Membrane Permeabilization by Oligomeric α-Synuclein: In Search of the Mechanism

Abstract

BackgroundThe question of how the aggregation of the neuronal protein α-synuclein contributes to neuronal toxicity in Parkinson's disease has been the subject of intensive research over the past decade. Recently, attention has shifted from the amyloid fibrils to soluble oligomeric intermediates in the α-synuclein aggregation process. These oligomers are hypothesized to be cytotoxic and to permeabilize cellular membranes, possibly by forming pore-like complexes in the bilayer. Although the subject of α-synuclein oligomer-membrane interactions has attracted much attention, there is only limited evidence that supports the pore formation by α-synuclein oligomers. In addition the existing data are contradictory.Methodology/Principal FindingsHere we have studied the mechanism of lipid bilayer disruption by a well-characterized α-synuclein oligomer species in detail using a number of in vitro bilayer systems and assays. Dye efflux from vesicles induced by oligomeric α-synuclein was found to be a fast all-or-none process. Individual vesicles swiftly lose their contents but overall vesicle morphology remains unaltered. A newly developed assay based on a dextran-coupled dye showed that non-equilibrium processes dominate the disruption of the vesicles. The membrane is highly permeable to solute influx directly after oligomer addition, after which membrane integrity is partly restored. The permeabilization of the membrane is possibly related to the intrinsic instability of the bilayer. Vesicles composed of negatively charged lipids, which are generally used for measuring α-synuclein-lipid interactions, were unstable to protein adsorption in general.Conclusions/SignificanceThe dye efflux from negatively charged vesicles upon addition of α-synuclein has been hypothesized to occur through the formation of oligomeric membrane pores. However, our results show that the dye efflux characteristics are consistent with bilayer defects caused by membrane instability. These data shed new insights into potential mechanisms of toxicity of oligomeric α-synuclein species.

Fluorescence as a Tool to Study Lipid-Protein Interactions: The Case of α-Synuclein

“…The images show that the leakage proces is fast and that the vesicles appear morphologically unchanged. Results from DLS experiments confirm that LUVs stay largely intact upon oligomer interaction [43]. These results, combined with work form other groups suggest that α-synuclein oligomers do not necessarily form pore-like sructures.…”

How Epigallocatechin Gallate Can Inhibit α-Synuclein Oligomer Toxicity in Vitro

“…(a) The oligomer acts as a pore, as suggested in the literature, and EGCG directly blocks the pore. (b) A critical concentration of oligomer in the membrane is required to permeabilize the membrane as suggested by Subramaniam and co-workers (38), and EGCG reduces the concentration of membrane-bound oligomers. Thus EGCG simply shifts the equilibrium away from the bound state, and we would expect lysis or permeabilization to occur (albeit at lower frequency) at sufficiently high oligomer concentrations.…”

“…These oligomers have not been chemically modified and on average consist of ϳ30 monomers (31,32), forming a rather compact ␤-sheet core with a disordered outer shell. ␣SN oligomers interact with and perturb membranes by a combination of electrostatic interactions between the N terminus of ␣SN and lipid head groups combined with hydrophobic interactions (33)(34)(35)(36)(37)(38).…”

α-Synuclein Senses Lipid Packing Defects and Induces Lateral Expansion of Lipids Leading to Membrane Remodeling

“…The binding of ␣-Syn to membranes might induce the permeabilization of lipid bilayers via pore or conductive ion channel formation (22)(23)(24)(25). Alternatively, ␣-Syn has been suggested to induce membrane remodeling events, such as the nascent budding and tubulation of vesicles (26 -28).…”