Calcium(II) selectively induces alpha-synuclein annular oligomers via interaction with the C-terminal domain

Alpha-synuclein filaments are the major component of intracytoplasmic inclusion bodies characteristic of Parkinson's disease and related disorders. The process of alpha-synuclein filament formation proceeds via intermediate or protofibrillar species, each of which may be cytotoxic. Because high levels of calcium(II) and other metal ions may play a role in disease pathogenesis, we investigated the influence of calcium and other metals on alpha-synuclein speciation. Here we report that calcium(II) and cobalt(II) selectively induce the rapid formation of discrete annular alpha-synuclein oligomeric species. We used atomic force microscopy to monitor the aggregation state of alpha-synuclein after 1 d at 4 degrees C in the presence of a range of metal ions compared with the filament formation pathway in the absence of metal ions. Three classes of effect were observed with different groups of metal ions: (1) Copper(II), iron(III), and nickel(II) yielded 0.8-4 nm spherical particles, similar to alpha-synuclein incubated without metal ions; (2) magnesium(II), cadmium(II), and zinc(II) gave larger, 5-8 nm spherical oligomers; and, (3) cobalt(II) and calcium(II) gave frequent annular oligomers, 70-90 nm in diameter with calcium(II) and 22-30 nm in diameter with cobalt(II). In the absence of metal ions, annular oligomers ranging 45-90 nm in diameter were observed after 10 d incubation, short branched structures appeared after a further 3 wk and extended filaments after 2-3 mo. Previous studies have shown that alpha-synuclein calcium binding is mediated by the acidic C terminus. We found that truncated alpha-synuclein (1-125), lacking the C-terminal 15 amino acids, did not form annular oligomers upon calcium addition, indicating the involvement of the calcium-binding domain.     

Certain metals trigger fibrillation of methionine-oxidized alpha-synuclein

The aggregation and fibrillation of alpha-synuclein has been implicated as a key step in the etiology of Parkinson's disease and several other neurodegenerative disorders. In addition, oxidative stress and certain environmental factors, including metals, are believed to play an important role in Parkinson's disease. Previously, we have shown that methionine-oxidized human alpha-synuclein does not fibrillate and also inhibits fibrillation of unmodified alpha-synuclein (Uversky, V. N., Yamin, G., Souillac, P. O., Goers, J., Glaser, C. B., and Fink, A. L. (2002) FEBS Lett. 517, 239-244). Using dynamic light scattering, we show that the inhibition results from stabilization of the monomeric form of Met-oxidized alpha-synuclein. We have now examined the effect of several metals on the structural properties of methionine-oxidized human alpha-synuclein and its propensity to fibrillate. The presence of metals induced partial folding of both oxidized and non-oxidized alpha-synucleins, which are intrinsically unstructured under conditions of neutral pH. Although the fibrillation of alpha-synuclein was completely inhibited by methionine oxidation, the presence of certain metals (Ti3+, Zn2+, Al3+, and Pb2+) overcame this inhibition. These findings indicate that a combination of oxidative stress and environmental metal pollution could play an important role in triggering the fibrillation of alpha-synuclein and thus possibly Parkinson's disease.     

Metal-triggered structural transformations, aggregation, and fibrillation of human alpha-synuclein. A possible molecular NK between Parkinson's disease and heavy metal exposure.

Parkinson's disease involves the aggregation of alpha-synuclein to form fibrils, which are the major constituent of intracellular protein inclusions (Lewy bodies and Lewy neurites) in dopaminergic neurons of the substantia nigra. Occupational exposure to specific metals, especially manganese, copper, lead, iron, mercury, zinc, aluminum, appears to be a risk factor for Parkinson's disease based on epidemiological studies. Elevated levels of several of these metals have also been reported in the substantia nigra of Parkinson's disease subjects. We examined the effect of various metals on the kinetics of fibrillation of recombinant alpha-synuclein and in inducing conformational changes, as monitored by biophysical techniques. Several di- and trivalent metal ions caused significant accelerations in the rate of alpha-synuclein fibril formation. Aluminum was the most effective, along with copper(II), iron(III), cobalt(III), and manganese(II). The effectiveness correlated with increasing ion charge density. A correlation was noted between efficiency in stimulating fibrillation and inducing a conformational change, ascribed to formation of a partially folded intermediate. The potential for ligand bridging by polyvalent metal ions is proposed to be an important factor in the metal-induced conformational changes of alpha-synuclein. The results indicate that low concentrations of some metals can directly induce alpha-synuclein fibril formation.     

PA700, the regulatory complex of the 26S proteasome, interferes with alpha-synuclein assembly

Parkinson's disease is characterized by the loss of dopaminergic neurons in the nigrostriatal pathway accompanied by the presence of intracellular cytoplasmic inclusions, termed Lewy bodies. Fibrillized alpha-synuclein forms the major component of Lewy bodies. We reported a specific interaction between rat alpha-synuclein and tat binding protein 1, a subunit of PA700, the regulatory complex of the 26S proteasome. It has been demonstrated that PA700 prevents the aggregation of misfolded, nonubiquinated substrates. In this study, we examine the effect of PA700 on the aggregation of wild-type and A53T mutant alpha-synuclein. PA700 inhibits both wild-type and A53T alpha-synuclein fibril formation as measured by Thioflavin T fluorescence. Using size exclusion chromatography, we present evidence for a stable PA700-alpha-synuclein complex. Sedimentation analyses reveal that PA700 sequesters alpha-synuclein in an assembly incompetent form. Analysis of the morphology of wild-type and A53T alpha-synuclein aggregates during the course of fibrillization by electron microscopy demonstrate the formation of amyloid-like fibrils. Secondary structure analyses of wild-type and A53T alpha-synuclein assembled in the presence of PA700 revealed a decrease in the overall amount of assembled alpha-synuclein with no significant change in protein conformation. Thus, PA700 acts on alpha-synuclein assembly and not on the structure of fibrils. We hypothesize that PA700 sequesters alpha-synuclein oligomeric species that are the precursors of the fibrillar form of the protein, thus preventing its assembly into fibrils.     

The effect of macromolecular crowding on protein aggregation and amyloid fibril formation

Macromolecular crowding is expected to have several significant effects on protein aggregation; the major effects will be those due to excluded volume and increased viscosity. In this report we summarize data demonstrating that macromolecular crowding may lead to a dramatic acceleration in the rate of protein aggregation and formation of amyloid fibrils, using the protein alpha-synuclein. The aggregation of alpha-synuclein has been implicated as a critical factor in development of Parkinson's disease. Various types of polymers, from neutral polyethylene glycols and polysaccharides (Ficolls, dextrans) to inert proteins, are shown to accelerate alpha-synuclein fibrillation. The stimulation of fibrillation increases with increasing length of polymer, as well as increasing polymer concentration. At lower polymer concentrations (typically up to approximately 100 mg/ml) the major effect is ascribed to excluded volume, whereas at higher polymer concentrations evidence of opposing viscosity effects become apparent. Pesticides and metals, which are linked to increased risk of Parkinson's disease by epidemiological studies, are shown to accelerate alpha-synuclein fibrillation under conditions of molecular crowding.     

Methionine oxidation, alpha-synuclein and Parkinson's disease

The aggregation of normally soluble alpha-synuclein in the dopaminergic neurons of the substantia nigra is a crucial step in the pathogenesis of Parkinson's disease. Oxidative stress is believed to be a contributing factor in this disorder. Because it lacks Trp and Cys residues, mild oxidation of alpha-synuclein in vitro with hydrogen peroxide selectively converts all four methionine residues to the corresponding sulfoxides. Both oxidized and non-oxidized alpha-synucleins have similar unfolded conformations; however, the fibrillation of alpha-synuclein at physiological pH is completely inhibited by methionine oxidation. The inhibition results from stabilization of soluble oligomers of Met-oxidized alpha-synuclein. Furthermore, the Met-oxidized protein also inhibits fibrillation of unmodified alpha-synuclein. The degree of inhibition of fibrillation by Met-oxidized alpha-synuclein is proportional to the number of oxidized methionines. However, the presence of metals can completely overcome the inhibition of fibrillation of the Met-oxidized alpha-synuclein. Since oligomers of aggregated alpha-synuclein may be cytotoxic, these findings indicate that both oxidative stress and environmental metal pollution could play an important role in the aggregation of alpha-synuclein, and hence possibly Parkinson's disease. In addition, if the level of Met-oxidized alpha-synuclein was under the control of methionine sulfoxide reductase (Msr), then this could also be factor in the disease.     

Membrane association and protein conformation of alpha-synuclein in intact neurons. Effect of Parkinson's disease-linked mutations

Two missense mutations (Ala-30 --> Pro and Ala-53 --> Thr) in the gene encoding alpha-synuclein are associated with rare autosomal dominant forms of familial Parkinson's disease. In addition, alpha-synuclein is an abundant component of Lewy bodies in sporadic Parkinson's disease and diffuse Lewy body disease. However, the normal conformation of alpha-synuclein, its cellular localization in neurons, and the effects of the mutations remain to be determined. In the present study, we examine these questions using sensitive fluorescence resonance energy transfer techniques. Transient transfection of alpha-synuclein expression constructs into primary cortical neurons and counterstaining with the lipophilic fluorescent marker, DiI, demonstrates a close association between alpha-synuclein and cellular membranes. Both the N- and C-terminal regions of alpha-synuclein are tightly associated with membranes. A weak interaction also occurs between the N and C termini themselves. The Parkinson's disease-associated mutations have no effect on membrane interaction; however, the Ala-30 --> Pro mutation alters the three-dimensional conformation of alpha-synuclein, as measured by significantly increased fluorescence resonance energy transfer between the N and C termini.     

Alpha-synuclein adopts an alpha-helical conformation in the presence of polyunsaturated fatty acids to hinder micelle formation

Alpha-synuclein is a small cytosolic protein involved in the pathogenesis of Parkinson's disease and other neurodegenerative disorders. Recent studies suggested a lipid-related function for this brain-enriched protein. Since the brain carries a high level of docosahexaenoic acid (DHA) and since the extent of alpha-synuclein gene expression increases in response to DHA intake, we have investigated the interaction of alpha-synuclein with this essential omega-3 fatty acid. We show that alpha-synuclein allows DHA to be present in a soluble rather than micellar form. Upon interaction with DHA, the normally unstructured alpha-synuclein rapidly adopts an alpha-helical conformation. Prolonged exposure to DHA, however, gradually converts alpha-synuclein into amyloid-like fibrils. These results identify a potential biological function for alpha-synuclein and define an omega-3-linked pathway leading to alpha-synuclein aggregation.     

DNA induces folding in alpha-synuclein: understanding the mechanism using chaperone property of osmolytes

Alpha-synuclein conformational modulation leading to fibrillation has been centrally implicated in Parkinson's disease. Previously, we have shown that alpha-synuclein has DNA binding property. In the present study, we have characterized the effect of DNA binding on the conformation and fibrillation kinetics of alpha-synuclein. It was observed that single-stranded circular DNA induce alpha-helix conformation in alpha-synuclein while plasmid supercoiled DNA has dual effect inducing a partially folded conformation and alpha-helix under different experimental conditions. Interestingly, alpha-synuclein showed a specificity for GC* nucleotide sequence in its binding ability to DNA. The aggregation kinetics data showed that DNA which induced partially folded conformation in alpha-synuclein promoted the fibrillation while DNA which induced alpha-helix delayed the fibrillation, indicating that the partially folded intermediate conformation is critical in the aggregation process. Further, the mechanism of DNA-induced folding/aggregation of alpha-synuclein was studied using effect of osmolytes on alpha-synuclein as a model system. Among the five osmolytes used, Glycerol, trimethylamine-N-oxide, Betaine, and Taurine induced partially folded conformation and in turn enhanced the aggregation of alpha-synuclein. The ability of DNA and osmolytes in inducing conformational transition in alpha-synuclein, indicates that two factors are critical in modulating alpha-synuclein folding: (i) electrostatic interaction as in the case of DNA, and (ii) hydrophobic interactions as in the case of osmolytes. The property of DNA inducing alpha-helical conformation in alpha-synuclein and inhibiting the fibrillation may be of significance in engineering DNA-chip based therapeutic approaches to PD and other amyloid disorders.     

Membrane binding and self-association of alpha-synucleins

Although its function is unknown, alpha-synuclein is widely distributed in neural tissue and is the major component in the pathological aggregates found in patients with Parkinson's disease, Alzheimer's disease, Down's syndrome, and multiple system atrophy. In this report, we have quantified the binding alpha-synucleins to lipid membranes. In contrast to previous studies, we find, using real time equilibrium fluorescence methods, that alpha-synuclein binds strongly to large, unilamellar vesicles with either anionic or zwitterionic headgroups. Membrane binding is also strong for beta-synuclein, phosphorylated alpha-synuclein, and a synuclein mutant that is associated with familial Parkinson's disease. In solution at less than 400 nM, synuclein has a tendency to undergo concentration-dependent oligomerization as determined by changes in intrinsic fluorescence and fluorescence resonance energy transfer. Above this concentration, the protein begins to aggregate into structures visible by light scattering. Although membrane binding does not affect the secondary structure of alpha-synuclein, it greatly inhibits the ability of this protein to self-associate. Taken together, our results indicate that pathological conditions may be associated with a disruption in synuclein-membrane interactions.     

Nuclear localization of alpha-synuclein and its interaction with histones

The aggregation of alpha-synuclein is believed to play an important role in the pathogenesis of Parkinson's disease as well as other neurodegenerative disorders ("synucleinopathies"). However, the function of alpha-synuclein under physiologic and pathological conditions is unknown, and the mechanism of alpha-synuclein aggregation is not well understood. Here we show that alpha-synuclein forms a tight 2:1 complex with histones and that the fibrillation rate of alpha-synuclein is dramatically accelerated in the presence of histones in vitro. We also describe the presence of alpha-synuclein and its co-localization with histones in the nuclei of nigral neurons from mice exposed to a toxic insult (i.e., injections of the herbicide paraquat). These observations indicate that translocation into the nucleus and binding with histones represent potential mechanisms underlying alpha-synuclein pathophysiology.     

Alpha-synuclein up-regulation and aggregation during MPP+-induced apoptosis in neuroblastoma cells: intermediacy of transferrin receptor iron and hydrogen peroxide

1-Methyl-4-phenylpyridinium (MPP(+)) is a neurotoxin that causes Parkinson's disease in experimental animals and humans. Despite the fact that intracellular iron was shown to be crucial for MPP(+)-induced apoptotic cell death, the molecular mechanisms for the iron requirement remain unclear. We investigated the role of transferrin receptor (TfR) and iron in modulating the expression of alpha-synuclein (alpha-syn) in MPP(+)-induced oxidative stress and apoptosis. Results show that MPP(+) inhibits mitochondrial complex-1 and aconitase activities leading to enhanced H(2)O(2) generation, TfR expression and alpha-syn expression/aggregation. Pretreatment with cell-permeable iron chelators, TfR antibody (that inhibits TfR-mediated iron uptake), or transfection with glutathione peroxidase (GPx1) enzyme inhibits intracellular oxidant generation, alpha-syn expression/aggregation, and apoptotic signaling as measured by caspase-3 activation. Cells overexpressing alpha-syn exacerbated MPP(+) toxicity, whereas antisense alpha-syn treatment totally abrogated MPP(+)-induced apoptosis in neuroblastoma cells without affecting oxidant generation. The increased cytotoxic effects of alpha-syn in MPP(+)-treated cells were attributed to inhibition of mitogen-activated protein kinase and proteasomal function. We conclude that MPP(+)-induced iron signaling is responsible for intracellular oxidant generation, alpha-syn expression, proteasomal dysfunction, and apoptosis. Relevance to Parkinson's disease is discussed.     

Structural and functional implications of C-terminal regions of alpha-synuclein

Aggregation of alpha-synuclein is thought to play a major role in the pathogenesis of Parkinson's disease (PD), which is characterized by the presence of intracytoplasmic Lewy bodies (LB) in the brain. alpha-Synuclein and its deletion mutants are largely unfolded proteins with random coil structures as revealed by CD spectra, fluorescence spectra, gel filtration chromatography, and ultracentrifugation. On the basis of its highly unfolded and flexible conformation, we have investigated the chaperone-like activity of alpha-synuclein in vitro. In our experiments, alpha-synuclein inhibited the aggregation of model substrates and protected the catalytic activity of alcohol dehydrogenase and rhodanese during heat stress. In addition, alpha-synuclein inhibited the initial aggregation of reduced/denatured lysozyme on the refolding pathway. Interestingly, deletion of the C-terminal regions led to the abolishment of chaperone activity, although largely unstructured conformations are maintained. Moreover, alpha-synuclein could inhibit the aggregation of various Escherichia coli cellular proteins during heat stress, and C-terminal deletion mutants could not provide any protection to these cellular proteins. Results with synthetic C-terminal peptides and C-terminal deletion mutants suggest that the second acidic repeat, (125)YEMPSEEGYQDYEPEA(140), is important for the chaperone activity of alpha-synuclein, and C-terminal deletion leads to the facilitated aggregation with the elimination of chaperone activity.     

Interaction of alpha-synuclein and synphilin-1: effect of Parkinson's disease-associated mutations

alpha-Synuclein is a major component of Lewy bodies, a neuropathological feature of Parkinson's disease. Two alpha-synuclein mutations, Ala53Thr and Ala30Pro, are associated with early onset, familial forms of the disease. Recently, synphilin-1, a protein found to interact with alpha-synuclein by yeast two hybrid techniques, was detected in Lewy bodies. In this study we report the interaction of alpha-synuclein and synphilin-1 in human neuroglioma cells using a sensitive fluorescence resonance energy transfer technique. We demonstrate that the C-terminus of alpha-synuclein is closely associated with the C-terminus of synphilin-1. A weak interaction occurs between the N-terminus of alpha-synuclein and synphilin-1. The familial Parkinson's disease associated mutations of alpha-synuclein (Ala53Thr and Ala30Pro) also demonstrate a strong interaction between their C-terminal regions and synphilin-1. However, compared with wild-type alpha-synuclein, significantly less energy transfer occurs between the C-terminus of Ala53Thr alpha-synuclein and synphilin-1, suggesting that the Ala53Thr mutation alters the conformation of alpha-synuclein in relation to synphilin-1.     

Role of cytochrome c as a stimulator of alpha-synuclein aggregation in Lewy body disease.

alpha-Synuclein is a major component of aggregates forming amyloid-like fibrils in diseases with Lewy bodies and other neurodegenerative disorders, yet the mechanism by which alpha-synuclein is intracellularly aggregated during neurodegeneration is poorly understood. Recent studies suggest that oxidative stress reactions might contribute to abnormal aggregation of this molecule. In this context, the main objective of the present study was to determine the potential role of the heme protein cytochrome c in alpha-synuclein aggregation. When recombinant alpha-synuclein was coincubated with cytochrome c/hydrogen peroxide, alpha-synuclein was concomitantly induced to be aggregated. This process was blocked by antioxidant agents such as N-acetyl-L-cysteine. Hemin/hydrogen peroxide similarly induced aggregation of alpha-synuclein, and both cytochrome c/hydrogen peroxide- and hemin/hydrogen peroxide-induced aggregation of alpha-synuclein was partially inhibited by treatment with iron chelator deferoxisamine. This indicates that iron-catalyzed oxidative reaction mediated by cytochrome c/hydrogen peroxide might be critically involved in promoting alpha-synuclein aggregation. Furthermore, double labeling studies for cytochrome c/alpha-synuclein showed that they were colocalized in Lewy bodies of patients with Parkinson's disease. Taken together, these results suggest that cytochrome c, a well known electron transfer, and mediator of apoptotic cell death may be involved in the oxidative stress-induced aggregation of alpha-synuclein in Parkinson's disease and related disorders.     

Peculiarities of copper binding to alpha-synuclein

Heavy metals have been implicated as the causative agents for the pathogenesis of the most prevalent neurodegenerative disease. Various mechanisms have been proposed to explain the toxic effects of metals ranging from metal-induced oxidation of protein to metal-induced changes in the protein conformation. Aggregation of a-synuclein is implicated in Parkinson's disease (PD), and various metals, including copper, constitute a prominent group of alpha-synuclein aggregation enhancers. In this study, we have systematically characterized the a-synuclein-Cu21 binding sites and analyzed the possible role of metal binding in a-synuclein fibrillation using a set of biophysical techniques, such as electron paramagnetic resonance (EPR), electron spin-echo envelope modulation (ESEEM), circular dichroism (CD), and size exclusion chromatography (SEC). Our analyses indicated that a-synuclein possesses at least two binding sites for Cu21. We have been able to locate one of the binding sites in the N-terminal region. Furthermore, based on the EPR studies of model peptides and Beta-synuclein, we concluded that the suspected His residue did not appear to participate in strong Cu21 binding.     

Characterization of oligomers during alpha-synuclein aggregation using intrinsic tryptophan fluorescence

The aggregation of the presynaptic protein alpha-synuclein is associated with Parkinson's disease (PD). The details of the mechanism of aggregation, as well as the cytotoxic species, are currently not well understood. alpha-Synuclein has four tyrosine and no tryptophan residues. We introduced a tyrosine to tryptophan mutation at position 39 to create an intrinsic fluorescence probe and allow additional characterization of the aggregation process. Y39W alpha-synuclein had similar fibrillation kinetics (2-fold slower), pH-induced conformational changes, and fibril morphology to wild-type alpha-synuclein. In addition to intrinsic Trp fluorescence, acrylamide quenching, fluorescence anisotropy, ANS binding, dynamic light scattering, and FTIR were employed to monitor the kinetics of aggregation. These biophysical probes revealed the significant population of two classes of oligomeric intermediates, one formed during the lag period of fibrillation and the other present at the completion of fibrillation. As expected for a natively unfolded protein, Trp 39 was highly solvent-exposed in the monomer and is solvent-exposed in the two oligomeric intermediates; however, it is partially, but not fully, buried in the fibrils. These observations demonstrate the utility of Trp fluorescence labeled alpha-synuclein and demonstrate the existence of an oligomeric intermediate that exists as a transient reservoir of alpha-synuclein for fibrillation.     

NMR of alpha-synuclein-polyamine complexes elucidates the mechanism and kinetics of induced aggregation

The aggregation of alpha-synuclein is characteristic of Parkinson's disease (PD) and other neurodegenerative synucleinopathies. The 140-aa protein is natively unstructured; thus, ligands binding to the monomeric form are of therapeutic interest. Biogenic polyamines promote the aggregation of alpha-synuclein and may constitute endogenous agents modulating the pathogenesis of PD. We characterized the complexes of natural and synthetic polyamines with alpha-synuclein by NMR and assigned the binding site to C-terminal residues 109-140. Dissociation constants were derived from chemical shift perturbations. Greater polyamine charge (+2 --> +5) correlated with increased affinity and enhancement of fibrillation, for which we propose a simple kinetic mechanism involving a dimeric nucleation center. According to the analysis, polyamines increase the extent of nucleation by approximately 10(4) and the rate of monomer addition approximately 40-fold. Significant secondary structure is not induced in monomeric alpha-synuclein by polyamines at 15 degrees C. Instead, NMR reveals changes in a region (aa 22-93) far removed from the polyamine binding site and presumed to adopt the beta-sheet conformation characteristic of fibrillar alpha-synuclein. We conclude that the C-terminal domain acts as a regulator of alpha-synuclein aggregation.     

Characterization of oligomeric intermediates in alpha-synuclein fibrillation: FRET studies of Y125W/Y133F/Y136F alpha-synuclein

The aggregation of alpha-synuclein is believed to be a critical step in the etiology of Parkinson's disease. A variety of biophysical techniques were used to investigate the aggregation and fibrillation of alpha-synuclein in which one of the four intrinsic Tyr residues was replaced by Trp, and two others by Phe, in order to permit fluorescence resonance energy transfer (FRET) between residues 39 (Tyr) and 125 (Trp). The mutant Y125W/Y133F/Y136F alpha-synuclein (one Tyr, one Trp) showed fibrillation kinetics similar to that of the wild-type, as did the Y125F/Y133F/Y136F (one Tyr, no Trp) and Y39F/Y125W/Y133F/Y136F (no Tyr, one Trp) mutants. Time-dependent changes in FRET, Fourier transform infrared, Trp fluorescence, dynamic light-scattering and other probes, indicate the existence of a transient oligomer, whose population reaches a maximum at the end of the lag time. This oligomer, in which the alpha-synuclein is in a partially folded conformation, is subsequently converted into fibrils, and has physical properties that are distinct from those of the monomer and fibrils. In addition, another population of soluble oligomers was observed to coexist with fibrils at completion of the reaction. The average distance between Tyr39 and Trp125 decreases from 24.9A in the monomer to 21.9A in the early oligomer and 18.8A in the late oligomer. Trp125 remains solvent-exposed in both the oligomers and fibrils, indicating that the C-terminal domain is not part of the fibril core. No FRET was observed in the fibrils, due to quenching of Tyr39 fluorescence in the fibril core. Thus, aggregation of alpha-synuclein involves multiple oligomeric intermediates and competing pathways.     

Ubiquitination of alpha-synuclein and autophagy in Parkinson's disease

alpha-Synuclein is mutated in Parkinson's disease (PD) and is found in cytosolic inclusions, called Lewy bodies, in sporadic forms of the disease. A fraction of alpha-synuclein purified from Lewy bodies is monoubiquitinated, but the role of this monoubiquitination has been obscure. We now review recent data indicating a role of alpha-synuclein monoubiquitination in Lewy body formation and implicating the autophagic pathway in regulating these processes. The E3 ubiquitin-ligase SIAH is present in Lewy bodies and monoubiquitinates alpha-synuclein at the same lysines that are monoubiquitinated in Lewy bodies. Monoubiquitination by SIAH promotes the aggregation of alpha-synuclein into amorphous aggregates and increases the formation of inclusions within dopaminergic cells. Such effect is observed even at low monoubiquitination levels, suggesting that monoubiquitinated alpha-synuclein may work as a seed for aggregation. Accumulation of monoubiquitinated alpha-synuclein and formation of cytosolic inclusions is promoted by autophagy inhibition and to a lesser extent by proteasomal and lysosomal inhibition. Monoubiquitinated alpha-synuclein inclusions are toxic to cells and recruit PD-related proteins, such as synphilin-1 and UCH-L1. Altogether, the new data indicate that monoubiquitination might play an important role in Lewy body formation. Decreasing alpha- synuclein monoubiquitination, by preventing SIAH function or by stimulating autophagy, constitutes a new therapeutic strategy for Parkinson's disease.