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Single‐molecule approach to find out toxic oligomeric alpa‐synuclein species formation

The formation and accumulation of misfolded protein aggregates is a central hallmark of several neurodegenerative diseases. In Parkinson’s disease (PD), the aggregation-prone protein, alpha-synuclein (a-Syn) is the culprit. To identify the pathological species of a-Syn involved in the perturbation of cellular function is a hot topic of current PD research. In the past few years, substantial progress has been made in elucidating how a-Syn undergoes spontaneous self-aggregation, as well as the process of aggregation of a-Syn from monomers, by oligomeric forms, into fibrils. These studies established that misfolded a-Syn, especially in its oligomeric forms, is probably the toxic species. Cremades et al., in a recent article in Cell, have now sifted through the different states of a-Syn aggregation and examined the functional consequences of aggregate-associated toxicity. To identify the pathological species of a-Syn responsible for neurotoxicity, the investigators developed a new technique to focus on individual oligomers. They used a-Syn labeled with extrinsic fluorophores, either red or green. They mixed the monomer populations and observed oligomer formation up to 10 to 150 monomers in size as time progressed. Using single-molecule analysis, they found that a-Syn takes shape as two distinct oligomer intermediates, named type A or B. To better characterize the structures, the investigators examined proteinase K resistance of the two oligomers. Type A oligomers were no more resistant than monomers, suggesting a loose conformation, whereas type B oligomers exhibited some resistance as fibrils, implying a b-sheet stucture. To evaluate the deleterious consequences of these two oligomers, they examined the effects on the rate of reactive oxygen species (ROS) production in primary dopaminergic cultures, given that protein aggregates are known to induce ROS. Neither monomers nor fibrils affected ROS generation, but the larger proteinase K–resistant oligomer appeared to be toxic. The investigators propose a pathogenic model for fibril formation and oligomer-induced toxicity: In this model, aSyn monomers initially nucleate in type A oligomers, which can either grow into larger A oligomers or convert to the B version, which takes longer to form at approximately 35 hours. Type B oligomers then grow into fibrils. Of importance, the A to B conversion would be the critical step in fibril formation. Furthermore, direct or indirect disaggregation of the fibrils would cause toxic B-type oligomer release. Together, this work elegantly identifies the exact source and distinct aggregation states of a-Syn, and shows that structural interconversion of a-Syn aggregated from an ahelical to high b-sheet content is highly damaging to cells. This study also sheds further light upon how to abort this process, for instance, by stabilizing fibrils, rather than disaggregating them, and by taking advantage of the A to B switch period of time to boost cellular quality-control machinery. This important discovery not only advances our current understanding of PD, but also lays the foundation for the development of novel approaches and drug therapies, which can specifically target this pathological species of aSyn and thus prevent neurotoxicity from occurring.