A distinct subfraction of Aβ is responsible for the high-affinity Pittsburgh compound B-binding site in Alzheimer's disease brain

Abstract

The positron emission tomography (PET) ligand (11) C-labeled Pittsburgh compound B (PIB) is used to image β-amyloid (Aβ) deposits in the brains of living subjects with the intent of detecting early stages of Alzheimer's disease (AD). However, deposits of human-sequence Aβ in amyloid precursor protein transgenic mice and non-human primates bind very little PIB. The high stoichiometry of PIB:Aβ binding in human AD suggests that the PIB-binding site may represent a particularly pathogenic entity and/or report local pathologic conditions. In this study, (3) H-PIB was employed to track purification of the PIB-binding site in > 90% yield from frontal cortical tissue of autopsy-diagnosed AD subjects. The purified PIB-binding site comprises a distinct, highly insoluble subfraction of the Aβ in AD brain with low buoyant density because of the sodium dodecyl sulfate-resistant association with a limited subset of brain proteins and lipids with physical properties similar to lipid rafts and to a ganglioside:Aβ complex in AD and Down syndrome brain. Both the protein and lipid components are required for PIB binding. Elucidation of human-specific biological components and pathways will be important in guiding improvement of the animal models for AD and in identifying new potential therapeutic avenues. A lipid-associated subpopulation of Aβ accounts for the high-affinity binding of Pittsburgh compound B (PIB) in Alzheimer's disease brain. Mass spectrometry of the isolated PIB-binding site from frontal cortex identified Aβ peptides and a set of plaque-associated proteins in AD but not age-matched normal brain. The PIB-binding site may represent a particularly pathogenic entity and/or report local pathologic conditions.

Keywords: ELISA; amyloid; lipids; plaque; radioreceptor assay; tau.

Figure 1

Purification scheme for the AD brain PIB binding site.

Figure 2

Equilibrium buoyant density separation indicates that the PIB binding site has a density suggesting associated lipid. Control (pool of 3) and AD brain (pool of 6) showing protein, Aβ, and PIB binding. Error bars show standard deviations across 3 gradients for each pool.

Figure 3

PIB binding site is immunoprecipitated by anti-Aβ monoclonal antibody 4G8. ³H-PIB prebound to purified PIB binding site is depleted by incubation with anti-Aβ 4G8 (but not by pre-immune mouse IgG or anti-Aβ 6E10) followed by isolation of the immune complexes on Protein G-Dynabead magnetic beads. See Methods for details.

Figure 4

Silver stain and western blot analysis of PIB binding site purification. Duplicate samples, each representing 1.33 mg wet weight of AD brain tissue, were separated on a Novex 12% acrylamide gel in Mes buffer. The gel was cut, and one set of samples was transferred to a nitrocellulose membrane. Aβ was detected with a mixture of the monoclonal antibodies 6E10 and 4G8 (panel A); the other set of samples was stained with silver (panel B). See Methods for details. Lane (1) SDS-extracted brain homogenate 100 kG pellet; lane (2) SDS-extracted 15–100 kG pellet; lane (3) equilibrium buoyant density-purified PIB binding site; lane (4) rPeptide Aβ(1–40) (20 ng western blot, 60 ng silver stain).