Amyloid-β and α-Synuclein Aggregate in Live Cells at Concentrations Far Below their in vitro Solubility Limits

Abstract

Amyloid protein aggregates are believed to be the key cause of neurodegenerative diseases such as Alzheimer's and Parkinson's, but the mechanism of their biological action is far from understood. Different studies have implicated different aggregated forms of these proteins as the key toxic elements. Significantly, these studies typically use concentrations in the μM range to get the proteins to aggregate, but this is at least an order of magnitude higher than the concentrations observed in vivo. Here we examine the aggregation of amyloid beta (Aβ) and α-synuclein on cell membranes and inside live cells at sub-μM concentrations, using coupled fluorescence correlation spectroscopy, confocal microscopy and time correlated single photon counting techniques. For Aβ, we find that only small aggregates (<6 nm hydrodynamic radius) form at 350 nM concentration in vitro, but large aggregates of Aβ are present on the cell membrane, together with much smaller species (monomers or small oligomers). When the in vitro concentration is lowered to 150 nM, the solution structures are even smaller (<4 nm, but significantly, still at least dimeric) and no large multimers form on the membrane, though the smaller species are still present. We conclude that Aβ aggregates at much lower concentrations on the membrane, possibly because of the lower free energy of association required for such aggregation in a two dimensional system. For α -synuclein, we find that a 550 nM solution which does not form any aggregates in the extra- cellular medium, develops large aggregates in the cytoplasm. Our results challenge the hypotheses that specific aggregate structures (e.g. donut shaped, spherical or proto-fibrillar aggregates) that form in the solution phase are responsible for the physiological effects of Aβ or α -synuclein.