- Email: [email protected]
Synergistic Effects of Pesticides and Metals on the Fibrillation of α-Synuclein: Implications for Parkinson’s Disease
NeuroToxicology 23 (2002) 527–536
Synergistic Effects of Pesticides and Metals on the Fibrillation of a-Synuclein: Implications for Parkinson’s Disease Vladimir N. Uversky, Jie Li, Kiowa Bower, Anthony L. Fink* Department of Chemistry and Biochemistry, University of California, Santa Cruz, CA 95064, USA Received 30 November 2001; accepted 22 April 2002
Abstract Aggregation of a-synuclein has been implicated in the formation of proteinaceous inclusions in the brain (Lewy bodies, Lewy neurites) that are characteristic of neurodegenerative diseases, such as Parkinson’s disease (PD) and dementia with Lewy bodies (DLBs). The etiology of PD is unknown, but recent work has shown that except in rare cases, there appears to be no direct genetic basis. However, several studies have implicated environmental factors, especially pesticides and metals. Here we show that certain pesticides and metals induce a conformational change in a-synuclein and directly accelerate the rate of formation of a-synuclein fibrils in vitro. In addition, the simultaneous presence of metal and pesticide led to synergistic effects on the rate of fibrillation. We propose a model in which environmental factors in conjunction with genetic susceptibility may form the underlying molecular basis for idiopathic PD. # 2002 Elsevier Science Inc. All rights reserved.
Keywords: a-Synuclein; Aggregation; Fibrils; Pesticides; Metals
INTRODUCTION The aggregation of a-synuclein has been implicated in the formation of inclusions in the brain, Lewy bodies and Lewy neurites, that are characteristic of neurodegenerative diseases, such as Parkinson’s disease (PD) and dementia with Lewy bodies (DLBs), as well as intraglial inclusions in multiple system atrophy (MSA) (Spillantini et al., 1998; Iwatsubo et al., 1996; Goedert, 2001). PD is the second most common neurodegenerative disease, affecting an estimated five million people worldwide. Lewy bodies are intraneuronal cytoplasmic inclusions composed of filaments of a-synuclein that radiate out from a central core. The two pathological hallmarks of PD are the loss of dopaminergic neurons in the substantia nigra region of the brain, and the presence of Lewy bodies in different * Corresponding author. Tel.: þ1-831-459-2744; fax: þ1-831-459-2744. E-mail address: [email protected] (A.L. Fink).
regions of the brain. The cause of the disease is unknown, but considerable evidence suggests a multifactorial etiology involving genetic and environmental factors. Recent work has shown that except in extremely rare cases, there appears to be no direct genetic basis (Tanner et al., 1999). However, several studies have implicated environmental factors, especially pesticides and metals (Tanner, 1989; Hashimoto et al., 1999; Hertzman et al., 1994; Hirsch et al., 1991; Good et al., 1992; Yasui et al., 1992; Gorell et al., 1999; Hellenbrand et al., 1996), including iron and aluminum (Altschuler, 1999; Good et al., 1992; Hirsch et al., 1991; Yasui et al., 1992). a-Synuclein is a relatively abundant brain protein of 140 amino acids and of unknown function. a-Synuclein belongs to the class of proteins known as natively unfolded (Uversky et al., 2000); i.e. the purified protein at neutral pH is substantially disordered (Weinreb et al., 1996; Eliezer et al., 2001; Uversky et al., 2001c). Fibrils of a-synuclein have been reported in Lewy bodies from individuals with Lewy body diseases, as
0161-813X/02/$ – see front matter # 2002 Elsevier Science Inc. All rights reserved. PII: S 0 1 6 1 - 8 1 3 X ( 0 2 ) 0 0 0 6 7 - 0
528
V.N. Uversky et al. / NeuroToxicology 23 (2002) 527–536
well as in vitro (El-Agnaf et al., 1998; Conway et al., 1998; Crowther et al., 1998; Jakes etal., 1999; Hashimoto et al., 1998; Narhi et al., 1999). Recently, we reported preliminary studies indicating that certain pesticides and metals could accelerate the formation of a-synuclein fibrils (Uversky et al., 2001c,d). Here we show that specific pesticides and metals can directly accelerate the formation of a-synuclein fibrils, both individually and synergistically. Since these agents also induce a conformational change in a-synuclein, it is likely that this partiallyfolded conformation is a critical precursor to association and fibrillation. These observations suggest a possible underlying molecular basis for PD and related Lewy body diseases.
A 100 ml aliquot of 71 mM a-synuclein in 25 mM Tris– HCl, 0.1 M NaCl, pH 8.0 and 20 mM TFT, with or without additives, was added into each well of 96-well plate (plastic from Dynex Technology, Chantilly, VA) with a Teflon bead (3 mm diameter from McMasterCarr, Los Angeles). The plate was incubated at 37 8C, shaking at 300–600 rpm with 1 mm diameter radius. The TFT fluorescence was measured at 30 min intervals with excitation at 450 nm and emission at 485 nm. Experiments were run in at least triplicate and averaged. All data were processed using DataMax/GRAMS software. The presence of fibrils was confirmed by electron microscopy (EM; negative staining with uranyl acetate) and atomic force microscopy (AFM). Circular Dichroism Measurements
MATERIALS AND METHODS Materials a-Synuclein was expressed and purified as described previously (Uversky et al., 2001b). Solutions of metal chlorides and pesticides (dissolved in acetone if necessary) were made by dissolving in pH 7.5 PBS buffer. For aluminum chloride, the initial solution was made at low pH in sodium acetate buffer and adjusted to pH 7.5 with NaOH. Fibril Formation Solutions of 71 mM a-synuclein at pH 7.5 in 25 mM Tris, 100 mM NaCl buffer were stirred or shaken at 37 8C. Fibril formation was monitored with thioflavin T (TFT) fluorescence (Naiki et al., 1989, 1990). Two different procedures were used in monitoring the kinetics of fibril formation: in cases where samples were incubated in glass vials, 0.5 ml samples of 1.0 mg/ml (71 mM) a-synuclein at pH 8.0 in 25 mM Tris buffer were stirred (600 rpm) at 37 8C in glass vials (2 ml; Fisher, PA, USA) with Teflon-coated micro stirrer bars (8 mm length 1:5 mm diameter, Fisher, PA, USA). The TFT fluorescence was recorded immediately after adding an aliquot of 5 ml of the sample to 1.0 ml of 10 mM TFT in 20 mM Tris–HCl, 0.1 M NaCl with excitation at 450 nm, and emission at 482 nm using a FluoroMax-2 spectrofluorometer from Jobin Yvon-Spex. For each sample, the signal was obtained as the TFT intensity at 482 nm less the blank measurement recorded prior to addition of a-synuclein. Alternately, TFT fluorescence was monitored using a 96-well plate reader (Labsystems Fluoroskan Ascent).
Circular dichroism (CD) spectra were obtained with an AVIV 60DS spectrophotometer (Lakewood, NJ) using a-synuclein concentration of 35 mM. Spectra were recorded in a 0.01 cm pathlength cell from 250 to 190 nm with a step size of 0.5 nm, a bandwidth of 1.5 nm, and an averaging time of 10 s. For all spectra, an average of five scans was obtained. CD spectra of the appropriate buffers were recorded and subtracted from the protein spectra. Fluorescence Measurements Fluorescence measurements were performed in semimicro quartz cuvettes (Hellma) with a 1 cm excitation light path using a FluoroMax-2 spectrofluorometer (Instruments SA, Inc., Jobin Yvon-Spex). The light source was a 150 W xenon lamp. Tyrosine fluorescence was excited at 275 nm and monitored in the range from 290 to 350 nm. Protein concentration for the intrinsic fluorescence measurements was kept at about 3.5 mM, unless otherwise mentioned.
RESULTS We have recently shown that the fibrillation of a-synuclein involves formation of a critical partially folded intermediate species (Uversky et al., 2001b), and that the minimum kinetic scheme for fibrillation is as follows: Monomer ! Intermediate ! Nucleus ! Fibrils It is likely that there may be additional oligomeric intermediates on the pathway, as well (Conway et al., 2000). There are a number of factors that accelerate the
V.N. Uversky et al. / NeuroToxicology 23 (2002) 527–536
rate of a-synuclein aggregation and fibril formation: these include increasing protein concentration (Uversky et al., 2001b) and mutations (Conway et al., 1998; Uversky et al., 2001b; Li et al., 2001), both of which may be implicated in PD, as well as agitation and increased ionic strength. Solutions of a-synuclein at pH 7.5, stirred at 37 8C, formed fibrils over a period of days to weeks, depending on the protein concentration, rate of agitation and ionic strength. As observed for other aggregating systems, the kinetics of a-synuclein aggregation exhibit an initial lag, followed by an exponential growth period, followed by a leveling off as fibril formation comes to a halt.
529
Pesticide-Stimulated a-Synuclein Fibril Formation In order to determine whether the observed epidemiological studies indicating a correlation between pesticide exposure and increased risk of PD could be due to a direct effect of the pesticide on a-synuclein fibrillation, we examined the effect of selected pesticides on the kinetics of in vitro fibrillation. In accordance with our recent report (Uversky et al., 2001d), certain pesticides representing different chemical classes, significantly stimulate the rate of formation of fibrils when incubated with a-synuclein at pH 7.5 and 37 8C (Fig. 1a and b). These experiments used low concentrations of a-synuclein and 10–100 mM pesticide.
Fig. 1. Kinetics of a-synuclein fibril formation in the presence of pesticides. Panel a: a-synuclein (70 mM) was incubated with shaking at 37 8C in Tris buffer, pH 7.5, 100 mM NaCl. Fibril formation was monitored by the increase in thioflavin T (TFT) fluorescence using a plate reader. The data shown are the averages of at least three separate experiments. The pesticides DDT and rotenone were 100 mM. The control was in the absence of pesticide. The different final TFT signals may reflect different amounts of fibrillar and amorphous aggregates, or effects of the pesticides on the TFT signal. Panel b: a similar experiment, using 25 mM dieldrin and 140 mM a-synuclein. Panel c: a similar experiment showing that 100 mM glypohosate or iprodione have similar fibrillation kinetics to the control (no pesticide). Panel d: 100 mM MPPþ also does not significantly affect the kinetics of a-synuclein fibrillation under these conditions.
530
V.N. Uversky et al. / NeuroToxicology 23 (2002) 527–536
Fig. 2. Pesticides that accelerate a-synuclein fibrillation do so in a dose-dependent fashion. Kinetics of a-synuclein fibril formation in the presence of 0–1000 mM dieldrin. a-Synuclein (70 mM) was incubated with shaking at 37 8C in Tris buffer, pH 7.5 and 100 mM NaCl. Fibril formation was monitored by the increase in thioflavin T (TFT) fluorescence using a plate reader.
Of the pesticides examined, those having the most significant accelerating effects were rotenone, DDT, 2,4-D, dieldrin, diethyldithiocarbamate, paraquat, maneb, trifluralin, parathion, and imidazoldinethione. A significant number of the pesticides examined had no significant effect on the kinetics of a-synuclein fibrillation (Fig. 1c; including iprodione, glyphosate, methomyl, thiuram, mevinphos, carbaryl, alachlor, thiobencarb). These also included MPPþ, the active metabolite of MPTP that causes PD-like symptoms in humans and other primates (Fig. 1d). Although the mechanism of action of MPPþ is unknown, it is believed to involve inhibition of Complex I of the mitochondria and free radicals or other oxidative stress. In contrast to MPPþ, rotenone, which is also thought to induce Parkinsonism in rats by inhibition of Complex I of the mitochondria and oxidative stress, accelerated a-synuclein fibrillation (Fig. 1a). Interestingly, given the apparent negative association of smoking and caffeine with PD, the presence of nicotine and caffeine had no effect on the kinetics of a-synuclein fibrillation. The effect of varying the concentration of pesticide was investigated for paraquat (Manning-Bog et al., 2002b), rotenone and dieldrin (Fig. 2). In each case, increasing the concentration of pesticide led to increased rates of fibrillation. For rotenone the lagtimes decreased from 74.5 h for the control to 41.8 h for 10 mM rotenone, to 35.2 h for 100 mM and to 2.7 h for 1000 mM. That pesticides may accelerate a-synuclein fibrillation at quite low concentrations was demon-
strated with rotenone and dieldrin (Fig. 2). For example, the kinetics of a-synuclein fibrillation were doubled in the presence of 25 mM dieldrin with 140 mM a-synuclein (Fig. 1b). Metal Ion-Stimulated a-Synuclein Fibril Formation We have recently shown that a variety of divalent and trivalent metal ions also can directly affect the fibril formation of a-synuclein (Uversky et al., 2001a,c). In those experiments, we used quite low concentrations of a-synuclein, possibly as much as 10-fold lower than in neurons, and high metal ion concentrations (5 mM). In experiments using higher a-synuclein concentrations, micromolar amounts of aluminum stimulated the rate of fibril formation (Fig. 3). There was a good correlation between charge density and effectiveness in stimulating a-synuclein fibrillation: thus, trivalent metals were most effective in general. In addition, low pH also stimulated the rate of fibril assembly (Uversky et al., 2001b). The most effective ions were Al3þ, Fe3þ, Co3þ, Cd2þ, Mn2þ, Cu2þ, Co2þ, in that order. Pesticide and Metal-Induced Conformational Changes in a-Synuclein We have previously shown that a conformational change corresponding to formation of a partially folded
V.N. Uversky et al. / NeuroToxicology 23 (2002) 527–536
531
Fig. 3. The amount of AlCl3 required to accelerate the fibrillation of a-synuclein decreases at higher a-synuclein concentrations. a-synuclein (200 mM) was incubated with shaking at 37 8C in Tris buffer, pH 7.5, 100 mM NaCl. Fibril formation was monitored by the increase in thioflavin T (TFT) fluorescence using a plate reader. The presence of 200 mM Al3þ (&) significantly increases the rate of fibrillation over the control (5) (no Al3þ).
intermediate is a critical step in the formation of a-synuclein fibrils (Uversky et al., 2001b). We confirmed that those pesticides and metals that caused a significant increase in the kinetics of fibrillation also caused a conformational change detectable by circular dichroism and tyrosine fluorescence. Fig. 4 shows the
changes induced in the far-UV circular dichroism spectra of a-synuclein by DDC and AlCl3. The spectra in the presence of DDC and aluminum are typical of the partially-folded intermediate induced by other metals and pesticides. Synergistic Effects of Pesticides and Metals on a-Synuclein Fibrillation
Fig. 4. Metal ion and pesticide-induced conformational changes in a-synuclein. Far-UV circular dichroism spectra of 35 mM a-synuclein (*); in the presence of 100 mM DDC (&); 4 mM AlCl3 (5); and both 100 mM DDC and 4 mM AlCl3 (^). Spectra were measured within minutes of mixing the samples.
Since many individuals at risk due to pesticide and/ or metal exposure are expected to accumulate both metals and pesticides in their brains, it is important to know whether the effects are additive. In fact, the simultaneous presence of Al3þ and the pesticide DDC led to fibril formation kinetics that were greater than expected from non-cooperative effectors. For example, in experiments in which a-synuclein was incubated in the simultaneous presence of both DDC and AlCl3, fibril formation was more rapid than that expected from the sum of the effects of the two agents alone, Fig. 5, indicating a synergistic effect between the pesticide and the metal ion. For example, with AlCl3 (4 mM) and DDC (100 mM) added in that order the observed tm (time to half-maximum signal) was 49.5 h, whereas the calculated time based on the product of the percentage decrease in lag time for each ligand, was 63.6 h. The values for tm were 198 h (no additions), 125 h (100 mM DDC) and 98 h (4 mM AlCl3). The simultaneous presence of DDC and AlCl3 led to enhanced conformational change, as detected by
532
V.N. Uversky et al. / NeuroToxicology 23 (2002) 527–536
Fig. 5. Synergistic interactions between pesticide and metal in the stimulation of a-synuclein fibril formation. The kinetics of fibril formation were monitored as in the legend to Fig. 1. a-Synuclein (28 mM) alone (&); with 100 mM DDC (*); with 4 mM AlCl3 (*); with DDC (100 mM) and AlCl3 (4 mM) added in that order (!); and with AlCl3 (4 mM) and DDC (100 mM) added in that order (5).
CD, over each additive individually, Fig. 4. In addition, the order of addition of metal and pesticide affected the kinetics (Fig. 5): the addition of the metal prior to the pesticide, probably because the effect of metals on the conformational change was significantly more rapid than that of pesticides, caused a larger effect, compared to the initial addition of pesticide. Interestingly, the effects of pesticides and metals on the tyrosine fluorescence of a-synuclein varied significantly, depending, in part, on the effect of the environmental agent on free tyrosine. For example, for AlCl3, plots of the change in fluorescence as a function of increasing Al3þ concentration gave a sigmoidal increase in fluorescence (Fig. 6a). On the other hand for the pesticide DDC, such plots showed an exponential decrease with increasing pesticide (Fig. 6b). The synergistic effects of DDC and AlCl3 are also seen in their effects on a-synuclein fluorescence (Fig. 6). The presence of DDC causes a major shift in the position of the curve for the effect of Al3þ on fluorescence (Fig. 6a). Similarly, the presence of AlCl3 causes substantial changes in the curves for the effect of DDC on a-synuclein fluorescence (Fig. 6b).
DISCUSSION Parkinsonism has been associated with long term occupational exposure to pesticides and certain metals. Currently, about one billion pound of pesticides used
annually in the US, specific pesticides that have been implicated include: paraquat, organochlorine compounds, dieldrin, 1,10 -(2,2-dichloroethenyldiene)bis(4-chlorobenzene), hexachlorocyclohexane (lindane). There are many possible mechanisms whereby pesticides and metals could lead to a-synuclein aggregation. Oxidative damage, especially involving inhibition of mitochondrial Complex I and dopamine oxidation, via free radicals is often considered to be the causative ‘‘metabolic’’ factor in PD. Alternative hypotheses are that environmental factors (and other chemicals) can directly affect the aggregation of a-synuclein or adversely affect proteasomal function leading to accumulation of a-synuclein and its subsequent aggregation. It is most likely that the cause of PD is multi-factorial (Fig. 7), with genetic factors at one extreme and environmental agents at the other. In most cases, the potential effects of environmental factors will be modulated by the genetic susceptibility of the individual, for example, the efficiency of liver enzymes to eliminate the pesticide and the efficiency of redox systems to minimize oxidative damage. The data for rotenone, paraquat and MPPþ are interesting in that they show that rotenone and paraquat have a direct effect on a-synuclein fibrillation, whereas MPPþ does not, even though the structure of MPPþ and paraquat are very similar. In addition, it is known that both rotenone and MPPþ are Complex I inhibitors, thus it is likely that the in vivo effects of these compounds are complex, probably interacting with
V.N. Uversky et al. / NeuroToxicology 23 (2002) 527–536
533
Fig. 6. Synergistic interactions between pesticide and metal in the induction of structure in a-synuclein. Structural changes were measured by tyrosine fluorescence (there are no tryptophan residues in a-synuclein). Panel a shows the effect of increasing AlCl3 concentration on the fluorescence in the presence of 0 (*) and 100 mM DDC (5). Panel b shows the effect of increasing DDC concentration on the intrinsic fluorescence of a-synuclein as a function of AlCl3. The normalized fluorescence signal was obtained by scaling the observed fluorescence intensity changes to start at 0 and finish at 1.0.
different cellular components and leading to different effects, which may culminate, however, in the aggregation of a-synuclein (Fig. 7). Our results are consistent with a model in which both the cations and pesticides interact with a-synuclein to bring about a conformational change to a partiallyfolded state with a high propensity to aggregate. Thus, the lag in the kinetics of fibril formation actually reflects the association of the partially-folded intermediate conformation, rather than the ‘‘native’’ conformation. Consequently, there are three major steps leading to fibril formation: a structural change that leads to the
critical intermediate conformation, association of this intermediate to form a fibril nucleus, and finally template-directed growth of the fibril. Comparison of the structures of the pesticide that are most effective in accelerating fibril formation, suggests that increasing hydrophobicity of the pesticide is an important factor in stimulating aggregation. This is consistent with the idea that the pesticide binds to the partially-folded intermediate conformation (which is in equilibrium with the natively unfolded state), thereby increasing the population of the intermediate. The driving force for this is the existence of contiguous
534
V.N. Uversky et al. / NeuroToxicology 23 (2002) 527–536
Fig. 7. Proposed relationships illustrating the multi-factorial basis of Parkinson’s disease. In the majority of cases it is proposed that environmental agents, modulated by the individual’s genetic susceptibility (see Discussion), result in the aggregation of a-synuclein. Some aspect of the aggregation process is neurotoxic (possibilities include oligomeric intermediates, amorphous aggregates or fibrils), leading to the death of dopaminergic neurons and eventually Parkinson’s disease.
patches of hydrophobic surface in the intermediate (and which are absent in the natively unfolded conformation). Precedence for such a ligand bindinginduced shift in equilibrium to favor a partially-folded intermediate conformation has previously been reported (Shi et al., 1994). Based on our observation that 8-anilinonaphthalene-1-sulfonic acid (ANS) also stimulates the aggregation of a-synuclein (data not shown), we predict that many compounds that are relatively hydrophobic and water-soluble will induce a-synuclein fibril formation. The C-terminal region of a-synuclein (about 40 amino acids) is very rich in acidic residues, and thus highly negatively charged at neutral pH. The resulting repulsive interactions are a major factor leading to the natively unfolded conformation, since the unfolded conformation of natively unfolded proteins is due to a combination of high net charge and low intrinsic hydrophobicity (Uversky et al., 2000). Although the results with the metal ion-stimulated a-synuclein fibrillation and conformational change indicate that there are definite cation-specific effects, we believe that the dominant effect of the metal ions is due to masking of the Coulombic charge–charge repulsion (Goto et al., 1990b). For polyvalent cations, an additional important factor is the potential for cross-linking or bridging between two or more carboxylates. With the apparent exception of cadmium, there is an excellent correlation between the size of the cation and the degree of fibril stimulation. The correlation is even more marked in
terms of the charge density. Thus, smaller polyvalent cations are able to screen the unfavorable electrostatic interactions and at the same time have minimal perturbation of the resulting protein structure due to their small size. The high net charge and unfolded conformation of a-synuclein (pI ¼ 4:7) at neutral pH means that it has many features that parallel those of acid-unfolded proteins. The addition of anions to acid-unfolded proteins leads to conformations that are much more folded (Goto et al., 1990a,b; Fink et al., 1994, 1998; Uversky et al., 1998), due to the screening effect of the anion on the electrostatic repulsion from the positively charged amines. A noteworthy point is that different anions differ dramatically in their effectiveness in inducing these conformational changes (Goto et al., 1990b; Fink et al., 1998; Uversky et al., 1998). Thus, anion effects at low pH are a good model for the effects of cations on a-synuclein at neutral pH. Thus, environmental factors that stimulate a-synuclein aggregation do so by increasing the concentration of the critical partially-folded intermediate conformation that leads to aggregation. For cations the effect is due to the positively charged metal ions masking the negatively charged groups mostly responsible for the natively unfolded conformation. For pesticides the effect is due to their preferential binding to the partially-folded intermediate conformation. Consequently, there are at least two different pathways by which environmental factors may stimulate the formation of a-synuclein fibrils at a fundamental molecular level. The synergy between cations and pesticides may arise from the different natures of their interactions with the partially folded intermediate as well as dimers or higher oligomers of the partially-folded intermediate. Most importantly, however, the rates of cation- or pesticide-induced aggregation of a-synuclein may be increased in the presence of other molecules due to synergistic effects. This observation is particularly relevant to PD because it suggests that when pesticides, herbicides or other small soluble hydrophobic molecules (including some products of oxidative stress) are in the presence of certain metal ions, the concentrations of either required to cause rapid formation of a-synuclein fibrils may be substantially reduced. These interactions between a-synuclein and environmental agents could play a role in the pathogenesis of nigrostriatal degeneration, and thus in the etiology of sporadic PD. The synergistic effects observed between pesticide and metal suggests that the total brain load of pesticides and metals, rather than individual levels, is a
V.N. Uversky et al. / NeuroToxicology 23 (2002) 527–536
very important contributor to the potential effect on a-synuclein fibrillation. Thus, although the levels necessary for significant accelerations of a-synuclein fibrillation in our experiments are in the micromolar range, they decrease with increasing a-synuclein concentration. From the overall abundance of a-synuclein in the brain it is reasonable to assume that the cellular concentration in neurons in the substantia nigra could be several hundred micromolar (Hashimoto et al., 2001). Experiments in which mice were treated with paraquat and develop a-synuclein deposits demonstrate that sufficient paraquat can enter the dopaminergic neurons in the substantia nigra, to cause a-synuclein to aggregate in vivo (ManningBog et al., 2002a). Thus, our in vitro observations are paralleled by corresponding in vivo effects. Similar observations were made in which mice were treated with a combination of DDC and AlCl3 (data not shown).
ACKNOWLEDGEMENTS We thank Drs. J. Gillespie, J.W, Langston and D. Di Monte for valuable discussions. This research was supported by grant RO1 NS39985 from the National Institutes of Health.
REFERENCES Altschuler E. Aluminum-containing antacids as a cause of idiopathic Parkinson’s disease. Med Hypotheses 1999;53:22–3. Conway KA, Harper JD, Lansbury PT. Accelerated in vitro fibril formation by a mutant a-synuclein linked to early-onset Parkinson disease. Nat Med 1998;4:1318–20. Conway KA, Lee SJ, Rochet JC, Ding TT, Williamson RE, Lansbury Jr, PT. Acceleration of oligomerization, not fibrillization, is a shared property of both a-synuclein mutations linked to early-onset Parkinson’s disease: implications for pathogenesis and therapy. Proc Natl Acad Sci USA 2000; 97:571–6[in process citation]. Crowther RA, Jakes R, Spillantini MG, Goedert M. Synthetic filaments assembled from C-terminally truncated a-synuclein. FEBS Lett 1998;436:309–12. El-Agnaf OMA, Jakes R, Curran MD, Wallace A. Effects of the mutations Ala(30) to Pro and Ala(53) to Thr on the physical and morphological properties of a-synuclein protein implicated in Parkinson’s disease. FEBS Lett 1998;440:67–70. Eliezer D, Kutluay E, Bussell Jr, R, Browne G. Conformational properties of a-synuclein in its free and lipid-associated states. J Mol Biol 2001;307:1061–73. Fink AL, Calciano LJ, Goto Y, Kurotsu T, Palleros DR. Classification of acid denaturation of proteins—intermediates and unfolded states. Biochemistry 1994;33:12504–11.
535
Fink AL, Oberg KA, Seshadri S. Discrete intermediates versus molten globule models for protein folding: characterization of partially folded intermediates of apomyoglobin. Fold Des 1998;3:19–25. Goedert M. Parkinson’s disease and other a-synucleinopathies. Clin Chem Lab Med 2001;39:308–12. Good PF, Olanow CW, Perl DP. Neuromelanin-containing neurons of the substantia nigra accumulate iron and aluminum in Parkinson’s disease: a LAMMA study. Brain Res 1992;593: 343–6. Gorell JM, Johnson CC, Rybicki BA, Peterson EL, Kortsha GX, Brown GG, Richardson RJ. Occupational exposure to manganese, copper, lead, iron, mercury and zinc and the risk of Parkinson’s disease. Neurotoxicology 1999;20:239–47. Goto Y, Calciano LJ, Fink AL. Acid-induced folding of proteins. Proc Natl Acad Sci USA 1990a;87:573–7. Goto Y, Takahashi N, Fink AL. Mechanism of acid-induced folding of proteins. Biochemistry 1990b;29:3480–8. Hashimoto M, Hsu LJ, Sisk A, Xia Y, Takeda A, Sundsmo M, Masliah E. Human recombinant NACP/a-synuclein is aggregated and fibrillated in vitro: Relevance for Lewy body disease. Brain Res 1998;799:301–6. Hashimoto M, Hsu LJ, Xia Y, Takeda A, Sisk A, Sundsmo M, Masliah E. Oxidative stress induces amyloid-like aggregate formation of NACP/a-synuclein in vitro. Neuroreport 1999;10: 717–21. Hashimoto M, Rockenstein E, Mante M, Mallory M, Masliah E. b-Synuclein inhibits a-synuclein aggregation. A possible role as an anti-Parkinsonian factor. Neuron 2001;32:213– 23. Hellenbrand W, Boeing H, Robra BP, Seidler A, Vieregge P, Nischan P, Joerg J, Oertel WH, Schneider E, Ulm G. Diet and Parkinson’s disease. II. A possible role for the past intake of specific nutrients. Results from a self-administered foodfrequency questionnaire in a case–control study. Neurology 1996;47:644–50[see comments]. Hertzman C, Wiens M, Snow B, Kelly S, Calne D. A case-control study of Parkinson’s disease in a horticultural region of British Columbia. Mov Disord 1994;9:69–75. Hirsch EC, Brandel JP, Galle P, Javoy-Agid F, Agid Y. Iron and aluminum increase in the substantia nigra of patients with Parkinson’s disease: an X-ray microanalysis. J Neurochem 1991;56:446–51. Iwatsubo T, Yamaguchi H, Fujimuro M, Yokosawa H, Ihara Y, Trojanowski JQ, Lee VMY. Purification and characterization of Lewy bodies from the brains of patients with diffuse Lewy body disease. Am J Pathol 1996;148:1517–29. Jakes R, Crowther RA, Lee VM, Trojanowski JQ, Iwatsubo T, Goedert M. Epitope mapping of LB509, a monoclonal antibody directed against human a-synuclein. Neurosci Lett 1999;269: 13–6. Li J, Uversky VN, Fink AL. Effect of familial Parkinson’s disease point mutations A30P and A53T on the structural properties, aggregation, and fibrillation of human a-synuclein. Biochemistry 2001;40:11604–13. Manning-Bog AB, McCormack AL, Li J, Uversky VN, Fink AL, Di Monte DA. The herbicide paraquat causes up-regulation and aggregation of a-synuclein in mice. Paraquat and a-synuclein. J Biol Chem 2002a;277:1641–4. Manning-Bog AB, McCormack AL, Li J, Uversky VN, Fink AL, Di Monte DA. The herbicide paraquat causes up-regulation and
536
V.N. Uversky et al. / NeuroToxicology 23 (2002) 527–536
aggregation of a-synuclein in mice. Paraquat and a-synuclein. J Biol Chem 2002b;277:1641–4. Naiki H, Higuchi K, Hosokawa M, Takeda T. Fluorometric determination of amyloid fibrils in vitro using the fluorescent dye, thioflavin T1. Anal Biochem 1989;177:244–9. Naiki H, Higuchi K, Matsushima K, Shimada A, Chen WH, Hosokawa M, Takeda T. Fluorometric examination of tissue amyloid fibrils in murine senile amyloidosis: use of the fluorescent indicator, thioflavine T. Lab Invest 1990;62:768–73. Narhi L, Wood SJ, Steavenson S, Jiang Y, Wu GM, Anafi D, Kaufman SA, Martin F, Sitney K, Denis P, Louis JC, Wypych J, Biere AL, Citron M. Both familial Parkinson’s disease mutations accelerate a-synuclein aggregation. J Biol Chem 1999;274:9843–6. Shi L, Palleros DR, Fink AL. Protein conformational changes induced by 1,10 -bis(4-anilino-5-naphthalenesulfonic acid)— preferential binding to the molten globule of dnak. Biochemistry 1994;33:7536–46. Spillantini MG, Crowther RA, Jakes R, Hasegawa M, Goedert M. a-Synuclein in filamentous inclusions of Lewy bodies from Parkinson’s disease and dementia with Lewy bodies. Proc Natl Acad Sci USA 1998;95:6469–73. Tanner CM. The role of environmental toxins in the etiology of Parkinson’s disease. Trends Neurosci 1989;12:49–54. Tanner CM, Ottman R, Goldman SM, Ellenberg J, Chan P, Mayeux R, Langston JW. Parkinson disease in twins: an etiologic study. JAMA 1999;281:341–6.
Uversky VN, Karnoup AS, Segel DJ, Seshadri S, Doniach S, Fink AL. Anion-induced folding of staphylococcal nuclease: characterization of multiple equilibrium partially folded intermediates. J Mol Biol 1998;278:879–94. Uversky VN, Gillespie JR, Fink AL. Why are ‘‘natively unfolded’’ proteins unstructured under physiologic conditions? Proteins 2000;41:415–27. Uversky VN, Lee HJ, Li J, Fink AL, Lee SJ. Stabilization of partially folded conformation during a-synuclein oligomerization in both purified and cytosolic preparations. J Biol Chem 2001a;276:43495–8. Uversky VN, Li J, Fink AL. Evidence for a partially folded intermediate in a-synuclein fibril formation. J Biol Chem 2001b;276:10737–44. Uversky VN, Li J, Fink AL. Metal-triggered structural transformations, aggregation, and fibrillation of human a-synuclein. A possible molecular link between Parkinson’s disease and heavy metal exposure. J Biol Chem 2001c;276:44284–96. Uversky VN, Li J, Fink AL. Pesticides directly accelerate the rate of a-synuclein fibril formation: a possible factor in Parkinson’s disease. FEBS Lett 2001d;500:105–8. Weinreb PH, Zhen WG, Poon AW, Conway KA, Lansbury Jr, PT. NACP, a protein implicated in Alzheimer’s disease and learning, is natively unfolded. Biochemistry 1996;35:13709–15. Yasui M, Kihira T, Ota K. Calcium, magnesium and aluminum concentrations in Parkinson’s disease. Neurotoxicology 1992; 13:593–600.
Synergistic Effects of Pesticides and Metals on the Fibrillation of a-Synuclein: Implications for Parkinson’s Disease Vladimir N. Uversky, Jie Li, Kiowa Bower, Anthony L. Fink* Department of Chemistry and Biochemistry, University of California, Santa Cruz, CA 95064, USA Received 30 November 2001; accepted 22 April 2002
Abstract Aggregation of a-synuclein has been implicated in the formation of proteinaceous inclusions in the brain (Lewy bodies, Lewy neurites) that are characteristic of neurodegenerative diseases, such as Parkinson’s disease (PD) and dementia with Lewy bodies (DLBs). The etiology of PD is unknown, but recent work has shown that except in rare cases, there appears to be no direct genetic basis. However, several studies have implicated environmental factors, especially pesticides and metals. Here we show that certain pesticides and metals induce a conformational change in a-synuclein and directly accelerate the rate of formation of a-synuclein fibrils in vitro. In addition, the simultaneous presence of metal and pesticide led to synergistic effects on the rate of fibrillation. We propose a model in which environmental factors in conjunction with genetic susceptibility may form the underlying molecular basis for idiopathic PD. # 2002 Elsevier Science Inc. All rights reserved.
Keywords: a-Synuclein; Aggregation; Fibrils; Pesticides; Metals
INTRODUCTION The aggregation of a-synuclein has been implicated in the formation of inclusions in the brain, Lewy bodies and Lewy neurites, that are characteristic of neurodegenerative diseases, such as Parkinson’s disease (PD) and dementia with Lewy bodies (DLBs), as well as intraglial inclusions in multiple system atrophy (MSA) (Spillantini et al., 1998; Iwatsubo et al., 1996; Goedert, 2001). PD is the second most common neurodegenerative disease, affecting an estimated five million people worldwide. Lewy bodies are intraneuronal cytoplasmic inclusions composed of filaments of a-synuclein that radiate out from a central core. The two pathological hallmarks of PD are the loss of dopaminergic neurons in the substantia nigra region of the brain, and the presence of Lewy bodies in different * Corresponding author. Tel.: þ1-831-459-2744; fax: þ1-831-459-2744. E-mail address: [email protected] (A.L. Fink).
regions of the brain. The cause of the disease is unknown, but considerable evidence suggests a multifactorial etiology involving genetic and environmental factors. Recent work has shown that except in extremely rare cases, there appears to be no direct genetic basis (Tanner et al., 1999). However, several studies have implicated environmental factors, especially pesticides and metals (Tanner, 1989; Hashimoto et al., 1999; Hertzman et al., 1994; Hirsch et al., 1991; Good et al., 1992; Yasui et al., 1992; Gorell et al., 1999; Hellenbrand et al., 1996), including iron and aluminum (Altschuler, 1999; Good et al., 1992; Hirsch et al., 1991; Yasui et al., 1992). a-Synuclein is a relatively abundant brain protein of 140 amino acids and of unknown function. a-Synuclein belongs to the class of proteins known as natively unfolded (Uversky et al., 2000); i.e. the purified protein at neutral pH is substantially disordered (Weinreb et al., 1996; Eliezer et al., 2001; Uversky et al., 2001c). Fibrils of a-synuclein have been reported in Lewy bodies from individuals with Lewy body diseases, as
0161-813X/02/$ – see front matter # 2002 Elsevier Science Inc. All rights reserved. PII: S 0 1 6 1 - 8 1 3 X ( 0 2 ) 0 0 0 6 7 - 0
528
V.N. Uversky et al. / NeuroToxicology 23 (2002) 527–536
well as in vitro (El-Agnaf et al., 1998; Conway et al., 1998; Crowther et al., 1998; Jakes etal., 1999; Hashimoto et al., 1998; Narhi et al., 1999). Recently, we reported preliminary studies indicating that certain pesticides and metals could accelerate the formation of a-synuclein fibrils (Uversky et al., 2001c,d). Here we show that specific pesticides and metals can directly accelerate the formation of a-synuclein fibrils, both individually and synergistically. Since these agents also induce a conformational change in a-synuclein, it is likely that this partiallyfolded conformation is a critical precursor to association and fibrillation. These observations suggest a possible underlying molecular basis for PD and related Lewy body diseases.
A 100 ml aliquot of 71 mM a-synuclein in 25 mM Tris– HCl, 0.1 M NaCl, pH 8.0 and 20 mM TFT, with or without additives, was added into each well of 96-well plate (plastic from Dynex Technology, Chantilly, VA) with a Teflon bead (3 mm diameter from McMasterCarr, Los Angeles). The plate was incubated at 37 8C, shaking at 300–600 rpm with 1 mm diameter radius. The TFT fluorescence was measured at 30 min intervals with excitation at 450 nm and emission at 485 nm. Experiments were run in at least triplicate and averaged. All data were processed using DataMax/GRAMS software. The presence of fibrils was confirmed by electron microscopy (EM; negative staining with uranyl acetate) and atomic force microscopy (AFM). Circular Dichroism Measurements
MATERIALS AND METHODS Materials a-Synuclein was expressed and purified as described previously (Uversky et al., 2001b). Solutions of metal chlorides and pesticides (dissolved in acetone if necessary) were made by dissolving in pH 7.5 PBS buffer. For aluminum chloride, the initial solution was made at low pH in sodium acetate buffer and adjusted to pH 7.5 with NaOH. Fibril Formation Solutions of 71 mM a-synuclein at pH 7.5 in 25 mM Tris, 100 mM NaCl buffer were stirred or shaken at 37 8C. Fibril formation was monitored with thioflavin T (TFT) fluorescence (Naiki et al., 1989, 1990). Two different procedures were used in monitoring the kinetics of fibril formation: in cases where samples were incubated in glass vials, 0.5 ml samples of 1.0 mg/ml (71 mM) a-synuclein at pH 8.0 in 25 mM Tris buffer were stirred (600 rpm) at 37 8C in glass vials (2 ml; Fisher, PA, USA) with Teflon-coated micro stirrer bars (8 mm length 1:5 mm diameter, Fisher, PA, USA). The TFT fluorescence was recorded immediately after adding an aliquot of 5 ml of the sample to 1.0 ml of 10 mM TFT in 20 mM Tris–HCl, 0.1 M NaCl with excitation at 450 nm, and emission at 482 nm using a FluoroMax-2 spectrofluorometer from Jobin Yvon-Spex. For each sample, the signal was obtained as the TFT intensity at 482 nm less the blank measurement recorded prior to addition of a-synuclein. Alternately, TFT fluorescence was monitored using a 96-well plate reader (Labsystems Fluoroskan Ascent).
Circular dichroism (CD) spectra were obtained with an AVIV 60DS spectrophotometer (Lakewood, NJ) using a-synuclein concentration of 35 mM. Spectra were recorded in a 0.01 cm pathlength cell from 250 to 190 nm with a step size of 0.5 nm, a bandwidth of 1.5 nm, and an averaging time of 10 s. For all spectra, an average of five scans was obtained. CD spectra of the appropriate buffers were recorded and subtracted from the protein spectra. Fluorescence Measurements Fluorescence measurements were performed in semimicro quartz cuvettes (Hellma) with a 1 cm excitation light path using a FluoroMax-2 spectrofluorometer (Instruments SA, Inc., Jobin Yvon-Spex). The light source was a 150 W xenon lamp. Tyrosine fluorescence was excited at 275 nm and monitored in the range from 290 to 350 nm. Protein concentration for the intrinsic fluorescence measurements was kept at about 3.5 mM, unless otherwise mentioned.
RESULTS We have recently shown that the fibrillation of a-synuclein involves formation of a critical partially folded intermediate species (Uversky et al., 2001b), and that the minimum kinetic scheme for fibrillation is as follows: Monomer ! Intermediate ! Nucleus ! Fibrils It is likely that there may be additional oligomeric intermediates on the pathway, as well (Conway et al., 2000). There are a number of factors that accelerate the
V.N. Uversky et al. / NeuroToxicology 23 (2002) 527–536
rate of a-synuclein aggregation and fibril formation: these include increasing protein concentration (Uversky et al., 2001b) and mutations (Conway et al., 1998; Uversky et al., 2001b; Li et al., 2001), both of which may be implicated in PD, as well as agitation and increased ionic strength. Solutions of a-synuclein at pH 7.5, stirred at 37 8C, formed fibrils over a period of days to weeks, depending on the protein concentration, rate of agitation and ionic strength. As observed for other aggregating systems, the kinetics of a-synuclein aggregation exhibit an initial lag, followed by an exponential growth period, followed by a leveling off as fibril formation comes to a halt.
529
Pesticide-Stimulated a-Synuclein Fibril Formation In order to determine whether the observed epidemiological studies indicating a correlation between pesticide exposure and increased risk of PD could be due to a direct effect of the pesticide on a-synuclein fibrillation, we examined the effect of selected pesticides on the kinetics of in vitro fibrillation. In accordance with our recent report (Uversky et al., 2001d), certain pesticides representing different chemical classes, significantly stimulate the rate of formation of fibrils when incubated with a-synuclein at pH 7.5 and 37 8C (Fig. 1a and b). These experiments used low concentrations of a-synuclein and 10–100 mM pesticide.
Fig. 1. Kinetics of a-synuclein fibril formation in the presence of pesticides. Panel a: a-synuclein (70 mM) was incubated with shaking at 37 8C in Tris buffer, pH 7.5, 100 mM NaCl. Fibril formation was monitored by the increase in thioflavin T (TFT) fluorescence using a plate reader. The data shown are the averages of at least three separate experiments. The pesticides DDT and rotenone were 100 mM. The control was in the absence of pesticide. The different final TFT signals may reflect different amounts of fibrillar and amorphous aggregates, or effects of the pesticides on the TFT signal. Panel b: a similar experiment, using 25 mM dieldrin and 140 mM a-synuclein. Panel c: a similar experiment showing that 100 mM glypohosate or iprodione have similar fibrillation kinetics to the control (no pesticide). Panel d: 100 mM MPPþ also does not significantly affect the kinetics of a-synuclein fibrillation under these conditions.
530
V.N. Uversky et al. / NeuroToxicology 23 (2002) 527–536
Fig. 2. Pesticides that accelerate a-synuclein fibrillation do so in a dose-dependent fashion. Kinetics of a-synuclein fibril formation in the presence of 0–1000 mM dieldrin. a-Synuclein (70 mM) was incubated with shaking at 37 8C in Tris buffer, pH 7.5 and 100 mM NaCl. Fibril formation was monitored by the increase in thioflavin T (TFT) fluorescence using a plate reader.
Of the pesticides examined, those having the most significant accelerating effects were rotenone, DDT, 2,4-D, dieldrin, diethyldithiocarbamate, paraquat, maneb, trifluralin, parathion, and imidazoldinethione. A significant number of the pesticides examined had no significant effect on the kinetics of a-synuclein fibrillation (Fig. 1c; including iprodione, glyphosate, methomyl, thiuram, mevinphos, carbaryl, alachlor, thiobencarb). These also included MPPþ, the active metabolite of MPTP that causes PD-like symptoms in humans and other primates (Fig. 1d). Although the mechanism of action of MPPþ is unknown, it is believed to involve inhibition of Complex I of the mitochondria and free radicals or other oxidative stress. In contrast to MPPþ, rotenone, which is also thought to induce Parkinsonism in rats by inhibition of Complex I of the mitochondria and oxidative stress, accelerated a-synuclein fibrillation (Fig. 1a). Interestingly, given the apparent negative association of smoking and caffeine with PD, the presence of nicotine and caffeine had no effect on the kinetics of a-synuclein fibrillation. The effect of varying the concentration of pesticide was investigated for paraquat (Manning-Bog et al., 2002b), rotenone and dieldrin (Fig. 2). In each case, increasing the concentration of pesticide led to increased rates of fibrillation. For rotenone the lagtimes decreased from 74.5 h for the control to 41.8 h for 10 mM rotenone, to 35.2 h for 100 mM and to 2.7 h for 1000 mM. That pesticides may accelerate a-synuclein fibrillation at quite low concentrations was demon-
strated with rotenone and dieldrin (Fig. 2). For example, the kinetics of a-synuclein fibrillation were doubled in the presence of 25 mM dieldrin with 140 mM a-synuclein (Fig. 1b). Metal Ion-Stimulated a-Synuclein Fibril Formation We have recently shown that a variety of divalent and trivalent metal ions also can directly affect the fibril formation of a-synuclein (Uversky et al., 2001a,c). In those experiments, we used quite low concentrations of a-synuclein, possibly as much as 10-fold lower than in neurons, and high metal ion concentrations (5 mM). In experiments using higher a-synuclein concentrations, micromolar amounts of aluminum stimulated the rate of fibril formation (Fig. 3). There was a good correlation between charge density and effectiveness in stimulating a-synuclein fibrillation: thus, trivalent metals were most effective in general. In addition, low pH also stimulated the rate of fibril assembly (Uversky et al., 2001b). The most effective ions were Al3þ, Fe3þ, Co3þ, Cd2þ, Mn2þ, Cu2þ, Co2þ, in that order. Pesticide and Metal-Induced Conformational Changes in a-Synuclein We have previously shown that a conformational change corresponding to formation of a partially folded
V.N. Uversky et al. / NeuroToxicology 23 (2002) 527–536
531
Fig. 3. The amount of AlCl3 required to accelerate the fibrillation of a-synuclein decreases at higher a-synuclein concentrations. a-synuclein (200 mM) was incubated with shaking at 37 8C in Tris buffer, pH 7.5, 100 mM NaCl. Fibril formation was monitored by the increase in thioflavin T (TFT) fluorescence using a plate reader. The presence of 200 mM Al3þ (&) significantly increases the rate of fibrillation over the control (5) (no Al3þ).
intermediate is a critical step in the formation of a-synuclein fibrils (Uversky et al., 2001b). We confirmed that those pesticides and metals that caused a significant increase in the kinetics of fibrillation also caused a conformational change detectable by circular dichroism and tyrosine fluorescence. Fig. 4 shows the
changes induced in the far-UV circular dichroism spectra of a-synuclein by DDC and AlCl3. The spectra in the presence of DDC and aluminum are typical of the partially-folded intermediate induced by other metals and pesticides. Synergistic Effects of Pesticides and Metals on a-Synuclein Fibrillation
Fig. 4. Metal ion and pesticide-induced conformational changes in a-synuclein. Far-UV circular dichroism spectra of 35 mM a-synuclein (*); in the presence of 100 mM DDC (&); 4 mM AlCl3 (5); and both 100 mM DDC and 4 mM AlCl3 (^). Spectra were measured within minutes of mixing the samples.
Since many individuals at risk due to pesticide and/ or metal exposure are expected to accumulate both metals and pesticides in their brains, it is important to know whether the effects are additive. In fact, the simultaneous presence of Al3þ and the pesticide DDC led to fibril formation kinetics that were greater than expected from non-cooperative effectors. For example, in experiments in which a-synuclein was incubated in the simultaneous presence of both DDC and AlCl3, fibril formation was more rapid than that expected from the sum of the effects of the two agents alone, Fig. 5, indicating a synergistic effect between the pesticide and the metal ion. For example, with AlCl3 (4 mM) and DDC (100 mM) added in that order the observed tm (time to half-maximum signal) was 49.5 h, whereas the calculated time based on the product of the percentage decrease in lag time for each ligand, was 63.6 h. The values for tm were 198 h (no additions), 125 h (100 mM DDC) and 98 h (4 mM AlCl3). The simultaneous presence of DDC and AlCl3 led to enhanced conformational change, as detected by
532
V.N. Uversky et al. / NeuroToxicology 23 (2002) 527–536
Fig. 5. Synergistic interactions between pesticide and metal in the stimulation of a-synuclein fibril formation. The kinetics of fibril formation were monitored as in the legend to Fig. 1. a-Synuclein (28 mM) alone (&); with 100 mM DDC (*); with 4 mM AlCl3 (*); with DDC (100 mM) and AlCl3 (4 mM) added in that order (!); and with AlCl3 (4 mM) and DDC (100 mM) added in that order (5).
CD, over each additive individually, Fig. 4. In addition, the order of addition of metal and pesticide affected the kinetics (Fig. 5): the addition of the metal prior to the pesticide, probably because the effect of metals on the conformational change was significantly more rapid than that of pesticides, caused a larger effect, compared to the initial addition of pesticide. Interestingly, the effects of pesticides and metals on the tyrosine fluorescence of a-synuclein varied significantly, depending, in part, on the effect of the environmental agent on free tyrosine. For example, for AlCl3, plots of the change in fluorescence as a function of increasing Al3þ concentration gave a sigmoidal increase in fluorescence (Fig. 6a). On the other hand for the pesticide DDC, such plots showed an exponential decrease with increasing pesticide (Fig. 6b). The synergistic effects of DDC and AlCl3 are also seen in their effects on a-synuclein fluorescence (Fig. 6). The presence of DDC causes a major shift in the position of the curve for the effect of Al3þ on fluorescence (Fig. 6a). Similarly, the presence of AlCl3 causes substantial changes in the curves for the effect of DDC on a-synuclein fluorescence (Fig. 6b).
DISCUSSION Parkinsonism has been associated with long term occupational exposure to pesticides and certain metals. Currently, about one billion pound of pesticides used
annually in the US, specific pesticides that have been implicated include: paraquat, organochlorine compounds, dieldrin, 1,10 -(2,2-dichloroethenyldiene)bis(4-chlorobenzene), hexachlorocyclohexane (lindane). There are many possible mechanisms whereby pesticides and metals could lead to a-synuclein aggregation. Oxidative damage, especially involving inhibition of mitochondrial Complex I and dopamine oxidation, via free radicals is often considered to be the causative ‘‘metabolic’’ factor in PD. Alternative hypotheses are that environmental factors (and other chemicals) can directly affect the aggregation of a-synuclein or adversely affect proteasomal function leading to accumulation of a-synuclein and its subsequent aggregation. It is most likely that the cause of PD is multi-factorial (Fig. 7), with genetic factors at one extreme and environmental agents at the other. In most cases, the potential effects of environmental factors will be modulated by the genetic susceptibility of the individual, for example, the efficiency of liver enzymes to eliminate the pesticide and the efficiency of redox systems to minimize oxidative damage. The data for rotenone, paraquat and MPPþ are interesting in that they show that rotenone and paraquat have a direct effect on a-synuclein fibrillation, whereas MPPþ does not, even though the structure of MPPþ and paraquat are very similar. In addition, it is known that both rotenone and MPPþ are Complex I inhibitors, thus it is likely that the in vivo effects of these compounds are complex, probably interacting with
V.N. Uversky et al. / NeuroToxicology 23 (2002) 527–536
533
Fig. 6. Synergistic interactions between pesticide and metal in the induction of structure in a-synuclein. Structural changes were measured by tyrosine fluorescence (there are no tryptophan residues in a-synuclein). Panel a shows the effect of increasing AlCl3 concentration on the fluorescence in the presence of 0 (*) and 100 mM DDC (5). Panel b shows the effect of increasing DDC concentration on the intrinsic fluorescence of a-synuclein as a function of AlCl3. The normalized fluorescence signal was obtained by scaling the observed fluorescence intensity changes to start at 0 and finish at 1.0.
different cellular components and leading to different effects, which may culminate, however, in the aggregation of a-synuclein (Fig. 7). Our results are consistent with a model in which both the cations and pesticides interact with a-synuclein to bring about a conformational change to a partiallyfolded state with a high propensity to aggregate. Thus, the lag in the kinetics of fibril formation actually reflects the association of the partially-folded intermediate conformation, rather than the ‘‘native’’ conformation. Consequently, there are three major steps leading to fibril formation: a structural change that leads to the
critical intermediate conformation, association of this intermediate to form a fibril nucleus, and finally template-directed growth of the fibril. Comparison of the structures of the pesticide that are most effective in accelerating fibril formation, suggests that increasing hydrophobicity of the pesticide is an important factor in stimulating aggregation. This is consistent with the idea that the pesticide binds to the partially-folded intermediate conformation (which is in equilibrium with the natively unfolded state), thereby increasing the population of the intermediate. The driving force for this is the existence of contiguous
534
V.N. Uversky et al. / NeuroToxicology 23 (2002) 527–536
Fig. 7. Proposed relationships illustrating the multi-factorial basis of Parkinson’s disease. In the majority of cases it is proposed that environmental agents, modulated by the individual’s genetic susceptibility (see Discussion), result in the aggregation of a-synuclein. Some aspect of the aggregation process is neurotoxic (possibilities include oligomeric intermediates, amorphous aggregates or fibrils), leading to the death of dopaminergic neurons and eventually Parkinson’s disease.
patches of hydrophobic surface in the intermediate (and which are absent in the natively unfolded conformation). Precedence for such a ligand bindinginduced shift in equilibrium to favor a partially-folded intermediate conformation has previously been reported (Shi et al., 1994). Based on our observation that 8-anilinonaphthalene-1-sulfonic acid (ANS) also stimulates the aggregation of a-synuclein (data not shown), we predict that many compounds that are relatively hydrophobic and water-soluble will induce a-synuclein fibril formation. The C-terminal region of a-synuclein (about 40 amino acids) is very rich in acidic residues, and thus highly negatively charged at neutral pH. The resulting repulsive interactions are a major factor leading to the natively unfolded conformation, since the unfolded conformation of natively unfolded proteins is due to a combination of high net charge and low intrinsic hydrophobicity (Uversky et al., 2000). Although the results with the metal ion-stimulated a-synuclein fibrillation and conformational change indicate that there are definite cation-specific effects, we believe that the dominant effect of the metal ions is due to masking of the Coulombic charge–charge repulsion (Goto et al., 1990b). For polyvalent cations, an additional important factor is the potential for cross-linking or bridging between two or more carboxylates. With the apparent exception of cadmium, there is an excellent correlation between the size of the cation and the degree of fibril stimulation. The correlation is even more marked in
terms of the charge density. Thus, smaller polyvalent cations are able to screen the unfavorable electrostatic interactions and at the same time have minimal perturbation of the resulting protein structure due to their small size. The high net charge and unfolded conformation of a-synuclein (pI ¼ 4:7) at neutral pH means that it has many features that parallel those of acid-unfolded proteins. The addition of anions to acid-unfolded proteins leads to conformations that are much more folded (Goto et al., 1990a,b; Fink et al., 1994, 1998; Uversky et al., 1998), due to the screening effect of the anion on the electrostatic repulsion from the positively charged amines. A noteworthy point is that different anions differ dramatically in their effectiveness in inducing these conformational changes (Goto et al., 1990b; Fink et al., 1998; Uversky et al., 1998). Thus, anion effects at low pH are a good model for the effects of cations on a-synuclein at neutral pH. Thus, environmental factors that stimulate a-synuclein aggregation do so by increasing the concentration of the critical partially-folded intermediate conformation that leads to aggregation. For cations the effect is due to the positively charged metal ions masking the negatively charged groups mostly responsible for the natively unfolded conformation. For pesticides the effect is due to their preferential binding to the partially-folded intermediate conformation. Consequently, there are at least two different pathways by which environmental factors may stimulate the formation of a-synuclein fibrils at a fundamental molecular level. The synergy between cations and pesticides may arise from the different natures of their interactions with the partially folded intermediate as well as dimers or higher oligomers of the partially-folded intermediate. Most importantly, however, the rates of cation- or pesticide-induced aggregation of a-synuclein may be increased in the presence of other molecules due to synergistic effects. This observation is particularly relevant to PD because it suggests that when pesticides, herbicides or other small soluble hydrophobic molecules (including some products of oxidative stress) are in the presence of certain metal ions, the concentrations of either required to cause rapid formation of a-synuclein fibrils may be substantially reduced. These interactions between a-synuclein and environmental agents could play a role in the pathogenesis of nigrostriatal degeneration, and thus in the etiology of sporadic PD. The synergistic effects observed between pesticide and metal suggests that the total brain load of pesticides and metals, rather than individual levels, is a
V.N. Uversky et al. / NeuroToxicology 23 (2002) 527–536
very important contributor to the potential effect on a-synuclein fibrillation. Thus, although the levels necessary for significant accelerations of a-synuclein fibrillation in our experiments are in the micromolar range, they decrease with increasing a-synuclein concentration. From the overall abundance of a-synuclein in the brain it is reasonable to assume that the cellular concentration in neurons in the substantia nigra could be several hundred micromolar (Hashimoto et al., 2001). Experiments in which mice were treated with paraquat and develop a-synuclein deposits demonstrate that sufficient paraquat can enter the dopaminergic neurons in the substantia nigra, to cause a-synuclein to aggregate in vivo (ManningBog et al., 2002a). Thus, our in vitro observations are paralleled by corresponding in vivo effects. Similar observations were made in which mice were treated with a combination of DDC and AlCl3 (data not shown).
ACKNOWLEDGEMENTS We thank Drs. J. Gillespie, J.W, Langston and D. Di Monte for valuable discussions. This research was supported by grant RO1 NS39985 from the National Institutes of Health.
REFERENCES Altschuler E. Aluminum-containing antacids as a cause of idiopathic Parkinson’s disease. Med Hypotheses 1999;53:22–3. Conway KA, Harper JD, Lansbury PT. Accelerated in vitro fibril formation by a mutant a-synuclein linked to early-onset Parkinson disease. Nat Med 1998;4:1318–20. Conway KA, Lee SJ, Rochet JC, Ding TT, Williamson RE, Lansbury Jr, PT. Acceleration of oligomerization, not fibrillization, is a shared property of both a-synuclein mutations linked to early-onset Parkinson’s disease: implications for pathogenesis and therapy. Proc Natl Acad Sci USA 2000; 97:571–6[in process citation]. Crowther RA, Jakes R, Spillantini MG, Goedert M. Synthetic filaments assembled from C-terminally truncated a-synuclein. FEBS Lett 1998;436:309–12. El-Agnaf OMA, Jakes R, Curran MD, Wallace A. Effects of the mutations Ala(30) to Pro and Ala(53) to Thr on the physical and morphological properties of a-synuclein protein implicated in Parkinson’s disease. FEBS Lett 1998;440:67–70. Eliezer D, Kutluay E, Bussell Jr, R, Browne G. Conformational properties of a-synuclein in its free and lipid-associated states. J Mol Biol 2001;307:1061–73. Fink AL, Calciano LJ, Goto Y, Kurotsu T, Palleros DR. Classification of acid denaturation of proteins—intermediates and unfolded states. Biochemistry 1994;33:12504–11.
535
Fink AL, Oberg KA, Seshadri S. Discrete intermediates versus molten globule models for protein folding: characterization of partially folded intermediates of apomyoglobin. Fold Des 1998;3:19–25. Goedert M. Parkinson’s disease and other a-synucleinopathies. Clin Chem Lab Med 2001;39:308–12. Good PF, Olanow CW, Perl DP. Neuromelanin-containing neurons of the substantia nigra accumulate iron and aluminum in Parkinson’s disease: a LAMMA study. Brain Res 1992;593: 343–6. Gorell JM, Johnson CC, Rybicki BA, Peterson EL, Kortsha GX, Brown GG, Richardson RJ. Occupational exposure to manganese, copper, lead, iron, mercury and zinc and the risk of Parkinson’s disease. Neurotoxicology 1999;20:239–47. Goto Y, Calciano LJ, Fink AL. Acid-induced folding of proteins. Proc Natl Acad Sci USA 1990a;87:573–7. Goto Y, Takahashi N, Fink AL. Mechanism of acid-induced folding of proteins. Biochemistry 1990b;29:3480–8. Hashimoto M, Hsu LJ, Sisk A, Xia Y, Takeda A, Sundsmo M, Masliah E. Human recombinant NACP/a-synuclein is aggregated and fibrillated in vitro: Relevance for Lewy body disease. Brain Res 1998;799:301–6. Hashimoto M, Hsu LJ, Xia Y, Takeda A, Sisk A, Sundsmo M, Masliah E. Oxidative stress induces amyloid-like aggregate formation of NACP/a-synuclein in vitro. Neuroreport 1999;10: 717–21. Hashimoto M, Rockenstein E, Mante M, Mallory M, Masliah E. b-Synuclein inhibits a-synuclein aggregation. A possible role as an anti-Parkinsonian factor. Neuron 2001;32:213– 23. Hellenbrand W, Boeing H, Robra BP, Seidler A, Vieregge P, Nischan P, Joerg J, Oertel WH, Schneider E, Ulm G. Diet and Parkinson’s disease. II. A possible role for the past intake of specific nutrients. Results from a self-administered foodfrequency questionnaire in a case–control study. Neurology 1996;47:644–50[see comments]. Hertzman C, Wiens M, Snow B, Kelly S, Calne D. A case-control study of Parkinson’s disease in a horticultural region of British Columbia. Mov Disord 1994;9:69–75. Hirsch EC, Brandel JP, Galle P, Javoy-Agid F, Agid Y. Iron and aluminum increase in the substantia nigra of patients with Parkinson’s disease: an X-ray microanalysis. J Neurochem 1991;56:446–51. Iwatsubo T, Yamaguchi H, Fujimuro M, Yokosawa H, Ihara Y, Trojanowski JQ, Lee VMY. Purification and characterization of Lewy bodies from the brains of patients with diffuse Lewy body disease. Am J Pathol 1996;148:1517–29. Jakes R, Crowther RA, Lee VM, Trojanowski JQ, Iwatsubo T, Goedert M. Epitope mapping of LB509, a monoclonal antibody directed against human a-synuclein. Neurosci Lett 1999;269: 13–6. Li J, Uversky VN, Fink AL. Effect of familial Parkinson’s disease point mutations A30P and A53T on the structural properties, aggregation, and fibrillation of human a-synuclein. Biochemistry 2001;40:11604–13. Manning-Bog AB, McCormack AL, Li J, Uversky VN, Fink AL, Di Monte DA. The herbicide paraquat causes up-regulation and aggregation of a-synuclein in mice. Paraquat and a-synuclein. J Biol Chem 2002a;277:1641–4. Manning-Bog AB, McCormack AL, Li J, Uversky VN, Fink AL, Di Monte DA. The herbicide paraquat causes up-regulation and
536
V.N. Uversky et al. / NeuroToxicology 23 (2002) 527–536
aggregation of a-synuclein in mice. Paraquat and a-synuclein. J Biol Chem 2002b;277:1641–4. Naiki H, Higuchi K, Hosokawa M, Takeda T. Fluorometric determination of amyloid fibrils in vitro using the fluorescent dye, thioflavin T1. Anal Biochem 1989;177:244–9. Naiki H, Higuchi K, Matsushima K, Shimada A, Chen WH, Hosokawa M, Takeda T. Fluorometric examination of tissue amyloid fibrils in murine senile amyloidosis: use of the fluorescent indicator, thioflavine T. Lab Invest 1990;62:768–73. Narhi L, Wood SJ, Steavenson S, Jiang Y, Wu GM, Anafi D, Kaufman SA, Martin F, Sitney K, Denis P, Louis JC, Wypych J, Biere AL, Citron M. Both familial Parkinson’s disease mutations accelerate a-synuclein aggregation. J Biol Chem 1999;274:9843–6. Shi L, Palleros DR, Fink AL. Protein conformational changes induced by 1,10 -bis(4-anilino-5-naphthalenesulfonic acid)— preferential binding to the molten globule of dnak. Biochemistry 1994;33:7536–46. Spillantini MG, Crowther RA, Jakes R, Hasegawa M, Goedert M. a-Synuclein in filamentous inclusions of Lewy bodies from Parkinson’s disease and dementia with Lewy bodies. Proc Natl Acad Sci USA 1998;95:6469–73. Tanner CM. The role of environmental toxins in the etiology of Parkinson’s disease. Trends Neurosci 1989;12:49–54. Tanner CM, Ottman R, Goldman SM, Ellenberg J, Chan P, Mayeux R, Langston JW. Parkinson disease in twins: an etiologic study. JAMA 1999;281:341–6.
Uversky VN, Karnoup AS, Segel DJ, Seshadri S, Doniach S, Fink AL. Anion-induced folding of staphylococcal nuclease: characterization of multiple equilibrium partially folded intermediates. J Mol Biol 1998;278:879–94. Uversky VN, Gillespie JR, Fink AL. Why are ‘‘natively unfolded’’ proteins unstructured under physiologic conditions? Proteins 2000;41:415–27. Uversky VN, Lee HJ, Li J, Fink AL, Lee SJ. Stabilization of partially folded conformation during a-synuclein oligomerization in both purified and cytosolic preparations. J Biol Chem 2001a;276:43495–8. Uversky VN, Li J, Fink AL. Evidence for a partially folded intermediate in a-synuclein fibril formation. J Biol Chem 2001b;276:10737–44. Uversky VN, Li J, Fink AL. Metal-triggered structural transformations, aggregation, and fibrillation of human a-synuclein. A possible molecular link between Parkinson’s disease and heavy metal exposure. J Biol Chem 2001c;276:44284–96. Uversky VN, Li J, Fink AL. Pesticides directly accelerate the rate of a-synuclein fibril formation: a possible factor in Parkinson’s disease. FEBS Lett 2001d;500:105–8. Weinreb PH, Zhen WG, Poon AW, Conway KA, Lansbury Jr, PT. NACP, a protein implicated in Alzheimer’s disease and learning, is natively unfolded. Biochemistry 1996;35:13709–15. Yasui M, Kihira T, Ota K. Calcium, magnesium and aluminum concentrations in Parkinson’s disease. Neurotoxicology 1992; 13:593–600.