Single Molecule Fluorescence Microscopy Reveals Neurite-Bound Amyloid-Beta Oligomers

Oligomers of the 39- to 42- residue amyloid-beta peptide are implicated as synaptotoxic factors in Alzheimer's disease. However, the stoichiometric identity of the species which damages neurons and the mechanism by which this species interacts with cells remain unknown. Particles ranging in size from small oligomers to protofibrils may be causative factors in loss of normal synaptic function, a harbinger of Alzheimer's. While these changes may be mediated through interaction with a specific receptor, significant evidence also points to pore formation and catastrophic calcium leakage as a possible mechanism for the peptide's toxicity. Using single molecule microscopy, we demonstrated that monomers and small oligomers bind to living cells even at nanomolar concentrations and that these oligomers can grow following binding (Johnson, et al. 2011. PLoS ONE 6(8): e23970). Similar methods are being applied to determine the size distribution of amyloid-beta oligomers which bind to the neurites of primary hippocampal neurons and to explore how changes in local environment affect this distribution. At low (1-10) nanomolar concentrations, amyloid-beta binds to neurites primarily as monomers to hexamers. A small number of larger neurite-bound oligomers, constituting less than 10% of the total population, is also present. Like oligomers previously observed on the somas of neuroblastoma cells, these neurite-bound species are immobile on a time scale of minutes and are significantly larger than oligomers adsorbed to the coverslip at these concentrations. Studies on the effects of these oligomers on intracellular calcium fluctuations and spine density provide insight into how cell-bound amyloid-beta oligomers impact neuronal electrophysiology and synapse integrity over time. Finally, oligomer binding location on the neuronal process is explored by fluorescence colocalization studies, in an effort to determine whether oligomers exhibit preferences for specific binding sites on the membrane.