ApoE facilitates the microglial response to amyloid plaque pathology

Abstract

One of the hallmarks of Alzheimer's disease is the presence of extracellular diffuse and fibrillar plaques predominantly consisting of the amyloid-β (Aβ) peptide. Apolipoprotein E (ApoE) influences the deposition of amyloid pathology through affecting the clearance and aggregation of monomeric Aβ in the brain. In addition to influencing Aβ metabolism, increasing evidence suggests that apoE influences microglial function in neurodegenerative diseases. Here, we characterize the impact that apoE has on amyloid pathology and the innate immune response in APPPS1ΔE9 and APPPS1-21 transgenic mice. We report that Apoe deficiency reduced fibrillar plaque deposition, consistent with previous studies. However, fibrillar plaques in Apoe-deficient mice exhibited a striking reduction in plaque compaction. Hyperspectral fluorescent imaging using luminescent conjugated oligothiophenes identified distinct Aβ morphotypes in Apoe-deficient mice. We also observed a significant reduction in fibrillar plaque-associated microgliosis and activated microglial gene expression in Apoe-deficient mice, along with significant increases in dystrophic neurites around fibrillar plaques. Our results suggest that apoE is critical in stimulating the innate immune response to amyloid pathology.

Figure 1.

Apoe deficiency increases Aβ plaque size but decreases amyloid burden. (A) Representative images of APPPS1 and Apoe^−/−;APPPS1 mice stained with an N-terminal Aβ antibody. (B) The percentage of cortical area immunopositive for Aβ was increased in Apoe^−/−;APPPS1 mice (28.0 ± 1.4%, n = 7 mice) relative to APPPS1 (8.8 ± 0.9%, n = 10 mice); t(15) = 12.1, P < 0.0001, Student’s t test. (C) The mean number of plaques was increased in Apoe^−/−;APPPS1 mice (230.6 ± 6.5, n = 7 mice) relative to APPPS1 (172.8 ± 9.6, n = 9); t(14) = 4.7, P = 0.0003. (D) The mean plaque size was increased in Apoe^−/−;APPPS1 mice (1,159 ± 72.9 µm², n = 7 mice) relative to APPPS1 (555.1 ± 23.2 µm², n = 10); t(15) = 9.1, P < 0.0001, Student’s t test. (E) Representative images of APPPS1ΔE9 and Apoe^−/−;APPPS1ΔE9 mice stained with an N-terminal Aβ antibody. (F) The percentage of cortical area immunopositive for Aβ was not significantly different between APPPS1ΔE9 (7.0 ± 0.7%, n = 9 mice) and Apoe^−/−;APPPS1ΔE9 (5.6 ± 1.0%, n = 7 mice); t(14) = 1.2, P = 0.25, Student’s t test. (G) The number of cortical plaques was significantly reduced in Apoe^−/−;APPPS1ΔE9 mice (105.2 ± 13.0, n = 7 mice) compared with APPPS1ΔE9 mice (183.5 ± 12.4, n = 9 mice); t(14) = 4.3, P = 0.0007, Student’s t test. (H) The mean plaque size was increased in Apoe^−/−;APPPS1ΔE9 mice (529.8 ± 48.6 µm², n = 7 mice) compared with APPPS1ΔE9 mice (377.9 ± 18.5 µm², n = 9 mice); t(14) = 3.2, P = 0.0064, Student’s t test. (I) Representative images of APPPS1 and Apoe^−/−;APPPS1 mice stained with X-34. (J) The percentage of cortical area stained with X-34 was significantly decreased in Apoe^−/−;APPPS1 mice (0.27 ± 0.08, n = 5 mice) compared with APPPS1 (1.2 ± 0.07, n = 4 mice); t(7) = 8.411, P < 0.0001, Student’s t test. (K) The percentage of hippocampal area stained with X-34 was significantly decreased in Apoe^−/−;APPPS1 mice (0.18 ± 0.04, n = 4 mice) compared with APPPS1 (0.85 ± 0.1, n = 5 mice); t(7) = 5.9, P = 0.0006, Student’s t test. (L) Representative images of APPPS1ΔE9 and Apoe^−/−;APPPS1ΔE9 mice stained with X-34. (M) The percentage of cortical area stained with X-34 was significantly decreased in Apoe^−/−;APPPS1ΔE9 mice (0.54 ± 0.11%, n = 7 mice) compared with APPPS1ΔE9 (2.5 ± 0.19%, n = 9 mice); t(14) = 8.0, P < 0.0001, Student’s t test. (N) The percentage of hippocampal area stained with X-34 was significantly decreased in Apoe^−/−;APPPS1ΔE9 mice (0.12 ± 0.05%, n = 7 mice) compared with APPPS1ΔE9 (2.0 ± 0.15, n = 9 mice); t(14) = 10.4, P < 0.0001, Student’s t test. All values given and plotted as mean ± SEM. Bars, 500 µm. **, P < 0.01; ***, P < 0.001.

Figure 2.

Apoe deficiency alters amyloid morphology and conformation. (A) Representative, high-magnification images of X-34⁺ plaques from APPPS1 and Apoe^−/−;APPPS1 mice and APPPS1ΔE9 and Apoe^−/−;APPPS1ΔE9 mice. Bars, 10 µm. (B) Significant decrease in the shape compactness of Apoe^−/−;APPPS1 mice (11.18 ± 0.38, n = 28 plaques) compared with APPPS1 (13.14 ± 0.50, n = 25 plaques); t(51) = 3.2, P < 0.001, Student’s t test. (C) Significant decrease in the shape compactness of Apoe^−/−;APPPS1ΔE9 (10.12 ± 0.47, n = 29 plaques) compared with APPPS1ΔE9 (15.19 ± 0.44, n = 43 plaques); t(70) = 7.6, P < 0.001, Student’s t test. (D) Representative pixel intensity heat maps for APPPS1 and Apoe^−/−;APPPS1. Same scale bars as in A. (E and F) The Gini coefficient for pixel intensity was decreased in Apoe^−/−;APPPS1 mice (0.37 ± 0.02, n = 28 plaques) compared with APPPS1 (0.47 ± 0.01, n = 25 plaques); t(51) = 4.3, P < 0.0001, Student’s t test (E) and the Gini coeffecient for Apoe^−/−;APPPS1ΔE9 mice (0.26 ± 0.01, n = 29) compared with APPPS1ΔE9 (0.48 ± 0.01, n = 43 plaques); t(70) = 15.53, P < 0.0001, Student’s t test (F). Values are given and plotted as mean ± SEM. (G) Fluorescence images of Aβ-deposits costained by the tetrameric LCO, q-FTAA (blue), and the heptameric LCO, h-FTAA (red). The images are rendered from z-stacks recorded with the dimensions of x = 250 µm (red line), y = 250 µm (blue line), and z = 90 µm (green line; bar, 75 µm). (H and I) Fluorescence lifetime images (H) and lifetime decay curves (I) for h-FTAA stained Aβ-deposits in brain tissue sections. The color bar represents lifetimes from 300 ps (orange) to 800 ps (blue) and the images are color-coded according to the representative lifetime. The fluorescence lifetimes were collected with excitation at 490 nm. Fluorescence decays were collected from 5 to 10 different plaques in tissue sections from five individual APPPS1- or Apoe^−/−;APPPS1 mice. Bars, 20 µm. **, P < 0.01; ***, P < 0.001.

Figure 3.

Decreased plaque-associated microgliosis in Apoe-deficient mice. (A) Representative images of APPPS1 and Apoe^−/−;APPPS1 mice stained with Iba1 to label microglia and X-34 to label amyloid plaques. (B) Significant reduction in the number of microglia in Apoe^−/−;APPPS1 (0.60 ± 0.04, n = 7 mice) mice compared with APPPS1 (1.7 ± 0.12, n = 7) within 15 µm (t[12] = 8.3, P < 0.0001) and 30 µm (2.5 ± 0.18, n = 7 mice versus 4.5 ± 0.28, n = 7 mice); t(12) = 5.9, P < 0.0001, Student’s t test. The number of microglia >30 µm from a plaque in APPPS1 (154.3 ± 7.1, n = 7 mice) compared with Apoe^−/−;APPPS1 (150.7 ± 14.9, n = 7 mice) was not significantly different; t(12) = 0.22, P = 0.83, Student’s t test. (C) Representative images of APPPS1ΔE9 and Apoe^−/−;APPPS1ΔE9 mice stained with Iba1 and X-34. (D) Significant reduction in the number of microglia within 15 µm of Apoe^−/−;APPPS1ΔE9 (1.2 ± 0.17, n = 7 mice), compared with APPPS1ΔE9 (2.2 ± 0.1, n = 11 mice); t(16) =5.6, P < 0.0001, Student’s t test, and within 30 µm (2.8 ± 0.24, n = 7 mice) compared with (4.6 ± 0.16, n = 11 mice); t(16) = 6.5, P < 0.0001, Student’s t test. The number of microglia >30 µm from a plaque in APPPS1ΔE9 (160.3 ± 14.7, n = 11 mice) and Apoe^−/−;APPPS1ΔE9 (133.2 ± 8.5, n = 7 mice) were not significantly different; t(16) = 1.4, P = 0.19, Student’s t test. (E) Selected differentially expressed inflammatory gene expression from APPPS1 (n = 6 mice) and Apoe^−/−;APPPS1 mice (n = 7 mice). See Table S1 for p-values. (F) Analysis of cytokine levels in the hippocampus of APPPS1 and Apoe^−/−;APPPS1 mice. Apoe^−/−;APPPS1 mice (n = 13 mice) exhibited a significant reduction in CCL3 compared with APPPS1 mice (n = 11 mice). P < 0.0001, Student’s t test. Bars, 100 µm. ***, P < 0.001.

Figure 4.

Significant increase in plaque-associated neuritic dystrophy in Apoe-deficient mice. (A) Representative image of dystrophic neurites labeled with APP (green) and amyloid labeled with X-34 (red). Nuclei are labeled with Topro-3 (blue). (B) Significant increase in the number of dystrophic neurites per plaque in Apoe^−/−;APPPS1 mice (7.3 ± 0.66, n = 40 plaques) compared with APPPS1 (5.5 ± 0.39, n = 95 plaques); t(133) = 2.48, P = 0.01, Student’s t test. (C) Representative image of neuritic dystrophy in APPPS1ΔE9 and Apoe^−/−;APPPS1ΔE9 mice. (D) Significant increase in the number of dystrophic neurites per plaque in Apoe^−/−;APPPS1ΔE9 mice (8.2 ± 0.86, n = 27 plaques) compared with APPPS1ΔE9 (4.5 ± 0.42, n = 26 plaques); t(51) = 3.8, P = 0.0004, Student’s t test. All values given and plotted as mean ± SEM. Bars, 20 µm. **, P < 0.01; ***, P < 0.001.