Abstract
Aggregation of alpha-synuclein (αSyn) is a crucial event underlying the pathophysiology of synucleinopathies. The existence of various intracellular and extracellular αSyn species, including cleaved αSyn, complicates the quest for an appropriate therapeutic target. Hence, to develop efficient disease-modifying strategies, it is fundamental to achieve a deeper understanding of the relevant spreading and toxic αSyn species. Here, we describe comparative and proof-of-principle approaches to determine the involvement of αSyn fragments in intercellular spreading. We demonstrate that two different αSyn fragments (1–95 and 61–140) fulfill the criteria of spreading species. They efficiently instigate formation of proteinase-K-resistant aggregates from cell-endogenous full-length αSyn, and drive it into different aggregation pathways. The resulting aggregates induce cellular toxicity. Strikingly, these aggregates are only detectable by specific antibodies. Our results suggest that αSyn fragments might be relevant not only for spreading, but also for aggregation-fate determination and differential strain formation.
Highlights
Synucleinopathies are a group of neurodegenerative diseases collectively characterized by intracellular protein inclusions containing alpha-synuclein. αSyn is a predominantly presynaptic, intrinsically unfolded protein of 140 amino acids[1,2,3]
Cells were transduced with adenoviral (AV) vectors at day in vitro (DIV) 2 and toxicity, intracellular and extracellular αSyn were monitored over time (Fig. 1a). αSyn-mediated toxicity was assessed with lactate dehydrogenase (LDH) activity in culture medium (Fig. 1b). αSyn overexpression induced increased toxicity over time in comparison to GFPoverexpressing and untreated control cells, and reached approximately 50% at DIV8
Several forms of αSyn appeared in the conditioned medium (CM) at DIV6; an oligomeric species at approximatively 37 kDa, monomeric FL-αSyn at 15 kDa and several αSyn fragments ranging between 13 and 6 kDa
Summary
Synucleinopathies are a group of neurodegenerative diseases collectively characterized by intracellular protein inclusions containing alpha-synuclein (αSyn). αSyn is a predominantly presynaptic, intrinsically unfolded protein of 140 amino acids (aa)[1,2,3]. Synucleinopathies are a group of neurodegenerative diseases collectively characterized by intracellular protein inclusions containing alpha-synuclein (αSyn). Its primary structure comprises three distinct domains: (i) an N-terminal domain (aa 1–60) that binds lipids and adopts alpha helical structures, (ii) a central domain known as non-amyloid component (NAC) (aa 61–95) involved in aggregation, and (iii) a C-terminal acidic tail (aa 96–140) accountable for most interactions with other proteins and small molecules[4]. Since aggregation of αSyn appears as a pivotal event in the pathogenesis of synucleinopathies, understanding the specific mechanisms that lead to accelerated aggregation is crucial. Truncation of αSyn seems to correlate with accelerated aggregation and pathology in cell and mouse models[13,14,15]. Fragments are emerging as a potential common feature observed for other neurodegeneration-related proteins, such as amyloid precursor protein and tau[16]
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