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J. Biol. Chem., Vol. 277, Issue 18, 16116-16123, May 3, 2002

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Magnesium Inhibits Spontaneous and Iron-induced Aggregation of -Synuclein^*

Natalie Golts, Heather Snyder, Mark Frasier, Catherine Theisler, Peter Choi, and Benjamin Wolozin

From the Department of Pharmacology, Loyola University Medical Center, Maywood, Illinois 60153

Received for publication, August 15, 2001, and in revised form, February 13, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Multiple studies implicate metals in the pathophysiology of neurodegenerative diseases. Disturbances in brain iron metabolism are linked with synucleinopathies. For example, in Parkinson's disease, iron levels are increased and magnesium levels are reduced in the brains of patients. To understand how changes in iron and magnesium might affect the pathophysiology of Parkinson's disease, we investigated binding of iron to -synuclein, which accumulates in Lewy bodies. Using fluorescence of the four tyrosines in -synuclein as indicators of metal-related conformational changes in -synuclein, we show that iron and magnesium both interact with -synuclein. -Synuclein exhibits fluorescence peaks at 310 and 375 nm. Iron lowers both fluorescence peaks, while magnesium increases the fluorescence peak only at 375 nm, which suggests that magnesium affects the conformation of -synuclein differently than iron. Consistent with this hypothesis, we also observe that magnesium inhibits -synuclein aggregation, measured by immunoblot, cellulose acetate filtration, or thioflavine-T fluorescence. In each of these studies, iron increases -synuclein aggregation, while magnesium at concentrations >0.75 mM inhibits the aggregation of -synuclein induced either spontaneously or by incubation with iron. These data suggest that the conformation of -synuclein can be modulated by metals, with iron promoting aggregation and magnesium inhibiting aggregation.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Parkinson's disease (PD)¹ is a common motor disorder that affects about 1% of population over the age of 65 (1). The disease is characterized by progressive neurodegeneration predominantly affecting dopaminergic neurons in the nigrostriatal system (2). The degenerating neurons develop intracellular inclusions, termed Lewy bodies, which are composed of a dense core of filamentous and granular material (3). Recent studies indicate that -synuclein is a major filamentous component of Lewy bodies (3, 4). Genetic studies suggest that -synuclein plays a key role in the pathophysiology of PD, because mutations in -synuclein, at A53T or A30P, are associated with early-onset familial PD (5, 6).

The accumulation of aggregated protein underlies the pathophysiology of many neurodegenerative disorders, and increasing evidence suggests that aggregated -synuclein plays a key role in the pathophysiology of PD. -Synuclein has a strong tendency to aggregate and does so spontaneously in vitro at a slow rate (7-9). Both the A53T and the A30P mutations in PD increase the tendency of -synuclein to aggregate. Many studies in cultured neurons, and some studies in transgenic animals, suggest that -synuclein aggregation is linked to cellular toxicity and neurodegeneration (10-12). In cell culture, formation of -synuclein aggregates correlates with cell injury (10). Overexpressing -synuclein in Drosophila leads to an age-dependent accumulation of aggregated -synuclein and associated neurodegeneration (12). Masliah and colleagues also observed that aggregated -synuclein is associated with loss of markers in dopaminergic neurons, although other studies of -synuclein overexpression in transgenic mice have been less conclusive (11, 13, 14). Thus, increasing lines of evidence suggest that aggregation of -synuclein is associated with the degeneration of dopaminergic neurons and suggest that -synuclein contributes to the neurodegenerative processes occurring in PD.

Recombinant -synuclein aggregates spontaneously following prolonged incubation in vitro. Recently, we and others have shown that -synuclein also aggregates rapidly following exposure to Fe(II) (10, 15). In vitro, Fe(II) accelerates the rate of -synuclein aggregation. For example, similar amounts of aggregation are induced in vitro by incubating 23 µM -synuclein alone for 30 days or with 50 µM FeCl₂ for only 3 days, suggesting that 50 µM Fe(II) increases the rate of -synuclein aggregation about 10-fold (see discussion below). These observations suggest that interaction with iron could greatly accelerate -synuclein aggregation.

The factors regulating -synuclein aggregation in the brain are poorly understood. Some studies suggest that neurotoxins, such as the pesticide rotenone or paraquat, stimulate -synuclein aggregation (16). The involvement of metals in PD suggests that metals might also play a role in the aggregation of -synuclein and the pathophysiology of PD. Epidemiological studies have shown that exposure to metals is associated with PD. For instance, individuals with industrial exposure to iron, copper, and/or lead have high rates of PD (17). Neuropathological studies show that synucleinopathies are generally associated with iron accumulation, which is consistent with a pathological link between iron and -synuclein (18). Brains from patients with PD, type I iron storage disease (Hallorvorden-Spatz disorder), and multiple systems atrophy all show increased iron content (19). In PD the levels of iron are increased over controls, and Fe(II) has been identified as a major component of Lewy bodies (20-26). How iron contributes to Lewy body formation and the pathophysiology of PD, though, is not understood. In the experiments described below, we examine the regulation of -synuclein aggregation using both spontaneous and iron-induced -synuclein aggregation in vitro and show contrasting actions of iron and magnesium on -synuclein aggregation. These studies have important implications for the pathophysiology of PD and other synucleionopathies.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- -Synuclein (wild-type, A53T, and A30P) was cloned into the NcoI/NotI site of the Pro-Ex His 6 vector (Invitrogen). To generate recombinant -synuclein, BPer (Pierce) reagent was used to solubilize the recombinant -synuclein from the isopropyl-1-thio--D-galactopyranoside-induced bacterial lysates, which were then passed over a nickel-agarose column for purification. All spectrophotometric analysis were repeated three to five times.

Immunoblotting-- Cells were harvested with SDS lysis solution (2% SDS, 10 mM Tris, pH 7.4, 2 mM -glycerol phosphate, 1 µM AEBSF). The amount of protein was determined using the BCA assay (Pierce), 5-30 µg per lane was run on 14% SDS-polyacrylamide gels and transferred to nitrocellulose (200 mA, 12 h). The nitrocellulose was then incubated 1 h in 5% I-block (Tropix)/phosphate-buffered saline, washed, incubated overnight in primary antibody, washed, then incubated 3 h in peroxidase-coupled secondary antibody and developed with chemiluminescent reagent (PerkinElmer Life Sciences).

Thioflavine-T Measurements-- For analysis of aggregation using thioflavine-T, 23 nM -synuclein was incubated in 10 µM thioflavine-T (in 50 mM glycine, pH 8.5) and measured by fluorescence (_ex = 440, _em = 450-600 nm).

Cellulose Acetate Assay-- To analyze aggregation of -synuclein by filtration, samples were diluted into 100 µl of water, filtered through cellulose acetate (0.2 µm pore size), washed with 200 µl phosphate-buffered saline, and then immunoblotted as described above.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Iron Quenches Tyrosine Fluorescence of -Synuclein: Evidence of Association-- To understand factors regulating -synuclein aggregation, we investigated the interaction of different metals with -synuclein using tyrosine fluorescence (27, 28). Tyrosine fluorescence has been used to monitor the association of various metals with a number of proteins, including A, -transducin and, more recently, -synuclein (27-29). In these studies tyrosine fluorescence is used as an indicator of changes in protein conformation or binding of metals. Exciting tyrosine at 280 nm elicits fluorescence that peaks at 310 nm for monomeric tyrosine and at 350-400 nm for tyrosinate (30). The fluorescence spectrum of -synuclein yielded fluorescence peaks at 310 and 375 nm (Fig. 1, A and B). Tyrosinate reactivity occurs when the phenolic hydroxyl group of tyrosine forms hydrogen bonds with carboxyl groups in nearby aspartates or glutamates. The fluorescence peak of -synuclein at 375 nm showed a pH dependence similar to that of tyrosinate (Fig. 1C), which is consistent with the pH dependence of fluorescence due to tyrosinate. The peak at 375 nm had the highest intrinsic fluorescence at low pH and showed little change in fluorescence at pH > 7.0 (Fig. 1C). Further studies confirmed that the peak at 375 nm is not due to tyrosine dimerization, because both gel electrophoresis and mass spectrometry of the -synuclein showed that the -synuclein was monomeric (Fig. 1D, Coomassie gel of recombinant -synuclein shown), and in addition, tyrosine dimerization of -synuclein reduced its intrinsic fluorescence (Fig. 1, E and F, described further below). These results suggest that the peak at 375 nm is due to tyrosinate, which could result from proton transfer from the phenolic hydroxyl to aspartic or glutamic acid protein acceptors (30).

View larger version (28K): [in this window] [in a new window]   Fig. 1.   Interaction of iron with recombinant -synuclein. A, excitation of wild-type -synuclein at ex 280 nm produces a biphasic fluorescence spectrum with peaks at 310 and 375 nm. Incubating -synuclein with increasing doses of FeCl2 yields a progressive quenching of both peaks. B, quantification of the relative fluorescence from Fig. 1A of 1 µM -synuclein at 310 nm during quenching by Fe(II). C, pH dependence of wild-type -synuclein fluorescence using ex 280 nm and em 290-450 nm. The pH sensitivity of the fluorescence peak at 375 indicates that this fluorescence is due to the presence of tyrosinate. D, identification of recombinant -synuclein following PAGE electrophoresis by staining with Coomassie Blue. The presence of a single -synuclein band at 16 kDa shows that there is no dimerization. E, reduction in -synuclein fluorescence following ultraviolet (UV) irradiation (2 h). The reduction in fluorescence of -synuclein occurred mainly around the peak at 375 nm (ex = 280 nm; em 290-450 nm), which contrasts with the changes in fluorescence induced by iron. F, UV irradiation (2 h) also reduces -synuclein fluorescence following excitation at ex = 315 nm; em 350-450 nm. UV-induced inhibition of -synuclein fluorescence contrasts with the increase in fluorescence induced by iron or spontaneous aggregation of -synuclein.

Metals Show Three Patterns of Interaction with -Synuclein-- Next we used the fluorescence to examine the interaction of -synuclein with metals. We observed three classes of interaction with -synuclein. Class I metals included iron (Fe(II) and Fe(III)) and copper (Cu(II)) and decreased the fluorescence at both 310 and 375 nm (Fig. 1A). Class II metals included magnesium, zinc, and calcium, and increased the fluorescence at 375 nm, but did not affect the fluorescence at 310 nm (Figs. 2A and 3A). Class III metals included nickel and manganese and did not affect -synuclein fluorescence (data not shown). We proceeded to examine the fluorescence of -synuclein in more detail to determine whether the metal induced changes -synuclein fluorescence reflected interaction with metals or some other process, such as tyrosine dimerization. To examine whether the changes in fluorescence could be explained by tyrosine dimerization, we exposed monomeric -synuclein to 312 nm light for 2 h (which is a process that induces tyrosine dimerization) and analyzed the emission fluorescence spectrum with excitation at either 280 or 315 nm. The emission spectrum derived using excitation at 280 nm showed that ultraviolet-irradiation reduced -synuclein fluorescence strongly at 375 nm, but only weakly at 310 nm (Fig. 1E). The contrast between the changes in fluorescence induced by ultraviolet irradiation and by metals suggests that the changes in -synuclein fluorescence induced by metals are not due to tyrosine dimerization. Analysis of the emission fluorescence spectrum of -synuclein following excitation at 315 nm showed a reduced fluorescence at 380 nm for the ultraviolet-irradiated -synuclein (Fig. 1F). In contrast, iron increased, rather than decreased, the fluorescence of -synuclein as measured using the 315 nm excitation. These data indicate that the iron-induced quenching of -synuclein fluorescence is not due to cross-linking of -synuclein mediated by tyrosine dimerization.

View larger version (11K): [in this window] [in a new window]   Fig. 2.   A, fluorescence of wild-type -synuclein in the presence of increasing doses of MgCl2 shows increased fluorescence emission at 375 nm and no change at 310 nm, using an excitation wavelength of 280 nm (ex = 280 nm; em 290-450 nm). B, a representative plot showing that magnesium increases the affinity of -synuclein for iron. -Synuclein shows an affinity for iron that is 5-fold lower when incubated in the presence of 100 µM MgCl2. C, fluorescence emissions of A53T -synuclein in the presence of increasing doses of MgCl2 shows no change at 375 or 310 nm, using an excitation of 280 nm.

Dose Dependence of Metal Binding-- Plotting of the dose dependence of iron-induced fluorescence quenching showed a dose-dependent decrease in fluorescence, with an IC₅₀ = 173 µM and a Hill coefficient of 1.0 (R2 = 1.0, p < 0.0001) (Fig. 1B), indicating one binding site or multiple binding sites with the same affinity and no cooperativity (Fig. 1, A and B). There is a small amount of binding of iron to -synuclein between 1-10 µM Fe(II), which is a range that could be physiologically relevant (intracellular free iron is about 1.5 µM) (31).

The effect of magnesium on -synuclein differed dramatically from that of iron. Magnesium increased the fluorescence at 375 nm but did not affect the fluorescence at 310 nm (Fig. 2A). Binding of magnesium to -synuclein was also striking because the tyrosine fluorescence showed a sharp stepwise increase between 60 and 80 µM of magnesium indicating cooperative binding (Fig. 2A). The cooperative regulation of tyrosine fluorescence, specifically at 375 nm, suggests that magnesium causes a conformational change in synuclein differing from that induced by iron. Co-incubating 80 µM magnesium with iron did not prevent iron-induced quenching of -synuclein tyrosine fluorescence and in fact increased affinity of -synuclein for iron from 173 to 50 µM (Fig. 2B). These data suggest that iron and magnesium bind to different sites on -synuclein.

Zinc and calcium also increased the fluorescence of -synuclein at 375 nm, but showed a more graded pattern of interaction (Fig. 3A). Jensen and colleagues recently noted a similar pattern of binding of calcium to -synuclein (29). Interestingly, immunoblots of recombinant -synuclein following incubation with zinc showed that zinc induced formation of a prominent band at 32 kDa, consistent with formation of an SDS-resistant -synuclein dimer (Fig. 3B). Neither magnesium nor calcium induced formation of SDS-resistant dimers identifiable by immunoblot (data not shown).

View larger version (35K): [in this window] [in a new window]   Fig. 3.   The effects of zinc on -synuclein fluorescence and dimerization. A, incubating zinc or calcium with recombinant -synuclein increases the peak of -synuclein emission fluorescence at 375 nm in a graded manner, using an excitation wavelength of 280 nm (ex = 280 nm; em 290-450 nm). B, immunoblot of recombinant -synuclein after incubation with zinc shows formation of a 32-kDa band consistent with formation of an SDS-resistant -synuclein dimer. Lane 1, 0 nM ZnCl2; lane 2, 100 nM ZnCl2; lane 3, 200 nM ZnCl2; lane 4, 400 nM ZnCl2.

We also examined how the A53T mutation in human -synuclein affected binding of iron and magnesium. The A53T mutation did not change the apparent affinity of iron for -synuclein (data not shown), but did abolish the interaction between magnesium and -synuclein (Fig. 2C). Previous studies have shown that the A53T mutation changes the conformation of -synuclein by increasing its helical content (5). These conformational changes might either reduce binding of magnesium to -synuclein or prevent the conformational change associated with binding of magnesium to -synuclein.

Magnesium Inhibits -Synuclein Aggregation-- The differing effects of magnesium and iron on the fluorescence spectrum of -synuclein suggested to us that magnesium and iron might also induce different conformational states. We hypothesized that the conformational changes induced by binding of magnesium to -synuclein might inhibit -synuclein aggregation. To test this, we examined whether magnesium could inhibit the spontaneous aggregation of -synuclein. -Synuclein (23 µM) was incubated for 30 days at 37° ± MgCl₂ (500 µM). To measure the amount of aggregation, the -synuclein was diluted to 23 nM in the presence of 10 µM thioflavine-T (in 50 mM glycine pH 8.5), and the fluorescence spectrum was measured. The solution of aged -synuclein showed a strong fluorescence peak at 480, indicating the presence of abundant -pleated sheet structures (Fig. 4A). Prior experiments have shown that the spontaneous aggregation of -synuclein proceeds through a mechanism involving -pleated sheet formation, and that thioflavine-T, which binds to proteins with -pleated sheet structure, accurately measures -synuclein aggregation (9). Using thioflavine-T we observed that samples incubated in the presence of magnesium showed only base-line levels of fluorescence, indicating that magnesium prevented the formation of -pleated sheet structures and the aggregation of -synuclein (Fig. 4A). To verify that the magnesium was inhibiting -synuclein aggregation, we measured the amount of aggregated -synuclein in each sample by capturing the aggregates with 0.2-µm cellulose acetate filters, measuring the amount of retained -synuclein by dot blot, and quantitating the resulting optical density. The results of the cellulose acetate assay paralleled the thioflavine-T assay and showed that magnesium prevented the spontaneous aggregation of -synuclein (Fig. 4, B and C). Thus, two independent methods show that magnesium inhibits the spontaneous aggregation of -synuclein in vitro.

View larger version (13K): [in this window] [in a new window]   Fig. 4.   Magnesium inhibits the spontaneous aggregation of -synuclein. A, -synuclein was prepared freshly (Mono) or aged 30 days at 37 °C to induce spontaneous aggregation (Ag) ± 0.8 mM Mg (Ag/Mg). Then the samples were then analyzed by Thioflavine-T fluorescence. The spontaneously aggregated -synuclein gave strong fluorescence, while the sample aged in the presence of magnesium had a fluorescence curve identical to that of fresh -synuclein. B, -synuclein was prepared freshly (M, monomeric) or aged 30 days at 37 °C to induce spontaneous aggregation (Ag, aged) or aged 30 days at 37 °C in the presence of 0.8 mM MgCl2 (Ag/Mg, aged plus Mg2+). Then the samples were filtered through a cellulose acetate membrane and immunoblotted with rabbit anti--synuclein antibody. Incubating the -synuclein with magnesium prevented formation of the large aggregates that are captured by the membrane. C, quantification of the optical density of the dot blots using the NIH Image program (n = 6, *, p < 0.001, analysis of variance factorial).

Magnesium was also able to prevent iron-induced -synuclein aggregation. -Synuclein (8 µM) was incubated with 50 µM FeCl₂ for 72 h and then analyzed by thioflavine-T fluorescence or cellulose acetate. In both cases, -synuclein samples co-incubated with 500 µM magnesium chloride showed little aggregation (Fig. 5, A and B). The amount of thioflavine-T fluorescence induced by iron was less than that induced by spontaneously aggregated -synuclein, which likely indicates that spontaneously induced -synuclein contains more -pleated sheet structure. Indeed analysis of iron-induced -synuclein aggregates by circular dichroism did not show formation of -pleated sheet structures, which suggests formation of a more amorphous aggregate (Fig. 5, C and D). These data suggest that magnesium inhibits the formation of -synuclein aggregates containing either -pleated sheet structure (via spontaneous aggregation) or amorphous structure (via iron-induced aggregation).

View larger version (24K): [in this window] [in a new window]   Fig. 5.   Magnesium inhibits iron-induced -synuclein aggregation. A, thioflavine-T fluorescence of -synuclein (8 µM) following a 3-day incubation in the presence of 50 µM FeCl2 ± 0.8 mM MgCl2. B, analysis of the affects of magnesium on iron induced -synuclein aggregation ± magnesium. -Synuclein was prepared freshly (Mono) or aged 3 days at 37 °C in the presence of 50 µM FeCl2 ± 0.8 mM MgCl2. Then the samples were filtered through a cellulose acetate membrane and immunoblotted with rabbit anti--synuclein antibody. Cellulose acetate assay of spontaneously aggregated synuclein ± Mg2+. C, circular dichroism spectrum of native -synuclein (160 µg/ml). D, circular dichroism spectrum of -synuclein (160 µg/ml) following incubation with 500 µM FeSO4 for 5 days.

We also examined aggregation by immunoblot analysis, which has been successfully used to examine aggregation of -synuclein, as well as aggregation of other proteins implicated in neurodegenerative disease, such as the huntingtin and PrP proteins (29, 32-35). In these assays, wild-type recombinant -synuclein (8 µM) was incubated with 0-3 mM FeCl₂ and 0 or 100 µM MgCl₂ for 24 h at 37 °C. The samples were immunoblotted with anti--synuclein antibody, and the total amount of -synuclein reactivity above 46 kDa (which includes structures larger than a dimers) was quantified by video densitometry (Fig. 6, A and B). We observed a reduction in formation of high molecular weight immunoreactivity of -synuclein at all but the highest dose of iron (n = 3, p < 0.0001). These results support the hypothesis that magnesium inhibits aggregation of -synuclein induced following treatment with iron.

View larger version (52K): [in this window] [in a new window]   Fig. 6.   Immunoblotting of -synuclein following incubation with varying doses of Fe(II) ± 100 µM MgCl2. A, immunoblotting of recombinant wild-type -synuclein following treatment with 0-3 mM FeCl2 plus 0.1 mM MgCl2 for 1 day. The mean optical density above 46 kDa was quantified and used as an index of aggregation. Increasing doses of MgCl2 reduced the formation of high molecular weight aggregates of -synuclein. Concentrations (µM) of iron salts: 0 (lanes 1 and 7), 30 (lanes 2 and 8), 100 (lanes 3 and 9), 300 (lanes 4 and 10), 1000 (lanes 5 and 11); 3000 (lanes 6 and 12). Concentrations (µM) of MgCl2: 0 (lanes 1-6), 100 (lanes 7-12). B, quantification of the aggregate formation by video densitometry showed a dose dependent decrease in aggregate formation that was statistically significant at 0.1 mM MgCl2 (n = 3 for each point). D, immunoblotting of recombinant A53T -synuclein following treatment with 0-3 mM FeCl2 plus 0.1 mM MgCl2 for 1 day. The mean optical density above 46 kDa was quantified and used as an index of aggregation. Increasing doses of MgCl2 did not consistently inhibit formation of high molecular weight aggregates of -synuclein. E, quantification of aggregate formation by video densitometry, showing that magnesium did not inhibit aggregation of A53T -synuclein (n = 3 for each point).

Since we did not observe interaction between magnesium and A53T -synuclein using tyrosine fluorescence, we tested whether aggregation of recombinant A53T -synuclein was also insensitive to magnesium. We incubated recombinant A53T -synuclein with 0-3 mM FeCl₂ and 0 or 100 µM MgCl₂ for 24 h, then immunoblotted the -synuclein and quantified aggregation by video densitometry, as described above (Fig. 6, C and D). We did not observe consistent inhibition of iron-induced aggregation of A53T -synuclein by magnesium. This suggests that magnesium cannot inhibit iron-induced aggregation of A53T -synuclein.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

These data demonstrate that iron (II), magnesium, zinc, and calcium all interact with -synuclein. Our data primarily rely on tyrosine fluorescence as a measure of the interaction of -synuclein with metals. The K_i of -synuclein for iron is 173 µM, and the K_i of magnesium for -synuclein is between 60 and 80 µM. These affinities are consistent with an affinity of -synuclein for calcium, determined by Nielson and colleagues (29). Nielson and colleagues also confirmed their tyrosine fluorescence studies using equilibrium dialysis; our attempts at using equilibrium dialysis were stymied by extensive binding of -synuclein to dialysis membranes. However, the studies of Nielson and colleagues show that tyrosine fluorescence provides an accurate indication of metal-synuclein binding interactions. The apparent affinity of -synuclein for magnesium is strong enough to allow interaction of -synuclein with magnesium in living cells, where the average intracellular concentration of magnesium is about 0.5 mM. This suggests that this interaction could have physiological significance.

Although binding of magnesium to -synuclein occurs at a concentration range that is physiologically significant, the concentration of free iron in the cell is much lower (<1.5 µM), which is far below the affinity of -synuclein for iron that we observed (173 µM) (36). However, studies using cell culture and neuropathology both suggest that -synuclein interacts with iron. Incubating cells with iron induces -synuclein aggregation in viable cells, which suggests that the concentration of iron in a cell is sufficient to induce -synuclein aggregation under some conditions. In addition, -synuclein aggregates in iron type I storage disease, and iron co-localizes with -synuclein in Lewy bodies (19). Although it is unclear how -synuclein might interact with iron in the living cell, it is possible that cofactors increase the affinity of -synuclein for iron sufficient to allow a physiological interaction. Many other factors might also affect the behavior of -synuclein. For instance, binding to lipids and phosphorylation or binding to -synuclein have all been shown to change the biochemistry of -synuclein, and these agents might increase its affinity for iron (44). Our preliminary studies examining magnesium already provide a hint of modulation. The K_i of iron (II) drops to 50 µM in the presence of magnesium. Future studies might unravel the biochemistry of -synuclein further.

Although binding of magnesium appears to introduce a conformation that promotes binding of iron, this same conformational change inhibits aggregation of -synuclein. We hypothesize that magnesium either changes the conformation of -synuclein to one that resists aggregation or induces dimerization to a structure that resists aggregation (Fig. 7). The ability of zinc to induce SDS-resistant -synuclein dimers, coupled with the similarity the changes in tyrosine fluorescence observed with magnesium and zinc, suggest that magnesium might induce dimerization of -synuclein in a manner similar to that of zinc. Future studies using nuclear magnetic resonance spectroscopy will need to be performed to investigate further how magnesium affects the conformation of -synuclein.

View larger version (17K): [in this window] [in a new window]   Fig. 7.   Model of interaction of iron and magnesium with -synuclein. In this model, iron binds to -synuclein and promotes aggregation. Magnesium appears to bind to a different site than does iron and therefore does not inhibit binding of iron. We hypothesize that binding of magnesium to -synuclein induces a conformational change that prevents formation of large -synuclein aggregates.

The most important observation made in this paper is that magnesium inhibits the aggregation of -synuclein. This observation is supported by our use of four independent lines of investigation (immunoblot, cellulose acetate filtration, and thioflavine-T fluorescence). The type of aggregate measured by each assay likely differs slightly. Immunoblotting detects aggregates that are stable enough to resist both heating and SDS. Cellulose acetate filtration and thioflavine-T are more gentle methods that can detect both stable aggregates and also aggregates that might be re-dissolved by SDS. Thioflavine-T recognizes aggregate with a -pleated sheet structure. Interestingly, spontaneously aggregated -synuclein shows much more fluorescence by thioflavine-T than iron-induced aggregate, suggesting that the former has more -pleated sheet structure. We have also taken care to examine two forms of -synuclein aggregation: spontaneous and iron-induced aggregation. Many studies show that -synuclein has a strong tendency to spontaneously aggregate, and this is the most widely accepted method for inducing -synuclein aggregation (7-9). Metal-induced aggregation has only been investigated by a small number of groups, but is perhaps the only method currently available for inducing -synuclein aggregation in cultured cells (10, 15, 37). The ability of magnesium to inhibit -synuclein aggregation induced by both protocols (spontaneous and iron-induced) suggests that this is a robust phenomenon.

Increasing evidence suggests that metals play a pivotal role in the pathophysiology of neurodegenerative disorders. Zinc and copper greatly accelerate aggregation of -amyloid and might play a critical role in neurotoxicity induced by -amyloid (38, 39). Copper and manganese both bind to the prion protein and appear to influence the clinical course of prion-induced neurodegeneration (40, 41). Iron levels are increased in brains of patients with PD, and iron is present in Lewy bodies. Neuromelanin selectively binds Fe(III) and might liberate Fe(II) as the Fe(III) is reduced to Fe(II) (via the Haber Weiss reaction) by free radicals produced in response to the oxidative stress associated with PD, providing a potential source of Fe(II) to accelerate -synuclein aggregation (42, 43). On the other hand, magnesium levels are reduced in brains of patients with PD (21-26). If iron accelerates -synuclein aggregation, then the abundance of iron in the substantia nigra could increase the tendency of Lewy bodies to accumulate in this region. In a companion paper,² we extend our studies from the test tube to neurons, and show that magnesium also inhibits aggregation of -synuclein in neurons. Together, these data raise the possibility that iron and magnesium might also modulate -synuclein aggregation in the brain.

	ACKNOWLEDGEMENTS

We acknowledge the assistance of Alan Frankfater (Loyola University) and Brian Shoichet (Northwestern) and thank Matthew Farrer and John Hardy (Mayo Clinic) for providing the -synuclein cDNA.

	FOOTNOTES

^* This work was supported by NINDS Grant NS41786-01, United States Army Medical Research and Materiel Command Grant DAMD17-01-1-0781, Retirement Research Foundation, and Panacea Pharmaceuticals (to B. W.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Pharmacology, Loyola University Medical Center, Bldg. 102, Rm. 3634, 2160 S. 1^st Ave., Maywood, IL 60153. Tel.: 708-216-6195; Fax: 708-216-6596; E-mail: bwolozi@lumc.edu.

Published, JBC Papers in Press, February 15, 2002, DOI 10.1074/jbc.M107866200

² N. Golts, H. Snyder, M. Frasier, C. Theisler, P. Choi, and B. Wolozin, submitted for publication.

	ABBREVIATIONS

The abbreviation used is: PD, Parkinson's disease.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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