Differential inhibition of α-synuclein oligomeric and fibrillar assembly in parkinson's disease model by cinnamon extract

Background

The oligomeriztion of α-synuclein (α-syn) into ordered assemblies is associated with the symptoms of Parkinson's Disease (PD). Yet, it is still debatable whether oligomers are formed as part of a multistep process towards amyloid fibril formation or alternatively as "off-pathway" aggregates.

Methods

100 μM α-syn was incubated with decreasing amounts of cinnamon extract precipitation (CEppt). The fibril formation was measured using spectroscopy and microscopy analyses and oligomers were detected using western blot analysis. The secondary structure of the protein was analyzed using CD. Drosophila brains were studied using immunostaining and confocal microscopy.

Results

Here we probed the inhibition pattern of oligomeric and fibrillar forms of α-syn, using a natural substance, CEppt which was previously shown to effectively inhibit aggregation of β-amyloid polypeptide. We demonstrated that CEppt has a differential inhibitory effect on the formation of soluble and insoluble aggregates of α-synuclein in vitro. This inhibition pattern revokes the possibility of redirection to "off-pathway" oligomers. When administering to Drosophila fly model expressing mutant A53T α-syn in the nervous system, a significant curative effect on the behavioral symptoms of the flies and on α-syn aggregation in their brain was observed.

Conclusions

We conclude that CEppt affects the process of aggregation of α-syn without changing its secondary structure and suggest that increasing amounts of CEppt slow this process by stabilizing the soluble oligomeric phase. When administered to Drosophila fly model, CEppt appears to have a curative effect on the defective flies.

General significance

Our results indicate that CEppt can be a potential therapeutic agent for PD.     

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.     

Stimulatory effect of α-synuclein on the tau-phosphorylation by GSK-3β

Hyperphosphorylation of tau protein (tau) causes neurodegenerative diseases such as Alzheimer's disease (AD). Recent studies of the physiological correlation between tau and α-synuclein (α-SN) have demonstrated that: (a) phosphorylated tau is also present in Lewy bodies, which are cytoplasmic inclusions formed by abnormal aggregation of α-SN; and (b) the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) increases the phosphorylation of tau as well as the protein level of α-SN in cultured neuronal cells, and also in mice. However, the molecular mechanism responsible for the α-SN-mediated hyperphosphorylation of tau remains to be elucidated. In this in vitro study, we found that: (a) α-SN directly stimulates the phosphorylation of tau by glycogen synthase kinase-3β (GSK-3β), (b) α-SN forms a heterotrimeric complex with tau and GSK-3β, and (c) the nonamyloid beta component (NAC) domain and an acidic region of α-SN are responsible for the stimulation of GSK-3β-mediated tau phosphorylation. Thus, it is concluded that α-SN functions as a connecting mediator for tau and GSK-3β, resulting in GSK-3β-mediated tau phosphorylation. Because the expression of α-SN is promoted by oxidative stress, the accumulation of α-SN induced by such stress may directly induce the hyperphosphorylation of tau by GSK-3β. Furthermore, we found that heat shock protein 70 (Hsp70) suppresses the α-SN-induced phosphorylation of tau by GSK-3β through its direct binding to α-SN, suggesting that Hsp70 acts as a physiological suppressor of α-SN-mediated tau hyperphosphorylation. These results suggest that the cellular level of Hsp70 may be a novel therapeutic target to counteract α-SN-mediated tau phosphorylation in the initial stage of neurodegenerative disease.     

Depth of α-synuclein in a bilayer determined by fluorescence, neutron reflectometry, and computation

α-Synuclein (α-syn) membrane interactions are implicated in the pathogenesis of Parkinson's disease. Fluorescence and neutron reflectometry (NR) measurements reveal that α-syn penetrates ～9–14 Å into the outer leaflet of the bilayer, with a substantial portion of the membrane-bound polypeptide extending into the aqueous solvent. For the first time, to our knowledge, we used NR to obtain direct quantitative evidence of α-syn-induced membrane thinning. To examine the effect of specific residues on membrane penetration depths, we used a series of W4-containing N-terminal peptides. We identified that the first 15 residues (P15) nearly recapitulate the features of the full-length protein (i.e., partition constants, molecular mobility, and insertion of the W4 side chain into the bilayer), and found that as few as the first four N-terminal residues are sufficient for vesicle binding. Although at least one imperfect amphipathic repeat sequence (KAKEGV) is required for α-helical formation, secondary structural formation has little effect on membrane affinity. To develop an N-terminal α-syn model for bilayer interactions, we performed molecular-dynamics simulations of the P15 peptide submerged in a bilayer. The simulation results are highly consistent with experimental data indicating a broad low-energy region (8.5–14.5 Å) for W4 insertion.     

The mode of α-synuclein binding to membranes depends on lipid composition and lipid to protein ratio

Interactions of the presynaptic protein α-synuclein with membranes are involved in its physiological action as well as in the pathological misfolding and aggregation related to Parkinsons's disease. We studied the conformation and orientation of α-synuclein bound to model vesicular membranes using multiparametric response polarity-sensitive fluorescent probes together with CD and EPR measurements. At low lipid to α-synuclein ratio the protein binds membranes through its N-terminal domain. When lipids are in excess, the α-helical content and the role of the C-terminus in binding increase. Highly rigid membranes also induce a greater α-helical content and a lower polarity of the protein microenvironment.     

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.     

Compact conformations of α-synuclein induced by alcohols and copper

The intrinsically disordered protein α-synuclein aggregates into amyloid fibrils, a process known to be implicated in several neurodegenerative states. Partially folded forms of the protein are thought to trigger the aggregation process. Here, α-synuclein conformers are characterized by analysis of the charge-state distributions observed in electrospray-ionization mass spectrometry under negative-ion mode. It is found that, even at neutral pH, a small fraction of the molecular population is in a compact conformation. Several distinct partially folded forms are then identified under conditions that promote α-synuclein aggregation, such as solutions of simple and fluorinated alcohols. Specific intermediates accumulate at increasing concentrations of ethanol, hexafluoro-2-propanol, and trifluoroethanol. Finally, extensive folding induced by Cu(2+) binding is revealed by titrations in the presence of Cu(2+)-glycine. The data confirm the existence of a single, high-affinity binding site for Cu(2+). Because accumulation of this partially folded form correlates with enhancement of fibrillation kinetics, it is likely to represent an amyloidogenic intermediate in α-synuclein conformational transitions.     

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.     

Membrane interactions and fibrillization of α-synuclein play an essential role in membrane disruption

We studied α-synuclein (αS) aggregation in giant vesicles, and observed dramatic membrane disintegration, as well as lipid incorporation into micrometer-sized suprafibrillar aggregates. In the presence of dye-filled vesicles, dye leakage and fibrillization happen concurrently. However, growing fibrils do not impair the integrity of phospholipid vesicles that have a low affinity for αS. Seeding αS aggregation accelerates dye leakage, indicating that oligomeric species are not required to explain the observed effect. The evolving picture suggests that fibrils that appear in solution bind membranes and recruit membrane-bound monomers, resulting in lipid extraction, membrane destabilization and the formation of lipid-containing suprafibrillar aggregates.     

Conserved core of amyloid fibrils of wild type and A30P mutant α-synuclein

The major component of neural inclusions that are the pathological hallmark of Parkinson's disease are amyloid fibrils of the protein α-synuclein (aS). Here we investigated if the disease-related mutation A30P not only modulates the kinetics of aS aggregation, but also alters the structure of amyloid fibrils. To this end we optimized the method of quenched hydrogen/deuterium exchange coupled to NMR spectroscopy and performed two-dimensional proton-detected high-resolution magic angle spinning experiments. The combined data indicate that the A30P mutation does not cause changes in the number, location and overall arrangement of β-strands in amyloid fibrils of aS. At the same time, several residues within the fibrillar core retain nano-second dynamics. We conclude that the increased pathogenicity related to the familial A30P mutation is unlikely to be caused by a mutation-induced change in the conformation of aS aggregates.     

Inhibition and disaggregation of α-synuclein oligomers by natural polyphenolic compounds

Aggregation of alpha-synuclein (αS) into oligomers is critically involved in the pathogenesis of Parkinson's disease (PD). Using confocal single-molecule fluorescence spectroscopy, we have studied the effects of 14 naturally-occurring polyphenolic compounds and black tea extract on αS oligomer formation. We found that a selected group of polyphenols exhibited potent dose-dependent inhibitory activity on αS aggregation. Moreover, they were also capable of robustly disaggregating pre-formed αS oligomers. Based upon structure-activity analysis, we propose that the key molecular scaffold most effective in inhibiting and destabilizing self-assembly by αS requires: (i) aromatic elements for binding to the αS monomer/oligomer and (ii) vicinal hydroxyl groups present on a single phenyl ring. These findings may guide the design of novel therapeutic drugs in PD.     

Disintegration of amyloid fibrils of α-synuclein by dequalinium

α-Synuclein is the major amyloidogenic component observed in the Lewy bodies of Parkinson's disease. Amyloid fibrils of α-synuclein prepared in vitro were instantaneously disintegrated by dequalinium (DQ). Double-headed cationic amphipathic structure of DQ with two aminoquinaldinium rings at both ends turned out to be crucial to exert the disintegration activity. The defibrillation activity was shown to be selective toward the fibrils of α-synuclein and Aβ40 while the other β2-microglobulin amyloid fibrils were not susceptible so much. Besides the common cross β-sheet conformation of amyloid fibrils, therefore, additional specific molecular interactions with the target amyloidogenic proteins have been expected to be involved for DQ to exhibit its defibrillation activity. The disintegrating activity of DQ was also evaluated in vivo with the yeast system overexpressing α-synuclein-GFP. With the DQ treatment, the intracellular green inclusions turned into green smears, which resulted in the enhanced cell death. Based on the data, the previous observation that DQ led to the predominant protofibril formation of α-synuclein could be explained by the dual function of DQ showing both the facilitated self-oligomerization of α-synuclein and the instantaneous defibrillation of its amyloid fibrils. In addition, amyloidosis-related cytotoxicity has been demonstrated to be amplified by the fragmentation of mature amyloid fibrils by DQ.     

Modulation of α-synuclein phase separation by biomolecules

Liquid-liquid phase separation (LLPS) is currently recognized as a common mechanism involved in the regulation of a number of cellular functions. On the other hand, aberrant phase separation has been linked to the biogenesis of several neurodegenerative disorders since many proteins that undergo LLPS are also found in pathological aggregates. The formation of mixed protein coacervates may constitute a risk factor in overlapping neuropathologies, such as Parkinson's (PD) and Alzheimer's (AD) diseases. In this work, we evaluated the homotypic and heterotypic phase behaviour of the PD-related protein α-synuclein (AS) in the presence of the biologically relevant molecules ATP, polyamines, and the AD-related protein Tau. We found that AS exhibits a low propensity to form homotypic liquid droplets, yet phase separates into liquid-like or solid-like phases depending on the interacting biomolecule. We further demonstrated the synergistic droplet formation of AS and Tau providing support for a mechanism in which mixed condensates might contribute to the biogenesis of AS/Tau pathologies.     

Metalloproteomics and metal toxicology of α-synuclein

Significant evidence has been accumulated linking exposure to heavy metals and/or distortion of metal homeostasis with the development of various neurodegenerative diseases. α-Synuclein is known to be involved in pathogenesis of a subset of neurodegenerative diseases collectively known as synucleinopathies. Therefore the interplay between metals, α-synuclein and neurodegeneration has attracted significant attention of researchers. This review discusses some of the aspects of the α-synuclein metalloproteomics and represents the peculiarities and consequences of α-synuclein interaction with various metal ions. Both non-specific and specific binding of this protein to metals is considered together with the analysis of the effects of such interactions on α-synuclein structure and aggregation propensity.     

Direct observation of the interconversion of normal and toxic forms of α-synuclein

Here, we use single-molecule techniques to study the aggregation of α-synuclein, the protein whose misfolding and deposition is associated with Parkinson's disease. We identify a conformational change from the initially formed oligomers to stable, more compact proteinase-K-resistant oligomers as the key step that leads ultimately to fibril formation. The oligomers formed as a result of the structural conversion generate much higher levels of oxidative stress in rat primary neurons than do the oligomers formed initially, showing that they are more damaging to cells. The structural conversion is remarkably slow, indicating a high kinetic barrier for the conversion and suggesting that there is a significant period of time for the cellular protective machinery to operate and potentially for therapeutic intervention, prior to the onset of cellular damage. In the absence of added soluble protein, the assembly process is reversed and fibrils disaggregate to form stable oligomers, hence acting as a source of cytotoxic species.     

Assembly of α-synuclein aggregates on phospholipid bilayers

The spontaneous self-assembly of α-synuclein (α-syn) into aggregates of different morphologies is associated with the development of Parkinson's disease. However, the mechanism behind the spontaneous assembly remains elusive. The current study shows a novel effect of phospholipid bilayers on the assembly of the α-syn aggregates. Using time-lapse atomic force microscopy, it was discovered that α-syn assembles into aggregates on bilayer surfaces, even at the nanomolar concentration range. The efficiency of the aggregation process depends on the membrane composition, with the greatest efficiency observed for of 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-l-serine (POPS). Importantly, assembled aggregates can dissociate from the surface, suggesting that on-surface aggregation is a mechanism by which pathological aggregates may be produced. Computational modeling revealed that dimers of α-syn assembled rapidly, through the membrane-bound monomer on POPS bilayer, due to an aggregation-prone orientation of α-syn. Interaction of α-syn with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) leads to a binding mode that does not induce a fast assembly of the dimer. Based on these findings, we propose a model in which the interaction of α-syn with membranes plays a critical role initiating the formation of α-syn aggregates and the overall aggregation process.     

Electrostatic and hydrophobic interactions of lipid-associated α-synuclein: The role of a water-limited interfaces in amyloid fibrillation

Human α‑synuclein (αSyn) is an intrinsically disordered protein (IDP) whose biological and pathological functions in brain neuronal cells have not yet been fully elucidated. αSyn intrinsically participates in aiding neurotransmitter trafficking through αSyn the association with lipid membranes. However, lipid-associated states of αSyn also induce amyloid self-assembly that is linked to the pathogenesis of various synucleinopathies. These contradicting actions arise from the limited water content near lipid-water interfaces that controls αSyn electrostatic and hydrophobic interactions. Thus, understanding the molecular interactions between αSyn and lipid membranes in the presence of water molecules is critical in elucidating the pivotal role of lipid-associated αSyn in amyloid self-assembly. In this review, we describe how the membrane interface controls electrostatic and hydrophobic interactions of lipid-associated αSyn. Moreover, membrane amyloid self-assembly of αSyn will be further discussed with regards to the structural dynamics of lipid-associated αSyn and water molecules near the interface.