AD synapses contain abundant Aβ monomer and multiple soluble oligomers, including a 56-kDa assembly

Abstract

Much evidence indicates that soluble amyloid beta (Aβ) oligomers are key mediators of early cognitive loss, but the localization and key peptide species remain unclear. We have used flow cytometry analysis to demonstrate that surviving Alzheimer's disease (AD) synapses accumulate both Aβ and p-tau. The present experiments use peptide-specific xMAP assays and Western blotting to identify the Aβ peptide species in synaptosome-enriched samples from normal human subjects, neurologic controls, and AD cases. Aβ40 peptide levels did not vary, but both Aβ42 and Aβ oligomers were increased in soluble AD extracts, with oligomer levels 20-fold higher in aqueous compared to detergent extracts. In Western blots, a ladder of SDS-stable oligomers was observed in AD cases, varying in size from monomer, the major peptide observed, to larger assemblies up to about 200 kD and larger. Multiple oligomers, including monomer, small oligomers, a 56 kD assembly, and APP were correlated with the Aβ level measured in flow cytomety-purified synaptosomes. These results suggest that multiple APP processing pathways are active in AD synapses and multiple soluble oligomeric assemblies may contribute to synaptic dysfunction.

Synaptic Amyloid-β Oligomers Precede p-Tau and Differentiate High Pathology Control Cases

“…ajp.amjpathol.org -The American Journal of Pathology anti-Ab antibodies are sensitive to conformation and differentially label aggregated Ab preparations. 46 As previously reported, synaptic terminals from control cases show little to no Ab immunolabeling ( Figure 1C) 26,33,42,47 ; representative plots from AD cases illustrate the rise in synaptic Ab with increasing neuropathologic disease stage and the degree to which synaptic terminal Ab increases between plaque stage 0 (no plaque deposition) and stage C (end-stage plaque deposition) (Figure 1, D and E). 48 Figure 2A shows group differences in aggregate flow cytometric data [F(3,30) Z 6.13, P Z 0.002] in both early-stage (Braak II to IV) and late-stage (Braak V to VI) AD cases.…”

“…Like 10G4, MOAB2, a new monoclonal antibody specific for intraneuronal Ab that does not label APP, 45 shows a strong specific signal for synaptic Ab with flow cytometric analysis (Supplemental Figure S2, CeE). Taken together with evidence from Western blot analysis that 10G4 strongly labels high molecular weight aggregates, SDS-stable oligomers, and monomer in a pattern that resembles labeling by an Ab42-specific antibody, 26 these results indicate that, for flow cytometry, 10G4 is selective for aggregated Ab in situ. This interpretation is also supported by recent work showing that sequence-specific Figure 2 Synaptic Ab pathology across disease stage.…”

“…In contrast, the oAb assay measures only oligomeric Ab extracted from synaptosome-enriched P-2 fractions into buffer. Because Western blot analysis of P-2 samples shows a strong signal for postsynaptic density protein 95, 26 it is possible that some of the Ab signal in P-2 samples arises from extracellular Ab bound to postsynaptic surface receptors, particularly heparan sulfate proteoglycan. 66 However, prior electron microscopic studies reported by our group and others document the spherical and primarily presynaptic structure of synaptosomes, 33,40 and incubation of AD synaptosomes with a combination of trypsin and heparinase does not reduce Ab labeling.…”

“…8,18e21 In humans, electron microscopic studies have documented synapse-associated Ab and tau, 22,23 and much work documents activity-dependent release of synaptic Ab into interstitial fluid, which drives local Ab deposition in human subjects and in rodents. 4,24,25 Of importance, most synapse-associated Ab in cortical synapses of AD patients consists of soluble oligomeric species, 26 and synaptic tau pathology in AD also includes accumulations of SDS-stable tau oligomers. 27e31 With the use of synaptosomes (resealed nerve terminals) from the cortex of postmortem human subjects and a transgenic rat model of AD, the present experiments were aimed at determining the sequence of appearance of Ab and hyperphosphorylated tau (p-tau) pathology in synaptic terminals.…”

Levels of Soluble Apolipoprotein E/Amyloid-β (Aβ) Complex Are Reduced and Oligomeric Aβ Increased with APOE4 and Alzheimer Disease in a Transgenic Mouse Model and Human Samples*

“…However, the effect of APOE genotype on soluble synaptic apoE/A␤ levels is less clear. Soluble A␤, soluble oA␤, p-Tau, and SDS-stable p-Tau oligomers (37)(38)(39)68) are detected in AD synaptosomes, and data presented herein also demonstrate the presence of soluble apoE/A␤ in AD synaptosomes, with levels reduced compared with controls. Soluble apoE/A␤ levels were also lower in synaptosomes from AD patients with APOE4 compared with APOE3.…”