In vitro aggregation assays for the characterization of α-synuclein prion-like properties

Aggregation of α-synuclein plays a crucial role in the pathogenesis of synucleinopathies, a group of neurodegenerative diseases including Parkinson disease (PD), dementia with Lewy bodies (DLB), diffuse Lewy body disease (DLBD) and multiple system atrophy (MSA). The common feature of these diseases is a pathological deposition of protein aggregates, known as Lewy bodies (LBs) in the central nervous system. The major component of these aggregates is α-synuclein, a natively unfolded protein, which may undergo dramatic structural changes resulting in the formation of β-sheet rich assemblies. In vitro studies have shown that recombinant α-synuclein protein may polymerize into amyloidogenic fibrils resembling those found in LBs. These aggregates may be uptaken and propagated between cells in a prion-like manner. Here we present the mechanisms and kinetics of α-synuclein aggregation in vitro, as well as crucial factors affecting this process. We also describe how PD-linked α-synuclein mutations and some exogenous factors modulate in vitro aggregation. Furthermore, we present a current knowledge on the mechanisms by which extracellular aggregates may be internalized and propagated between cells, as well as the mechanisms of their toxicity.     

Modulation effect of filamentous phage on α-synuclein aggregation

Conversion of soluble peptides and proteins into amyloid fibrils and/or intermediate oligomers is believed to be the central event in the pathogenesis of most human neurodegenerative diseases, including Parkinson’s disease (PD). Here we describe the modulating effect of filamentous phages on aggregation of α-synuclein (AS) in vitro and in a PD cellular model. Filamentous phages, well understood at both structural and genetic levels, have a nanotubular appearance, showing conformational similarities to amyloid fibrils. Since filamentous phages can infect only bacteria and have no tropism to mammalian cells, we utilized the f88 system to present a peptide containing a cyclic RGD (arg-gly-asp), which enabled phage internalization into the cells. Detection of intracellular AS oligomers, in differentiated SH-SY5Y cells, stably transfected with wild type AS gene, was performed using Western blot and ELISA measurements. Data presented here show reduced levels of AS soluble aggregates in phage treated cells compared to non-treated cells, suggesting new therapeutics for PD.     

N-homocysteinylation of α-synuclein promotes its aggregation and neurotoxicity

The aggregation of α-synuclein plays a pivotal role in the pathogenesis of Parkinson's disease (PD). Epidemiological evidence indicates that high level of homocysteine (Hcy) is associated with an increased risk of PD. However, the molecular mechanisms remain elusive. Here, we report that homocysteine thiolactone (HTL), a reactive thioester of Hcy, covalently modifies α-synuclein on the K80 residue. The levels of α-synuclein K80Hcy in the brain are increased in an age-dependent manner in the TgA53T mice, correlating with elevated levels of Hcy and HTL in the brain during aging. The N-homocysteinylation of α-synuclein stimulates its aggregation and forms fibrils with enhanced seeding activity and neurotoxicity. Intrastriatal injection of homocysteinylated α-synuclein fibrils induces more severe α-synuclein pathology and motor deficits when compared with unmodified α-synuclein fibrils. Increasing the levels of Hcy aggravates α-synuclein neuropathology in a mouse model of PD. In contrast, blocking the N-homocysteinylation of α-synuclein ameliorates α-synuclein pathology and degeneration of dopaminergic neurons. These findings suggest that the covalent modification of α-synuclein by HTL promotes its aggregation. Targeting the N-homocysteinylation of α-synuclein could be a novel therapeutic strategy against PD.     

Inhibition of α-synuclein aggregation by small heat shock proteins

The fibrillization of α-synuclein (α-syn) is a key event in the pathogenesis of α-synucleinopathies. Mutant α-syn (A53T, A30P, or E46K), each linked to familial Parkinson's disease, has altered aggregation properties, fibril morphologies, and fibrillization kinetics. Besides α-syn, Lewy bodies also contain several associated proteins including small heat shock proteins (sHsps). Since α-syn accumulates intracellularly, molecular chaperones like sHsps may regulate α-syn folding and aggregation. Therefore, we investigated if the sHsps αB-crystallin, Hsp27, Hsp20, HspB8, and HspB2B3 bind to α-syn and affect α-syn aggregation. We demonstrate that all sHsps bind to the various α-syns, although the binding kinetics suggests a weak and transient interaction only. Despite this transient interaction, the various sHsps inhibited mature α-syn fibril formation as shown by a Thioflavin T assay and atomic force microscopy. Interestingly, HspB8 was the most potent sHsp in inhibiting mature fibril formation of both wild-type and mutant α-syn. In conclusion, sHsps may regulate α-syn aggregation and, therefore, optimization of the interaction between sHsps and α-syn may be an interesting target for therapeutic intervention in the pathogenesis of α-synucleinopathies.     

Curcumin prevents aggregation in α-synuclein by increasing reconfiguration rate

α-Synuclein is a protein that is intrinsically disordered in vitro and prone to aggregation, particularly at high temperatures. In this work, we examined the ability of curcumin, a compound found in turmeric, to prevent aggregation of the protein. We found strong binding of curcumin to α-synuclein in the hydrophobic non-amyloid-β component region and complete inhibition of oligomers or fibrils. We also found that the reconfiguration rate within the unfolded protein was significantly increased at high temperatures. We conclude that α-synuclein is prone to aggregation because its reconfiguration rate is slow enough to expose hydrophobic residues on the same time scale that bimolecular association occurs. Curcumin rescues the protein from aggregation by increasing the reconfiguration rate into a faster regime.     

PAMAM G4 dendrimers affect the aggregation of α-synuclein

α-Synuclein (ASN) aggregation plays a key role in neurodegenerative disorders including Parkinson's disease, and inhibition of fibril formation is a potential therapeutic strategy for these conditions. The aim of the present study was to investigate polyamidoamine (PAMAM) dendrimers (generations 4 and 3.5) as inhibitors of fibril formation in vitro by examining their interaction with ASN intrinsic tyrosine fluorescence. Furthermore, the effect of dendrimers on ASN aggregation was studied using circular dichroism (CD) spectroscopy and CD studies were complemented by a fluorescence assays using the dye thioflavin T (ThT). The PAMAM G4 dendrimer caused an increase in tyrosine residue fluorescence, and inhibited fibrillation of ASN; inhibited fibrillation was not observed with PAMAM G3.5 dendrimers.     

Gallic acid interacts with α-synuclein to prevent the structural collapse necessary for its aggregation

The accumulation of protein aggregates containing amyloid fibrils, with α-synuclein being the main component, is a pathological hallmark of Parkinson's disease (PD). Molecules which prevent the formation of amyloid fibrils or disassociate the toxic aggregates are touted as promising strategies to prevent or treat PD. In the present study, in vitro Thioflavin T fluorescence assays and transmission electron microscopy imaging results showed that gallic acid (GA) potently inhibits the formation of amyloid fibrils by α-synuclein. Ion mobility-mass spectrometry demonstrated that GA stabilises the extended, native structure of α-synuclein, whilst NMR spectroscopy revealed that GA interacts with α-synuclein transiently.     

Supramolecular non-amyloid intermediates in the early stages of α-synuclein aggregation

The aggregation of α-synuclein is associated with progression of Parkinson's disease. We have identified submicrometer supramolecular structures that mediate the early stages of the overall mechanism. The sequence of structural transformations between metastable intermediates were captured and characterized by atomic force microscopy guided by a fluorescent probe sensitive to preamyloid species. A novel ~0.3-0.6 μm molecular assembly, denoted the acuna, nucleates, expands, and liberates fibers with distinctive segmentation and a filamentous fuzzy fringe. These fuzzy fibers serve as precursors of mature amyloid fibrils. Cryo-electron tomography resolved the acuna inner structure as a scaffold of highly condensed colloidal masses interlinked by thin beaded threads, which were perceived as fuzziness by atomic force microscopy. On the basis of the combined data, we propose a sequential mechanism comprising molecular, colloidal, and fibrillar stages linked by reactions with disparate temperature dependencies and distinct supramolecular forms. We anticipate novel diagnostic and therapeutic approaches to Parkinson's and related neurodegenerative diseases based on these new insights into the aggregation mechanism of α-synuclein and intermediates, some of which may act to cause and/or reinforce neurotoxicity.     

GM1 oligosaccharide efficacy against α-synuclein aggregation and toxicity in vitro

Fibrillary aggregated α-synuclein represents the neurologic hallmark of Parkinson's disease and is considered to play a causative role in the disease. Although the causes leading to α-synuclein aggregation are not clear, the GM1 ganglioside interaction is recognized to prevent this process. How GM1 exerts these functions is not completely clear, although a primary role of its soluble oligosaccharide (GM1-OS) is emerging. Indeed, we recently identified GM1-OS as the bioactive moiety responsible for GM1 neurotrophic and neuroprotective properties, specifically reverting the parkinsonian phenotype both in in vitro and in vivo models.Here, we report on GM1-OS efficacy against the α-synuclein aggregation and toxicity in vitro. By amyloid seeding aggregation assay and NMR spectroscopy, we demonstrated that GM1-OS was able to prevent both the spontaneous and the prion-like α-synuclein aggregation. Additionally, circular dichroism spectroscopy of recombinant monomeric α-synuclein showed that GM1-OS did not induce any change in α-synuclein secondary structure. Importantly, GM1-OS significantly increased neuronal survival and preserved neurite networks of dopaminergic neurons affected by α-synuclein oligomers, together with a reduction of microglia activation.These data further demonstrate that the ganglioside GM1 acts through its oligosaccharide also in preventing the α-synuclein pathogenic aggregation in Parkinson's disease, opening a perspective window for GM1-OS as drug candidate.     

γ-Synuclein: seeding of α-synuclein aggregation and transmission between cells

Protein misfolding and aggregation is a ubiquitous phenomenon associated with a wide range of diseases. The synuclein family comprises three small naturally unfolded proteins implicated in neurodegenerative diseases and some forms of cancer. α-Synuclein is a soluble protein that forms toxic inclusions associated with Parkinson's disease and several other synucleinopathies. However, the triggers inducing its conversion into noxious species are elusive. Here we show that another member of the family, γ-synuclein, can be easily oxidized and form annular oligomers that accumulate in cells in the form of deposits. Importantly, oxidized γ-synuclein can initiate α-synuclein aggregation. Two amino acid residues in γ-synuclein, methionine and tyrosine located in neighboring positions (Met(38) and Tyr(39)), are most easily oxidized. Their oxidation plays a key role in the ability of γ-synuclein to aggregate and seed the aggregation of α-synuclein. γ-Synuclein secreted from neuronal cells into conditioned medium in the form of exosomes can be transmitted to glial cells and cause the aggregation of intracellular proteins. Our data suggest that post-translationally modified γ-synuclein possesses prion-like properties and may induce a cascade of events leading to synucleinopathies.     

Tau enhances α-synuclein aggregation and toxicity in cellular models of synucleinopathy

Background

The simultaneous accumulation of different misfolded proteins in the central nervous system is a common feature in many neurodegenerative diseases. In most cases, co-occurrence of abnormal deposited proteins is observed in different brain regions and cell populations, but, in some instances, the proteins can be found in the same cellular aggregates. Co-occurrence of tau and α-synuclein (α-syn) aggregates has been described in neurodegenerative disorders with primary deposition of α-syn, such as Parkinson''s disease and dementia with Lewy bodies. Although it is known that tau and α-syn have pathological synergistic effects on their mutual fibrillization, the underlying biological effects remain unclear.

Methodology/Principal Findings

We used different cell models of synucleinopathy to investigate the effects of tau on α-syn aggregation. Using confocal microscopy and FRET–based techniques we observed that tau colocalized and interacted with α-syn aggregates. We also found that tau overexpression changed the pattern of α-syn aggregation, reducing the size and increasing the number of aggregates. This shift was accompanied by an increase in the levels of insoluble α-syn. Furthermore, co-transfection of tau increased secreted α-syn and cytotoxicity.

Conclusions/Significance

Our data suggest that tau enhances α-syn aggregation and toxicity and disrupts α-syn inclusion formation. This pathological synergistic effect between tau and α-syn may amplify the deleterious process and spread the damage in neurodegenerative diseases that show co-occurrence of both pathologies.     

Design,synthesis, and in vitro evaluation of quinolinyl analogues for α-synuclein aggregation

Here we report the synthesis and in vitro evaluation of 25 new quinolinyl analogues for α-synuclein aggregates. Three lead compounds were subsequently labeled with carbon-11 or fluorine-18 to directly assess their potency in a direct radioactive competitive binding assay ng both α-synuclein fibrils and tissue homogenates from Alzheimer’s disease (AD) cases. The modest binding affinities of these three radioligands toward α-synuclein were comparable with results from the Thioflavin T fluorescence assay. However, all three ligand also showed modest binding affinity to the AD homogenates and lack selectivity for α-synuclein. The structure–activity relationship data from these 25 analogues will provide useful information for design and synthesis of new compounds for imaging α-synuclein aggregation.     

Lipid vesicles affect the aggregation of 4-hydroxy-2-nonenal-modified α-synuclein oligomers

Parkinson's disease (PD) and other synucleinopathies are characterized by accumulation of misfolded aggregates of α-synuclein (α-syn). The normal function of α-syn is still under investigation, but it has been generally linked to synaptic plasticity, neurotransmitter release and the maintenance of the synaptic pool. α-Syn localizes at synaptic terminals where it can bind to synaptic vesicles as well as to other cellular membranes. It has become clear that these interactions have an impact on both α-syn functional role and its propensity to aggregate. In this study, we investigated the aggregation process of α-syn covalently modified with 4-hydroxy-2-nonenal (HNE). HNE is a product of lipid peroxidation and has been implicated in the pathogenesis of different neurodegenerative diseases by modifying the kinetics of soluble toxic oligomers. Although HNE-modified α-syn has been reported to assemble into stable oligomers, we found that slightly acidic conditions promoted further protein aggregation. Lipid vesicles delayed the aggregation process in a concentration-dependent manner, an effect that was observed only when they were added at the beginning of the aggregation process. Co-aggregation of lipid vesicles with HNE-modified α-syn also induced cytotoxic effects on differentiated SHSY-5Y cells. Under conditions in which the aggregation process was delayed cell viability was reduced. By exploring the behavior and potential cytotoxic effects of HNE-α-syn under acidic conditions in relation to protein-lipid interactions our study gives a framework to examine a possible pathway leading from a physiological setting to the pathological outcome of PD.     

Gelsolin co-occurs with Lewy bodies in vivo and accelerates α-synuclein aggregation in vitro

Deposition of fibrillar α-synuclein as Lewy bodies is the neuropathological hallmark of Parkinson’s disease (PD) and dementia with Lewy bodies (DLB). Apart from α-synuclein, these intraneuronal inclusions contain over 250 different proteins. The actin binding protein gelsolin, has previously been suggested to be part of the Lewy body, but its potential role in α-synuclein aggregation remains unknown. Here, we studied the association between gelsolin and α-synuclein in brain tissue from PD and DLB patients as well as in a cell model for α-synuclein aggregation. Moreover, the potential effect of gelsolin on α-synuclein fibrillization was also investigated. Our data demonstrate that gelsolin co-occured with α-synuclein in Lewy bodies from affected human brain as well as with Lewy body-like inclusions in α-synuclein over expressing cells. Furthermore, in the presence of calcium chloride, gelsolin was found to enhance the aggregation rate of α-synuclein in vitro. Moreover, no apparent structural differences could be observed between fibrils formed in the presence or absence of gelsolin. Further studies on gelsolin and other Lewy body associated proteins are warranted to learn more about their potential role in the α-synuclein aggregation process.     

Conserved C-terminal charge exerts a profound influence on the aggregation rate of α-synuclein

α-Synuclein (α-syn) is the major component of filamentous Lewy bodies found in the brains of patients diagnosed with Parkinson's disease (PD). Recent studies demonstrate that, in addition to the wild-type sequence, α-syn is found in several modified forms, including truncated and phosphorylated species. Although the mechanism by which the neuronal loss in PD occurs is unknown, aggregation and fibril formation of α-syn are considered to be key pathological features. In this study, we analyze the rates of fibril formation and the monomer-fibril equilibrium for eight disease-associated truncated and phosphorylated α-syn variants. Comparison of the relative rates of aggregation reveals a strong monotonic relationship between the C-terminal charge of α-syn and the lag time prior to the observation of fibril formation, with truncated species exhibiting the fastest aggregation rates. Moreover, we find that a decrease in C-terminal charge shifts the equilibrium to favor the fibrillar species. An analysis of these findings in the context of linear growth theories suggests that the loss of the charge-mediated stabilization of the soluble state is responsible for the enhanced aggregation rate and increased extent of fibril fraction. Therefore, C-terminal charge is kinetically and thermodynamically protective against α-syn polymerization and may provide a target for the treatment of PD.     

N-Terminal acetylation is critical for forming α-helical oligomer of α-synuclein

The aggregation of the protein α-synuclein (AS) is critical to the pathogenesis of Parkinson's disease. Although generally described as an unstructured monomer, recent evidence suggests that the native form of AS may be an α-helical tetramer which resists aggregation. Here, we show that N-terminal acetylation in combination with a mild purification protocol results in an oligomeric form of AS with partial α-helical structure. N-terminal acetylation of AS could have important implications for both the native and pathological structures and functions of AS. Through our demonstration of a recombinant expression system, our results represent an important step toward biochemical and biophysical characterization of this potentially important form of AS.     

Preventing α-synuclein aggregation: The role of the small heat-shock molecular chaperone proteins

Protein homeostasis, or proteostasis, is the process of maintaining the conformational and functional integrity of the proteome. The failure of proteostasis can result in the accumulation of non-native proteins leading to their aggregation and deposition in cells and in tissues. The amyloid fibrillar aggregation of the protein α-synuclein into Lewy bodies and Lewy neuritis is associated with neurodegenerative diseases classified as α-synucleinopathies, which include Parkinson's disease and dementia with Lewy bodies. The small heat-shock proteins (sHsps) are molecular chaperones that are one of the cell's first lines of defence against protein aggregation. They act to stabilise partially folded protein intermediates, in an ATP-independent manner, to maintain cellular proteostasis under stress conditions. Thus, the sHsps appear ideally suited to protect against α-synuclein aggregation, yet these fail to do so in the context of the α-synucleinopathies. This review discusses how sHsps interact with α-synuclein to prevent its aggregation and, in doing so, highlights the multi-faceted nature of the mechanisms used by sHsps to prevent the fibrillar aggregation of proteins. It also examines what factors may contribute to α-synuclein escaping the sHsp chaperones in the context of the α-synucleinopathies.     

Modulation of α-synuclein aggregation by dopamine in the presence of MPTP and its metabolite

The neurotransmitter dopamine has been shown to inhibit fibrillation of α-synuclein by promoting the formation of nonamyloidogenic oligomers. Fibrillation of α-synuclein is accelerated in the presence of pesticides and the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). The aim of this study was to determine whether dopamine continues to have an adverse effect on the fibrillation of α-synuclein in the presence of MPTP and its metabolite 1-methyl-4-phenylpyridinum ion (MPP(+) ). We also attempted to answer the ambiguous question of whether conversion of MPTP to MPP(+) is required for the fibrillation of α-synuclein. For this, α-synuclein was incubated in the presence of MPTP and MPP(+) along with dopamine. The fibrillation of α-synuclein was monitored by Thioflavin T fluorescence and immunoblotting. The morphology of the aggregates formed was observed using scanning electron microscopy. The concentrations of the neurotoxin and its metabolite were estimated by reverse phase HPLC. We found definitive evidence that the conversion of MPTP to MPP(+) is not required for aggregation of α-synuclein. MPP(+) was found to accelerate the rate of α-synuclein aggregation even in the absence of components of mitochondrial complex I. In contrast to the effect of dopamine on the aggregation of α-synuclein alone, in the presence of MPTP or MPP(+) , the aggregates formed are Thioflavin T-positive and amyloidogenic. Thus, the effect of dopamine on the nature of aggregates formed in case of α-synuclein alone and in the presence of MPTP/MPP(+) is different.