Nanoscopic insights into seeding mechanisms and toxicity of α-synuclein species in neurons

Abstract

New strategies for visualizing self-assembly processes at the nanoscale give deep insights into the molecular origins of disease. An example is the self-assembly of misfolded proteins into amyloid fibrils, which is related to a range of neurodegenerative disorders, such as Parkinson's and Alzheimer's diseases. Here, we probe the links between the mechanism of α-synuclein (AS) aggregation and its associated toxicity by using optical nanoscopy directly in a neuronal cell culture model of Parkinson's disease. Using superresolution microscopy, we show that protein fibrils are taken up by neuronal cells and act as prion-like seeds for elongation reactions that both consume endogenous AS and suppress its de novo aggregation. When AS is internalized in its monomeric form, however, it nucleates and triggers the aggregation of endogenous AS, leading to apoptosis, although there are no detectable cross-reactions between externally added and endogenous protein species. Monomer-induced apoptosis can be reduced by pretreatment with seed fibrils, suggesting that partial consumption of the externally added or excess soluble AS can be significantly neuroprotective.T he proliferation of α-synuclein (AS) aggregates (1-6), including the existence of distinct "prion-like" strains (7, 8) as well as their spatial propagation throughout the brain (9), has been proposed to occur in the brains of patients suffering from Parkinson's disease, but their links to pathology and to neuronal death have remained elusive (10-13). In this study, we use optical nanoscopy (14-18) to observe neurons directly and to assay AS internalization, as well as fibril-induced templating reactions involving endogenous AS at the molecular level. We further correlate this information with toxic phenotypes.We have previously shown that the presence of AS short fibrils (seed fibrils or seeds) preformed in vitro (Materials and Methods and Fig. 1 and Fig. S1) favors elongation reactions over spontaneous nucleation (term used for seed-independent aggregation hereafter) in vitro (18). Furthermore, the seed fibrils were found to display highly inhomogeneous growth kinetics, with a significant fraction showing little or no growth at all (18). Here, we use two-color direct stochastic optical reconstruction microscopy (dSTORM), a superresolution imaging method (14), to investigate how such processes may be modified in the cellular environment. The results show the potential of this technique for studying the mechanisms of aggregation events in vivo and provide evidence for the neuroprotective role of reducing the concentration of free AS in the cellular environment. Results and DiscussionExternally Added Seed Fibrils of AS Act as Templates for Exogenously Added Monomeric AS and Prevent Its Nucleation. We first incubated neuronal cells with AS seed fibrils [50 nM, 5% covalently labeled with Alexa Fluor 568 (AF568), green] and AS monomers [500 nM, 10% covalently labeled with Alexa Fluor 647 (AF647), red], either each individually or both in sequence. Either seed fibrils and...

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“…Clearance of higher order structures of α-Syn have been shown to require autophagy [46]. A recent publication showed that relative concentrations of the different forms of α-Syn, including extracellular forms of the monomer, may play important roles in PD [47]. It was especially interesting that when the monomeric form was added exogenously to cells, it could trigger aggregation of the endogenous α-Syn, leading to apoptosis.…”

“…It was especially interesting that when the monomeric form was added exogenously to cells, it could trigger aggregation of the endogenous α-Syn, leading to apoptosis. Furthermore, reduction in the levels of monomer (in this case by fibrillar seeding) was protective [47]. …”

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