Abstract
Protein aggregation plays a central role in numerous neurodegenerative diseases. The key proteins in these diseases are of significant importance, but their investigation can be challenging due to unique properties of protein misfolding and oligomerization. Alpha-synuclein protein (α-Syn) is the predominant component of Lewy Bodies in Parkinson’s disease (PD) and is a member of this class of proteins. Many α-Syn studies are limited by the inability to separate various monomeric, oligomeric, and fibrillar forms of the protein from heterogeneous mixtures. This Editorial Highlight summarizes the impact of a study published in the current issue of Journal of Neurochemistry, in which Lashuel and colleagues developed a simple, rapid centrifugation- and filter-based method for separating, isolating, and quantifying different forms of α-Syn. The researchers used electron microscopy, SDS-PAGE, circular dichroism, and protein assays to carefully validate the method and quantitate α-Syn yields and loss. The publication of this new method will not only aid in future studies of α-Syn, but will likely extend to other proteins that underlie a variety of neurodegenerative diseases.
Keywords: Alpha-synuclein, protein purification, amyloid fibrils, oligomers
Graphical Abstract:
Protein aggregation plays a central role in numerous neurodegenerative diseases, yet investigation of these proteins is difficult due to their unique properties. Alpha-synuclein protein (α-Syn) is the predominant component of Lewy Bodies in Parkinson’s disease (PD) and well known for its ability to form higher molecular weight species. Thus, many α-Syn studies are limited by the inability to separate monomers, oligomers, and fibrils from heterogeneous α-Syn solutions. This Editorial Highlight showcases a study by Lashuel and colleagues in the current issue of Journal of Neurochemistry whereby the researchers developed a rapid centrifugation- and filter-based method for separating, isolating, and quantifying different forms of α-Syn.
Protein aggregation is a key step in numerous neurodegenerative diseases including Alzheimer’s (amyloid-β, tau), Parkinson’s (alpha-synuclein), Huntington’s (huntingtin, polyglutamine), Creutzfeldt-Jakob (prion), amyotrophic lateral sclerosis (superoxide dismutase 1, TDP-43), and others (Jucker and Walker 2018). In order to fully understand the protein aggregation process both biophysically and biochemically, it is necessary to apply rigorous protein purification methods. Failure to do so complicates the interpretation of not only in vitro results, but in vivo findings as well. Overall, protein aggregation studies are fraught with problems if appropriate steps are not taken to isolate monomeric, oligomeric, and fibrillar species.
Alpha-synuclein (α-Syn) is a member of the aggregation-prone, neurodegenerative class of proteins, and plays a critical role in Parkinson’s disease (PD) etiology (Sveinbjornsdottir 2016; Sulzer and Edwards 2019). A characteristic feature of PD is the presence of Lewy Body inclusions that contain a significant fraction of aggregated α-Syn (Delenclos et al. 2019). There is an ongoing debate among neurodegenerative disease researchers on which species of protein aggregate is the most toxic or pathogenic during disease progression. Naturally, this question has been posed for α-Syn. Yet, in order to investigate key mechanistic or functional processes, successful isolation of different α-Syn species is imperative. As with some other amyloid-forming proteins, α-Syn possesses a unique structural polydispersity whereupon the unstructured monomer can adopt a helical conformation when interacting with membranes, or β-sheet structure when forming fibrils (Meade et al. 2019).
In the study by Kumar et al. (2020), a simple rapid centrifugation- and filter-based method for isolating and quantifying α-Syn monomers, oligomers and fibrils was presented. 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 researchers also utilized multiple complementary methods for quantitation of each α-Syn species. The first step involves sedimentation and separation of fibrils from monomers and oligomers by ultracentrifugation of the sample at 100000g for 30 min. The second step separates monomers and mixtures of oligomers by centrifugation through a 100 kDa molecular weight cut-off (MWCO) filter. The study validated the successful separation of α-Syn species with a variety of analytical techniques including electron microscopy (EM), SDS-PAGE and circular dichroism (CD). Quantitative assessment of each α-Syn species using UV absorbance, bicinchoninic acid (BCA) assay and amino acid analysis significantly strengthened the findings.
Isolation of α-Syn fibrils by centrifugation yielded assemblies with a classical long, straight amyloid morphology by EM and highly enriched β-sheet structure by CD. Oligomers of α-Syn had a predominantly spherical morphology yet also produced a CD spectra rich in β-sheet structure. Separation of unstructured α-Syn monomers from oligomers was confirmed by observation of a random coil CD spectra. The most common and effective method for purification of soluble monomers and oligomers of amyloid-forming proteins is size exclusion chromatography (SEC) (Walsh et al. 1997; Bitan and Teplow 2005; Hellstrand et al. 2009; Jan et al. 2010; Paranjape et al. 2012). The new method reported by Lashuel and colleagues was comparable to SEC in the ability to separate α-Syn oligomers of different sizes from monomers and looks to be an appropriate substitute for SEC-based methods.
The new purification method reported by Kumar et al. (2020) 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 (PFF) preparations; effective ways to determine concentration of monomers and oligomers; quantitation of protein loss and yield during a separation process; and reconstitution of lyophilized PFF samples that may be shared and shipped for a collaborative study.
The development of alternative, yet effective, purification methods for aggregation-prone proteins is vital to maintain consistency between laboratories within the research field. The findings by Lashuel and colleagues have added to the repertoire of methods available for separation of α-Syn monomers, oligomers, and fibrils. It is likely that these methods extend to other proteins that are central to a variety of neurodegenerative diseases.
Figure 1.
Effective separation of α-Syn fibrils, oligomers, and monomers. A new centrifugation- and filter-based method for purification of distinct α-Syn species (Kumar et al. 2020). 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.
Acknowledgement and Conflict of Interest Disclosure
This work was in part supported by the National Institute of General Medical Sciences of the National Institutes of Health under award number R15GM119070 (MRN). The author declares that he has no conflicts of interests.
Abbreviations
- α-Syn
Alpha-synuclein
- CD
circular dichroism
- EM
electron microscopy
- MWCO
molecular weight cut-off
- PD
Parkinson’s disease
- PFF
pre-formed fibril
- SEC
size exclusion chromatography
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