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.     

Biophysical properties of the synucleins and their propensities to fibrillate: inhibition of alpha-synuclein assembly by beta- and gamma-synucleins.

The pathological hallmark of Parkinson's disease is the presence of intracellular inclusions, Lewy bodies, and Lewy neurites, in the dopaminergic neurons of the substantia nigra and several other brain regions. Filamentous alpha-synuclein is the major component of these deposits and its aggregation is believed to play an important role in Parkinson's disease and several other neurodegenerative diseases. Two homologous proteins, beta- and gamma-synucleins, are also abundant in the brain. The synucleins are natively unfolded proteins. beta-Synuclein, which lacks 11 central hydrophobic residues compared with its homologs, exhibited the properties of a random coil, whereas alpha- and gamma-synucleins were slightly more compact and structured. gamma-Synuclein, unlike its homologs, formed a soluble oligomer at relatively low concentrations, which appears to be an off-fibrillation pathway species. Here we show that, although they have similar biophysical properties to alpha-synuclein, beta- And gamma-synucleins inhibit alpha-synuclein fibril formation. Complete inhibition of alpha-synuclein fibrillation was observed at 4:1 molar excess of beta- and gamma-synucleins. No significant incorporation of beta-synuclein into the fibrils was detected. The lack of fibrils formed by beta-synuclein is most readily explained by the absence of a stretch of hydrophobic residues from the middle region of the protein. A model for the inhibition is proposed.     

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 overexpression and aggregation exacerbates impairment of mitochondrial functions by augmenting oxidative stress in human neuroblastoma cells

Overexpression of alpha-synuclein and oxidative stress has been implicated in the neuronal cell death in Parkinson's disease. Alpha-synuclein associates with mitochondria and excessive accumulation of alpha-synuclein causes impairment of mitochondrial functions. However, the mechanism of mitochondrial impairment caused by alpha-synuclein is not fully understood. We recently reported that alpha-synuclein associates with mitochondria and that overexpression of alpha-synuclein causes nitration of mitochondrial proteins and release of cytochrome c from the mitochondria [Parihar M.S., Parihar A., Fujita M., Hashimoto M., Ghafourifar P. Mitochondrial association of alpha-synuclein causes oxidative stress. Cell Mol Life Sci. 2008a;65:1272–1284]. The present study shows that overexpression of alpha-synuclein A53T or A30P mutants or wild-type in human neuroblastoma cells augmented aggregation of alpha-synuclein. Immunoblotting and immuno-gold electron transmission microscopy show localization of alpha-synuclein aggregates within the mitochondria of overexpressing cells. Overexpressing cells show increased mitochondrial reactive oxygen species, increased protein tyrosine nitration, decreased mitochondrial transmembrane potential, and hampered cellular respiration. These findings suggest an important role for mitochondria in cellular responses to alpha-synuclein.     

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.     

G209A mutant alpha synuclein expression specifically enhances dopamine induced oxidative damage

Alpha synuclein protein may play an important role in familial and sporadic Parkinson's disease pathology. We have induced G209A mutant or wild-type alpha-synuclein expression in stable HEK293 cell models to determine if this influences markers of oxidative stress and damage under normal conditions or in the presence of dopamine or paraquat. Induced wild-type or mutant alpha-synuclein expression alone had no effect upon levels of oxidative stress or damage, as measured by glutathione levels or aconitase activity. Both wild-type and mutant alpha-synuclein expression decreased the oxidative damage induced by paraquat, although the protection was less marked with mutant alpha-synuclein expression. This suggests that alpha-synuclein expression may either have anti-oxidant properties or may upregulate cellular antioxidant levels, a function that was diminished by the G209A mutation. However, mutant but not wild-type alpha-synuclein expression specifically enhanced dopamine associated oxidative damage. Non-expressing cells treated with reserpine to inhibit the vesicular monoamine compartmentalisation produced similar results. However, consistent with the hypothesis that mutant alpha-synuclein disrupts vesicular dopamine compartmentalization, this effect was diminished in cells expressing mutant alpha-synuclein. This may result in increased dopamine metabolism and cause selective oxidative damage to dopaminergic cells.     

Impact of Tyr to Ala mutations on alpha-synuclein fibrillation and structural properties

Substantial evidence suggests that the fibrillation of alpha-synuclein is a critical step in the development of Parkinson's disease. In vitro, alpha-synuclein forms fibrils with morphologies and a staining characteristic similar to those extracted from disease-affected brain. Monomeric alpha-synuclein is an intrinsically disordered protein, with three Tyr residues in the C-terminal region, one in the N-terminus, and lacking Trp. It is thought that interactions between the C-terminus and the central portion of the molecule may prevent or minimize aggregation/fibrillation. To test this hypothesis we examined the importance of the Tyr residues on the propensity for alpha-synuclein to fibrillate in vitro. Fibril formation of alpha-synuclein was completely inhibited, in the timescale over which measurements were made, by replacing the three C-terminal Tyr residues with Ala. In addition, substitution of Tyr133 by Ala also resulted in the absence of fibrillation, whereas the individual Y125A and Y136A mutants showed limited inhibition. Replacement of Tyr39 by Ala also resulted in substantial inhibition of fibrillation. Structural analysis showed that the Y133A mutant had a substantially different conformation, rich in alpha-helical secondary structure, as compared with the wild-type and other mutants, although the formation of any tertiary structure has not been observed as can be judged from near-UV-CD spectra. These observations suggest that the long-range intramolecular interactions between the N- and C-termini of alpha-synuclein are likely to be crucial to the fibrillation process.     

Conformational behavior and aggregation of alpha-synuclein in organic solvents: modeling the effects of membranes

Intracellular proteinaceous inclusions (Lewy bodies and Lewy neurites) of alpha-synuclein are pathological hallmarks of neurodegenerative diseases such as Parkinson's disease, dementia with Lewy bodies (DLB), and multiple systemic atrophy. The molecular mechanisms underlying the aggregation of alpha-synuclein into such filamentous inclusions remain unknown, although many factors have been implicated, including interactions with lipid membranes. To model the effects of membrane fields on alpha-synuclein, we analyzed the structural and fibrillation properties of this protein in mixtures of water with simple and fluorinated alcohols. All solvents that were studied induced folding of alpha-synuclein, with the common first stage being formation of a partially folded intermediate with an enhanced propensity to fibrillate. Protein fibrillation was completely inhibited due to formation of beta-structure-enriched oligomers with high concentrations of methanol, ethanol, and propanol and moderate concentrations of trifluoroethanol (TFE), or because of the appearance of a highly alpha-helical conformation at high TFE and hexafluoro-2-propanol concentrations. At least to some extent, these conformational effects mimic those observed in the presence of phospholipid vesicles, and can explain some of the observed effects of membranes on alpha-synuclein fibrillation.     

Effect of reparation of repeat sequences in the human alpha-synuclein on fibrillation ability

The aggregation and fibrillation of alpha-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 alpha-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 alpha-helix intermediate. Resistance to formation in the partially folded intermediate may repress the folding of alpha-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 alpha-synuclein.     

Forcing nonamyloidogenic beta-synuclein to fibrillate

The fibrillation and aggregation of alpha-synuclein is a key process in the formation of intracellular inclusions, Lewy bodies, in substantia nigral neurons and, potentially, in the pathology of Parkinson's disease and several other neurodegenerative disorders. Alpha-synuclein and its homologue beta-synuclein are both natively unfolded proteins that colocalize in presynaptic terminals of neurons in many regions of the brain, including those of dopamine-producing cells of the substantia nigra. Unlike its homologue, beta-synuclein does not form fibrils and has been shown to inhibit the fibrillation of alpha-synuclein. In this study, we demonstrate that fast and efficient aggregation and fibrillation of beta-synuclein can be induced in the presence of a variety of factors. Certain metals (Zn(2+), Pb(2+), and Cu(2+)) induce a partially folded conformation of beta-synuclein that triggers rapid fibrillation. In the presence of these metals, mixtures of alpha- and beta-synucleins exhibited rapid fibrillation. The metal-induced fibrillation of beta-synuclein was further accelerated by the addition of glycosaminoglycans or high concentrations of macromolecular crowding agents. Beta-synuclein also rapidly formed soluble oligomers and fibrils in the presence of pesticides, whereas the addition of low concentrations of organic solvents induced formation of amorphous aggregates. These new findings demonstrate the potential effect of environmental pollutants in generating an amyloidogenic, and potentially neurotoxic, conformation, in an otherwise benign protein.     

alpha-Synuclein interacts with phospholipase D isozymes and inhibits pervanadate-induced phospholipase D activation in human embryonic kidney-293 cells

alpha-Synuclein has been implicated in the pathogenesis of many neurodegenerative diseases, including Parkinson's disease and Alzheimer's disease. Although the function of alpha-synuclein remains largely unknown, recent studies have demonstrated that this protein can interact with phospholipids. To address the role of alpha-synuclein in neurodegenerative disease, we have investigated whether it binds phospholipase D (PLD) and affects PLD activity in human embryonic kidney (HEK)-293 cells overexpressing wild type alpha-synuclein or the mutant forms of alpha-synuclein (A53T, A30P) associated with Parkinson's disease. Tyrosine phosphorylation of alpha-synuclein appears to play a modulatory role in the inhibition of PLD, because mutation of Tyr(125) to Phe slightly increases inhibitory effect of alpha-synuclein on PLD activity. Treatment with pervanadate or phorbol myristate acetate inhibits PLD more in HEK 293 cells overexpressing alpha-synuclein than in control cells. Binding of alpha-synuclein to PLD requires phox and pleckstrin homology domain of PLD and the amphipathic repeat region and non-Abeta component of alpha-synuclein. Although biologically important, co-transfection studies indicate that the interaction of alpha-synuclein with PLD does not influence the tendency of alpha-synuclein to form pathological inclusions. These results suggest that the association of alpha-synuclein with PLD, and modulation of PLD activity, is biologically important, but PLD does not appear to play an essential role in the pathophysiology of alpha-synuclein.     

Pesticides directly accelerate the rate of alpha-synuclein fibril formation: a possible factor in Parkinson's disease.

Parkinson's disease involves intracellular deposits of alpha-synuclein in the form of Lewy bodies and Lewy neurites. The etiology of the disease is unknown, however, several epidemiological studies have implicated environmental factors, especially pesticides. Here we show that several pesticides, including rotenone, dieldrin and paraquat, induce a conformational change in alpha-synuclein and significantly accelerate the rate of formation of alpha-synuclein fibrils in vitro. We propose that the relatively hydrophobic pesticides preferentially bind to a partially folded intermediate conformation of alpha-synuclein, accounting for the observed conformational changes, and leading to association and subsequent fibrillation. These observations suggest one possible underlying molecular basis for Parkinson's disease.     

Heparin and other glycosaminoglycans stimulate the formation of amyloid fibrils from alpha-synuclein in vitro

Parkinson's disease is the second most common neurodegenerative disease and results from loss of dopaminergic neurons in the substantia nigra. The aggregation and fibrillation of alpha-synuclein have been implicated as a causative factor in the disease. Glycosaminoglycans (GAGs) are routinely found associated with amyloid deposits in most amyloidosis diseases, and there is evidence to support an active role of GAGs in amyloid fibril formation in some cases. In contrast to the extracellular amyloid deposits, the alpha-synuclein deposits in Lewy body diseases are intracellular, and thus it is less clear whether GAGs may be involved. To determine whether the presence of GAGs does affect the fibrillation of alpha-synuclein, the kinetics of fibril formation were investigated in the presence of a number of GAGs and other charged polymers. Certain GAGs (heparin, heparan sulfate) and other highly sulfated polymers (dextran sulfate) were found to significantly stimulate the formation of alpha-synuclein fibrils. Interestingly, the interaction of GAGs with alpha-synuclein is quite specific, since some GAGs, e.g., keratan sulfate, had negligible effect. Heparin not only increased the rate of fibrillation but also apparently increased the yield of fibrils. The molar ratio of heparin to alpha-synuclein and the incorporation of fluorescein-labeled heparin into the fibrils demonstrate that the heparin is integrated into the fibrils and is not just a catalyst for fibrillation. The apparent dissociation constant for heparin in stimulating alpha-synuclein fibrillation was 0.19 microM, indicating a strong affinity. Similar effects of heparin were observed with the A53T and A30P mutants of alpha-synuclein. Since there is some evidence that Lewy bodies may contain GAGs, these observations may be very relevant in the context of the etiology of Parkinson's disease.     

Role of individual methionines in the fibrillation of methionine-oxidized alpha-synuclein

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. We have previously established that oxidation of all four methionine residues in alpha-synuclein (to the sulfoxide, MetO) inhibits fibrillation of this protein in vitro and that the MetO protein also inhibits fibrillation of unmodified alpha-synuclein. Here we show that the degree of inhibition of fibrillation by MetO alpha-synuclein is proportional to the number of oxidized methionines. This was accomplished be selectively converting Met residues into Leu, prior to Met oxidation. The results showed that with one oxidized Met the kinetics of fibrillation were comparable to those for the control (nonoxidized), and with increasing numbers of methionine sulfoxides the kinetics of fibrillation became progressively slower. Electron microscope images showed that the fibril morphology was similar for all species examined, although fewer fibrils were observed with the oxidized forms. The presence of zinc was shown to overcome the Met oxidation-induced inhibition. Interestingly, substitution of Met by Leu led to increased propensity for aggregation (soluble oligomers) but slower formation of fibrils.     

Overexpression of alpha-synuclein in rat substantia nigra results in loss of dopaminergic neurons, phosphorylation of alpha-synuclein and activation of caspase-9: resemblance to pathogenetic changes in Parkinson's disease

To elucidate the role of alpha-synuclein in the pathogenesis of Parkinson's disease, both human alpha-synuclein transgenic mice and targeted overexpression of human alpha-synuclein in rat substantia nigra using viral vector-based methods have been studied, however, little is known about the pathogenetic changes of dopaminergic neuron loss. Therefore, it is necessary to address whether the pathogenetic changes in brains with Parkinson's disease are recapitulated in these models. Here, we used the recombinant adeno-associated viral (rAAV) vector system for human alpha-synuclein gene transfer to rat substantia nigra and observed approximately 50% loss of dopaminergic neurons at 13 weeks after infection, which was comparably slower than the progression of neurodegeneration reported in other studies. In the slower progression of neurodegeneration, we identified several important features in common with the pathogenesis of Parkinson's disease, such as phosphorylation of alpha-synuclein at Ser129 and activation of caspase-9. Both findings were also evident in cortical tissues overexpressing alpha-synuclein via rAAV. Our results indicate that overexpression of alpha-synuclein via rAAV apparently recapitulates several important features of brains with Parkinson's disease and dementia with Lewy bodies, and thus alpha-synucleinopathy described here is likely to be an ideal model for the study of the pathogenesis of Parkinson's disease and dementia with Lewy bodies.     

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.     

Polycation-induced oligomerization and accelerated fibrillation of human alpha-synuclein in vitro

The aggregation and fibrillation of alpha-synuclein has been implicated as a causative factor in Parkinson's disease and several other neurodegenerative disorders known as synucleinopathies. The effect of different factors on the process of fibril formation has been intensively studied in vitro. We show here that alpha-synuclein interacts with different unstructured polycations (spermine, polylysine, polyarginine, and polyethyleneimine) to form specific complexes. In addition, the polycations catalyze alpha-synuclein oligomerization. The formation of alpha-synuclein-polycation complexes was not accompanied by significant structural changes in alpha-synuclein. However, alpha-synuclein fibrillation was dramatically accelerated in the presence of polycations. The magnitude of the accelerating effect depended on the nature of the polymer, its length, and concentration. The results illustrate the potential critical role of electrostatic interactions in protein aggregation, and the potential role of naturally occurring polycations in modulating alpha-synuclein aggregation.     

Overexpression of Parkinson's disease-associated alpha-synucleinA53T by recombinant adeno-associated virus in mice does not increase the vulnerability of dopaminergic neurons to MPTP

Mutations in the alpha-synuclein gene are linked to a rare dominant form of familial Parkinson's disease, and alpha-synuclein is aggregated in Lewy bodies of both sporadic and dominant Parkinson's disease. It has been proposed that mutated alpha-synuclein causes dopaminergic neuron loss by enhancing the vulnerability of these neurons to a variety of insults, including oxidative stress, apoptotic stimuli, and selective dopaminergic neurotoxins, such as 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). To test this hypothesis in vivo, we overexpressed human alpha-synuclein(A53T) in the substantia nigra of normal and MPTP-treated mice by rAAV-mediated gene transfer. Determination of dopaminergic neuron survival, striatal tyrosine hydroxylase fiber density, and striatal content of dopamine and its metabolites in rAAV-injected and uninjected hemispheres demonstrated that alpha-synuclein(A53T) does not increase the susceptibility of dopaminergic neurons to MPTP. Our findings argue against a direct detrimental role for (mutant) alpha-synuclein in oxidative stress and/or apoptotic pathways triggered by MPTP, but do not rule out the possibility that alpha-synuclein aggregation in neurons exposed to oxidative stress for long periods of time may be neurotoxic.     

Oxidative dimer formation is the critical rate-limiting step for Parkinson's disease alpha-synuclein fibrillogenesis

Intraneuronal deposition of alpha-synuclein as fibrils and oxidative stress are both implicated in the pathogenesis of Parkinson's disease. We found that the critical rate-limiting step in nucleation of alpha-synuclein fibrils under physiological conditions is the oxidative formation and accumulation of a dimeric, dityrosine cross-linked prenucleus. Dimer formation is accelerated for the pathogenic A30P and A53T mutant alpha-synucleins, because of their greater propensity to self-interact, which is reflected in the smaller values of the osmotic second virial coefficient compared to that of wild-type synuclein. Our finding that oxidation is an essential step in alpha-synuclein aggregation supports a mechanism of Parkinson's disease pathogenesis in which the separately studied pathogenic factors of oxidative stress and alpha-synuclein aggregation converge at the critical step of alpha-synuclein dimer formation.     

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.