Structural and Biophysical Characterization of Stable Alpha-Synuclein Oligomers

Abstract

The aggregation of α-synuclein (α-syn) into neurotoxic oligomers and fibrils is an important pathogenic feature of synucleinopatheis, including Parkinson’s disease (PD). A further characteristic of PD is the oxidative stress that results in the formation of aldehydes by lipid peroxidation. It has been reported that the brains of deceased patients with PD contain high levels of protein oligomers that are cross-linked to these aldehydes. Increasing evidence also suggests that prefibrillar oligomeric species are more toxic than the mature amyloid fibrils. However, due to the heterogenous and metastable nature, characterization of the α-syn oligomeric species has been challenging. Here, we generated and characterized distinct α-syn oligomers in vitro in the presence of DA and lipid peroxidation products 4-hydroxy-2-nonenal (HNE) and 4-oxo-2-nonenal (ONE). HNE and ONE oligomer were stable towards the treatment with SDS, urea, and temperature. The secondary structure analysis revealed that only HNE and ONE oligomers contain β-sheet content. In the seeding assay, both DA and ONE oligomers significantly accelerated the aggregation. Furthermore, all oligomeric preparations were found to seed the aggregation of α-syn monomers in vitro and found to be cytotoxic when added to SH-SY5Y cells. Finally, both HNE and ONE α-syn oligomers can be used as a calibrator in an α-syn oligomers-specific ELISA.

Assessment of Aggregated and Exosome-Associated α-Synuclein in Brain Tissue and Cerebrospinal Fluid Using Specific Immunoassays

“…To develop a conformation-specific immunoassay for aggregated α-synuclein, we used the mouse monoclonal Syn-F2 antibody to capture fibrillar and oligomeric α-synuclein assemblies. The Syn-F2 antibody has been extensively characterized by a variety of biochemical techniques, including dot blot, immunohistochemistry and ELISA, and has been shown to preferentially bind to mature amyloid fibrils and high molecular weight oligomers, either prepared in vitro or found in post-mortem brain sections [ 30 , 32 ]. To ensure that our immunoassay would selectively detect the aggregated α-synuclein species and not monomeric α-synuclein, we used the aggregate-specific rabbit monoclonal antibody MJFR-14-6-4-2 (Abcam) as the detection antibody.…”

“…After two cycles of freezing and thawing, PFF aliquots were discarded. The production and characterization of the stable α-synuclein oligomers in the presence of the lipid peroxidation products, 4-oxo-2-nonenal (ONE oligomers) and 4-hydroxy-2-nonenal (HNE oligomers), have been recently established [ 32 ].…”

Effective Inhibition of TDP‐43 Aggregation by Native State Stabilization

“…For example, curcumin derivatives were used to modulate and reshape disease‐relevant tau oligomeric strains and rescue neurons from oligomer‐associated toxicity [30] . Furthermore, αSyn oligomers generated in the presence of several compounds have facilitated biophysical and mechanistic studies as well as the generation of new tools to study oligomers in vitro and in vivo [31] . Therefore, the advances we report here pave the way for conducting large‐scale HTS and drug discovery studies to identify novel drug‐like and native‐state stabilizers of TDP‐43 or modulators of TDP‐43 aggregation and toxicity.…”

Effect of Electric Field on α-Synuclein Fibrils: Revealed by Molecular Dynamics Simulations

“…The formation of the α-synuclein inclusions occurs by a generic process of misfolding, by which an ordinarily soluble protein converts into fibrillar aggregates via a series of oligomeric intermediates and, ultimately, the insoluble fibrils are deposited in the brain. Soluble oligomeric species generated during the formation of fibrils are the most neurotoxic species linked with the development of PD [ 72 , 73 , 74 ]. The kinetic of α-synuclein fibril formation can often be dominated by secondary nucleation events, such as fibril fragmentation, adding further elements of complexity to the kinetic process [ 75 ].…”