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Liquid-liquid phase separation (LLPS) has emerged as an important phenomenon associated with formation of membraneless organelles. Recently, LLPS has been shown to act as nucleation centers for disease-associated protein aggregation and amyloid fibril formation. Phase-separated α-synuclein droplets gradually rigidify during the course of protein aggregation, and it is very challenging to understand the biomolecular interactions that lead to liquid-like to solid-like transition using conventional ensemble measurements. Here, we describe a spectrally-resolved fluorescence microscopy based Förster resonance energy transfer (FRET) imaging to probe interactions of α-synuclein in individual droplets during LLPS-mediated aggregation. By acquiring entire emission spectral profiles of individual droplets upon sequential excitation of acceptors and donors therein, this technique allows for the quantification of sensitized emission proportional to the extent of FRET, which enables interrogation of the evolution of local interactions of donor-/acceptor-labeled α-synuclein molecules within each droplet. The present study on single droplets is not only an important development for studying LLPS but can also be used to investigate self-assembly or aggregation in biomolecular systems and soft materials.

α-Synuclein (α-Syn) is a natively unstructured protein, which self-assembles into higher-order aggregates possessing serious pathophysiological implications. α-Syn aberrantly self-assembles into protein aggregates, which have been widely implicated in Parkinson’s disease (PD) pathogenesis and other synucleinopathies. The self-assembly of α-Syn involves the structural conversion of soluble monomeric protein into oligomeric intermediates and eventually fibrillar aggregates of amyloids with cross-β-sheet rich conformation. These aggregated α-Syn species majorly constitute the intraneuronal inclusions, which is a hallmark of PD neuropathology. Self-assembly/aggregation of α-Syn is not a single-state conversion process as unfolded protein can access multiple conformational states through the formation of metastable, transient pre-fibrillar intermediate species. Recent studies have indicated that soluble oligomers are the potential neurotoxic species responsible for cell death in PD pathogenesis. The heterogeneous and transient nature of oligomers formed during the early stage of aggregation pathway limit their detailed study in understanding the structure–toxicity relationship. Moreover, the precise molecular events occurring in the early stage of α-Syn aggregation process majorly remain unsolved. Recently, liquid–liquid phase separation (LLPS) of α-Syn has been designated as an alternate nucleation mechanism, which occurs in the early lag phase of the aggregation pathway leading to the formation of dynamic supramolecular assemblies. The stronger self-association among the protein molecules triggers the irreversible liquid-to-solid transition of these supramolecular assemblies into the amyloid-like hydrogel, which may serve as a reservoir entrapping toxic oligomeric intermediates and fibrils. This review strives to provide insights into different modes of α-Syn self-assemblies including LLPS-mediated self-assembly and its recent advancements.

The size of amyloid seeds is known to modulate their autocatalytic amplification and cellular toxicity. However, the seed size-dependent secondary nucleation mechanism, toxicity, and disease-associated biological processes mediated by α-synuclein (α-Syn) fibrils are largely unknown. Using the cellular model and

Protein aggregation into amyloid fibrils is a key feature of a multitude of neurodegenerative diseases such as Alzheimer's, Parkinson's, and Prion disease. To detect amyloid fibrils, fluorophores with high sensitivity and better efficiency coupled with the low toxicity are in high demand even to date. In this pursuit, we have unveiled two benzimidazole-based fluorescence sensors ([C15 H15 N3 ] (C1) and [C16 H16 N3 O2 ] (C2), which possess exceptional affinity toward different amyloid fibrils in its submicromolar concentration (8 × 10-9 M), whereas under a similar concentration, the gold standard Thioflavin-T (ThT) fails to bind with amyloid fibrils. These fluorescent markers bind to α-Syn amyloid fibrils as well as amyloid fibrils forming other proteins/peptides including Aβ42 amyloid fibrils. The 1 H-15 N heteronuclear quantum correlation spectroscopy nuclear magnetic resonance data collected on wild-type α-Syn monomer with and without the fluorophores (C1 and C2) reveal that there is weak or no interactions between C1 or C2 with residues in α-Syn monomer, which indirectly reflects the specific binding ability of C1 and C2 to the α-Syn amyloid fibrils. Detailed studies further suggest that C1 and C2 can detect/bind with the α-Syn amyloid fibril as low as 100 × 10-9 M. Extremely low or no cytotoxicity is observed for C1 and C2 and they do not interfere with α-Syn fibrillation kinetics, unlike ThT. Both C1/C2 not only shows selective binding with amyloid fibrils forming various proteins/peptides but also displays excellent affinity and selectivity toward α-Syn amyloid aggregates in SH-SY5Y cells and Aβ42 amyloid plaques in animal brain tissues. Overall, our data show that the developed dyes could be used for the detection of amyloid fibrils including α-Syn and Aβ42 amyloids with higher sensitivity as compared to currently used ThT.

Synucleinopathies are a class of neurodegenerative diseases, including Parkinson's disease (PD), Dementia with Lewy bodies (DLB), and Multiple System Atrophy (MSA). The common pathological hallmark of synucleinopathies is the filamentous α-synuclein (α-Syn) aggregates along with membrane components in cytoplasmic inclusions in the brain. β-Synuclein (β-Syn), an isoform of α-Syn, inhibits α-Syn aggregation and prevents its neurotoxicity, suggesting the neuroprotective nature of β-Syn. However, this notion changed with the discovery of disease-associated β-Syn mutations, V70M and P123H, in patients with DLB. It is still unclear how these missense mutations alter the structural and amyloidogenic properties of β-Syn, leading to neurodegeneration. Here, we characterized the biophysical properties and investigated the effect of mutations on β-Syn fibrillation under different conditions. V70M and P123H show high membrane binding affinity compared to wild-type β-Syn, suggesting their potential role in membrane interactions. β-Syn and its mutants do not aggregate under normal physiological conditions; however, the proteins undergo self-polymerization in a slightly acidic microenvironment and/or in the presence of an inducer, forming long unbranched amyloid fibrils similar to α-Syn. Strikingly, V70M and P123H mutants exhibit accelerated fibrillation compared to native β-Syn under these conditions. NMR study further revealed that these point mutations induce local perturbations at the site of mutation in β-Syn. Overall, our data provide insight into the biophysical properties of disease-associated β-Syn mutations and demonstrate that these mutants make the native protein more susceptible to aggregation in an altered microenvironment.

α-Synuclein (α-Syn) amyloids in synucleinopathies are suggested to be structurally and functionally diverse, reminiscent of prion-like strains. The mechanism of how the aggregation of the same precursor protein results in the formation of fibril polymorphs remains elusive. Here, we demonstrate the structure-function relationship of two polymorphs, pre-matured fibrils (PMFs) and helix-matured fibrils (HMFs), based on α-Syn aggregation intermediates. These polymorphs display the structural differences as demonstrated by solid-state NMR and mass spectrometry studies and also possess different cellular activities such as seeding, internalization, and cell-to-cell transfer of aggregates. HMFs, with a compact core structure, exhibit low seeding potency but readily internalize and transfer from one cell to another. The less structured PMFs lack transcellular transfer ability but induce abundant α-Syn pathology and trigger the formation of aggresomes in cells. Overall, the study highlights that the conformational heterogeneity in the aggregation pathway may lead to fibril polymorphs with distinct prion-like behavior.

Aberrant aggregation of the misfolded presynaptic protein, α-Synuclein (α-Syn) into Lewy body (LB) and Lewy neuritis (LN) is a major pathological hallmark of Parkinson's disease (PD) and other synucleinopathies. Numerous studies have suggested that prefibrillar and fibrillar species of the misfolded α-Syn aggregates are responsible for cell death in PD pathogenesis. However, the precise molecular events during α-Syn aggregation, especially in the early stages, remain elusive. Emerging evidence has demonstrated that liquid-liquid phase separation (LLPS) of α-Syn occurs in the nucleation step of α-Syn aggregation, which offers an alternate non-canonical aggregation pathway in the crowded microenvironment. The liquid-like α-Syn droplets gradually undergo an irreversible liquid-to-solid phase transition into amyloid-like hydrogel entrapping oligomers and fibrils. This new mechanism of α-Syn LLPS and gel formation might represent the molecular basis of cellular toxicity associated with PD. This review aims to demonstrate the recent development of α-Syn LLPS, the underlying mechanism along with the microscopic events of aberrant phase transition. This review further discusses how several intrinsic and extrinsic factors regulate the thermodynamics and kinetics of α-Syn LLPS and co-LLPS with other proteins, which might explain the pathophysiology of α-Syn in various neurodegenerative diseases.

Cytoplasmic deposition of aberrantly misfolded α-synuclein (α-Syn) is a common feature of synucleinopathies, including Parkinson's disease (PD). However, the precise pathogenic mechanism of α-Syn in synucleinopathies remains elusive. Emerging evidence has suggested that α-Syn may contribute to PD pathogenesis in several ways; wherein the contribution of fibrillar species, for exerting toxicity and disease transmission, cannot be neglected. Further, the oligomeric species could be the most plausible neurotoxic species causing neuronal cell death. However, understanding the structural and molecular insights of these oligomers are very challenging due to the heterogeneity and transient nature of the species. In this review, we discuss the recent advancements in understanding the formation and role of α-Syn oligomers in PD pathogenesis. We also summarize the different types of α-Syn oligomeric species and potential mechanisms to exert neurotoxicity. Finally, we address the possible ways to target α-Syn as a promising approach against PD and the possible future directions.

Abnormal accumulation of aggregated α-synuclein (α-Syn) is seen in a variety of neurodegenerative diseases, including Parkinson’s disease (PD), multiple system atrophy (MSA), dementia with Lewy body (DLB), Parkinson’s disease dementia (PDD), and even subsets of Alzheimer’s disease (AD) showing Lewy-body-like pathology. These synucleinopathies exhibit differences in their clinical and pathological representations, reminiscent of prion disorders. Emerging evidence suggests that α-Syn self-assembles and polymerizes into conformationally diverse polymorphs in vitro and in vivo, similar to prions. These α-Syn polymorphs arising from the same precursor protein may exhibit strain-specific biochemical properties and the ability to induce distinct pathological phenotypes upon their inoculation in animal models. In this review, we discuss clinical and pathological variability in synucleinopathies and several aspects of α-Syn fibril polymorphism, including the existence of high-resolution molecular structures and brain-derived strains. The current review sheds light on the recent advances in delineating the structure–pathogenic relationship of α-Syn and how diverse α-Syn molecular polymorphs contribute to the existing clinical heterogeneity in synucleinopathies.

Liquid-liquid phase separation (LLPS) is a crucial phenomenon for the formation of functional membraneless organelles. However, LLPS is also responsible for protein aggregation in various neurodegenerative diseases such as amyotrophic lateral sclerosis, Alzheimer's disease, and Parkinson's disease (PD). Recently, several reports, including ours, have shown that α-synuclein (α-Syn) undergoes LLPS and a subsequent liquid-to-solid phase transition, which leads to amyloid fibril formation. However, how the environmental (and experimental) parameters modulate the α-Syn LLPS remains elusive. Here, we show that in vitro α-Syn LLPS is strongly dependent on the presence of salts, which allows charge neutralization at both terminal segments of protein and therefore promotes hydrophobic interactions supportive for LLPS. Using various purification methods and experimental conditions, we showed, depending upon conditions, α-Syn undergoes either spontaneous (instantaneous) or delayed LLPS. Furthermore, we delineate that the kinetics of liquid droplet formation (i.e., the critical concentration and critical time) is relative and can be modulated by the salt/counterion concentration, pH, presence of surface, PD-associated multivalent cations, and N-terminal acetylation, which are all known to regulate α-Syn aggregation in vitro. Together, our observations suggest that α-Syn LLPS and subsequent liquid-to-solid phase transition could be pathological, which can be triggered only under disease-associated conditions (high critical concentration and/or conditions promoting α-Syn self-assembly). This study will significantly improve our understanding of the molecular mechanisms of α-Syn LLPS and the liquid-to-solid transition.

Amyloid formation of α-synuclein (α-Syn) and its familial mutations are directly linked with Parkinson's disease (PD) pathogenesis. Recently, a new familial α-Syn mutation (A53E) was discovered, associated with an early onset aggressive form of PD, which delays α-Syn aggregation. When we overexpressed wild-type (WT) and A53E proteins in cells, showed neither toxicity nor aggregate formation, suggesting merely overexpression may not recapitulate the PD phenotype in cell models. We hypothesized that cells expressing the A53E mutant might possess enhanced susceptibility to PD-associated toxicants compared to that of the WT. When cells were treated with PD toxicants (dopamine and rotenone), cells expressing A53E showed more susceptibility to cell death along with compromised mitochondrial potential and an increased production of reactive oxygen species. The higher toxicity of A53E could be due to more oligomers being formed in cells as confirmed by a dot blot assay using amyloid specific OC and A11 antibody and using an in vitro aggregation study. The cellular model presented here suggests that along with familial mutation, environmental and other cellular factors might play a crucial role in dictating PD pathogenesis.

α-Synuclein (α-Syn) aggregation is directly associated with Parkinson's disease (PD) pathogenesis. In vitro aggregation and in vivo animal model studies of α-Syn recapitulate many features of the disease pathogenesis. Six familial PD associated mutations of α-Syn have been discovered; many of which are associated with early onset PD. Three of PD associated mutations have been shown to accelerate the α-Syn aggregation, whereas other three are shown to delay the aggregation kinetics. The membrane binding studies also suggest that few of these PD mutants strongly bind to synthetic membrane vesicles, while others are shown to have attenuated membrane binding ability. Furthermore, the PD mutations do not drastically alter the toxicity of α-Syn oligomers/fibrils. Although according to recent suggestions that early formed oligomers are the most potent toxic species responsible for PD, only p.A30P mutant is shown to form faster oligomers and delayed conversion from oligomers to fibrils. Therefore, it is difficult to establish a unifying mechanism of how familial PD associated mutations affect the α-Syn structure, aggregation and function for their disease association. It is possible that each PD associated mutation alters α-Syn biology in a unique way, which might be responsible for disease pathogenesis. In this review, we discuss the structure function of α- Syn and how these are altered due to the PD associated mutations and their relationship to disease pathogenesis.

α-Synuclein (α-Syn) aggregation and amyloid formation is directly linked with Parkinson's disease pathogenesis. However, the early events involved in this process remain unclear. Here, using the in vitro reconstitution and cellular model, we show that liquid-liquid phase separation of α-Syn precedes its aggregation. In particular, in vitro generated α-Syn liquid-like droplets eventually undergo a liquid-to-solid transition and form an amyloid hydrogel that contains oligomers and fibrillar species. Factors known to aggravate α-Syn aggregation, such as low pH, phosphomimetic substitution and familial Parkinson's disease mutations, also promote α-Syn liquid-liquid phase separation and its subsequent maturation. We further demonstrate α-Syn liquid-droplet formation in cells. These cellular α-Syn droplets eventually transform into perinuclear aggresomes, the process regulated by microtubules. This work provides detailed insights into the phase-separation behaviour of natively unstructured α-Syn and its conversion to a disease-associated aggregated state, which is highly relevant in Parkinson's disease pathogenesis.

Altered intestinal permeability has been correlated with Parkinson's pathophysiology in the enteric nervous system, before manifestations in the central nervous system (CNS). The inflammatory endotoxin or lipopolysaccharide (LPS) released by gut bacteria is known to modulate α-synuclein amyloidogenesis through the formation of intermediate nucleating species. Here, biophysical techniques in conjunction with microscopic images revealed the molecular interaction between lipopolysaccharide and α-synuclein that induce rapid nucleation events. This heteromolecular interaction stabilizes the α-helical intermediates in the α-synuclein aggregation pathway. Multitude NMR studies probed the residues involved in the LPS-binding structural motif that modulates the nucleating forms, affecting the cellular internalization and associated cytotoxicity. Collectively, our data characterizes this heteromolecular interaction associated with an alternative pathway in Parkinson's disease progression.

α-Synuclein (α-Syn) has been extensively studied for its structural and biophysical properties owing to its pathophysiological role in Parkinson's disease (PD). Lewy bodies and Lewy neurites are the pathological hallmarks of PD and contain α-Syn aggregates as their major component. It was therefore hypothesized that α-Syn aggregation is actively associated with PD pathogenesis. The central role of α-Syn aggregation in PD is further supported by the identification of point mutations in α-Syn protein associated with rare familial forms of PD. However, the correlation between aggregation propensities of α-Syn mutants and their association with PD phenotype is not straightforward. Recent evidence suggested that oligomers, formed during the initial stages of aggregation, are the potent neurotoxic species causing cell death in PD. However, the heterogeneous and unstable nature of these oligomers limit their detailed characterization. α-Syn fibrils, on the contrary, are shown to be the infectious agents and propagate in a prion-like manner. Although α-Syn is an intrinsically disordered protein, it exhibits remarkable conformational plasticity by adopting a range of structural conformations under different environmental conditions. In this review, we focus on the structural and functional aspects of α-Syn and role of potential factors that may contribute to the underlying mechanism of synucleinopathies. This information will help to identify novel targets and develop specific therapeutic strategies to combat Parkinson's and other protein aggregation related neurodegenerative diseases.

Parkinson's disease is mainly a sporadic disorder in which both environmental and cellular factors play a major role in the initiation of this disease. Glycosaminoglycans (GAG) are integral components of the extracellular matrix and are known to influence amyloid aggregation of several proteins, including α-synuclein (α-Syn). However, the mechanism by which different GAGs and related biological polymers influence protein aggregation and the structure and intercellular spread of these aggregates remains elusive. In this study, we used three different GAGs and related charged polymers to establish their role in α-Syn aggregation and associated biological activities of these aggregates. Heparin, a representative GAG, affected α-Syn aggregation in a concentration-dependent manner, whereas biphasic α-Syn aggregation kinetics was observed in the presence of chondroitin sulfate B. Of note, as indicated by 2D NMR analysis, different GAGs uniquely modulated α-Syn aggregation because of the diversity of their interactions with soluble α-Syn. Moreover, subtle differences in the GAG backbone structure and charge density significantly altered the properties of the resulting amyloid fibrils. Each GAG/polymer facilitated the formation of morphologically and structurally distinct α-Syn amyloids, which not only displayed variable levels of cytotoxicity but also exhibited an altered ability to internalize into cells. Our study supports the role of GAGs as key modulators in α-Syn amyloid formation, and their distinct activities may regulate amyloidogenesis depending on the type of GAG being up- or down-regulated in vivo.

α-Synuclein (α-Syn) aggregation and amyloid formation are associated with loss of dopaminergic neurons in Parkinson's disease (PD). In addition, familial mutations in α-Syn are shown to be one of the definite causes of PD. Here we have extensively studied familial PD associated α-Syn G51D, H50Q, and E46K mutations using Drosophila model system. Our data showed that flies expressing α-Syn familial mutants have a shorter lifespan and exhibit more climbing defects compared to wild-type (WT) flies in an age-dependent manner. The immunofluorescence studies of the brain from the old flies showed more dopaminergic neuronal cell death in all mutants compared to WT. This adverse effect of α-Syn familial mutations is highly correlated with the sustained population of oligomer production and retention in mutant flies. Furthermore, this was supported by our in vitro studies, where significantly higher amount of oligomer was observed in mutants compared to WT. The data suggest that the sustained population of oligomer formation and retention could be a major cause of cell death in α-Syn familial mutants.

The involvement of α-synuclein (α-Syn) amyloid formation in Parkinson's disease (PD) pathogenesis is supported by the discovery of α-Syn gene (SNCA) mutations linked with familial PD, which are known to modulate the oligomerization and aggregation of α-Syn. Recently, the A53V mutation has been discovered, which leads to late-onset PD. In this study, we characterized for the first time the biophysical properties of A53V, including the aggregation propensities, toxicity of aggregated species, and membrane binding capability, along with those of all familial mutations at the A53 position. Our data suggest that the A53V mutation accelerates fibrillation of α-Syn without affecting the overall morphology or cytotoxicity of fibrils compared to those of the wild-type (WT) protein. The aggregation propensity for A53 mutants is found to decrease in the following order: A53TA53VWTA53E. In addition, a time course aggregation study reveals that the A53V mutant promotes early oligomerization similar to the case for the A53T mutation. It promotes the largest amount of oligomer formation immediately after dissolution, which is cytotoxic. Although in the presence of membrane-mimicking environments, the A53V mutation showed an extent of helix induction capacity similar to that of the WT protein, it exhibited less binding to lipid vesicles. The nuclear magnetic resonance study revealed unique chemical shift perturbations caused by the A53V mutation compared to those caused by other mutations at the A53 site. This study might help to establish the disease-causing mechanism of A53V in PD pathology.

Aggregation of α-synuclein (α-Syn) into neurotoxic oligomers and amyloid fibrils is suggested to be the pathogenic mechanism for Parkinson's disease (PD). Recent studies have indicated that oligomeric species of α-Syn are more cytotoxic than their mature fibrillar counterparts, which are responsible for dopaminergic neuronal cell death in PD. Therefore, the effective therapeutic strategies for tackling aggregation-associated diseases would be either to prevent aggregation or to modulate the aggregation process to minimize the formation of toxic oligomers during aggregation. In this work, we showed that arginine-substituted α-Syn ligands, based on the most aggregation-prone sequence of α-Syn, accelerate the protein aggregation in a concentration-dependent manner. To elucidate the mechanism by which Arg-substituted peptides could modulate α-Syn aggregation kinetics, we performed surface plasmon resonance (SPR) spectroscopy, nuclear magnetic resonance (NMR) studies, and all-atom molecular dynamics (MD) simulation. The SPR analysis showed a high binding potency of these peptides with α-Syn but one that was nonspecific in nature. The two-dimensional NMR studies suggest that a large stretch within the C-terminus of α-Syn displays a chemical shift perturbation upon interacting with Arg-substituted peptides, indicating C-terminal residues of α-Syn might be responsible for this class of peptide binding. This is further supported by MD simulation studies in which the Arg-substituted peptide showed the strongest interaction with the C-terminus of α-Syn. Overall, our results suggest that the binding of Arg-substituted ligands to the highly acidic C-terminus of α-Syn leads to reduced charge density and flexibility, resulting in accelerated aggregation kinetics. This may be a potentially useful strategy while designing peptides, which act as α-Syn aggregation modulators.

α-Synuclein (α-Syn) aggregation is associated with Parkinson's disease (PD) pathogenesis. In PD, the role of oligomers versus fibrils in neuronal cell death is debatable, but recent studies suggest oligomers are a proximate neurotoxin. Herein, we show that soluble α-Syn monomers undergo a transformation from a solution to a gel state on incubation at high concentration. Detailed characterization of the gel showed the coexistence of monomers, oligomers, and short fibrils. In vitro, the gel was highly cytotoxic to human neuroblastoma cells. The individual constituents of the gel are short-lived species but toxic to the cells. They comprise a structurally heterogeneous population of α-helical and β-sheet-rich oligomers and short fibrils with the cross-β motif. Given the recent evidence of the gel-like state of the protein associated with neurodegenerative diseases, the gel state of α-Syn in this study represents a mechanistic and structural model for the in vivo toxicity of α-Syn in PD.

Aggregation of α-synuclein (α-Syn) into toxic oligomers and fibrils leads to Parkinson's disease (PD) pathogenesis. Molecules that can inhibit the fibrillization and oligomerization of α-Syn have potential therapeutic value. Here, we studied four selective amyloid inhibitors: dopamine (Dopa), amphotericin-B (Amph), epigallocatechingallate (EGCG), and quinacrinedihydrochloride (Quin) for their effect on oligomerization, fibrillization, and preformed fibrils of α-Syn. The aggregation kinetics of α-Syn using ThT fluorescence and conformational transition by circular dichroism (CD) in the presence and absence of these four compounds suggest that, except Quin, the remaining three molecules inhibit α-Syn aggregation in a concentration dependent manner. Consistent with the aggregation kinetics data, the morphological study of aggregates formed in the presence of these compounds showed corresponding decrease in fibrillar size. The analysis of cell viability using MTT assay showed reduction in toxicity of α-Syn aggregates formed in the presence of these compounds, which also correlates with reduction of exposed hydrophobic surface as studied by ANS binding. Additionally, these inhibitors, except Quin, demonstrated reduction in size as well as the toxicity of oligomeric/fibrillar aggregates of α-Syn. The residue specific interaction to low molecular weight (LMW) species of α-Syn by 2D NMR study revealed that, the region and extent of binding are different for all these molecules. Furthermore, fibril-binding data using SPR suggested that there is no direct relationship between the binding affinity and fibril inhibition by these compounds. The present study suggests that sequence based interaction of small molecules with soluble α-Syn might dictate their inhibition or modulation capacity, which might be helpful in designing modulators of α-Syn aggregation.

Parkinson's disease (PD) is a neurological disorder marked by the presence of cytoplasmic inclusions, Lewy bodies (LBs) and Lewy neurites (LNs) as well as the degeneration of dopamine producing neurons in the substantia nigra region of the brain. The LBs and LNs in PD are mainly composed of aggregated form of a presynaptic protein, alpha-synuclein (alpha-Syn). However, the mechanisms of alpha-Syn aggregation and actual aggregated species responsible for the degeneration of dopaminergic neurons have not yet been resolved. Despite the fact that alpha-Syn aggregation in LBs and LNs is crucial and mutations of alpha-Syn are associated with early onset PD, it is really a challenging task to establish a correlation between alpha-Syn aggregation rate and PD pathogenesis. Regardless of strong genetic contribution, PD is mostly sporadic and familial forms of the disease represent only a minor part (

It has been suggested that conjugated charged polymers are amyloid imaging agents and promising therapeutic candidates for neurological disorders. However, very less is known about their efficacy in modulating the amyloid aggregation pathway. Here, we studied the modulation of Parkinson's disease associated α-synuclein (AS) amyloid assembly kinetics using conjugated polyfluorene polymers (PF, cationic; PFS, anionic). We also explored the complexation of these charged polymers with the various AS aggregated species including amyloid fibrils and oligomers using multidisciplinary biophysical techniques. Our data suggests that both polymers irrespective of their different charges in the side chains increase the fibrilization kinetics of AS and also remarkably change the morphology of the resultant amyloid fibrils. Both polymers were incorporated/aligned onto the AS amyloid fibrils as evident from electron microscopy (EM) and atomic force microscopy (AFM), and the resultant complexes were structurally distinct from their pristine form of both polymers and AS supported by FTIR study. Additionally, we observed that the mechanism of interactions between the polymers with different species of AS aggregates were markedly different.

Background: Copper is an essential trace element required for the proper functioning of various enzymes present in the central nervous system. An imbalance in the copper homeostasis results in the pathology of various neurodegenerative disorders including Parkinson's Disease. Hence, residue specific interaction of Cu2+ to alpha-Syn along with the familial mutants H50Q and G51D needs to be studied in detail. Methods: We investigated the residue specific mapping of Cu2+ binding sites and binding strength using solution-state NMR and ITC respectively. The aggregation kinetics, secondary structural changes, and morphology of the formed fibrils in the presence and absence of Cu2+ were studied using fluorescence, CD, and AFM respectively. Results: Copper binding to alpha-Syn takes place at three different sites with a higher affinity for the region 48-53. While one of the sites got abolished in the case of H50Q the mutant G51D showed a binding pattern similar to WT. The aggregation kinetics of these proteins in the presence of Cu2+ showed an enhanced rate of fibril formation with a pronounced effect for G51D. Conclusion: Cu2+ binding results in the destabilization of long-range tertiary interactions in alpha-Syn leading to the exposure of highly amyloidogenic NAC region which results in the increased rate of fibril formation. Although the residues 48-53 have a stronger affinity for Cu2+ in case of WT and G51D, the binding is not responsible for enhancing the rate of fibril formation in case of H50Q. General Significance: These findings will help in the better understanding of Cu2+ catalyzed aggregation of synucleins. (C) 2016 Elsevier B.V. All rights reserved.

Alpha-synuclein (α-Syn) aggregation into oligomers and fibrils is associated with dopaminergic neuron loss occurring in Parkinson’s disease (PD) pathogenesis. Compounds that modulate α-Syn aggregation and interact with preformed fibrils/oligomers and convert them to less toxic species could have promising applications in the drug development efforts against PD. Curcumin is one of the Asian food ingredient which showed promising role as therapeutic agent against many neurological disorders including PD. However, the instability and low solubility makes it less attractive for the drug development. In this work, we selected various curcumin analogs and studied their toxicity, stability and efficacy to interact with different α-Syn species and modulation of their toxicity. We found a subset of curcumin analogs with higher stability and showed that curcumin and its various analogs interact with preformed fibrils and oligomers and accelerate α-Syn aggregation to produce morphologically different amyloid fibrils in vitro. Furthermore, these curcumin analogs showed differential binding with the preformed α-Syn aggregates. The present data suggest the potential role of curcumin analogs in modulating α-Syn aggregation.

Aggregation of alpha-Synuclein (alpha-Syn) into amyloid fibrils is known to be associated with the pathogenesis of Parkinson's disease (PD). Several missense mutations of the a-Syn gene have been associated with rare, early onset familial forms of PD. Despite several studies done so far, the local/residue-level structure and dynamics of alpha-Syn in its soluble and aggregated fibril form and how these are affected by the familial PD associated mutations are still not clearly understood. Here, we review studies performed by our group as well as other research groups, where time-resolved fluorescence spectroscopy has been used to understand the site-specific structure and dynamics of alpha-Syn under physiological conditions as well as under conditions that alter the aggregation properties of the protein such as low pH, high temperature, presence of membrane mimics and familial PD associated mutations. These studies have provided important insights into the critical structural properties of alpha-Syn that may govern its aggregation. The review also highlights time-resolved fluorescence as a promising tool to study the critical conformational transitions associated with early oligomerization of alpha-Syn, which are otherwise not accessible using other commonly used techniques such as thioflavin T (ThT) binding assay.

Protein aggregation and amyloid formation are associated with both pathological conditions in humans such as Alzheimer's disease and native functions such as peptide hormone storage in the pituitary secretory granules in mammals. Here, we studied amyloid fibrils formation by three neuropeptides namely physalaemin, kassinin and substance P of tachykinin family using biophysical techniques including circular dichroism, thioflavin T, congo red binding and microscopy. All these neuropeptides under study have significant sequence similarity with A beta(25-35) that is known to form neurotoxic amyloids. We found that all these peptides formed amyloid-like fibrils in vitro in the presence of heparin, and these amyloids were found to be nontoxic in neuronal cells. However, the extent of amyloid formation, structural transition, and morphology were different depending on the primary sequences of peptide. When A beta(25-35) and A beta 40 were incubated with each of these neuropeptides in 1: 1 ratio, a drastic increase in amyloid growths were observed compared to that of individual peptides suggesting that co-aggregation of A beta and these neuropeptides. The electron micrographs of these co-aggregates were dissimilar when compared with individual peptide fibrils further supporting the possible incorporation of these neuropeptides in A beta b amyloid fibrils. Further, the fibrils of these neuropeptides can seed the fibrils formation of A beta 40 and reduced the toxicity of preformed A beta fibrils. The present study of amyloid formation by tachykinin neuropeptides is not only providing an understanding of the mechanism of amyloid fibril formation in general, but also offering plausible explanation that why these neuropeptide might reduce the cytotoxicity associated with Alzheimer's disease related amyloids.

alpha-Synuclein (alpha-Syn) aggregation is directly implicated in both the initiation and spreading of Parkinson's Diseases (PD) pathogenesis. Although the familial PD-associated mutations (A53T, E46K, and A30P) are known to affect the aggregation kinetics of alpha-Syn in vitro, their structural differences in resultant fibrils are largely unknown. In this report we studied the site-specific dynamics of wild type (wt) alpha-Syn and its three PD mutant fibrils using time-resolved fluorescence intensity, anisotropy decay kinetics, and fluorescence quenching. Our data suggest that the N- and C-terminus are more flexible and exposed compared to the middle non-amyloid-beta component (NAC) region of wt and PD mutant alpha-Syn fibrils. Yet the N-terminus showed great conformational heterogeneity compared to the C-terminus for all these proteins. 71 position of E46K showed more flexibility and solvent exposure compared to other alpha-Syns, whereas both E46K and A53T fibrils possess a more rigid C-terminus compared to wt and A30P. The present data suggest that wt and PD mutant fibrils possess large differences in flexibility and solvent exposure at different positions, which may contribute to their different pathogenicity in PD.

The aggregation of proteins into amyloid fibrils is associated with several neurodegenerative diseases. In Parkinson's disease it is believed that the aggregation of alpha-synuclein (alpha-syn) from monomers by intermediates into amyloid fibrils is the toxic disease-causative mechanism. Here, we studied the structure of alpha-syn in its amyloid state by using various biophysical approaches. Quenched hydrogen/deuterium exchange NMR spectroscopy identified five beta-strands within the fibril core comprising residues 35-96 and solid-state NMR data from amyloid fibrils comprising the fibril core residues 30-110 confirmed the presence of beta-sheet secondary structure. The data suggest that beta1-strand interacts with beta2, beta2 with beta3, beta3 with beta4, and beta4 with beta5. High-resolution cryoelectron microscopy revealed the protofilament boundaries of approximately 2 x 3.5 nm. Based on the combination of these data and published structural studies, a fold of alpha-syn in the fibrils is proposed and discussed.

Background: Aggregation of -Syn is associated with PD pathogenesis. Results: Despite being natively unfolded, a site-specific structure exists in -Syn that is significantly altered by familial PD-associated E46K, A53T, and A30P mutations. Conclusion: Altered site-specific structure of the PD-associated mutants may attribute to their different aggregation propensity. Significance: This study contributes to understanding the relationship between structure and aggregation of -Syn. Human -synuclein (-Syn) is a natively unstructured protein whose aggregation into amyloid fibrils is associated with Parkinson disease (PD) pathogenesis. Mutations of -Syn, E46K, A53T, and A30P, have been linked to the familial form of PD. In vitro aggregation studies suggest that increased propensity to form non-fibrillar oligomers is the shared property of these familial PD-associated mutants. However, the structural basis of the altered aggregation propensities of these PD-associated mutants is not yet clear. To understand this, we studied the site-specific structural dynamics of wild type (WT) -Syn and its three PD mutants (A53T, E46K, and A30P). Tryptophan (Trp) was substituted at the N terminus, central hydrophobic region, and C terminus of all -Syns. Using various biophysical techniques including time-resolved fluorescence studies, we show that irrespective of similar secondary structure and early oligomerization propensities, familial PD-associated mutations alter the site-specific microenvironment, solvent exposure, and conformational flexibility of the protein. Our results further show that the common structural feature of the three PD-associated mutants is more compact and rigid sites at their N and C termini compared with WT -Syn that may facilitate the formation of a partially folded intermediate that eventually leads to their increased oligomerization propensities.

Amyloids are highly ordered protein/peptide aggregates associated with human diseases as well as various native biological functions. Given the diverse range of physiochemical properties of amyloids, we hypothesized that higher order amyloid self-assembly could be used for fabricating novel hydrogels for biomaterial applications. For proof of concept, we designed a series of peptides based on the high aggregation prone C-terminus of A beta 42, which is associated with Alzheimer's disease. These Fmoc protected peptides self assemble to beta sheet rich nanofibrils, forming hydrogels that are thermoreversible, non-toxic and thixotropic. Mechanistic studies indicate that while hydrophobic, pi-pi interactions and hydrogen bonding drive amyloid network formation to form supramolecular gel structure, the exposed hydrophobic surface of amyloid fibrils may render thixotropicity to these gels. We have demonstrated the utility of these hydrogels in supporting cell attachment and spreading across a diverse range of cell types. Finally, by tuning the stiffness of these gels through modulation of peptide concentration and salt concentration these hydrogels could be used as scaffolds that can drive differentiation of mesenchymal stem cells. Taken together, our results indicate that small size, ease of custom synthesis, thixotropic nature makes these amyloid-based hydrogels ideally suited for biomaterial/nanotechnology applications. (C) 2015 Elsevier Ltd. All rights reserved.

Mechanistic understanding of nucleation dependent polymerization by α-synuclein (α-Syn) into toxic oligomers and amyloids is important for the drug development against Parkinson's disease. However the structural and morphological characterization during nucleation and subsequent fibrillation process of α-Syn is not clearly understood. Using a variety of complementary biophysical techniques monitoring entire pathway of nine different synucleins, we found that transition of unstructured conformation into β-sheet rich fibril formation involves helix-rich intermediates. These intermediates are common for all aggregating synucleins, contain high solvent-exposed hydrophobic surfaces, are cytotoxic to SHSY-5Y cells and accelerate α-Syn aggregation efficiently. A multidimensional NMR study characterizing the intermediate accompanied with site-specific fluorescence study suggests that the N-terminal and central portions mainly participate in the helix-rich intermediate formation while the C-terminus remained in an extended conformation. However, significant conformational transitions occur at the middle and at the C-terminus during helix to β-sheet transition as evident from Trp fluorescence study. Since partial helix-rich intermediates were also observed for other amyloidogenic proteins such as Aβ and IAPP, we hypothesize that this class of intermediates may be one of the important intermediates for amyloid formation pathway by many natively unstructured protein/peptides and represent a potential target for drug development against amyloid diseases.

alpha-Synuclein (a-Syn) oligomerization and amyloid formation are associated with Parkinson's disease (PD) pathogenesis. Studying familial a-Syn mutants associated with early onset PD has therapeutic importance. Here we report the aggregation kinetics and other biophysical properties of a newly discovered PD associated Finnish mutation (A53E). Our in vitro study demonstrated that A53E attenuated a-Syn aggregation and amyloid formation without altering the major secondary structure and initial oligomerization tendency. Further, A53E showed reduced membrane binding affinity compared to A53T and WT. The present study would help to delineate the role of A53E mutation in early onset PD pathogenesis.

In human beings, Parkinson's disease (PD) is associated with the oligomerization and amyloid formation of alpha-synuclein (alpha-Syn). The polyphenolic Asian food ingredient curcumin has proven to be effective against a wide range of human diseases including cancers and neurological disorders. While curcumin has been shown to significantly reduce cell toxicity of alpha-Syn aggregates, its mechanism of action remains unexplored. Here, using a series of biophysical techniques, we demonstrate that curcumin reduces toxicity by binding to preformed oligomers and fibrils and altering their hydrophobic surface exposure. Further, our fluorescence and two-dimensional nuclear magnetic resonance (2D-NMR) data indicate that curcumin does not bind to monomeric alpha-Syn but binds specifically to oligomeric intermediates. The degree of curcumin binding correlates with the extent of alpha-Syn oligomerization, suggesting that the ordered structure of protein is required for effective curcumin binding. The acceleration of aggregation by curcumin may decrease the population of toxic oligomeric intermediates of alpha-Syn. Collectively; our results suggest that curcumin and related polyphenolic compounds can be pursued as candidate drug targets for treatment of PD and other neurological diseases.

alpha-Synuclein (alpha-Syn) aggregation is directly linked with Parkinson's disease (PD) pathogenesis. Here, we analyzed the aggregation of newly discovered a-Syn missense mutant H50Q in vitro and found that this mutation significantly accelerates the aggregation and amyloid formation of alpha-Syn. This mutation, however, did not alter the overall secondary structure as suggested by two-dimensional nuclear magnetic resonance and circular dichroism spectroscopy. The initial oligomerization study by cross-linking and chromatographic techniques suggested that this mutant oligomerizes to an extent similar to that of the wild-type alpha-Syn protein. Understanding the aggregation mechanism of this H50Q mutant may help to establish the aggregation and phenotypic relationship of this novel mutant in PD.

The aggregation of proteins into oligomers and amyloid fibrils is characteristic of several neurodegenerative diseases, including Parkinson disease (PD). In PD, the process of aggregation of α-synuclein (α-syn) from monomers, via oligomeric intermediates, into amyloid fibrils is considered the disease-causative toxic mechanism. We developed α-syn mutants that promote oligomer or fibril formation and tested the toxicity of these mutants by using a rat lentivirus system to investigate loss of dopaminergic neurons in the substantia nigra. The most severe dopaminergic loss in the substantia nigra is observed in animals with the α-syn variants that form oligomers (i.e., E57K and E35K), whereas the α-syn variants that form fibrils very quickly are less toxic. We show that α-syn oligomers are toxic in vivo and that α-syn oligomers might interact with and potentially disrupt membranes.

The aggregation of proteins into amyloid fibrils is associated with several neurodegenerative diseases. In Parkinson's disease it is believed that the aggregation of alpha-synuclein (alpha-syn) from monomers by intermediates into amyloid fibrils is the toxic disease-causative mechanism. Here, we studied the structure of alpha-syn in its amyloid state by using various biophysical approaches. Quenched hydrogen/deuterium exchange NMR spectroscopy identified five beta-strands within the fibril core comprising residues 35-96 and solid-state NMR data from amyloid fibrils comprising the fibril core residues 30-110 confirmed the presence of beta-sheet secondary structure. The data suggest that beta1-strand interacts with beta2, beta2 with beta3, beta3 with beta4, and beta4 with beta5. High-resolution cryoelectron microscopy revealed the protofilament boundaries of approximately 2 x 3.5 nm. Based on the combination of these data and published structural studies, a fold of alpha-syn in the fibrils is proposed and discussed.