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 phosphorylated tau (p-tau). The present experiments use peptide-specific X-map assays and Western blot analyses 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 with detergent extracts. In Western blot analysis, a ladder of sodium dodecyl sulfate (SDS)-stable oligomers was observed in AD cases, varying in size from monomer, the major peptide observed, to larger assemblies up to about 200 kDa and larger. Multiple oligomers, including monomer, small oligomers, a 56-kDa assembly, and amyloid precursor protein (APP) were correlated with the Aβ level measured in flow cytometry-purified synaptosomes. These results suggest that multiple amyloid precursor protein processing pathways are active in AD synapses and multiple soluble oligomeric assemblies may contribute to synaptic dysfunction.

Introduction

A number of studies have documented that levels of soluble amyloid beta (Aβ) peptides are superior to amyloid deposits as correlates of cognitive decline in Alzheimer's disease (AD; McLean et al., 1999, Näslund et al., 2000). Accordingly, the original amyloid cascade hypothesis has evolved to propose that soluble oligomeric Aβ assemblies precede deposition and are the proximal cause of synaptic dysfunction and early impairment in AD (see Walsh and Selkoe, 2007 for review). However, the size of the key assembly state and relevant downstream pathways remain the subject of intense study. Among natural low-n assemblies, dimers and/or trimers in particular have been isolated from AD brain and shown to impair cognition in vitro (Cleary et al., 2005, Klyubin et al., 2008, Shankar et al., 2008, Townsend et al., 2006). In the Tg2576 mouse model a larger assembly, (Aβ*56), possibly a multimer of smaller oligomers, was associated with cognitive decline in Tg2576 mice (Lesné et al., 2006).

Soluble Aβ peptides are associated with synaptic loss (Lue et al., 1999), and multiple studies have shown that soluble oligomers bind to dendritic spines in primary cultures (Lacor et al., 2004, Lacor et al., 2007). Recent evidence also suggests that brief passive immunotherapy has acute and extended benefits on synaptic density and plasticity (Rozkalne et al., 2009, Spires-Jones et al., 2009). Consistent with synaptic Aβ release, interstitial Aβ levels are increased by synaptic activity (Cirrito et al., 2005, Cirrito et al., 2008), and have been shown to correlate with neurological status in patients with brain injury (Brody et al., 2008). Reasoning that study of surviving synaptic terminals is critical for understanding the sources for synaptic Aβ production and release as well as pathways leading to loss of synapses, we have analyzed human synaptosomal preparations by flow cytometry analysis and have shown that Aβ accumulates in synaptic terminals in multiple regions of AD brain. Phosphorylated tau (p-tau) also accumulates in Aβ-bearing synapses, and the colocalization of Aβ and p-tau is accompanied by increased synaptosome size, modest losses of the postsynaptic density scaffold protein, PSD-95, and increased cholesterol and ganglioside GM1 (Gylys et al., 2004b, Gylys et al., 2007, 2008). With flow cytometry, the synaptosomal Aβ signal is best detected by an N-terminal antibody (10G4) that does not discriminate between peptides; the present study correlates the flow cytometry signal with a series of peptide and conformation-specific antibodies along with a series of Aβ peptide-specific assays on the Luminex platform. We report here that monomeric Aβ is prominent among multiple sodium dodecyl sulfate (SDS)-stable soluble Aβ species, including a 56-kDa assembly, in synaptic terminals from AD cortex.