α-Synuclein aggregation nucleates through liquid–liquid phase separation

Ray, Soumik; Singh, Nitu; Kumar, Rakesh; Patel, Komal; Pandey, Satyaprakash; Datta, Debalina; Mahato, Jaladhar; Panigrahi, Ranajit; Navalkar, Ambuja; Mehra, Surabhi; Gadhe, Laxmikant; Chatterjee, Debdeep; Sawner, Ajay Singh; Maiti, Sukumar; Bhatia, Sandhya; Gerez, Juan; Chowdhury, Arindam; Kumar, Ashutosh; Padinhateeri, Ranjith; Riek, Roland; Krishnamoorthy, G.; Maji, Somnath

doi:10.1038/s41557-020-0465-9

Cited by 598 publications

(924 citation statements)

References 127 publications

(144 reference statements)

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“…This phenomenon has been observed for FUS and TDP-43 in ALS [38,39] and more recently also for tau in Alzheimer's disease [40][41][42]. Our results show that a-synuclein can undergo a similar process by forming a dense liquid droplet state, which matures into a gel-like state rich in amyloid structure, consistently with very recent results [43].…”

supporting

confidence: 91%

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

Hardenberg

Sinnige

Casford

et al. 2020

Preprint

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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...

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supporting

confidence: 91%

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

Hardenberg

Sinnige

Casford

et al. 2020

Preprint

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“…Additionally, the study of the τ and correlation time of the pathogenic alpha-synuclein mutants, A30P and A53T, showed that they had distinct types of binding sites, which was in concordance with the difference in fibril morphology [ 120 ]. Additionally, the anisotropy decay profiles of fluorescein labelled-alpha synuclein have recently been used to study its liquid–liquid phase separation into droplets, an event that occurs before protein fibrillation [ 121 ].…”

Section: Fluorescence Spectroscopy Is a Versatile Tool For Evaluatmentioning

confidence: 99%

Evaluation of Peptide/Protein Self-Assembly and Aggregation by Spectroscopic Methods

2020

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The self-assembly of proteins is an essential process for a variety of cellular functions including cell respiration, mobility and division. On the other hand, protein or peptide misfolding and aggregation is related to the development of Parkinson’s disease and Alzheimer’s disease, among other aggregopathies. As a consequence, significant research efforts are directed towards the understanding of this process. In this review, we are focused on the use of UV-Visible Absorption Spectroscopy, Fluorescence Spectroscopy and Circular Dichroism to evaluate the self-organization of proteins and peptides in solution. These spectroscopic techniques are commonly available in most chemistry and biochemistry research laboratories, and together they are a powerful approach for initial as well as routine evaluation of protein and peptide self-assembly and aggregation under different environmental stimulus. Furthermore, these spectroscopic techniques are even suitable for studying complex systems like those in the food industry or pharmaceutical formulations, providing an overall idea of the folding, self-assembly, and aggregation processes, which is challenging to obtain with high-resolution methods. Here, we compiled and discussed selected examples, together with our results and those that helped us better to understand the process of protein and peptide aggregation. We put particular emphasis on the basic description of the methods as well as on the experimental considerations needed to obtain meaningful information, to help those who are just getting into this exciting area of research. Moreover, this review is particularly useful to those out of the field who would like to improve reproducibility in their cellular and biomedical experiments, especially while working with peptide and protein systems as an external stimulus. Our final aim is to show the power of these low-resolution techniques to improve our understanding of the self-assembly of peptides and proteins and translate this fundamental knowledge in biomedical research or food applications.

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“…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.…”

Section: Antiparallel -Sheet Aggregates Represent the Most Stable Stmentioning

confidence: 99%

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

Camino

Gracia

Chen

et al. 2020

Preprint

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α-Synuclein amyloid self-assembly is the hallmark of a number of neurodegenerative disorders, including Parkinson’s disease, although there is still very limited understanding about the factors and mechanisms that trigger this process. Primary nucleation has been observed to be initiated in vitro at hydrophobic/hydrophilic interfaces by heterogeneous nucleation generating parallel β-sheet aggregates, although no such interfaces have yet been identified in vivo. In this work, we have discovered that α-synuclein can self-assemble into amyloid aggregates by homogeneous nucleation, without the need of an active surface, and with a preference for an antiparallel β-sheet arrangement. This particular structure has been previously proposed to be distinctive of stable toxic oligomers and we here demonstrate that it indeed represents the most stable structure of the preferred amyloid pathway triggered by homogeneous nucleation under limited hydration conditions, including those encountered inside α-synuclein droplets generated by liquid-liquid phase separation. In addition, our results highlight the key role that water plays not only in modulating the transition free energy of amyloid nucleation, and thus governing the initiation of the process, but also in dictating the type of preferred primary nucleation and the type of amyloid polymorph generated depending on the extent of protein hydration. These findings are particularly relevant in the context of in vivo α-synuclein aggregation where the protein can encounter a variety of hydration conditions in different cellular microenvironments, including the vicinity of lipid membranes or the interior of membraneless compartments, which could lead to the formation of remarkably different amyloid polymorphs by either heterogeneous or homogeneous nucleation.

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α-Synuclein aggregation nucleates through liquid–liquid phase separation

Cited by 598 publications

References 127 publications

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

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

Evaluation of Peptide/Protein Self-Assembly and Aggregation by Spectroscopic Methods

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

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