Three-dimensional view of microglia-amyloid plaque interactions

Abstract

Recent gene expression studies have revealed about 10 different states of microglia, some of which are characteristic for Alzheimer-like amyloid plaque pathology. However, it is not presently known how these translate into morphological features that would reflect microglia interaction with amyloid plaques. With optimized conditions for confocal microscopy in amyloid plaque forming APP/PS1 transgenic mice we reveal new details of how microglia processes interact with amyloid plaques. The microglia contacts differed drastically between purely diffuse plaque and those with a fibrillar core. We identified microglia that extend their enlarged processes through the diffuse shell of the amyloid plaques and cover the fibrillar plaque core with snowplow-like expanded end-feet. These end-feet were filled with the lysosomal marker CD68, while both non-fibrillar and fibrillar amyloid was found in perinuclear vesicles of some "snowplower" microglia. In the organized dense-core plaques, we consistently saw a layer of Apolipoprotein E (ApoE) between the fibrillar core and the microglial end-feet. ApoE covered also loose fibrillar amyloid and diffuse amyloid plaques that were about 10 μm or larger in diameter. These findings are compatible with both amyloid plaque phagocytosis and compaction by microglia. Further, they support a chemotactic role of ApoE for microglia contacts with amyloid plaques.

Keywords: 3D rendering; ApoE; confocal imaging; dense‐core plaque; transgenic mice.

FIGURE 1

Composition of diffuse, fibrillar, and dense‐core amyloid plaques stained with three dyes binding to fibrillar amyloid, Thioflavin‐S, X34, and DAPI, and the N‐terminal anti‐Aβ D54D2 antibody. The diffuse plaques are well visible with anti‐Aβ D54D2 antibody but show no staining standing out from the background with Thio‐S, X34, or DAPI. The fibrillar plaques show a consistent signal in the plaque center that has fibrillar or cotton wool‐like texture radiating to all directions. Of the fibrillar amyloid dyes, X34 is the most avid to stain single fibril‐like structures. The dense‐core plaques display a clear round center that is surrounded by a D54D2+ cloud. Thio‐S stains the core very intensively with a sharp boundary. In contrast, X34 staining shows a ragged boundary between the core and surrounding fibrillar amyloid. DAPI staining, on the other hand, delineates the dense core with intensive staining and is surrounded by a less intense halo of fibrillar amyloid. Note that only DAPI stains the nuclear chromatin (white arrow). Scale bar = 10 μm.

FIGURE 2

(a) Correlation between amyloid plaque size and amyloid plaque composition in 6‐ and 13‐month‐old APP/PS1 mice. Composition categories (1 = diffuse, 2 = fibrillar, 3 = dense‐core). There was a significant shift from diffuse toward dense‐core plaques at both 6 months (R = 0.35, p = 0.02, n = 42) and 13 months (R = 0.53, p < 0.001, n = 60) of age. (b) The mean number of microglia around an amyloid plaque depends on the plaque size. Size categories based on diameter (cat 1 = 3–5 μm, cat 2 = 6–7 μm, cat 3 = 8–10 μm, cat 4 = 11–15 μm, cat 5 = 16–20 μm, cat 6 > 20 μm). All sizes of plaques were rounded up to full numbers. For 6‐month‐old: R = 0.59, p < 0.001, n = 44, for 13‐months‐old: R = 0.57, p < 0.001, n = 61; Spearman correlation.

FIGURE 3

Examples of microglia–amyloid plaque interaction categories. (a) Category 1: the amyloid plaque is contacted by processes of a single microglia (arrows). (b) Category 2: the amyloid plaque is contacted by processes of two microglia (arrows). Note that the body of the microglia at 4 o'clock may seem to contact the plaque but a side view shows it is on a different z‐level. (c) Category 3: two microglia encroach into the amyloid plaque with enlarged processes (arrows and arrowheads). (d) Category 4: the microglial body is directly in contact with the plaque. Scale bar = 10 μm.

FIGURE 4

(a) The intensity of microglia–amyloid plaque interaction increases as a function of amyloid plaque size. See Figure 2 for the size categories. Age 6 months: R = 0.39, p = 0.01, n = 44. Age 13 months: R = 0.45, p < 0.001, n = 61, Spearman rho. (b) The microglia–amyloid plaque interaction intensity tends to increase from diffuse plaques toward dense‐core plaques. Age 6 months: p = 0.10, n = 44; age 13 months: p < 0.001, n = 60, Kruskal–Wallis H.

FIGURE 5

3D IMARIS rendering showing the difference in the microglia interaction with the diffuse plaque shell stained with anti‐Aβ antibody D54D2 and the DAPI+ dense core. (a) Microglia processes (GFP) dive into the loose diffuse amyloid layer (D54D2). (c) Microglia processes (GFP) reach and cover the DAPI+ (blue) core. (b and d) The same images rendered so that the microglia appear transparent to uncover the hidden DAPI+ dense core. Scale bar = 10 μm.

FIGURE 6

CD68 positivity in the microglia clustered around amyloid plaques. (a) Dense meshwork of processes from two microglia surrounding a DAPI+ fibrillar plaque. (b) The same image without the green channel to better visualize the CD68+ vesicles (red) that in this case are only found in the processes in contact with the fibrillar plaque (DAPI, arrows). (c) A single optical slice through the center of a fibrillar plaque surrounded by three enlarged microglia processes and the soma of one of the microglia. Concentration of CD68+ vesicles is clearly seen in two of the processes. (d) DAPI channels alone to show the extent of the fibrillar amyloid deposit. Scale bar = 10 μm.

FIGURE 7

Images of two dense‐core amyloid plaque stained with D54D2 for diffuse amyloid and DAPI for nuclear chromatin and fibrillar amyloid. (a) A single z‐plane image of a plaque surrounded by three microglia, two of which contact the plaque core, while the third one at 4 o'clock is actually on a lower level without any contact with the shown plaque. All three microglia display small D54D2+/DAPI− blobs in the cell body, compatible with perinuclear lysosomes (arrows). (b) A merged z‐stack image of a small dense‐core plaque that is contacted from below by one microglia with a plate‐like extension. The microglia is abutted by two other microglia with no direct contact with the plaque. All three microglia display D54D2+/DAPI+ perinuclear deposits (arrows), presumably in an enlarged lysosome. Scale bar = 10 μm.

FIGURE 8

(a) An 18 μm thick single z‐stack plane showing the cross‐section through a small dense‐core plaque with surrounding thick microglia processes (GFP). The DAPI+ plaque core is seen with blue and the ring‐like layer of ApoE around it in red. A fibrillar extension of the amyloid plaque (white arrow) is covered with ApoE as well but is not in direct contact with any microglia process. (b) Red + blue channels, (c) blue (DAPI) channel only. (d) Another dense‐core plaque stained with X34 that highlights amyloid fibrils outside the dense plaque core better than DAPI. (e) ApoE (red) covers all fibrillar Aβ except the dense core. (f) The microglia coverage instead is only partial (white arrows). Scale bar = 10 μm.

FIGURE 9

Examples of determinants of ApoE immunopositivity in amyloid plaques. (a) Three small (< 10 μm) diffuse amyloid plaques with no evidence of ApoE immunopositivity. (b) A single diffuse amyloid plaque (<10 μm) with a trace ApoE immunopositivity without overlapping DAPI positivity. (c) A bit larger (> 10 μm) ostensibly diffuse amyloid plaque that is strongly ApoE positive. However, a strong excitation that already saturates the nuclei, reveals a central nidus of fibrillar amyloid that overlaps with the strongest ApoE signal.