In vivo detection of amyloid plaques in a mouse model of Alzheimer's disease

Abstract

Strategies for treating Alzheimer's disease (AD) include therapies designed to decrease senile plaque (SP) formation and/or promote clearance of SPs, but clinical trials of these treatments are limited by the lack of effective methods to monitor changes in plaque burden in the brains of living AD patients. However, because SPs are extracellular deposits of amyloid-beta peptides (Abeta), it may be possible to eventually develop radioligands that cross the blood-brain barrier (BBB) and label SPs so they can be visualized by current imaging methods. As a first step toward the generation of such a radioligand, we developed a probe, [(trans,trans)-1-bromo-2, 5-bis-(3-hydroxycarbonyl-4-hydroxy)styrylbenzene (BSB)], and we report here that BSB has the following properties essential for a probe that can detect SPs in vivo. First, BSB sensitively labels SPs in AD brain sections. Second, BSB permeates living cells in culture and binds specifically to intracellular Abeta aggregates. Third, after intracerebral injection in living transgenic mouse models of AD amyloidosis, BSB labels SPs composed of human Abeta with high sensitivity and specificity. Fourth, BSB crosses the BBB and labels numerous AD-like SPs throughout the brain of the transgenic mice after i.v. injection. Thus, we conclude that BSB is an appropriate starting point for future efforts to generate an antemortem diagnostic for AD.

Figure 1

BSB fluorescently labels AD SPs and NFTs with similar intensity as TF-S. Cortical (A and B) and hippocampal (C and D) sections from patients (n = 3) with postmortem-confirmed AD were stained with BSB (A and C) or TF-S (B and D). Postmortem sections from 12-mo-old Tg2576 mice were similarly stained with BSB (E) or TF-S (F). Shown are representative SP with accompanying neuritic pathology taken at ×400 (A and B), abundant NFTs (×400, C and D), or mouse cortical plaques taken at ×200 (arrows in E and F).

Figure 2

Aβ-treated human fibroblasts accumulate TF-S-positive/BSB-positive intracellular amyloid. Untreated fibroblasts (A and D), fibroblasts treated with 5 μg/ml Aβ (B and E), and fibroblasts treated with 10 μg/ml Aβ (C and F) were trypsinized, replated on cover slips, fixed, and stained with BSB (A–C) or with TF-S (D–F). Examples of small paranuclear intracellular amyloid aggregates are labeled with arrowheads. Arrows label examples of extracellular amyloid aggregates. Images shown (×400) are representative of three separate experiments.

Figure 3

BSB but not TF-S labels intracellular amyloid aggregates in living cells. Fibroblasts treated with 1 μg/ml Aβ (A), 5 μg/ml Aβ (B), 10 μg/ml Aβ (C and F), untreated with Aβ (D) or treated with 5 μg/ml of the reverse Aβ sequence peptide Aβ_42–1 (E), were trypsinized, replated, and incubated with for 2 h with the addition of 0.005% BSB (A–E) or 0.005% TF-S (F) to the culture medium. Cells were fixed and viewed directly under the fluorescence microscope. Intracellular amyloid aggregates were detected with BSB only and are labeled with arrowheads. Extracellular amyloid aggregates were detected with both BSB and TF-S and are labeled with arrows. Images shown (×400) are representative of three separate experiments.

Figure 4

Intrahippocampal injection of BSB in control mice detects no plaque-like structures. BSB was stereotactically injected into the hippocampus of a 12-mo-old control mouse. Eighteen hours later, frozen sections reveal diffuse hippocampal labeling (A) with occasional specific uptake by neurons (Inset, ×400) at the site of injection. On the contralateral side, no staining over background is apparent (B). Images shown are at ×100 and are representative of three mice.

Figure 5

Intrahippocampal injection of BSB in Tg2576 mice specifically labels abundant plaque pathology in the hippocampus and entorhinal cortex. BSB was stereotactically injected into the hippocampus of a 12-mo-old Tg2576 mouse. Eighteen hours later, frozen sections revealed prominent labeling of SPs in the ipsilateral (A Left) and contralateral (A Right) hippocampi. SPs are also clearly visible in the entorhinal cortex (C). After imaging for BSB staining, sections were stained with TF-S and reimaged (B and D). Images shown are at ×200 and are representative of two mice.

Figure 6

Injection of BSB into the lateral ventricle of living Tg2576 mice labels abundant plaques throughout the brain. BSB was stereotactically injected into the left lateral ventricle of a 12-mo-old Tg2576 mouse. Eighteen hours later, frozen sections revealed prominent labeling of SPs in the hippocampus, entorhinal cortex, cingulate gyrus, and other affected brain regions ipsilaterally (Right) and contralaterally (Left). (A) A composite of 12 × 20 fields is shown, with examples of SPs marked by arrows. A cluster of entorhinal cortical SPs (marked with an arrowhead) are shown in Inset (×400). Representative sections were first imaged for BSB fluorescence (B) and then immunostained for Aβ by using the mAb 4G8 (C). BSB labeled the majority of plaques detected by immunostaining (arrows). The intensity of BSB labeling allowed simultaneous visualization of fluorescence and 3,3′-diaminobenzidine tetrahydrochloride staining. Images shown are representative of two mice.

Figure 7

i.v. injection of BSB results in abundant labeling of SPs in Tg2576 mice. BSB was injected into the tail vein of a 12-mo-old Tg2576 mouse. Eighteen hours later, direct examination of frozen sections of the brain revealed prominent BSB labeling of plaques in the hippocampus (A, ×200), entorhinal cortex (D, ×400), cingulate gyrus, and other affected brain regions bilaterally. Sections were subsequently immunostained with the Aβ specific antiserum 2332 (B and E). Fluorescent and light microscopic images were digitally overlain (C and F), revealing the specificity of BSB plaque labeling (arrows). Arrowheads (B) show less intensely Aβ immunopositive plaques that were not labeled with BSB. Images shown are representative of three mice.