A simple, versatile and robust centrifugation-based filtration protocol for the isolation and quantification of α-synuclein monomers, oligomers and fibrils: towards improving experimental reproducibility in α-synuclein research

Abstract

Increasing evidence suggests that the process of alpha-synuclein (aSyn) aggregation from monomers into amyloid fibrils via oligomeric intermediates plays an essential role in the pathogenesis of different synucleinopathies, including Parkinson's disease (PD), multiple system atrophy and dementia with Lewy bodies. However, the nature of the toxic species and the mechanisms by which they contribute to neurotoxicity and disease progression remain elusive. Over the past two decades, significant efforts and resources have been invested in studies aimed at identifying the putative toxic species along the pathway of aSyn fibrillization, and to develop small molecule drugs or antibodies that target toxic aSyn oligomeric intermediates. Although this approach has helped to advance the field and provide insights into the biological properties and toxicity of different aSyn species, many of the fundamental questions regarding the role of aSyn aggregation in PD remain unanswered, and no therapeutic compounds targeting aSyn oligomers have passed clinical trials. Several factors have contributed to this slow progress, including the complexity of the aggregation pathways and the heterogeneity and dynamic nature of aSyn aggregates. In the majority of experiment, the aSyn samples used contain mixtures of aSyn species that exist in an equilibrium and their ratio changes upon modifying experimental conditions. The failure to quantitatively account for the distribution of different aSyn species in different studies has contributed not only to experimental irreproducibility but also to misinterpretation of results and misdirection of valuable resources. Towards addressing these challenges and improving experimental reproducibility in Parkinson's research, we describe here a simple centrifugation-based filtration protocol for the isolation, quantification and assessment of the distribution of of aSyn monomers, oligomers and fibrils, in heterogeneous aSyn samples of increasing complexity.The protocol is simple, does not require any special instrumentation and can be performed rapidly on multiple samples using small volumes. Here, we present and discuss several examples that illustrate the applications of this protocol and how it could contribute to improving the reproducibility of experiments aimed at elucidating the structural basis of aSyn aggregation, seeding activity, toxicity and pathology spreading. This protocol is applicable, with slight modifications, to other amyloid-forming proteins.

A simple, versatile and robust centrifugation‐based filtration protocol for the isolation and quantification of α‐synuclein monomers, oligomers and fibrils: Towards improving experimental reproducibility in α‐synuclein research

“…We also acknowledge the EPFL CIME central facility for their assistance during the EM imaging. The preprint version of this article, prior to peer review, was published on bioRxiv (Kumar et al ; https://www.biorxiv.org/content/10.1101/772160v1). The authors do not have any conflict of interest to disclose.…”

Pronounced α-Synuclein Pathology in a Seeding-Based Mouse Model Is Not Sufficient to Induce Mitochondrial Respiration Deficits in the Striatum and Amygdala

“…α-synuclein fibrils were prepared and characterized as described previously (Burtscher et al, 2019;Kumar et al, 2020). Briefly, a 325 μM solution of pure recombinant mouse α-synuclein protein (in PBS, pH 7.2) was centrifuged in 0.2 μM filter tubes (5 min, 5000 rpm).…”

“…To assess the amount of fibrils formed and the monomers released, we used sedimentation (100000 g, 30 min) and filtration assays (14000 g for 15 min through a 100 kD filter). Electron microscopy was applied for morphological characterizations, as described previously (Burtscher et al, 2019;Kumar et al, 2020).…”

Disentangling aggregation‐prone proteins: a new method for isolating α‐synuclein species

“…Effective separation of α‐Syn fibrils, oligomers, and monomers. A new centrifugation‐ and filter‐based method for purification of distinct α‐Syn species (Kumar, Donzelli, Chiki, Syed, & Lashuel, ). Schematic representation illustrates the primary steps used to isolate and recover distinct forms of α‐Syn by taking advantage of their differences in size and solubility…”

“…In the study by Kumar et al (), a simple rapid centrifugation‐ and filter‐based method for isolating and quantifying α‐Syn monomers, oligomers and fibrils was presented (Figure ). The carefully constructed protocol described by Lashuel and colleagues takes advantage of the size and solubility differences between α‐Syn monomers (14 kDa), oligomers, including dimers (28 kDa‐1 MDa) and fibrils (>2 MDa) for successful separation from heterogeneous mixtures.…”