Disaggregation of Alzheimer beta-amyloid by site-directed mAb - PubMed (original) (raw)

Disaggregation of Alzheimer beta-amyloid by site-directed mAb

B Solomon et al. Proc Natl Acad Sci U S A. 1997.

Abstract

In Alzheimer disease, beta-amyloid peptide accumulates in the brain as insoluble amyloid plaques. Amyloid filaments, similar to those found in amyloid plaques, can be assembled in vitro from chemically synthesized beta-peptides. In this study, we report that antibodies raised against the N-terminal region (1-28) of the beta-amyloid peptide bind to the in vitro-formed beta-amyloid assemblies, leading to disaggregation of the fibrils and partial restoration of the peptide's solubility. The concomitant addition of fibrillar beta-amyloid with these antibodies to PC 12 cells leads to the inhibition of the neurotoxic effects of beta-amyloid. Some of the mAbs raised against soluble beta-peptide (1-28) have been found to prevent in vitro fibrillar aggregation of beta-amyloid peptide. These experimental data suggest that site-directed mAbs interfere with the aggregation of beta-amyloid and trigger reversal to its nontoxic, normal components. The above findings give hints on how to convert in vivo senile plaques into nontoxic, diffuse components and may have therapeutic interest for those studying Alzheimer disease and other human diseases related to amyloidogenic properties of physiological peptides and proteins.

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Figures

Figure 1

Figure 1

Solubilization of β-amyloid in the presence of mAbs 6C6, 1C2 and 14C2. The amounts of immunocomplexes of the resolubilized Aβ obtained after 24-h incubation with the respective antibodies were measured by a sandwich ELISA assay using rabbit polyclonal antibodies raised against Aβ as coating antibodies. Measurement of the OD of A495 monitors antibody binding to β-amyloid using horseradish peroxidase-labeled goat anti-mouse antibody (Bio-Rad). The data represent the mean of three replicates. The SDs of the intraassay and interassay were 8%. The percentages are related to maximal OD measured for each mAb after the addition of 100 ng of soluble Aβ complexed with each of the studied mAb to the coated ELISA wells before incubation at 37°C.

Figure 2

Figure 2

ThT-based fluorometric assay. Estimation of the fluorescence of ThT, which correlates with the amount of fibrillar β-amyloid formed after incubation for 1 week at 37°C before and after additional incubation for 24 h with mAbs 6C6 and 1C2 and unrelated antibodies (at molar ratio 10:1 Aβ/mAb) using the ThT assay as described.

Figure 3

Figure 3

Dose-dependent neurotoxicity of β-amyloid aggregates on PC 12 cells measured by MTT assay. Increasing concentrations of β-amyloid aged 1 week were added to the same amount of cultured cells and assayed as described.

Figure 4

Figure 4

Selective inhibitory effect of various mAbs on the β-amyloid toxicity toward PC 12 cells using the MTT assay. The mAbs were added to 0.25 μM β-amyloid at molar ratios of 100:1 Aβ/mAb (left bars) and 10:1 Aβ/mAb (right bars), and the mixtures were added to the cells. The percentages are related to cell viability measured in the absence of the β-amyloid, which is considered 100%.

Figure 5

Figure 5

The effect of mAb 6C6 on the inhibition of fibrillar β-amyloid neurotoxicity toward PC12 cells using the MTT assay. The following preparations were added to the cells for 48 h: (i) Aβ (2.5 μM) preincubated for 7 days at 37°C; (ii) soluble Aβ (2.5 μM) incubated for 7 days at 37°C in the presence of mAb 6C6 (molar ratio mAb/Aβ 1:10); (iii) preformed β-amyloid (2.5 μM) incubated for a further 24 h with mAb 6C6 (molar ratio mAb/Aβ 1:10); and (iv) Aβ incubated with unrelated antibodies. The percentages are related to the cell viability measured in the absence of β-amyloid, which is considered as 100%.

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