A 56kD SDS-stable peptide assembly is prominent among multiple oligomers in Alzheimer synapses

A growing body of evidence indicates that early cognitive decline is linked to synaptic dysfunction that precedes the appearance of plaques and tangles, and that soluble amyloid beta oligomers are key mediators of early synaptic toxicity. We have used flow cytometry analysis of synaptosomes prepared from Alzheimer's disease (AD) brain in order to examine changes that occur in surviving synaptic terminals in AD, and have demonstrated that AD synapses accumulate both Aβ and p-tau. Synaptic Aβ accumulation occurs in the Tg2576 and 3XTg animal models of AD, and is accompanied by increased free cholesterol and ganglioside GM1 and decreased PSD-95. To date, the most robust synaptic Aβ signal in intact synaptosomes by flow cytometry has been obtained with the N-terminal antibody 10G4; the present experiments examine a series of N- and C-terminal APP and Aβ antibodies and conformation-specific antibodies in Western blots of synaptosome-enriched samples from normal cases (n=4), neurologic controls (n=3) and AD cases (n=7). In most AD cases, a ladder of SDS-stable oligomers is visible with several anti-Aβ antibodies, varying in size from dimers/trimers to larger assemblies up to about 200kD. 56kD dodecamers are prominent with an Aβ42-specific antibody, and this band (Aβ-star) correlates significantly (r=0.557, p<0.02) with the synaptic Aβ level measured by flow cytometry in size-purified synaptosomes. The N-terminal antibodies 6E10 and 10G4 have similar epitopes but differentially detect oliogomers and APP; 10G4 does not detect APP and labels primarily tetramers, oligomers in the range of 40-48kD, and larger aggregates that do not enter the gel. 6E10 detects smaller oligomers between 8-28kD, and species around 56kD; dimer/trimer bands are strong in 2-3 cases. The c-terminal antibody CT20 detects a c-terminal fragment at ∼17kD in AD cases. P-tau aggregates and fragments are detected in the same samples with a series of tau antibodies. These results indicate that multiple APP processing pathways are active in AD synapses and multiple oligomeric assemblies, particularly a 56kD assembly, may contribute to synaptic dysfunction.