Neuronal and axonal loss are selectively linked to fibrillar amyloid-{beta} within plaques of the aged primate cerebral cortex

Abstract

The amyloid-beta peptide (Abeta) deposited in plaques in Alzheimer's disease has been shown to cause degeneration of neurons in experimental paradigms in vivo and in vitro. However, it has been difficult to convincingly demonstrate toxicity of native amyloid deposits in the aged and Alzheimer brains. Here we provide evidence that the fibrillar conformation of Abeta (fAbeta) deposited in compact plaques is associated with the pathologies observed in Alzheimer brains. fAbeta containing compact but not diffuse plaques in the aged rhesus cortex contained activated microglia and clusters of phosphorylated tau-positive swollen neurites. Scholl's quantitative analysis revealed that the area adjacent to fAbeta, containing compact but not diffuse plaques in aged rhesus, aged human, and Alzheimer's disease cortex, displays significant loss of neurons and small but statistically significant reduction in the density of cholinergic axons. These observations suggest that fAbeta toxicity may not be restricted to cultured cells and animal injection models. Rather, fAbeta deposited in native compact plaques in aged and AD brains may exert selective toxic effects on its surrounding neural environment.

Figure 1

Microglia are colocalized with fibrillar amyloid-β (fAβ) in compact plaques of the aged rhesus cortex. A: Compact plaques in the aged rhesus cortex contained intense Aβ immunoreactivity and displayed well-defined borders. B: Diffuse plaques were lightly stained for Aβ and lacked clear borders. C: Thioflavin-S epifluorescence was present in compact plaques, indicative of the presence of fAβ. D: Immunoreactivity for HLA-DR, a marker of activated microglia, visualized clusters of microglia in plaque-like arrangements in the aged rhesus cortex. E: Double-staining for Aβ (blue) and the microglia marker CD68 (brown) indicated that nearly all such microglia clusters were within plaques. F: Double-staining for HLA-DR and thioflavin-S demonstrated that virtually all activated microglia clusters (arrows) in the aged rhesus cortex are within compact plaques containing fAβ. Magnification in A–F, ×400.

Figure 2

Abnormally phosphorylated tau (PHF-1) immunoreactivity in swollen neurites is present within fAβ-containing compact plaques in the aged rhesus cortex. A: Clusters of PHF-1 immunoreactive swollen neurites were present in plaque-like arrangements in the aged rhesus cortex. B: Double staining for Aβ and PHF-1 demonstrated that nearly all such swollen neurites (brown) were located within Aβ deposits. (blue) C: Light microscopy in PHF-1 and thioflavin-S double stained material demonstrated the presence of abnormal swollen neurites in plaque-like arrangements. D: Presence of thioflavin-S epifluorescence in the same section demonstrated that virtually all PHF-1 swollen neurites were within compact plaques containing fAβ. Magnification in A–D, ×400.

Figure 3

Neuronal and axonal loss is associated with compact plaques in the aged rhesus cortex. A: Compact Aβ immunoreactive plaques in the rhesus cortex were associated with significantly reduced numbers of Nissl stained neurons (arrows) in their vicinity. In contrast, the area around such plaques displayed increased density of glial cells (arrowhead). B: No visible reduction in the numbers of neurons was seen in the vicinity of diffuse plaques. Intact neurons were observed in the vicinity and within such plaques (arrows). C: Compact plaques in the human brain displayed even greater loss of neurons in their vicinity when compared with the rhesus. D: Similar to the rhesus, diffuse plaques in the human cortex were not associated with loss of neurons in their vicinity. E: The area around compact plaques in the aged rhesus cortex displayed significant loss of acetylcholinesterase-positive cholinergic axons. F: Cholinergic axons were intact in the area surrounding diffuse Aβ immunoreactive plaques. Magnification in A–F, ×200.

Figure 4

Results of Scholl’s analysis revealed neuronal and cholinergic axonal loss in the vicinity of compact plaques that were progressively less pronounced with distance away from plaques. A: The ring bound by two circles 20 μm apart in the immediate vicinity of compact plaques in the aged rhesus cortex contained significantly smaller numbers of neurons than the next four rings in the direction away form compact plaques (*P < 0.001). The same was true of the second ring (**P < 0.001) and the third ring (***P < 0.01) when compared with the rings away from plaques. There was no significant difference between the numbers of neurons in the fourth and fifth ring (P > 0.05). B: In the area next to diffuse plaques, only the numbers of neurons in the first ring showed a small but statistically significant lower number of neurons when compared with the fifth ring (*P < 0.01). No significant differences were found in the number of neurons among other rings. C: Similar but more pronounced loss of neurons was observed around compact plaques in the aged human and AD cortex. The numbers of neurons in the first ring next of compact plaques in the human cortex was significantly smaller than the number in the next four rings away from plaques (*P < 0.001), and this trend was progressively less pronounced with distance away from plaques (**P < 0.001 when compared with rings 3–5; ***P < 0.001 when compared with rings 4 and 5; P > 0.05 when comparing ring 4 and 5). D: In contrast to compact plaques, the numbers of neurons in five rings around diffuse plaques were not significantly different from each other (P > 0.05). E: The numbers of cholinergic axon intersects with the first circle around compact plaques in the aged rhesus cortex were significantly lower than intersects with the next four circles away from plaques (*P < 0.001). The number of intersects with the second circle was significantly lower when compared with circle 4 (**P < 0.001). There were no other significant differences between numbers of intersects with other circles. F: There were no significant differences between numbers of axon intersects with any of five circles around diffuse plaques in the aged rhesus cortex (P > 0.05).

Figure 5

Neuronal loss is prominent in the vicinity of microglia clusters in the aged rhesus cortex, virtually all of which are in compact thioflavin-S–positive plaques that contain fAβ. The number of neurons in the first ring around microglia clusters was significantly smaller that the next four rings away from clusters (*P < 0.001), and this trend became progressively less pronounced in subsequent rings around microglia clusters (**P < 0.001 when compared with the fifth ring; ***P < 0.01 when compared with the fifth ring).