Large Aggregates Are the Major Soluble Ab Species in AD Brain Fractionated with Density Gradient Ultracentrifugation

Soluble amyloid-beta (A beta) aggregates of various sizes, ranging from dimers to large protofibrils, have been associated with neurotoxicity and synaptic dysfunction in Alzheimer's Disease (AD). To investigate the properties of biologically relevant A beta species, brain extracts from amyloid beta protein precursor (A beta PP) transgenic mice and AD patients as well as synthetic A beta preparations were separated by size under native conditions with density gradient ultracentrifugation. The fractionated samples were then analyzed with atomic force microscopy (AFM), ELISA, and MTT cell viability assay. Based on AFM appearance and immunoreactivity to our protofibril selective antibody mAb158, synthetic A beta 42 was divided in four fractions, with large aggregates in fraction 1 and the smallest species in fraction 4. Synthetic A beta aggregates from fractions 2 and 3 proved to be most toxic in an MTT assay. In A beta PP transgenic mouse brain, the most abundant soluble A beta species were found in fraction 2 and consisted mainly of A beta 40. Also in AD brains, A beta was mainly found in fraction 2 but primarily as A beta 42. All biologically derived A beta from fraction 2 was immunologically discriminated from smaller species with mAb158. Thus, the predominant species of biologically derived soluble A beta, natively separated by density gradient ultracentrifugation, were found to match the size of the neurotoxic, 80-500 kDa synthetic A beta protofibrils and were equally detected with mAb158.