Structural characterization of toxic oligomers that are kinetically trapped during α-synuclein fibril formation

Abstract

We describe the isolation and detailed structural characterization of stable toxic oligomers of α-synuclein that have accumulated during the process of amyloid formation. Our approach has allowed us to identify distinct subgroups of oligomers and to probe their molecular architectures by using cryo-electron microscopy (cryoEM) image reconstruction techniques. Although the oligomers exist in a range of sizes, with different extents and nature of β-sheet content and exposed hydrophobicity, they all possess a hollow cylindrical architecture with similarities to certain types of amyloid fibril, suggesting that the accumulation of at least some forms of amyloid oligomers is likely to be a consequence of very slow rates of rearrangement of their β-sheet structures. Our findings reveal the inherent multiplicity of the process of protein misfolding and the key role the β-sheet geometry acquired in the early stages of the self-assembly process plays in dictating the kinetic stability and the pathological nature of individual oligomeric species.protein misfolding | amyloid aggregation | toxic oligomer | cryoelectron microscopy | neurodegeneration

A natural product inhibits the initiation of α-synuclein aggregation and suppresses its toxicity

“…We then extended this study by using SH-SY5Y cells treated with oligomers of α-synuclein previously shown to be toxic to cells in culture (23,24) and found that squalamine inhibits completely the mitochondrial dysfunction and the cellular ROS production induced by the oligomers. The degree of binding of toxic α-synuclein oligomers to neuronal cells also decreased with increasing squalamine concentration, and based on our results, we proposed a competitive binding model, where toxic oligomers of α-synuclein and squalamine compete for binding sites at the surface of neuronal cells.…”

“…S1), there was no evidence in the former experiments for the interaction of squalamine with the C-terminal residues. In addition, when the squalamine concentration was increased to 80 μM in the absence (23,24) were resuspended in the cell culture medium at a concentration of 0.3 μM, incubated with or without increasing concentrations (0.03 μM, 0.1 μM, 0.3 μM, 1.0 μM, and 3.0 μM) of squalamine for 1 h at 37°C under shaking conditions, and then added to the cell culture medium of SH-SY5Y cells for 24 h. The cells were also treated with squalamine preincubated in the absence of oligomers for 1 h at 37°C under shaking conditions. **P ≤ 0.01 and ***P ≤ 0.001, respectively, relative to untreated cells and°°P ≤ 0.01 relative to cells treated with α-synuclein oligomers.…”

“…We have recently developed a protocol to isolate oligomers of α-synuclein shown to be toxic to human cells (23,24). We exposed cultured human SH-SY5Y neuroblastoma culture cells to such oligomers at a concentration of 0.3 μM (monomer equivalent of α-synuclein).…”

“…We first investigated the possible mechanism of action of squalamine in this regard by detailed biophysical studies in vitro and extended those results by testing the effects of squalamine on the toxicity of α-synuclein oligomers, using human neuroblastoma cells in culture (23,24), and then carried out experiments in vivo, using a well-established C. elegans animal model of PD (25).…”

“…By carrying out standard We used a wide range of biophysical techniques, including chemical kinetics, to study quantitatively the effects of squalamine on α-synuclein aggregation in vitro and we observed that squalamine affects the binding of α-synuclein to lipid membranes and inhibits the initial events in its aggregation. (B and C) By using the protocols that we recently developed to isolate oligomers of α-synuclein toxic to human cells (23,24), we have shown that squalamine inhibits dramatically the mitochondrial dysfunction and cellular ROS production induced by the oligomers of α-synuclein (23,24). (D-F) We further validated our results in vivo by using a well-established C. elegans model of PD (25), and we found that administration of a single dose of squalamine at the larval stage L4 (D) abolishes α-synuclein aggregation and its associated toxicity in vivo over several days (E) and increases the total fitness of the PD worms (F).…”