Biochemical stages of amyloid-β peptide aggregation and accumulation in the human brain and their association with symptomatic and pathologically preclinical Alzheimer's disease.

Alzheimer's disease is characterized by the deposition of amyloid-beta peptide in the brain. N-terminal truncation resulting in the formation of A beta(N3pE) and phosphorylation at serine 8 have been reported to modify aggregation properties of amyloid-beta. Biochemically, soluble, dispersible, membrane-associated, and insoluble, plaque-associated amyloid-beta aggregates have been distinguished. Soluble and dispersible amyloid-beta aggregates are both in mixture with the extracellular or intracellular fluid but dispersible aggregates can be cleared from proteins in solution by ultracentrifugation. To clarify the role of phosphorylated amyloid-beta and A beta(N3pE) in soluble, dispersible, membrane-associated, and plaque-associated amyloid-beta aggregates in the pathogenesis of Alzheimer's disease we studied brains from 21 cases with symptomatic Alzheimer's disease, 33 pathologically preclinical Alzheimer's disease cases, and 20 control cases. Western blot analysis showed that soluble, dispersible, membrane-associated and plaque-associated amyloid-beta aggregates in the earliest preclinical stage of Alzheimer's disease did not exhibit detectable amounts of A beta(N3pE) and phosphorylated amyloid-beta. This stage was referred to as biochemical stage 1 of amyloid-beta aggregation and accumulation. In biochemical amyloid-beta stage 2, A beta(N3pE) was additionally found whereas phosphorylated amyloid-beta was restricted to biochemical amyloid-beta stage 3, the last stage of amyloid-beta aggregation. Phosphorylated amyloid-beta was seen in the dispersible, membrane-associated, and plaque-associated fraction. All cases with symptomatic Alzheimer's disease in our sample fulfilled biochemical amyloid-beta stage 3 criteria, i.e. detection of phosphorylated amyloid-beta. Most, but not all, cases with pathologically preclinical Alzheimer's disease had biochemical amyloid-beta stages 1 or 2. Immunohistochemistry confirmed the hierarchical occurrence of amyloid-beta, A beta(N3pE), and phosphorylated amyloid-beta in amyloid plaques. Phosphorylated amyloid-beta containing plaques were, thereby, seen in all symptomatic cases with Alzheimer's disease but only in a few non-demented control subjects. The biochemical amyloid-beta stages correlated with the expansion of amyloid-beta plaque deposition and with that of neurofibrillary tangle pathology. Taken together, we demonstrate that A beta(N3pE) and phosphorylated amyloid-beta are not only detectable in plaques, but also in soluble and dispersible amyloid-beta aggregates outside of plaques. They occur in a hierarchical sequence that allows the distinction of three stages. In light of our findings, it is tempting to speculate that this hierarchical, biochemical sequence of amyloid-beta aggregation and accumulation is related to disease progression and may be relevant for an increasing toxicity of amyloid-beta aggregates.