Selective targeting of perivascular macrophages for clearance of beta-amyloid in cerebral amyloid angiopathy

Abstract

Cerebral amyloid angiopathy (CAA), the deposition of beta-amyloid (Abeta) peptides in leptomeningeal and cortical blood vessels, affects the majority of patients with Alzheimer's disease (AD). Evidence suggests that vascular amyloid deposits may result from impaired clearance of neuronal Abeta along perivascular spaces. We investigated the role of perivascular macrophages in regulating CAA severity in the TgCRND8 mouse model of AD. Depletion of perivascular macrophages significantly increased the number of thioflavin S-positive cortical blood vessels. ELISA confirmed that this increase was underscored by elevations in total vascular Abeta(42) levels. Conversely, stimulation of perivascular macrophage turnover reduced cerebral CAA load, an effect that was not mediated through clearance by microglia or astrocytes. These results highlight a function for the physiological role of perivascular macrophages in the regulation of CAA and suggest that selective targeting of perivascular macrophage activation might constitute a therapeutic strategy to clear vascular amyloid.

Fig. 1.

Administration of liposome-encapsulated clodronate depletes perivascular macrophages. (A and B) TgCRND8 mice injected with PBS liposomes (A) showed more CD163-positive perivascular macrophages (green) associated with GLUT-1- immunoreactive (red) blood vessels in the caudate putamen than those that received clodronate-containing liposomes (B). (C) Immunoblotting of right brain homogenates (30 μg/lane) demonstrated a significant reduction in CD206 levels (P = 0.002, n = 5) in mice receiving clodronate. (D and E) Photomicrographs showing that CD206 immunoreactivity is expressed by perivascular macrophages (red) but not by GFAP-immunoreactive astrocytes (D, green) or Iba1-positive microglia (E, green) in TgCRND8 mice. (Scale bars: A and B, 10 μm; D and E, 20 μm.)

Fig. 2.

Depletion of perivascular macrophages increases CAA severity. (A–C) Naive TgCRND8 mice (A) and those treated with PBS solution (B) show fewer thioS-positive cortical blood vessels than clodronate-treated mice (C). (D) Total cortical area covered in thioS-positive blood vessels was increased 5-fold in clodronate-treated animals (P = 0.01, n = 10). (E and F) No colocalization was found between thioS (green) and anti-perlecan (red) staining in PBS solution- (E) or clodronate-treated mice (F). (G and H) No differences in Aβ₄₀-positive staining were noted between vehicle- (G) and clodronate-treated animals (H). (I and J) The number and intensity of Aβ₄₂-positive cortical blood vessels was increased in mice treated with clodronate (J) versus control animals (I). (K–M) Total human Aβ₄₂ levels in cortical blood vessels isolated from clodronate-treated mice (K) were significantly increased (P = 0.03, n = 12) compared with PBS solution-injected animals. Aβ₄₂ levels were significantly decreased in the cortical samples (L; P = 0.04, n = 12) of clodronate-treated animals, but were not altered in plasma samples (M; P = 0.29). (N–P) Iba-1-positive microglia were noted throughout the cortex of vehicle- (N) and clodronate-treated mice (O). No differences were noted in Iba-1 levels (P) between treatment groups (P = 0.54, n = 4). Values represent mean ± SEM of samples analyzed in triplicate; *P < 0.05 and **P < 0.01. (Scale bars: A–C, 75 μm; E and F, 10 μm; G–J, 20 μm; N and M, 70 μm.)

Fig. 3.

Chitin administration stimulates perivascular macrophage turnover and clears CAA. (A and B) Most cells in PBS-treated mice showed colocalization of red and green dextran dyes (A, yellow-labeled cells), whereas chitin-treated animals (B) showed numerous singly, red-labeled macrophages (P = 0.01, n = 4). (C–E) The number of thioS-positive cortical blood vessels was significantly decreased following chitin treatment (D) compared with PBS solution-injected mice (C) (E, P = 0.01, n = 10). Histograms represent mean ± SEM values obtained from 3 brain sections per animal; **P < 0.01. (Scale bars: A and B, 10 μm; C and D, 75 μm.)

Fig. 4.

Perivascular macrophages clear CAA. (A–C) Chitin-treated mice showed no colocalization between thioS (A–C, green) and GFAP-positive astrocytes (A, red), nor with Iba1-positive microglia (B, red), which were however associated with parenchymal amyloid plaques (B, arrows). However, CD163-immunoreactive macrophages (C, red) colocalized with thioS-labeled vascular amyloid (C, green) in these mice. (D–G) No differences were noted between vehicle- (D) and chitin-treated animals (E) in brain tissue sections processed for anti-Aβ₄₀ staining. A significant reduction in Aβ₄₂-positive staining (F and G) was noted in chitin-treated animals (G) compared with controls (F). (H–J) Total human Aβ₄₂ levels were significantly decreased in blood vessels (H, P = 0.04) and plasma samples (J, P = 0.04, n = 6) isolated from chitin-treated mice, but not in vessel-depleted cortical samples (I, P = 0.16). Values represent mean ± SEM of samples analyzed in triplicate; *P < 0.05. (Scale bars: A and B, 5 μm; C, 25 μm; D–G, 20 μm.)