Cytotoxic Oligomers and Fibrils Trapped in a Gel‐like State of α‐Synuclein Assemblies

Kumar, Rakesh; Das, Subhadeep; Mohite, Ganesh M.; Rout, Saroj Kumar; Halder, Saayak; Jha, Narendra Nath; Ray, Soumik; Mehra, Surabhi; Agarwal, Vipin; Maji, Samir K.

doi:10.1002/ange.201711854

Cited by 10 publications

(8 citation statements)

References 21 publications

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“…We have also shown that the droplet state can undergo a maturation process into a gel-like state rich in amyloid structure, which is reminiscent of the pathological state seen in Lewy body pathologies [14,15,60]. This phenomenon is expected on the grounds that the transition from the droplet state to the amyloid state takes place through a maturation process, known as Ostwald ripening [61,62], and it is consistent with the recent report that a-synuclein can form hydrogels [63,64]. From a thermodynamic point of view, it is important to note that the gellike assembly is not a stable state in addition to the native, the droplet and the amyloid states, but a slowly evolving conformation on pathway to the amyloid state ( Figure 6A).…”

Section: Discussionsupporting

confidence: 89%

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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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Section: Discussionsupporting

confidence: 89%

“…We can thus suggest that the effects of such membranous structures on the ageing process of the droplets involve a slowing down of the amyloid conversion of the gel-like a-synuclein assemblies. This process could create a metastable toxic state, as the gel-like assemblies can be a reservoir for cytotoxic a-synuclein oligomers [64].…”

Section: Discussionmentioning

confidence: 99%

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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“…For HMF and Syn fibrils, the first 80 and 72 residues out of the starting 98 residues were assigned, respectively ( Figure S4). Similar to previous reports 34 , we observed that for these two fibril forms, the region spanning residues 98-140 was flexible and could not be observed in the crosspolarization (CP) based experiments ( Figure 3, S4 and S5). For HMF, the flexibility of these residues was independently confirmed by INEPT experiments 35 .…”

Section: Structural Differences Between α-Syn Fibril Polymorphs Reveasupporting

confidence: 89%

α-Synuclein aggregation intermediates form fibril polymorphs with distinct prion-like properties

Mehra

Ahlawat

Kumar

et al. 2020

Preprint

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α-Synuclein (α-Syn) amyloid fibrils in synucleinopathies (such as Parkinson's disease (PD), multiple system atrophy (MSA)) are structurally and functionally different, reminiscent of prion-like strains. However, how a single protein can form different fibril polymorphs in various synucleinopathies is not known. Here, we demonstrate the structure-function relationship of two distinct α-Syn fibril polymorphs, the pre-matured fibrils (PMF) and helixmatured fibrils (HMF) based on α-Syn aggregation intermediates. These polymorphs not only display the structural differences, including their fibril core structure as demonstrated by solidstate nuclear magnetic resonance (NMR) spectroscopy and H/D-exchange coupled with mass spectrometry but also possess different cellular activities such as seeding, cellular internalization, and cell-to-cell transmission. The HMF with a compact core structure exhibits low seeding potency in cells but readily internalizes and transmits from one cell to another.Whereas the less structured PMF lacks the cell-to-cell transmission ability but induces abundant α-Syn pathology and triggers the formation of aggresomes in cells. Overall, the study highlights how the conformational heterogeneity in the aggregation pathway may lead to fibril polymorphs with distinct prion-like behavior in PD. IntroductionSynucleinopathies are the group of neurological disorders, which are characterized by the presence of intracellular inclusion bodies composed of α-synuclein (α-Syn) amyloid fibrils 1 . Although α-Syn amyloids in neuronal inclusions termed as Lewy bodies (LBs) and Lewy neurites (LNs) are the characteristic feature of Parkinson's disease (PD), the α-Syn inclusions in oligodendrocytes (glial cell inclusions; GCIs) are predominant in multiple system atrophy (MSA). Previous studies have suggested that amyloid fibrils associated with various neurodegenerative disorders are infectious and exhibit 'prion-like' behavior 2,3 . For example, exogenously added α-Syn fibrils readily internalize in cells and induce the aggregation of endogenous soluble α-Syn into pathogenic insoluble LB-like inclusions 4,5 . Moreover, the LB/LN-like inclusions are also observed in animals upon receiving intracerebral injections of synthetic α-Syn fibrils or brain homogenates from old transgenic (Tg) mice with α-Syn pathology 6,7 . This suggests prion-like cellular transmission and propagation of α-Syn amyloids in PD and related disorders 8 . In this context, it has been hypothesized that α-Syn assembles into polymorphs, which may account for distinct disease phenotypes observed amongst the synucleinopathies. α-Syn fibrils from GCIs in oligodendrocytes and LBs in neurons of diseased brain samples differ significantly in their structure and exhibit distinct seeding propensity 9 .Although α-Syn fibril polymorphs mostly differ in their fibril diameter, presence of twists, and the number of protofilaments 10,11 , they share a common cross-β-sheet structure with different packing and inter-protofilament interface 12,13 as shown by cryo-...

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“…Towards achieving these goals, we present here a straightforward protocol based on centrifugation and filtration procedures, which allows rapid quantitative and qualitative assessment of the different types of α-syn species such as monomers, oligomers and fibrils in samples of different complexities. This protocol is based on years of experience in our group (Fauvet, Mbefo, & Fares, 2012;Khalaf, Fauvet, & Oueslati, 2014) and the evidence that it has been successfully applied in α-syn research by other groups (Ghosh et al, 2015;Kumar, Das, & Mohite, 2018;Zhang, Griggs, Rochet, & Stanciu, 2013).…”

Section: Introductionmentioning

confidence: 99%

A simple, versatile and robust centrifugation‐based filtration protocol for the isolation and quantification of α‐synuclein monomers, oligomers and fibrils: Towards improving experimental reproducibility in α‐synuclein research

Kumar

Donzelli

Chiki

et al. 2020

Journal of Neurochemistry

102

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Increasing evidence suggests that the process of alpha‐synuclein (α‐syn) aggregation from monomers into amyloid fibrils and Lewy bodies, via oligomeric intermediates plays an essential role in the pathogenesis of different synucleinopathies, including Parkinson's disease (PD), multiple system atrophy and dementia with Lewy bodies (DLB). However, the nature of the toxic species and the mechanisms by which they contribute to neurotoxicity and disease progression remain elusive. Over the past two decades, significant efforts and resources have been invested in studies aimed at identifying and targeting toxic species along the pathway of α‐syn fibrillization. Although this approach has helped to advance the field and provide insights into the biological properties and toxicity of different α‐syn species, many of the fundamental questions regarding the role of α‐syn aggregation in PD remain unanswered, and no therapeutic compounds targeting α‐syn aggregates have passed clinical trials. Several factors have contributed to this slow progress, including the complexity of the aggregation pathways and the heterogeneity and dynamic nature of α‐syn aggregates. In the majority of experiment, the α‐syn samples used contain mixtures of α‐syn species that exist in equilibrium and their ratio changes upon modifying experimental conditions. The failure to quantitatively account for the distribution of different α‐syn species in different studies has contributed not only to experimental irreproducibility but also to misinterpretation of results and misdirection of valuable resources. Towards addressing these challenges and improving experimental reproducibility in Parkinson's research, we describe here a simple centrifugation‐based filtration protocol for the isolation, quantification and assessment of the distribution of α‐syn monomers, oligomers and fibrils, in heterogeneous α‐syn samples of increasing complexity. The protocol is simple, does not require any special instrumentation and can be performed rapidly on multiple samples using small volumes. Here, we present and discuss several examples that illustrate the applications of this protocol and how it could contribute to improving the reproducibility of experiments aimed at elucidating the structural basis of α‐syn aggregation, seeding activity, toxicity and pathology spreading. This protocol is applicable, with slight modifications, to other amyloid‐forming proteins.

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Cytotoxic Oligomers and Fibrils Trapped in a Gel‐like State of α‐Synuclein Assemblies

Cited by 10 publications

References 21 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

α-Synuclein aggregation intermediates form fibril polymorphs with distinct prion-like properties

A simple, versatile and robust centrifugation‐based filtration protocol for the isolation and quantification of α‐synuclein monomers, oligomers and fibrils: Towards improving experimental reproducibility in α‐synuclein research

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