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Fibrils of the intrinsically-disordered protein α-synuclein are hallmarks of Parkinson's disease. The fluorescent dye thioflavin T is often used to characterize fibrillation, but this assay may not provide quantitative information about structure and mechanism. To gain such information, we incorporated the 19F-labeled amino acid, 3-fluorotyrosine, into recombinant human α-synuclein at its endogenous tyrosine residues. Tyrosine 39 is in the positively-charged N-terminal region of this 140-residue protein. The other three, tyrosines, 125, 133, and 136, are near the C-terminus. 19F-nuclear magnetic resonance spectroscopy was used to study several properties of labeled α-synuclein, including its conformation; conformational changes induced by urea, spermine, and sodium dodecyl sulfate (SDS); its interaction with SDS micelles; and the kinetics of fibril formation. The results show that the tyrosines are in disordered regions but that there is some structure near position 39 that is disrupted by urea. SDS binding alters the conformation near position 39, but the C-terminal tyrosines are disordered under all conditions. The NMR data also indicate that SDS-micelle bound α-synuclein and the free protein exchange on the 10-ms time scale. Studies of fibrillation show the utility of 19F-labeled NMR. The data indicate that fibrillation is not accompanied by the formation of large quantities of low molecular-weight intermediates. Although dye-binding and 19F NMR data show that 1-mM SDS and 1-mM spermine accelerate aggregation compared to buffer alone, only the NMR data indicate that the species formed in SDS are smaller than those formed in buffer or buffer plus spermine. We conclude that 19F NMR spectroscopy is useful for obtaining residue-level, quantitative information about the structure, binding, and aggregation of α-synuclein.

α-synuclein is the major component of filamentous Lewy bodies found in the brains of patients diagnosed with Parkinson’s disease. Recent studies demonstrate that, in addition to the wild-type sequence, α-synuclein is found in several modified forms, including truncated and phosphorylated species. Although the mechanism by which the neuronal loss in Parkinson’s disease occurs is unknown, aggregation and fibril formation of α-synuclein is considered to be a key pathological feature. In this study we analyze the rates of fibril formation and the monomer-fibril equilibrium for eight disease-associated truncated and phosphorylated α-synuclein variants. Comparison of the relative rates of aggregation reveals a strong monotonic relationship between the C-terminal charge of α-synuclein 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-synuclein polymerization and may provide a target for the treatment of Parkinson’s Disease.

Oxidative stress and aggregation of the protein α-synuclein are thought to be key factors in Parkinson’s disease. Previous work shows that cytochrome c plus H2O2 causes tyrosine-dependent in vitro peroxidative aggregation of proteins, including α-synuclein. Here, we examine the role of each of α-synuclein’s four tyrosine residues and how the protein’s conformation affects covalent oxidative aggregation. When α-synuclein adopts a collapsed conformation, tyrosine 39 is essential for wild-type-like covalent aggregation. This lone N-terminal tyrosine, however, is not required for wild type-like covalent aggregation in the presence of a denaturant or when α-synuclein is present in non-covalent fibrils. We also show that pre-formed oxidative aggregates are not incorporated into non-covalent fibrils. These data provide insight as to how dityrosine may be formed in Lewy bodies seen in Parkinson’s disease.

Background

Parkinson's disease (PD) involves the selective damage of dopaminergic neuron cells resulting from the accumulation and fibril formation of alpha-synuclein. Recently, it has been shown that not only full-length alpha-synuclein, but also C-terminal truncated forms exist in the normal brain, as well as Lewy bodies, which are cytoplasmic inclusions in PD. It is known that truncated alpha-synuclein has a much higher ability to aggregate and fibrillate than full-length alpha-synuclein. Since the fibrils and precursor oligomers of alpha-synuclein are cytotoxic to the neuron, inhibitors that prevent the formation of oligomers and/or fibrils might open the way to a novel therapeutic approach to PD. However, no inhibitor for truncated alpha-synuclein has been reported yet.

Results

In this study, we first characterized the aggregation and cytotoxicity of C-truncated alpha-synuclein119 and alpha-synuclein133 which have been found in both the normal and the pathogenic brain. Alpha-synuclein119 aggregated more rapidly and enhanced significantly the fibril formation of alpha-synuclein. Although both of alpha-synuclein119 and alpha-synuclein133 showed a high cytotoxicity, alpha-synuclein133 showed a similar aggregation with full-length alpha-synuclein and no acceleration effect. We showed that PQQ dramatically inhibits the fibril formation of C-terminal truncated alpha-synuclein110119, and 133 as well as the mixtures of full-length alpha-synuclein with these truncated variants. Moreover, PQQ decreases the cytotoxicity of truncated alpha-synuclein.

Conclusions

Our results demonstrate that PQQ inhibits the amyloid fibril formation and cytotoxicity of the C-truncated alpha-synuclein variants. We believe that PQQ is a strong candidate for a reagent compound in the treatment of PD.

Indolic derivatives can affect fibril growth of amyloid forming proteins. The neurotransmitter serotonin (5-HT) is of particular interest, as it is an endogenous molecule with a possible link to neuropsychiatric symptoms of Parkinson disease. A key pathomolecular mechanism of Parkinson disease is the misfolding and aggregation of the intrinsically unstructured protein α-synuclein. We performed a biophysical study to investigate an influence between these two molecules. In an isolated in vitro system, 5-HT interfered with α-synuclein amyloid fiber maturation, resulting in the formation of partially structured, SDS-resistant intermediate aggregates. The C-terminal region of α-synuclein was essential for this interaction, which was driven mainly by electrostatic forces. 5-HT did not bind directly to monomeric α-synuclein molecules and we propose a model where 5-HT interacts with early intermediates of α-synuclein amyloidogenesis, which disfavors their further conversion into amyloid fibrils.

Research highlights

► The neurotransmitter serotonin (5-HT) suppresses amyloid fibril growth of alpha-synuclein (AS). ► 5-HT binds to intermediate aggregates of alpha-synuclein, not to monomeric AS. Consequently, 5-HT does not influence initial steps of amyloidogenesis. ► 5-HT promotes the accumulation of partially structured, SDS-resistant “on pathway” aggregates of AS. ► The C-terminal region of AS is essential for a charge dependent interaction. ► “On pathway” and “off-pathway” aggregations of AS might mechanistically overlap.

The aggregation of α-synuclein into amyloid fibrils constitutes a key step in the onset of Parkinson's disease. Amyloid fibrils of α-synuclein are the major component of Lewy bodies, histological hallmarks of the disease. Little is known about the mechanism of aggregation of α-synuclein. During this process, α-synuclein forms transient intermediates that are considered to be toxic species. The dimerization of α-synuclein could represent a rate-limiting step in the aggregation of the protein. Here, we analyzed four covalent dimers of α-synuclein, obtained by covalent link of the N-terms, C-terms, tandem cloning of two sequences and tandem juxtaposition in one protein of the 1–104 and 29–140 sequences. Their biophysical properties in solution were determined by CD, FT-IR and NMR spectroscopies. SDS-induced folding was also studied. The fibrils formation was analyzed by ThT and polarization fluorescence assays. Their morphology was investigated by TEM and AFM-based quantitative morphometric analysis. All dimers were found to be devoid of ordered secondary structure under physiological conditions and undergo α-helical transition upon interaction with SDS. All protein species are able to form amyloid-like fibrils. The reciprocal orientation of the α-synuclein monomers in the dimeric constructs affects the kinetics of the aggregation process and a scale of relative amyloidogenic propensity was determined. Structural investigations by FT IR spectroscopy, and proteolytic mapping of the fibril core did not evidence remarkable difference among the species, whereas morphological analyses showed that fibrils formed by dimers display a lower and diversified level of organization in comparison with α-synuclein fibrils. This study demonstrates that although α-synuclein dimerization does not imply the acquisition of a preferred conformation by the participating monomers, it can strongly affect the aggregation properties of the molecules. The results presented highlight a substantial role of the relative orientation of the individual monomer in the definition of the fibril higher structural levels.

In the Parkinson’s disease-associated state, α-synuclein (α-syn) undergoes large conformational changes forming ordered, β-sheet containing fibrils. To unravel the role of specific residues during the fibril assembly process, we prepared single-Cys mutants in the disordered (G7C and Y136C) and proximal (V26C and L100C) fibril core sites and derivatized them with environment sensitive dansyl (Dns) fluorophores. Dns fluorescence exhibits residue-specificity in spectroscopic properties as well as kinetic behavior; early kinetic events were revealed by probes located at positions 7 and 136 compared to those positioned at 26 and 100.

α-Synuclein is an abundant highly charged protein that is normally predominantly localized around synaptic vesicles in presynatic terminals. Although the function of this protein is still ill-defined, genetic studies have demonstrated that point mutations or genetic alteration (duplications or triplications) that increase the number of copies of the α-synuclein (SCNA) gene can cause Parkinson’s disease or the related disorder dementia with Lewy bodies. α-Synuclein can aberrantly polymerize into fibrils with typical amyloid properties, and these fibrils are the major component of many types of pathological inclusions, including Lewy bodies, which are associated with neurodegenerative diseases, such as Parkinson’s disease. Genetic studies have clearly established that alteration in the α-synuclein gene can lead to neuronal demise. Although there is substantial evidence supporting the toxic nature of α-synuclein inclusions, other modes of toxicity such as oligomers have been proposed. In this review, some of the evidence for the different mechanisms of α-synuclein toxicity is presented and discussed.

α-Synuclein participates in the Lewy body formation of Parkinson's disease. Elucidation of the underlying molecular mechanism of the amyloid fibril formation is crucial not only to develop a controlling strategy toward the disease, but also to apply the protein fibrils for future biotechnology. Discernable homogeneous granules of α-synuclein composed of approximately 11 monomers in average were isolated in the middle of a lag phase during the in vitro fibrillation process. They were demonstrated to experience almost instantaneous fibrillation during a single 12-min centrifugal membrane-filtration at 14,000×g. The granular assembly leading to the drastically accelerated fibril formation was demonstrated to be a result of the physical influence of shear force imposed on the preformed granular structures by either centrifugal filtration or rheometer. Structural rearrangement of the preformed oligomomeric structures is attributable for the suprastructure formation in which the granules act as a growing unit for the fibril formation. To parallel the prevailing notion of nucleation-dependent amyloidosis, we propose a double-concerted fibrillation model as one of the mechanisms to explain the in vitro fibrillation of α-synuclein, in which two consecutive concerted associations of monomers and subsequent oligomeric granular species are responsible for the eventual amyloid fibril formation.

Several studies have shown that catecholamines can inhibit the fibrillation of α-synuclein, a small presynaptic protein whose aggregation is believed to be a critical step in the etiology of Parkinson's disease and several other neurodegenerative disorders. However, the mechanism of this inhibition is uncertain. We show here that substoichiometric concentrations of DOPAC (3, 4-dihydroxyphenylacetic acid), a normal product of the metabolism of dopamine, can inhibit the fibrillation of α-synuclein (α-Syn), due to non-covalent binding of DOPAC to α-Syn monomer. Intriguingly, the presence of α-synuclein accelerates the spontaneous oxidation of DOPAC, and the oxidized form of DOPAC (the quinone) is responsible for the fibrillation inhibition. In addition, the presence of DOPAC leads to the oxidation of the methionine residues of α-Syn, probably due to the H2O2 production as a by-product of DOPAC oxidation. The lack of fibrillation results from the formation of stable oligomers, which are very similar to those observed transiently at early of the α-Syn fibrillation. A possible explanation for this phenomenon is that DOPAC stabilizes the normally transient oligomers and prevents them from subsequent fibril formation. The analysis of the α-synuclein Y39W variant suggests that DOPAC binds non-covalently to the same N-terminal region of α-Syn as lipid vesicles, probably in the vicinity of residue 39. In contrast to the compounds with 1,2-dihydroxyphenyl groups (DOPAC, catechol), their 1,4-dihydroxyphenyl isomers (hydroquinone, homogentisic acid) are able to modify α-Syn covalently, probably due to the less steric hindrance in the Michael addition.

The aggregation and fibrillation of α-synuclein has been implicated as a causative factor in the Parkinson's disease. The hexamer motif KTKEGV is found in each of the seven imperfect repeat sequences in the N-terminal half of α-synuclein. The motif is not fully conserved in the sixth and seventh repeats. We created mutants in which the motif was repaired to be fully conserved in either (Rep6 and Rep7) or both (Rep67) of these two repeats. The Rep6 and Rep67 mutants showed a greatly reduced propensity to aggregate and fibrillate while all three mutants showed greater resistance to HFIP-induced formation of the α-helix intermediate. Resistance to formation in the partially folded intermediate may repress the folding of α-synuclein, consequently interfering with the aggregation and fibril formation. These results demonstrated that KTKEGV repeats may have a significant role in keeping native unfolded status of α-synuclein.

The intracellular oligomerization of α-synuclein is associated with Parkinson's disease and appears to be an important target for disease-modifying treatment. Yet, to date, there is no specific inhibitor for this aggregation process. Using unbiased systematic peptide array analysis, we indentified molecular interaction domains within the β-synuclein polypeptide that specifically binds α-synuclein. Adding such peptide fragments to α-synuclein significantly reduced both amyloid fibrils and soluble oligomer formation in vitro. A retro-inverso analogue of the best peptide inhibitor was designed to develop the identified molecular recognition module into a drug candidate. While this peptide shows indistinguishable activity as compared to the native peptide, it is stable in mouse serum and penetrates α-synuclein over-expressing cells. The interaction interface between the D-amino acid peptide and α-synuclein was mapped by Nuclear Magnetic Resonance spectroscopy. Finally, administering the retro-inverso peptide to a Drosophila model expressing mutant A53T α-synuclein in the nervous system, resulted in a significant recovery of the behavioral abnormalities of the treated flies and in a significant reduction in α-synuclein accumulation in the brains of the flies. The engineered retro-inverso peptide can serve as a lead for developing a novel class of therapeutic agents to treat Parkinson's disease.

Summary

To investigate the α-synuclein protein and its role in Parkinson’s disease, we screened a library of random point mutants both in vitro and in yeast to find variants in an unbiased way that could help us understand the sequence-phenotype relationship. We developed a rapid purification method that allowed us to screen 59 synuclein mutants in vitro and discovered two double point mutants that fibrillized slowly relative to wild type, A30P, and A53T α-synucleins. The yeast toxicity of all of these proteins was measured and we found no correlation with fibrillization rate, suggesting that fibrillization is not necessary for synuclein-induced yeast toxicity. We also found that β-synuclein was of intermediate toxicity to yeast and γ-synuclein was non-toxic. Coexpression of Parkinson’s disease related genes DJ-1, parkin, Pink1, UCH-L1, or synphilin, with synuclein, did not affect synuclein toxicity. A second screen, of several thousand library clones in yeast, identified 25 non-toxic α-synuclein sequence variants. Most of these contained a mutation to either proline or glutamic acid that caused a defect in membrane binding. We hypothesize that yeast toxicity is caused by synuclein binding directly to membranes at levels sufficient to non-specifically disrupt homeostasis.

α-Synuclein (AS) fibrils are the major component of Lewy bodies, the pathological hallmark of Parkinson’s disease (PD). Here, we use results from an extensive investigation employing solid-state NMR to present a detailed structural characterization and conformational dynamics quantification of full-length AS fibrils. Our results show that the core extends with a repeated structural motif. This result disagrees with the previously proposed fold of AS fibrils obtained with limited solid-state NMR data. Additionally, our results demonstrate that the three single point mutations associated with early-onset PD—A30P, E46K and A53T—are located in structured regions. We find that E46K and A53T mutations, located in rigid β-strands of the wild-type fibrils, are associated with major and minor structural perturbations, respectively.

We have previously reported that pyrroloquinoline quinone (PQQ) prevents the amyloid formation of α-synuclein, amyloid β1–42 (Aβ1–42), and mouse prion protein. Moreover, PQQ-modified α-synuclein and a proteolytic fragment of the PQQ-modified α-synuclein are able to inhibit the amyloid formation of α-synuclein. Here, we identified the peptide sequences that play an important role as PQQ-modified specific peptide inhibitors of α-synuclein. We demonstrate that the PQQ-modified α-Syn36–46 peptide, which is a partial sequence of α-synuclein, prevented α-synuclein amyloid fibril formation but did not inhibit Aβ1–42 fibril formation. In addition, the α-synuclein partial peptide modified with other small-molecule inhibitors, Baicalein and epigallocatechin gallate (EGCG), prevented α-synuclein fibril formation. Currently reported quinone amyloid inhibitors do not have selectivity toward protein molecules. Therefore, our achievements provide a novel strategy for the development of targeted specific amyloid formation inhibitors: the combination of quinone compounds with specific peptide sequence from target proteins involved in amyloid formation.

Fibrillar αSynuclein is the major constituent of Lewy bodies and Lewy neurites, the protein deposits characteristic for Parkinson’s disease (PD). Multiplications of the αSynuclein gene, as well as point mutations cause familial PD. However, the exact role of αSynuclein in neurodegeneration remains uncertain. Recent research in invertebrates has suggested that oligomeric rather than fibrillizing αSynuclein mediates neurotoxicity. To investigate the impact of αSynuclein aggregation on the progression of neurodegeneration, we expressed variants with different fibrillation propensities in the rat substantia nigra (SN) by means of recombinant adeno-associated viral (AAV) vectors. The formation of proteinase K-resistant αSynuclein aggregates was correlated to the loss of nigral dopaminergic (DA) neurons and striatal fibers. Expression of two prefibrillar, structure-based design mutants of αSynuclein (i.e., A56P and A30P/A56P/A76P) resulted in less aggregate formation in nigral DA neurons as compared to human wild-type (WT) or the inherited A30P mutation. However, only the αSynuclein variants capable of forming fibrils (WT/A30P), but not the oligomeric αSynuclein species induced a sustained progressive loss of adult nigral DA neurons. These results demonstrate that divergent modes of αSynuclein neurotoxicity exist in invertebrate and mammalian DA neurons in vivo and suggest that fibrillation of αSynuclein promotes the progressive degeneration of nigral DA neurons as found in PD patients.

α-Synuclein (α-syn) is a 140-amino acid protein that plays a central role in the pathogenesis of Parkinson’s disease (PD) and other synucleinopathies. However, the molecular determinants that are responsible for triggering and/or propagating α-syn aggregation and toxicity remain poorly understood. Several studies have suggested that there are direct interactions between different metals and α-syn, but the role of metal ions and α-syn in the pathogenesis of PD is not firmly established. Interestingly, the majority of disease-associated post-translational modifications (PTMs) (e.g., truncation, phosphorylation, and nitration) of α-syn occur at residues within the C-terminal region (Y125, S129, Y133, and Y136) and in very close proximity to the putative metal binding sites. Therefore, we hypothesized that phosphorylation within this domain could influence the α-syn–metal interactions. In this paper, we sought to map the interactions between the di- and trivalent cations, Cu(II), Pb(II), Fe(II), and Fe(III), and the C-terminal region of α-syn encompassing residues 107–140 and to determine how phosphorylation at S129 or Y125 alters the specificity and binding affinity of metals using electrospray ionization-mass spectrometry (ESI-MS) and fluorescence spectroscopy. We demonstrate that D115-M116 and P128-S129 act as additional Cu(II) binding sites and show for the first time that the residues P128-S129 and D119 are also involved in Pb(II) and Fe(II) coordination, although D119 is not essential for binding to Fe(II) and Pb(II). Furthermore, we demonstrate that phosphorylation at either Y125 or S129 increases the binding affinity of Cu(II), Pb(II), and Fe(II), but not Fe(III). Additionally, we also show that phosphorylations at these residues lead to a shift in the binding sites of metal ions from the N-terminus to the C-teminus. Together, our findings provide critical insight into and expand our understanding of the molecular and structural bases underlying the interactions between α-syn and metal ions, including the identification of novel metal binding sites, and highlight the potential importance of cross-talk between post-translational modifications and metal ion binding in modulating α-syn functional and aggregation properties that are regulated by its C-terminal domain.

α-Synuclein (α-Syn) amyloid filaments are the major ultrastructural component of pathological inclusions that define several neurodegenerative disorders, including Parkinson disease and other disorders that are collectively termed synucleinopathies. Since the aggregation of α-syn is associated with the etiology of these diseases, defining the molecular elements that influence this process may have important therapeutics implication. The deletions of major portions of the hydrophobic region of α-syn (Δ74-79 and Δ71-82) impair the ability to form amyloid. However, mutating residue E83 to an A restored the ability of these proteins to form amyloid. Additionally supporting an inhibitory role of residue E83 on amyloid formation, mutating this residue to an A enhanced amyloid formation in the presence of small molecule inhibitors, such as dopamine and EGCG. Our data, therefore, suggest that the presence and placement of the highly charged E83 residue plays a significant inhibitory role in α-syn amyloid formation and these findings provide important insights in the planning of therapeutic agents that may be capable of preventing α-syn amyloid formation.

Intraneuronal depositions of α-synuclein have been implicated in the pathogenesis of Parkinsons's disease (PD). Previous reports have identified the crosslinking between α-synuclein and tTG (tissue transglutaminase) in both PD patients and the cellular model. However, no researches have been conducted to further investigate their interaction in physiological conditions. To address this question, we generated the SH-SY5Y cell line which stably expressed the wild-type or mutant (Ser129Ala) α-synuclein. After the treatment with okadaic acid, α-synuclein started forming aggregates upon the activation of tTG. Coimmunoprecipitation assays revealed a decreased interaction of the mutant α-synuclein S129A with tTG compared with the wild-type α-synuclein. Cells expressing the wild-type α-synuclein showed increased eosinophilic cytoplasmic inclusion bodies that resembled Lewy bodies compared with the mutant. Double immunofluorescence staining confirmed the colocalization of the phosphorylated α-synuclein and the tTG in the cells. The S129A mutant demonstrated a lesser degree of colocalization than the wild type.

α-Synuclein is a major component of Lewy bodies in Parkinson's disease. Although no signal sequence is apparent, α-synuclein expressed in Escherichia coli is mostly located in the periplasm. The possibilities that α-synuclein translocated into the periplasm across the inner membrane by the SecA or the Tat targeting route identified in bacteria and that α-synuclein was released through MscL were excluded. The signal recognition particle-dependent pathway is involved in the translocation of α-synuclein. The C-terminal 99-to-140 portion of the α-synuclein molecule plays a signal-like role for its translocation into the periplasm, cooperating with the central 61-to-95 section. The N-terminal 1-to-60 region is not required for this translocation.

Both oxidative stress and aggregation of the protein α-synuclein (aS) have been implicated as key factors in the etiology of Parkinson’s disease. Specifically, oxidative modifications to aS disrupt its binding to lipid membranes, an interaction considered critical to its native function. Here we seek to provide a mechanistic explanation for this phenomenon by investigating the effects of oxidative nitration of tyrosine residues on the structure of aS and its interaction with lipid membranes. Membrane binding is mediated by the first ~95 residues of aS. We find that nitration of the single tyrosine (Y39) in this domain disrupts binding due to electrostatic repulsion. Moreover, we observe that nitration of the three tyrosines (Y125/133/136) in the C-terminal domain is equally effective in perturbing binding, an intriguing result given that the C-terminus is not thought to interact directly with membranes. Our investigations show that tyrosine nitration results in a change of the conformational states populated by aS in solution, with the most prominent changes occurring in the C-terminal region. These results lead us to suggest that nitration of Y125/133/136 reduces the membrane binding affinity of aS through allosteric coupling by altering the ensemble of conformational states and depopulating those capable of membrane binding. While allostery is a well-established concept for structured proteins, it has only recently been discussed in the context of disordered proteins. We propose that allosteric regulation through modification of specific residues in, or ligand binding to, the C-terminus may even be a general mechanism for modulating aS function.

The function of α-synuclein, a soluble protein abundant in the brain and concentrated at presynaptic terminals, is still undefined. Yet, α-synuclein overexpression and the expression of its A30P mutant are associated with familial Parkinson's disease. Working in cell-free conditions, in two cell lines as well as in primary neurons we demonstrate that α-synuclein and its A30P mutant have different effects on actin polymerization. Wild-type α-synuclein binds actin, slows down its polymerization and accelerates its depolymerization, probably by monomer sequestration; A30P mutant α-synuclein increases the rate of actin polymerization and disrupts the cytoskeleton during reassembly of actin filaments. Consequently, in cells expressing mutant α-synuclein, cytoskeleton-dependent processes, such as cell migration, are inhibited, while exo- and endocytic traffic is altered. In hippocampal neurons from mice carrying a deletion of the α-synuclein gene, electroporation of wild-type α-synuclein increases actin instability during remodeling, with growth of lamellipodia-like structures and apparent cell enlargement, whereas A30P α-synuclein induces discrete actin-rich foci during cytoskeleton reassembly. In conclusion, α-synuclein appears to play a major role in actin cytoskeletal dynamics and various aspects of microfilament function. Actin cytoskeletal disruption induced by the A30P mutant might alter various cellular processes and thereby play a role in the pathogenesis of neurodegeneration.

Human α-Synuclein (αSyn) is a natively unfolded protein whose aggregation into amyloid fibrils is involved in the pathology of Parkinson disease. A full comprehension of the structure and dynamics of early intermediates leading to the aggregated states is an unsolved problem of essential importance to researchers attempting to decipher the molecular mechanisms of αSyn aggregation and formation of fibrils. Traditional bulk techniques used so far to solve this problem point to a direct correlation between αSyn's unique conformational properties and its propensity to aggregate, but these techniques can only provide ensemble-averaged information for monomers and oligomers alike. They therefore cannot characterize the full complexity of the conformational equilibria that trigger the aggregation process. We applied atomic force microscopy–based single-molecule mechanical unfolding methodology to study the conformational equilibrium of human wild-type and mutant αSyn. The conformational heterogeneity of monomeric αSyn was characterized at the single-molecule level. Three main classes of conformations, including disordered and “β-like” structures, were directly observed and quantified without any interference from oligomeric soluble forms. The relative abundance of the “β-like” structures significantly increased in different conditions promoting the aggregation of αSyn: the presence of Cu2+, the pathogenic A30P mutation, and high ionic strength. This methodology can explore the full conformational space of a protein at the single-molecule level, detecting even poorly populated conformers and measuring their distribution in a variety of biologically important conditions. To the best of our knowledge, we present for the first time evidence of a conformational equilibrium that controls the population of a specific class of monomeric αSyn conformers, positively correlated with conditions known to promote the formation of aggregates. A new tool is thus made available to test directly the influence of mutations and pharmacological strategies on the conformational equilibrium of monomeric αSyn.

Author Summary

Natively unstructured proteins defy the classical “one sequence–one structure” paradigm of protein science. In pathological conditions, monomers of these proteins can aggregate in the cell, a process that underlies neurodegenerative diseases such as Alzheimer and Parkinson. A key step in the aggregation process—the formation of misfolded intermediates—remains obscure. To shed light on this process, we characterized the folding and conformational diversity of αSyn, a natively unstructured protein involved in Parkinson disease, by mechanically stretching single molecules of this protein and recording their mechanical properties. These experiments permitted us to observe directly and quantify three main classes of conformations that, under in vitro physiological conditions, exist simultaneously in the αSyn sample. We found that one class of conformations, “β-like” structures, is directly related to αSyn aggregation. In fact, their relative abundance increases drastically in three different conditions known to promote the formation of αSyn fibrils. We expect that a critical concentration of αSyn with a “β-like” structure must be reached to trigger fibril formation. This critical concentration is therefore controlled by a chemical equilibrium. Novel pharmacological strategies can now be tailored to act upstream, before the aggregation process ensues, by targeting this equilibrium. To this end, single-molecule force spectroscopy can be an effective tool to tailor and test new pharmacological agents.

A single-molecule study detects structured and unstructured conformers in equilibrium in monomeric α-synuclein. The β-like conformers increase with pathological mutations and under other conditions known to promote aggregation.

Background

Oligomerization and aggregation of α-synuclein molecules play a major role in neuronal dysfunction and loss in Parkinson's disease [1]. However, α-synuclein oligomerization and aggregation have mostly been detected indirectly in cells using detergent extraction methods [2], [3], [4]. A number of in vitro studies showed that dopamine can modulate the aggregation of α-synuclein by inhibiting the formation of or by disaggregating amyloid fibrils [5], [6], [7].

Methodology/Principal Findings

Here, we show that α-synuclein adopts a variety of conformations in primary neuronal cultures using fluorescence lifetime imaging microscopy (FLIM). Importantly, we found that dopamine, but not dopamine agonists, induced conformational changes in α-synuclein which could be prevented by blocking dopamine transport into the cell. Dopamine also induced conformational changes in α-synuclein expressed in neuronal cell lines, and these changes were also associated with alterations in oligomeric/aggregated species.

Conclusion/Significance

Our results show, for the first time, a direct effect of dopamine on the conformation of α-synuclein in neurons, which may help explain the increased vulnerability of dopaminergic neurons in Parkinson's disease.

In Parkinson disease, wild-type α-synuclein accumulates during aging, whereas α-synuclein mutations lead to an early onset and accelerated course of the disease. The generation of new neurons is decreased in regions of neurogenesis in adult mice overexpressing wild-type human α-synuclein. We examined the subventricular zone/olfactory bulb neurogenesis in aged mice expressing either wild-type human or A53T mutant α-synuclein. Aging wild-type and mutant α-synuclein-expressing animals generated significantly fewer new neurons than their non-transgenic littermates. This decreased neurogenesis was caused by a reduction in cell proliferation within the subventricular zone of mutant α-synuclein mice. In contrast, no difference was detected in mice overexpressing the wild-type allele. Also, more TUNEL-positive profiles were detected in the subventricular zone, following mutant α-synuclein expression and in the olfactory bulb, following wild-type and mutant α-synuclein expression. The impaired neurogenesis in the olfactory bulb of different transgenic α-synuclein mice during aging highlights the need to further explore the interplay between olfactory dysfunction and neurogenesis in Parkinson disease.