Observation of an α-synuclein liquid droplet state and its maturation into Lewy body-like assemblies

Abstract

Misfolded a-synuclein is a major component of Lewy bodies, which are a hallmark of Parkinson's disease. A large body of evidence shows that a-synuclein can self-assemble into amyloid fibrils, but the relationship between amyloid formation and Lewy body formation still remains unclear. Here we show, both in vitro and in a C. elegans model of Parkinson's disease, that a-synuclein undergoes liquid-liquid phase separation by forming a liquid droplet state, which converts into an amyloid-rich hydrogel. This maturation process towards the amyloid state is delayed in the presence of model synaptic vesicles in vitro. Taken together, these results suggest that the formation of Lewy bodies is linked to the arrested maturation of a-synuclein condensates in the presence of lipids and other cellular components.Parkinson's disease (PD) is the most common neurodegenerative movement disorder, affecting over 2% of the world's population over 65 years of age [1,2]. The molecular origins of this disease are not fully understood, although they have been closely associated with dysregulation of the behaviour of a-synuclein [1, 3, 4], a disordered protein whose function appears to involve the regulation of synaptic vesicle trafficking [5][6][7][8]. This gap in our knowledge of the disease aetiology is no doubt partly responsible for the lack of disease modifying therapies for PD [9][10][11][12].The pathological hallmark of PD is the presence of Lewy bodies within dopaminergic neurons in the brains of affected patients [13]. Although misfolded a-synuclein is a major constituent of Lewy bodies [14,15], ultrastructural and proteome studies have indicated that these aberrant deposits contain a plethora of cellular components, including lipid membranes and organelle fragments [16][17][18][19][20]. These findings have led to the suggestion that the processes associated with the formation of Lewy bodies could be major drivers of neurotoxicity in PD [16,18,21].Since it has also been shown that a-synuclein can aggregate into ordered amyloid fibrils in vitro [22][23][24][25][26] and in vivo [27][28][29][30][31][32], we asked how such a-synuclein aggregation can be reconciled with the formation of highly complex and heterogeneous assemblies such as Lewy bodies. We hypothesised that a-synuclein may be capable of irreversibly capturing cellular components through liquid-liquid phase separation, as this mechanism has been shown to drive the self-assembly of various disease-associated proteins on-pathway to the formation of solid aggregates [33][34][35][36]. Under healthy conditions, the condensation of proteins into a dense liquid droplet state through liquid-liquid phase separation is normally reversible, and exploited in a variety of ways to carry out cellular functions, including RNA metabolism, ribosome biogenesis, DNA damage response and signal transduction [33,34,37]. Upon dysregulation, however, liquid droplets can mature into gel-like deposits, which irreversibly sequester important cellular components and lead to pathological processes [38...

The extent of protein hydration dictates the preference for heterogeneous or homogeneous nucleation generating either parallel or antiparallel β-sheet α-synuclein aggregates

“…With the aim of elucidating if homogeneous primary nucleation and thus the formation of antiparallel -sheet aggregates can be triggered inside αS droplets generated by LLPS, we have reproduced the conditions of reported in vitro αS droplet formation 68 and observed aggregation inside the droplets very rapidly (within 2h of incubation, not shown) ( Fig. 8B), with essentially all the protein being aggregated in less than 20 h (Fig.…”

“…Indeed, recent experimental evidences suggest a role of these protein droplets on the in vivo aggregation of these amyloidogenic proteins and the induction of pathology 66 . Very recently, it has been reported that αS droplet formation by LLPS precedes its aggregation in cellular 67 and animal models 68 and the liquid-to-solid transition of αS droplets has been recapitulated in vitro.…”

Widespread occurrence of the droplet state of proteins in the human proteome

“…We applied the FuzDrop method to predict the droplet-promoting propensities of two proteins reported to undergo liquid-liquid phase separation, TDP-43 (37) and a-synuclein (38,39). Our results indicate that the low-complexity (LC) region of TDP-43 (residues 262-414) mediates spontaneous phase separation.…”

Multiplicity of α-Synuclein Aggregated Species and Their Possible Roles in Disease

“…One of the cellular microenvironments with a particularly low content of water (by definition) is the interior of protein-rich droplets, generated by liquid-liquid phase separation (LLPS). These phase separated protein droplets likely play a role in the in vivo aggregation of a number of amyloidogenic proteins such as tau [104,105], TDP-43 [106,107] and αS [108,109]. Although the mechanism of the liquid-to-solid transition of these protein droplets is unclear, our recent data on the amyloid aggregation inside αS droplets generated in vitro suggest that the process is triggered by homogeneous primary nucleation resulting in the formation of amyloid aggregates with a preference for an intermolecular antiparallel β-sheet structure [92].…”

“…Water activity and, therefore, the protein hydration state have been recently demonstrated to be a key determinant not only for triggering αS self-assembly (maintaining αS monomeric and preventing it from misfolding and self-assembly under highly hydration conditions), but also for dictating the preference for the type of primary nucleation (heterogeneous vs homogeneous) and the type of structural amyloid polymorph generated (parallel vs antiparallel β-sheet structure). Conditions of very poor water activity such as those encountered inside protein-rich droplets, generated by LLPS, have been reported to be particularly efficient in triggering αS amyloid aggregation both in vitro and in vivo [105,108,109].…”