Stabilization of α-synuclein oligomers using formaldehyde

Abstract

The group of neurodegenerative diseases, Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA) all exhibit inclusions containing amyloid-type α-synuclein (α-syn) aggregates within degenerating brain cells. α-syn also exists as soluble oligomeric species that are hypothesized to represent intermediates between its native and aggregated states. These oligomers are present in brain extracts from patients suffering from synucleinopathies and hold great potential as biomarkers. Although easily prepared in vitro, oligomers are metastable and dissociate over time, thereby complicating α-syn oligomer research. Using the small amine-reactive cross-linker, formaldehyde (FA), we successfully stabilized α-syn oligomers without affecting their size, overall structure or antigenicity towards aggregate-conformation specific α-syn antibodies FILA and MJFR-14-6-4-2. Further, cross-linked α-syn oligomers show resistance towards denaturant like urea and SDS treatment and remain fully functional as internal standard in an aggregation-specific enzyme-linked immunosorbent assay (ELISA) despite prior incubation with urea. We propose that FA cross-linked α-syn oligomers could serve as important calibrators to facilitate comparative and standardized α-syn biomarker studies going forward.

Characterization and validation of 16 α-synuclein conformation-specific antibodies using well-characterized preparations of α-synuclein monomers, fibrils and oligomers with distinct structures and morphology: How specific are the conformation-specific α-synuclein antibodies?

“…However, it is possible to generate preparations that are enriched in specific oligomeric species by subfractionating these preparations using size exclusion chromatography or other separation methods (Lashuel et al 2002;Lashuel & Lansbury 2006). The protocols involve the generation of oligomers either by incubating recombinant α-syn monomers at high concentrations in buffers with or without additional components such as dopamine (Conway et al 2001;Cappai et al 2005;Norris et al 2005;Leong et al 2009;Rekas et al 2010;Choi et al 2013;Planchard et al 2014), lipids (Broersen et al 2006;Trostchansky et al 2006;Qin et al 2007;Nasstrom et al 2009;Näsström et al 2011a;Näsström et al 2011b;De Franceschi et al 2011;Diógenes et al 2012;Xiang et al 2013), metals (Lowe et al 2004;Cole et al 2005;Danzer et al 2007;Danzer et al 2009;Wright et al 2009;Schmidt et al 2012), alcohols (Danzer et al 2007;Ehrnhoefer et al 2008;Danzer et al 2009;Illes-Toth et al 2015) , or by using methods that are based on the use of chemical cross-linking agents (Ruesink et al 2019). In the absence of additional components, oligomers are found to exhibit heterogeneous morphologies, such as globular, spherical, annular pore-shaped, rectangular and tubular-shaped, and are usually but not always enriched in b-sheet structures (Lashuel et al 2002).…”

Low Dose DMSO Treatment Induces Oligomerization and Accelerates Aggregation of α-Synuclein

“…1) and cellular experiments (Fig. 3) which suggest that α-syn multimerization occurs in the presence of DMSO, as well as previous reports showing that α-syn adopt β-sheet structure upon oligomerization and fibrillation [15,34], we cannot exclude that α-syn is one of these cellular proteins adopting β-sheet structure upon DMSO treatment.…”

“…Based on this finding, we sought to examine if the increased particle size observed was caused by α-syn aggregation. Utilizing the aggregation-specific antibody MJFR-14-6-4-2 that preferentially recognizes oligomeric and fibrillar forms of α-syn [15], we performed dot-blots on recombinant α-syn treated with different concentrations of DMSO. To control for equal loading between samples we used the SYN-1 antibody, which measures total α-syn levels (Fig.…”

The amyloid concentric β-barrel hypothesis: models of Synuclein oligomers, annular protofibrils, lipoproteins, and transmembrane channels

“…Some medium to large α-Syn oligomers appear to contain aggregates of smaller components [51][52, 53]. The smallest of these components (other than monomers) appear as beads of ∼ 4 nm in diameter that cluster or string together (Fig.…”

“…We added yellow circles of 4.0 nm diameter around some beads. (a) An assembly in which oligomers were stabilized using formaldehyde cross-linking [51]. (b) Adapted with permission from Paslawski W, Andreasen M, Nielsen SB, et al High stability and cooperative unfolding of alpha-synuclein oligomers.…”