AD and Older DS Brains
In both single-labeled adjacent sections and in double-labeled sections, the vast majority of AMY 117 immunoreactive plaques were co-localized to some degree with Aβ immunoreactivity (IR) detected by antibodies R1282 (a general Aβ polyclonal antibody) and 21F12 (Aβ42-specific MAb) in both AD and older (≥29 years) DS brains. AMY 117 IR was always restricted to those cortical and hippocampal regions that contained Aβ deposits. Many, but not all, AMY IR plaques overlapped with thioflavin-S-labeled amyloid plaques. As exemplified in Figure 1
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, AMY 117 IR frequently co-localized (eg, overlapped) with Aβ IR (Figure 1, a–c)
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, was interspersed with it (Figure 1c)
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, or, in the more pathologically severe brains, surrounded it (Figure 1, d–f)
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within an individual plaque lesion. In the latter case, the two antigens were found to partially overlap or to segregate but abut each other. Subpial Aβ deposits, large diffuse Aβ42 IR bands, cerebellar Aβ deposits, and vessel wall Aβ were all devoid of any AMY 117 IR (see asterisks in Figure 1, d–f
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). Occasionally, small punctate AMY 117 immunoreactive deposits that did not appear to overlap with Aβ IR were observed; such deposits occurred only in brain regions bearing abundant Aβ IR plaques (see arrowheads in Figure 1, d and e
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). However, the presence of both antigens could often be detected in these same lesions in sections just above or below the plane of the initially stained section (see arrowhead in Figure 1f
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). Absorption of antibodies R1282 (Aβ) and AMY 117
asc with synthetic Aβ1–40 and Aβ1–42 peptides caused ablation of plaque staining by R1282 (absorption with Aβ1–40 peptide shown in Figure 2, a and b
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) but did not diminish AMY 117 IR (Figure 2, c and d)
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. Figure 2
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further illustrates the close co-occurrence of Aβ and AMY 117 within plaques, at both high and low magnification, in the brain of a 65-year-old DS patient. Regions of compacted Aβ42 IR plaques in hippocampus, parahippocampal gyrus, and temporal cortex were also AMY 117 IR, as shown at low magnification in Figure 2
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(eg, small arrowheads in e and f). However, AMY 117 IR was absent in Aβ42 IR diffuse plaques in the parahippocampal gyrus (arrows in Figure 2, e and f
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), in Aβ42 IR plaques in the subpial layers of temporal cortex (asterisks in Figure 2, e and f
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), and in Aβ42 IR plaques in deep cortical layers and white matter (large arrowheads in Figure 2, e and f
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) as judged in immediately adjacent sections. In AD and DS brains in general, Aβ IR, especially as detected with the Aβ42 MAb 21F12, was more abundant than AMY 117 IR.
![Figure 2. Figure 2.](picrender.fcgi?artid=1866646&blobname=jh0791767002.gif) | Figure 2.AMY 117 IR was not diminished by pre-absorption with Aβ peptide and was less abundant than Aβ42 in old DS brain. a and b: Adjacent 8-μm briefly formalin-fixed, paraffin sections of hippocampus from a 65-year-old DS patient with (more ...) |
Aged Human Control Brains
Of the 10 nondemented aged control brains examined, 2 had no Aβ or AMY 117 IR. In the remaining eight cases, varying amounts of Aβ deposition were detected in the cortex in each brain. Only three of the eight Aβ-bearing brains had any AMY 117 IR; Aβ IR was always much more abundant than that of AMY 117. In general, AMY 117 IR was associated with spherical, compacted plaques and plaques apparently undergoing compaction, but not with diffuse, thioflavin-negative plaques in these aged control brains (data not shown). As in AD brains, no vessel wall AMY 117 IR was detected, even when Aβ IR was present in the blood vessel.
Down Syndrome Brains
To determine the relative temporal sequence of deposition of Aβ and the AMY 117 antigen, we immunolabeled adjacent sections of DS brains from patients ranging from 12 to 73 years old using the highly sensitive Aβ42 MAb 21F12 and the AMY 117 MAb. Frontal cortex sections from 13 young DS patients (aged 12 to 29 years) were examined; in addition, large sections containing both temporal cortex and hippocampus were available for three of these young DS cases and were immunostained with both antibodies. Because the young DS brains had been subjected to long-term fixation in formalin, various pretreatments were tested and then employed to allow visualization of the AMY 117 antigen. A combination pretreatment involving antigen retrieval by microwaving the section in a citrate buffer solution followed by a brief proteinase K digestion allowed the unmasking of the AMY 117 antigen in the long-term fixed tissues. Aβ42 plaque IR was observed in 7 of the 13 young DS brains (aged 12, 15, 16, 17, 21, 27, and 29 years) (exemplified in Figure 3, a, c, e, and g
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). Quantitative analyses of Aβ deposition and other neuropathological characterization of these brains has been previously reported.
5 AMY 117 IR was detected in three of the seven young DS brains that had Aβ deposits (and in none of those that did not). Specifically, the frontal and temporal cortices and hippocampus of a 15-year-old DS patient having thioflavin-positive amyloid plaques and shown previously to have compacted and cored Aβ IR plaques, gliosis, and some neuritic changes had some AMY 117 IR plaques (not shown); the hippocampus of a 16-year-old DS patient that showed compacted Aβ IR plaques had some AMY 117 IR (Figure 3, e and f)
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, whereas the frontal and temporal cortices having only diffuse Aβ42 IR plaques did not (Figure 3, c and d)
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; and the frontal cortex of a 29-year-old DS patient (Figure 3, g and h)
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previously shown to have compacted and cored Aβ IR plaques, gliosis, and some neuritic changes had many plaques positive for both Aβ and AMY 117. In the brains of the four young DS patients (aged 12, 17, 21, and 27 years) that had almost exclusively diffuse Aβ42 IR plaques, AMY 117 IR was not detected (eg, Figure 3, a and b
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). Brains from middle-aged and older DS patients showed AMY 117 IR very similar to that described above for AD, as demonstrated in the brain of a 65-year-old DS patient in Figure 2
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. As in AD cases, no vascular or cerebellar AMY 117 IR was detected in DS brains at any age, even though abundant Aβ deposition occurred in each structure in these older DS brains.
![Figure 3. Figure 3.](picrender.fcgi?artid=1866646&blobname=jh0791767003.gif) | Figure 3.Aβ42 deposition precedes that of the AMY 117 antigen in young DS brain. Eight-micron, long-term formalin-fixed, paraffin sections from young DS brain were immunostained with Aβ42 antibody 21F12 and are shown in the left column (a, c, e, (more ...) |
Monkey Brains and PD-APP Transgenic Mouse Brains
To further characterize the temporal accrual of AMY 117 in Aβ plaque lesions, two animal models of AD pathogenesis were examined. Cortical sections bearing Aβ (R1282 and 21F12) immunoreactive plaques from the brains of eight monkeys ranging in age from 17 to 34 years were immunostained with the AMY 117 MAb. In addition, hemibrain sections from 30 Aβ (R1282) immunoreactive plaque-bearing PD-APP transgenic mice, aged 8 to 20 months, were examined for AMY 117 IR (using the HistoMouse kit (Zymed Laboratories, South San Francisco, CA) to avoid mouse IgG cross-reactivity). No AMY 117 IR was detected in any of these monkey or transgenic mouse brains, regardless of Aβ plaque deposition (data not shown). Even in the 18- and 20-month-old PD-APP transgenic mice that have very abundant compacted and cored Aβ IR and thioflavin-positive plaques, AMY 117 IR was not observed.