High‐molecular weight Aβ oligomers and protofibrils are the predominant Aβ species in the native soluble protein fraction of the AD brain

Ajeet Rijal Upadhaya,Dietmar Rudolf Thal,Marcus Fändrich,Haruyasu Yamaguchi,Irina Lungrin

doi:10.1111/j.1582-4934.2011.01306.x

Abstract

Alzheimer’s disease (AD) is characterized by the aggregation and deposition of amyloid β protein (Aβ) in the brain. Soluble Aβ oligomers are thought to be toxic. To investigate the predominant species of Aβ protein that may play a role in AD pathogenesis, we performed biochemical analysis of AD and control brains. Sucrose buffer-soluble brain lysates were characterized in native form using blue native (BN)-PAGE and also in denatured form using SDS-PAGE followed by Western blot analysis. BN-PAGE analysis revealed a high-molecular weight smear (>1000 kD) of Aβ42-positive material in the AD brain, whereas low-molecular weight and monomeric Aβ species were not detected. SDS-PAGE analysis, on the other hand, allowed the detection of prominent Aβ monomer and dimer bands in AD cases but not in controls. Immunoelectron microscopy of immunoprecipitated oligomers and protofibrils/fibrils showed spherical and protofibrillar Aβ-positive material, thereby confirming the presence of high-molecular weight Aβ (hiMWAβ) aggregates in the AD brain. In vitro analysis of synthetic Aβ40- and Aβ42 preparations revealed Aβ fibrils, protofibrils, and hiMWAβ oligomers that were detectable at the electron microscopic level and after BN-PAGE. Further, BN-PAGE analysis exhibited a monomer band and less prominent low-molecular weight Aβ (loMWAβ) oligomers. In contrast, SDS-PAGE showed large amounts of loMWAβ but no hiMWAβ40 and strikingly reduced levels of hiMWAβ42. These results indicate that hiMWAβ aggregates, particularly Aβ42 species, are most prevalent in the soluble fraction of the AD brain. Thus, soluble hiMWAβ aggregates may play an important role in the pathogenesis of AD either independently or as a reservoir for release of loMWAβ oligomers.

Highlights

Alzheimer’s disease (AD) is characterized by the extracellular deposition of amyloid ␤ protein (A␤) aggregates in the brain [1]
It Antibodies and antibody fragments have been developed to detect specific hiMWA␤ oligomers (A11) and protofibrillar/fibrillar conformations (B10AP) [2, 6]. These antibodies and antibody fragments allow isolation of oligomers, protofibrils and fibrils from soluble native protein lysates by immunoprecipitation for further protein analysis. We employed these antibodies and blue native (BN)-PAGE analysis to clarify whether soluble hiMWA␤ oligomers and A␤ protofibrils/fibrils or A␤ dimers and other loMWA␤ species represent the predominant A␤ aggregates in the native soluble fraction of the AD brain
A␤ monomers, dimers, or other loMWA␤ species were not observed in AD cases or in controls (Fig. 1A)

Summary

Introduction

Alzheimer’s disease (AD) is characterized by the extracellular deposition of amyloid ␤ protein (A␤) aggregates in the brain [1]. Antibodies and antibody fragments have been developed to detect specific hiMWA␤ oligomers (A11) and protofibrillar/fibrillar conformations (B10AP) [2, 6]. These antibodies and antibody fragments allow isolation of oligomers, protofibrils and fibrils from soluble native protein lysates by immunoprecipitation for further protein analysis. We employed these antibodies and BN-PAGE analysis to clarify whether soluble hiMWA␤ oligomers and A␤ protofibrils/fibrils or A␤ dimers and other loMWA␤ species represent the predominant A␤ aggregates in the native soluble fraction of the AD brain.

Methods

Results

Conclusion