Direct Observation of Single Amyloid-β(1-40) Oligomers on Live Cells: Binding and Growth at Physiological Concentrations

Abstract

Understanding how amyloid-β peptide interacts with living cells on a molecular level is critical to development of targeted treatments for Alzheimer's disease. Evidence that oligomeric Aβ interacts with neuronal cell membranes has been provided, but the mechanism by which membrane binding occurs and the exact stoichiometry of the neurotoxic aggregates remain elusive. Physiologically relevant experimentation is hindered by the high Aβ concentrations required for most biochemical analyses, the metastable nature of Aβ aggregates, and the complex variety of Aβ species present under physiological conditions. Here we use single molecule microscopy to overcome these challenges, presenting direct optical evidence that small Aβ(1-40) oligomers bind to living neuroblastoma cells at physiological Aβ concentrations. Single particle fluorescence intensity measurements indicate that cell-bound Aβ species range in size from monomers to hexamers and greater, with the majority of bound oligomers falling in the dimer-to-tetramer range. Furthermore, while low-molecular weight oligomeric species do form in solution, the membrane-bound oligomer size distribution is shifted towards larger aggregates, indicating either that bound Aβ oligomers can rapidly increase in size or that these oligomers cluster at specific sites on the membrane. Calcium indicator studies demonstrate that small oligomer binding at physiological concentrations induces only mild, sporadic calcium leakage. These findings support the hypothesis that small oligomers are the primary Aβ species that interact with neurons at physiological concentrations.

Amyloid-β(1–42) Aggregation Initiates Its Cellular Uptake and Cytotoxicity

“…It is tempting to speculate that small oligomers may have a higher affinity to the plasma membrane than the monomeric peptide, facilitating the conversion to ␤-sheet-rich structures and subsequent internalization. Our results demonstrate that, unlike monomers, preexisting A␤ aggregates are internalized at low nanomolar concentrations, which corresponds to previously observed binding of A␤ oligomers to neuronal plasma membranes at nanomolar concentration (31,32).…”

Aβ1-42 monomers or oligomers have different effects on autophagy and apoptosis

“…10 Indeed, the membrane-affinity of oligomers and monomers is very similar, 11,12 with a higher propensity to attach to the membrane by the oligomers, 13 and a lower affinity to bind to the cell membrane by the monomers. 14 There is the need to better differentiate the effects of the 2 Aβ species.…”

Weak Glycolipid Binding of a Microdomain-Tracer Peptide Correlates with Aggregation and Slow Diffusion on Cell Membranes

“…However, some evidence exists that higher-order oligomeric Aβ molecules or aggregates, rather than binding more tightly, lose the capacity to bind the membrane, and that primarily monomers or small clusters of the peptide associate with the membrane [29], [31], [32] and induce regions of immobility and/or decreased fluidity, depending on membrane composition [29], [33], [34]. It is not established whether unstructured aggregation would lead to membrane coalescence or stimulate uptake as does CtxB, which has a much greater affinity for the membrane than Aβ [25], [35], [36], [37].…”