Synapse-Binding Subpopulations of Aβ Oligomers Sensitive to Peptide Assembly Blockers and scFv Antibodies

Abstract

Amyloid β42 self-assembly is complex, with multiple pathways leading to large insoluble fibrils or soluble oligomers. Oligomers are now regarded as most germane to Alzheimer's pathogenesis. We have investigated the hypothesis that oligomer formation itself occurs through alternative pathways, with some leading to synapse-binding toxins. Immediately after adding synthetic peptide to buffer, solutions of Aβ42 were separated by a 50 kDa filter and fractions assessed by SDS-PAGE silver stain, Western blot, immunoprecipitation, and capacity for synaptic binding. Aβ42 rapidly assembled into aqueous-stable oligomers, with similar protein abundance in small (<50 kDa) and large (>50 kDa) oligomer fractions. Initially, both fractions were SDS-labile and resolved into tetramers, trimers, and monomers by SDS-PAGE. Upon continued incubation, the larger oligomers developed a small population of SDS-stable 10−16mers, and the smaller oligomers generated gel-impermeant complexes. The two fractions associated differently with neurons, with prominent synaptic binding limited to larger oligomers. Even within the family of larger oligomers, synaptic binding was associated with only a subset of these species, as a new scFv antibody (NUsc1) immunoprecipitated only a small portion of the oligomers while eliminating synaptic binding. Interestingly, low doses of the peptide KLVFFA blocked assembly of the 10−16mers, and this result was associated with loss of the smaller clusters of oligomers observed at synaptic sites. What distinguishes these smaller clusters from the unaffected larger clusters is not yet known. Results indicate that distinct species of Aβ oligomers are generated by alternative assembly pathways and that synapse-binding subpopulations of Aβ oligomers could be specifically targeted for Alzheimer's therapeutics.

A Study of Aβ Oligomers in the Temporal Cortex and Cerebellum of Patients with Neuropathologically Confirmed Alzheimer's Disease Compared to Aged Controls

“…5b). These three low-molecular signals were similar to those observed by Lesné et al [34], Lambert et al [21] and Velasco et al [22]. These signals, normalized with the standard DB71, revealed no significant group differences.…”

“…Unlike Aβ trimers that are thought to be secreted by neurons with a stable expression by the age of 70 years and cause no cognitive impairment, Aβ*56 and Aβ dimers increase steadily after 40 and 70 years of age, respectively, and impair brain function when injected into rats [18,19,20]. Besides these endogenous oligomeric Aβ assemblies, a variety of synthetic mid-range molecular weight oligomeric Aβ assemblies were identified as dodecamers to 24-mers using NU-1 antibodies [21,22]. …”

NLRP3 Inflammasome Inhibitor Ameliorates Amyloid Pathology in a Mouse Model of Alzheimer’s Disease

“…Antigen retrieval through pressure cooking was used for some experiments (Biocare Medical, Concord, MA, USA). The slides were blocked for 30 min in 10% normal goat serum (NGS) in PBS and then sections were incubated overnight at 4°C with the following primary antibodies: anti-caspase-1 p10 (1:100, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-amyloid oligomers (NU-2, 1:2000 [23]), anti-amyloid 1–16 (82E1, 1:250, Immunobiological Laboratories, Minneapolis, MN, USA), anti-Iba-1 (1:250, Wako, Osaka, Japan), anti-HNE (1:100, Alpha Diagnostics Intl. Inc., San Antonio, TX, USA) and anti-GFAP (1:100, Invitrogen, Carlsbad, CA, USA).…”

Amyloid β oligomers in Alzheimer’s disease pathogenesis, treatment, and diagnosis

“…An emerging concept is that self-association of Aβ proceeds along divergent paths, one toward oligomers and the other toward fibrils [173, 189]. Biophysical analysis shows there are structural similarities and differences between AβOs and fibrils and that, while common β-strand conformations form early, oligomers and fibrils differ in schemes of intermolecular organization [165].…”