 (4G8) (blue). a1, b2, and c3 are higher magnification images of the areas designated in a, b, and c. Abbreviations: N, non-Tg; T, Tg. Scale bars: a-f, 50 µm; a1-c3, 15 µm.