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 (α‐syn) aggregation from monomers into amyloid fibrils and Lewy bodies, 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 (DLB). 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 and targeting toxic species along the pathway of α‐syn fibrillization. Although this approach has helped to advance the field and provide insights into the biological properties and toxicity of different α‐syn species, many of the fundamental questions regarding the role of α‐syn aggregation in PD remain unanswered, and no therapeutic compounds targeting α‐syn aggregates 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 α‐syn aggregates. In the majority of experiment, the α‐syn samples used contain mixtures of α‐syn species that exist in equilibrium and their ratio changes upon modifying experimental conditions. The failure to quantitatively account for the distribution of different α‐syn 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 α‐syn monomers, oligomers and fibrils, in heterogeneous α‐syn 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 α‐syn aggregation, seeding activity, toxicity and pathology spreading. This protocol is applicable, with slight modifications, to other amyloid‐forming proteins.

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?

“…Oligomers can be broadly defined as all the soluble multimeric species that exist before the formation of α-syn fibrils, including a) dimers, trimers and low molecular weight assemblies, which are not easily discernable by electron microscopy (EM) and atomic force microscopy (AFM)), and b) higher molecular weight oligomers with different morphologies that are composed of >10 monomers, which are easily detectable by EM, AFM and other imaging techniques (Lashuel et al 2002;Lashuel & Lansbury 2006;Stöckl et al 2013;Cremades et al 2017;Ruggeri et al 2018;Kumar et al 2020a). Our current knowledge of the biophysical properties of α-syn oligomers has been shaped primarily by results obtained by investigating α-syn aggregation and fibril formation in vitro.…”

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). Brieflly, a 325 μM solution of pure mouse α-synuclein recombinant protein (in PBS, pH7.2) was centrifuged in filter tubes of 0.2 μM (5 min, 5000 rpm).…”

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.…”

“…The new purification method reported by Kumar et al () allowed the researchers to scrutinize and highlight some of the challenges and conditions routinely encountered by investigators during protein aggregation studies. These include reproducibility between replicate pre‐formed fibril preparations; effective ways to determine concentration of monomers and oligomers; quantitation of protein loss and yield during a separation process; and reconstitution of lyophilized pre‐formed fibril samples that may be shared and shipped for a collaborative study.…”