Tài liệu Báo cáo khoa học: Modulation of a-synuclein aggregation by dopamine in the presence of MPTP and its metabolite pptx

Modulation of a-synuclein aggregation by dopamine in the presence of MPTP and its metabolite Prashant N. Jethva, Jay R. Kardani and Ipsita Roy Department of Biotechnology, National Institute of Pharmaceutical Education and Research (NIPER), S.A.S. Nagar, India Introduction The inability of the cell to degrade various stable mis- folded proteins leads to the formation of aggregates and inclusion bodies in the cell. Parkinson’s disease, Alzheimer’s disease, Huntington’s disease, prion dis- ease, etc. are disorders in which aggregation of normal and ⁄ or mutant protein occurs and leads to neurode- generation. Whether the aggregate itself is cytotoxic or if it is a defence mechanism of the cell, remains a mat- ter of debate [1,2]. Although the proteins involved in such diseases do not have any similarity in their pri- mary sequence and ⁄ or structure, the aggregates formed do exhibit similarity in their topology. They exhibit crossed b-sheet structure and common properties regarding their binding with different staining dyes, e.g. Congo red and Thioflavin T (ThT). Parkinson’s disease is a progressive neurological dis- order and is the second most prevalent neurodegenera- tive disease after Alzheimer’s disease, affecting $ 1% of people beyond 65 years of age. The etiological factors that are involved in the development of Parkin- son’s disease include genetic factors, susceptibility to various drugs and environmental factors [3–5]. The pathological changes that occur in the brain include selective loss of dopaminergic neurons in substantia nigra pars compacta and appearance of Lewy bodies consisting of aggregated protein, mainly a-synuclein, in Keywords amyloid; fibrillation; Parkinson’s disease; synuclein; thioflavin T Correspondence I. Roy, Department of Biotechnology, National Institute of Pharmaceutical Education and Research (NIPER), Sector 67, S.A.S. Nagar, Punjab 160 062, India Fax: +91 172 221 4692 Tel: +91 172 229 2061 E-mail: ipsita@niper.ac.in (Received 28 September 2010, revised 24 February 2011, accepted 7 March 2011) doi:10.1111/j.1742-4658.2011.08093.x The neurotransmitter dopamine has been shown to inhibit fibrillation of a-synuclein by promoting the formation of nonamyloidogenic oligomers. Fibrillation of a-synuclein is accelerated in the presence of pesticides and the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). The aim of this study was to determine whether dopamine continues to have an adverse effect on the fibrillation of a-synuclein in the presence of MPTP and its metabolite 1-methyl-4-phenylpyridinum ion (MPP + ). We also attempted to answer the ambiguous question of whether conversion of MPTP to MPP + is required for the fibrillation of a-synuclein. For this, a-synuclein was incubated in the presence of MPTP and MPP + along with dopamine. The fibrillation of a-synuclein was monitored by Thioflavin T fluorescence and immunoblotting. The morphology of the aggregates formed was observed using scanning electron microscopy. The concentra- tions of the neurotoxin and its metabolite were estimated by reverse phase HPLC. We found definitive evidence that the conversion of MPTP to MPP + is not required for aggregation of a-synuclein. MPP + was found to accelerate the rate of a-synuclein aggregation even in the absence of com- ponents of mitochondrial complex I. In contrast to the effect of dopamine on the aggregation of a-synuclein alone, in the presence of MPTP or MPP + , the aggregates formed are Thioflavin T-positive and amyloidogenic. Thus, the effect of dopamine on the nature of aggregates formed in case of a-synuclein alone and in the presence of MPTP ⁄ MPP + is different. Abbreviations MPP, 1-methyl-4-phenylpyridinum; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; ThT, thioflavin T. 1688 FEBS Journal 278 (2011) 1688–1698 ª 2011 The Authors Journal compilation ª 2011 FEBS the surviving neurons. The axons of these nigral neu- rons face the striatum and employ dopamine as the neurotransmitter. Thus, reduction of dopamine levels in the striatum is a hallmark of Parkinson’s disease. A variety of pesticides including paraquat, rotenone and dielderin have been shown to be potential inducers of a-synuclein aggregation [3]. More insight into the role of environmental toxin as a cause of Parkinson’s disease came in the early 1980s, when young heroin addicts were seen with Parkinson’s disease-like symp- toms. The cause of this syndrome was found to be the use of homemade heroin which was contaminated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) [6]. Further studies showed that 1-methyl-4-phenylpy- ridinium ion (MPP + ), a metabolite of MPTP, was actually responsible for the neurotoxicity [7]. In humans and nonhuman primates, MPTP produces neurological, clinical and biochemical changes similar to those found in idiopathic Parkinson’s disease [6,8]. These patients also respond to levodopa therapy simi- lar to patients of idiopathic Parkinson’s disease and develop the same therapy-related complications. Post- mortem analysis of brains of patients with MPTP- induced Parkinson’s disease has disclosed important similarities and differences with idiopathic Parkinson’s disease [9]. Depletion of dopaminergic neural neurons and loss of tyrosine hydroxylase-positive termini were seen in both cases. This high selectivity of MPTP for dopaminergic neurons is due to the plasma membrane dopamine transporter which is also a carrier of MPP + , the active metabolite of MPTP. This leads to an increase in the concentration of MPP + in the dopa- minergic neurons, leading to selective damage to sub- stantia nigra, similar to idiopathic Parkinson’s disease. An important difference is the absence of Lewy bodies in MPTP-induced parkinsonism in humans. However, eosinophilic intraneuronal inclusions have been seen in the same region as Lewy bodies in squirrel monkeys injected with MPTP [10] although significant differ- ences in structure and morphology were seen. Admin- istration of MPTP has also been shown to form aggregates of a-synuclein in nigral neurons of baboons (Papio anubis) [11]. Depletion of a-synuclein was maxi- mum in the middle third region of substantia nigra where no neurons remained. In humans, Lewy bodies are also formed in other parts of the brain like locus ceruleus, cerebral cortex, sympathetic ganglia, etc. [12], which has not been observed in nonhuman primate models. Pesticides and MPTP have also been found to be mitochondrial toxins. A recent report, however, suggests that mitochondrial complex I inhibition is not required for MPP + , and other pesticides, to induce neurodegeneration [13]. Thus, confusion regarding the direct and ⁄ or indirect role of MPTP, and its conver- sion to MPP + , in inducing aggregation of a-synuclein still exists in the literature. Among the various factors that affect the kinetics of a-synuclein fibrillation, the role of dopamine is proba- bly one of the least understood [14]. As mentioned ear- lier, the loss of dopaminergic neurons in substantia nigra is a neuropathological hallmark of Parkinson’s disease. This leads to a decreased level of dopamine in the striatum. As a result, synaptic transmission is nega- tively affected in a-synuclein knockout mice [15]. How- ever, cells overexpressing a-synuclein have shown the formation of aggregates of the protein on exposure to dopamine [16]. In vitro experiments probably provide a better understanding of the role of various interacting components. The formation of dopamine–quinone adducts (because of auto-oxidation of the neurotrans- mitter), especially dopaminochrome, with a-synuclein, inhibited the conversion of the more-toxic a-synuclein protofibrils to the less-toxic mature fibrillar structures [17]. Also, dopamine has been shown to promote the initial aggregation of a-synuclein into off-pathway, sol- uble, SDS-resistant oligomers [18]. These nonamyloid- ogenic oligomers are sequestered together and do not form the less-toxic fibrils. Thus, dopamine promotes the accumulation of toxic protofibrils of a-synuclein, leading to cell death. In this study, we have determined the nature of aggregates formed in the presence of dopamine when a-synuclein is co-incubated with MPTP or MPP + and have shown that these are differ- ent from the aggregates that are formed when a-synuc- lein alone is exposed to dopamine. Results Expression and purification of a-synuclein Expression of a-synuclein was carried out using isopro- pyl thio-b-d-galactoside as an inducer, as described below. The expressed protein was isolated from the cells by lysis and subjected to purification using DEAE-Sepharose matrix-based anion-exchange chro- matography [18]. The target protein was eluted with 0.02 m Tris ⁄ HCl, pH 7.8 containing 0.5 m NaCl. The purified protein was used for further experiments. The eluted protein was concentrated to 7 mgÆ mL )1 (483 lm) for aggregation study. Aggregation of a-synuclein Purified a-synuclein [7 mgÆmL )1 (483 lm), 0.02 m Tris ⁄ HCl buffer, pH 7.8] was incubated at 37 °C [19]. Aliquots were withdrawn at different time intervals P. N. Jethva et al. Modulation of a-synuclein aggregation FEBS Journal 278 (2011) 1688–1698 ª 2011 The Authors Journal compilation ª 2011 FEBS 1689 and analysed by SDS ⁄ PAGE and immunoblotting. SDS ⁄ PAGE showed the formation of higher molecular mass species with time (Fig. 1A). For western blotting, samples were run on gradient SDS⁄ PAGE (5–15% cross-linking) and transferred to a nitrocellulose mem- brane, as described below. Figure 1B shows the pattern seen after the development of the blot. With increase in time of incubation, the intensity of the band for the monomeric protein decreased, whereas the bands for the higher molecular mass aggregates intensified. This confirmed the formation of SDS-insoluble aggregates of a-synuclein on incubation. Effect of MPTP and MPP + on the aggregation pattern of a-synuclein a-Synuclein was incubated with 100 and 200 lm MPTP as described below, along with a control sample (without MPTP). Aliquots were withdrawn at different time intervals and the fluorescence intensity of ThT in the presence of the protein samples was monitored at 482 nm (Fig. 2A). ThT, a cationic benzothiazole dye, has been used to identify amyloid aggregates since its fluorescence was first demonstrated to increase upon binding to amyloid fibrils [20]. It has been used to detect cross b-sheet fibril formation by a-synuclein [19,21,22] as well as b amyloid [23] and huntingtin [24], among other proteins. Because a-synuclein is reported to form amyloid-type aggregates [3,25], measurement of ThT fluorescence would be an important probe for characterization of the nature of the aggregates. Char- acteristic sigmoidal curves of amyloid-type aggregates, with three distinct phases of lag (nucleation), growth (fibrillation) and equilibrium (saturation) stages, were observed in all the cases (Fig. 2). The apparent rate constants (k app ) of fibrillation were calculated to be 0.058, 0.096 and 0.177 h )1 for a-synuclein incubated alone, and in the presence of 100 lm MPTP and 200 lm MPTP, respectively. Nota- bly, in the presence of the neurotoxin, there was a delay in the lag time for fibrillation. The lag time increased from 74.9 h in case of a -synuclein alone to 86.8 and 93.6 h in the presence of 100 and 200 lm MPTP, respectively. The rate of nucleation for protofi- bril formation was slower in the presence of MPTP, but the rate of fibrillation (protofibrils fi mature fibres) itself was faster. The presence of MPTP was sufficient to alter the fibrillation kinetics of a-synuc- lein. When a-synuclein was incubated with MPTP, the rate of formation of the more toxic protofibrils (mea- sured as lag time) was delayed, whereas the rate of conversion of protofibrils to the less toxic fibrils (mea- sured as apparent rate constant) was accelerated. Thus, when a-synuclein was exposed to increasing concentra- tions of the neurotoxin, the rate of fibrillation was enhanced. This may explain why acute exposure of MPTP is unable to reproduce the hallmark symptom of parkinsonism in mice [26], whereas continuous infu- sion of the neurotoxin results in the formation of Lewy bodies [27]. On intermittent exposure to MPTP, the lag time is not crossed and the protofibril to fibril tran- sition does not occur. Thus, a-synuclein fibrils and Lewy bodies are not formed. On continuous exposure, the lag time is overcome and the characteristic amyloid fibrils of a-synuclein are formed. a-Synuclein was incubated in the presence of two different concentrations of MPP + , the putative active metabolite of MPTP in the brain. Aliquots were with- drawn at different time intervals, added to a solution of ThT and the fluorescence intensity of the fluorescent probe was monitored at 482 nm (Fig. 2B). As can be seen, the presence of 100 lm MPP + accelerated the rate of fibrillation (0.103 h )1 compared with 0.058 h )1 for a-synuclein alone). This decreased to almost that of the original value of control a-synuclein (0.054 h )1 ) when the concentration of MPP + was increased to 200 lm. Interestingly, the lag time decreased from 82.3 to 48.2 h when the concentration of MPP + was increased from 100 to 200 lm. Thus, similar to MPTP, the presence of MPP + slowed the rate of nucleation of a-synuclein (82.3 h versus 74.9 h for a-synuclein alone) and the kinetics of fibrillation was slower at a higher concentration of the metabolite. Our results agree with earlier results with pesticides and MPP + [25]. The con- centration of MPP + used in the earlier study was Fig. 1. Aggregation of a-synuclein. (A) Samples were withdrawn after the indicated periods and SDS ⁄ PAGE was run 5–15% cross- linked polyacrylamide gel; lane M, molecular mass marker; lane 1, monomeric a-synuclein (control); lane 2, after 4 h; lane 3, after 9 h; lane 4, after 28 h; lane 5, after 55 h; lane 6, after 71 h; lane 7, after 95 h; and lane 8, after 120 h. (B) Gels were silver stained and wes- tern blotting of the samples was carried out; lane M, molecular mass marker; lane 1, 11 h; lane 2, 56 h; lane 3, 71 h; lane 4, 120 h; lane 5, 172 h; lane 6, monomeric a-synuclein (control). Modulation of a-synuclein aggregation P. N. Jethva et al. 1690 FEBS Journal 278 (2011) 1688–1698 ª 2011 The Authors Journal compilation ª 2011 FEBS 100 lm. At this concentration, MPP + showed only a marginal increase in the lag time for aggregation of a-synuclein, as observed in this case. At a higher concen- tration of MPP + , the lag time decreased significantly. In order to confirm that aggregation of a-synuclein was because of MPTP alone and not because of its con- version to MPP + , RP-HPLC of the samples was car- ried out. The incubated samples (a-synuclein alone and in the presence of 100 and 200 lm MPTP) were with- drawn after 250 h and centrifuged. The supernatants were injected directly into the RP-HPLC column [28]. As expected, no peak for MPTP was seen when a-syn- uclein was incubated alone (Fig. 3A). When a-synuclein was incubated in the presence of 100 lm MPTP (Fig. 3B) and 200 lm MPTP (Fig. 3C), peaks corre- sponding to the retention time of MPTP (6.4 min) could be seen at 245 nm. The peak areas, however, did not correspond to the concentration of MPTP origi- nally present in the reaction mixtures (100 and 200 lm, respectively), but were 80% of the original concentra- tions present in the original samples. The components of the reaction mixture did not dampen the signal of the neurotoxin (data not shown). To find the reason for this decrease, the a-synuclein aggregate formed after 250 h was dissolved in 8 m urea and centrifuged. The supernatant was injected into an RP-HPLC col- umn. No peak, corresponding to the retention time of MPTP, was observed at 245 nm (Fig. 3D). More Fig. 2. ThT fluorescence intensity of aggre- gated a-synuclein in the presence of (A) MPTP and (B) MPP + . Concentrations used are 0 l M (s, solid line), 100 lM ( • , dotted line) and 200 l M ( , dashed line) of neuro- toxins. Fig. 3. Chromatographic analysis of aggre- gated samples for the presence of MPTP or its metabolite after 240 h of incubation. a-Synuclein incubated (A) alone (k = 245 nm), (B) in the presence of 100 l M MPTP (k = 245 nm), (C) in the presence of 200 l M MPTP (k = 245 nm), (D) in the presence of 100 l M MPTP, dissolved in 8 M urea and centrifuged (k = 245 nm), (E) in the presence of 100 l M MPTP (k = 295 nm), and (F) in the presence of 100 l M MPP + , dissolved in 8 M urea and centrifuged (k = 295 nm). P. N. Jethva et al. Modulation of a-synuclein aggregation FEBS Journal 278 (2011) 1688–1698 ª 2011 The Authors Journal compilation ª 2011 FEBS 1691 interestingly, no peak corresponding to the formation of MPP + could be detected at 295 nm (Fig. 3E). To determine whether there was a direct interaction between a-synuclein and MPP + , the aggregate of a-synuclein obtained in the presence of 100 lm MPP + was dissolved in 8 m urea, centrifuged and injected into the C 18 column. The eluate was monitored at 295 nm. No peak for the presence of MPP + could be detected (Fig. 3F). It may be noted that the conversion of the unaccounted-for 20 lm MPTP (which is not detected in the reaction mixture) to MPP + is within the detection limit of our analytical method. Because some residual pellet remained after urea solubilization, the chaotrope may not have been able to solubilize the amyloid aggre- gate of a-synuclein completely. It is probable that in the case of MPTP-modulated a-synuclein fibrillation described here, MPTP is still entrapped in the residual pellet which is not solubilized by urea. Effect of dopamine on MPTP and MPP + induced changes in kinetics of the aggregation of a-synuclein a-Synuclein was incubated in the presence of 100 lm MPTP, along with 50 lm dopamine. Aliquots were withdrawn at different time intervals, added to a solu- tion of ThT and the fluorescence intensity of the solu- tion was measured at 482 nm. Figure 4A shows the kinetics of aggregation of a-synuclein in the presence of 100 lm MPTP and the effect of 50 lm dopamine on the aggregation process. Dopamine delayed the lag phase of aggregation marginally to 95.5 h from 86.8 h in the presence of MPTP alone. The apparent rate constant of aggregation in the presence of dopamine was significantly higher (0.25 h )1 ) than in the presence of MPTP alone (0.096 h )1 ). This indicates a faster rate of conversion of protofibrils to fibrillar structure. Thus, in the presence of MPTP, dopamine induces a-synuclein to form fibrillar structures which are prob- ably less cytotoxic than the protofibrils. Similar results were seen when a-synuclein was incubated in the pres- ence of 200 lm MPTP along with 50 lm dopamine (Fig. 4B). The lag phase (nucleation stage) remained unchanged (93.5 h versus 93.6 h in the presence of 200 lm MPTP alone), whereas the apparent rate constant was significantly higher in the presence of dopamine (0.21 h )1 versus 0.177 h )1 in the presence of 200 lm MPTP alone). The delay in the nucleation phase, coupled with a higher rate of fibrillation, is opposite to the results obtained when a-synuclein was incubated in the absence of MPTP. When a-synuclein was incubated alone in the presence of dopamine, it led to inhibition of fibrillation, probably by the accu- mulation of spherical oligomers which were nonamy- loidogenic but cytotoxic [14,18]. In the presence of MPTP, dopamine accelerated the rate of fibrillation, leading to a higher rate constant of aggregation. Because accumulation of the toxic protofibrils did not occur, cytotoxicity of this coexposure should be low. Once MPTP is oxidized to MPP + , however, the effect of dopamine proved to be deleterious. When a-synuclein was incubated in the presence of 100 and 200 lm MPP + , along with 50 lm dopamine, the lag time of protofibril formation decreased significantly. It was 24.9 h in the presence of 100 lm MPP + and 50 lm dopamine (cf. 82.3 h for 100 lm MPP + alone) (Fig. 4C), which decreased to 2.9 h in the presence of 200 lm MPP + and 50 lm DA (cf. 48.2 h for 200 lm Fig. 4. ThT fluorescence intensity of aggre- gated a-synuclein and 50 l M dopamine in the presence of (A) 100 l M MPTP, (B) 200 l M MPTP, (C) 100 lM MPP + and (D) 200 l M MPP + . Samples are a-synuclein alone (s, solid line), in the presence of 100 l M neurotoxin ( • , dotted line), in the presence of 200 l M neurotoxin ( , dotted line) and in the presence of neurotoxin and 50 l M dopamine (h, dashed line). Modulation of a-synuclein aggregation P. N. Jethva et al. 1692 FEBS Journal 278 (2011) 1688–1698 ª 2011 The Authors Journal compilation ª 2011 FEBS MPP + alone) (Fig. 4D). Because the presence of MPP + itself reduced the lag time of fibrillation signifi- cantly (Fig. 2B), this reduction is perhaps not surpris- ing. The apparent rate constant of fibrillation also followed a trend different from that with MPTP. The rate of fibrillation decreased significantly when a-synuc- lein was coincubated with 100 lm MPP + and 50 lm dopamine (0.045 h )1 ) compared with when a-synuclein was incubated with 100 lm MPP + alone (0.103 h )1 ). The presence of dopamine, along with MPP + , results in a faster rate of formation of protofibrils (nucleation phase) and a slower rate of conversion of protofibrils to mature fibrils (growth phase). This leads to accumula- tion of the more toxic oligomeric species which, in the cellular milieu, could translate into higher cytotoxicity. Electrophoretic and immunoblotting analyses In order to confirm that the increase in ThT fluores- cence intensity indeed denoted the formation of higher molecular mass aggregates, SDS ⁄ PAGE and immuno- blotting were carried out according to the procedure described in Materials and methods. a-Synuclein was incubated in the presence of MPTP (Fig. 5A) and MPP + (Fig. 5B) for 250 h and loaded on a 15% cross-linked denaturing polyacrylamide gel. Images showed the presence of higher molecular mass species in both cases. Western blot analysis confirmed that the higher molecular mass bands corresponded to aggre- gates of a-synuclein formed in the presence of MPTP (Fig. 5C) and MPP + (Fig. 5D). The aggregates formed are SDS-insoluble, as reported earlier in the case of fibrillation of a-synuclein alone [18]. Scanning electron microscopy Scanning electron microscopy of the aggregated sam- ples was carried out to understand the change in sur- face morphology of the protein following aggregation. Monomeric a-synuclein showed the presence of small particles corresponding to the expected diameter of the protein (< 20 nm) (Fig. 6A). In the presence of 100 lm (Fig. 6B) and 200 lm (Fig. 6C) MPTP and 100 lm (Fig. 6D) and 200 lm (Fig. 6E) MPP + , the size of the particle increased, as expected from the data of ThT fluorescence intensity and immunoblotting. In both cases, a mixture of fibrillar and globular particles could be seen, which indicated the existence of compet- ing pathways for aggregation. It has been reported ear- lier that any minute change in reaction conditions is enough to alter the morphology of aggregation prod- ucts [3,21,29]. The relative fractions of amorphous and fibrillar aggregates are decided by the different compo- nents of the reaction mixture [21]; in this case, the interaction between a-synuclein and MPTP or MPP + . In the interaction studies between pesticides and a-syn- uclein, it had been observed that although no soluble a-synuclein was left at the end of the aggregation period, the ThT fluorescence intensity of different samples was not the same [3]. The difference in ThT intensities indicated that the extent of fibrillation was different in the presence of different pesticides although the amount of aggregates formed was the same. Electron microscopy had confirmed the presence of both amorphous aggregates and fibrillar deposits. Fig. 5. Aggregation of a-synuclein after 240 h. Samples containing MPTP (A and C) and MPP + (B and D) were analysed by SDS ⁄ PAGE (A and B) on 5–15% crosslinked polyacrylamide gel and western blotting (C and D). (A, B) Lane M, prestained molecular mass mark- ers; lane 1, monomeric a-synuclein (control); lane 2, with 100 l M neurotoxin; lane 3, with 200 lM neurotoxin; lane 4, with 100 lM neurotoxin and 50 lM dopamine; lane 5, with 200 lM neurotoxin and 50 l M dopamine. Gels were silver stained. (C) Lane M, pre- stained molecular mass markers; lane 1, monomeric a-synuclein (control); lane 2, with 100 l M neurotoxin; lane 3, with 200 lM neu- rotoxin; lane 4, with 100 l M neurotoxin and 50 lM dopamine; lane 5, with 200 l M neurotoxin and 50 lM dopamine. (D) Lane M, prestained molecular mass markers; lane 1, a-synuclein with 100 l M neurotoxin; lane 2, a-synuclein with 200 lM neurotoxin; lane 3, a-synuclein with 100 l M neurotoxin and 50 lM dopamine; lane 4, a-synuclein with 200 l M neurotoxin and 50 lM dopamine. P. N. Jethva et al. Modulation of a-synuclein aggregation FEBS Journal 278 (2011) 1688–1698 ª 2011 The Authors Journal compilation ª 2011 FEBS 1693 Discussion MPTP infusion does not result in neuronal cell death or behavioural symptoms associated with Parkinson’s disease in a-synuclein-deleted mice [30]. Continuous infusion of the neurotoxin MPTP, however, has been shown to induce symptoms of parkinsonism in a mouse model [27]. Thus, a direct cause and effect rela- tionship between MPTP and a-synuclein has been established. MPTP is metabolized to MPP + in the brain. MPP + is an inhibitor of mitochondrial com- plex I and a substrate for dopamine transporter [27]. It thus selectively accumulates in cells that transport dopamine and is toxic to dopaminergic neurons. A number of contradictory reports exist in the literature regarding the role of MPTP and MPP + in producing parkinsonism-like symptoms. It has recently been reported that mitochondrial complex I-deleted mice show the same level of sensitivity to MPP + and pesti- cides as wild-type mice [13]. Thus, the aim of this study was to delineate any direct role of MPTP in the aggregation of a-synuclein and the effect of dopamine on this process. Toxicity of MPTP is believed to be due to its conversion to MPP + [31], but its toxic func- tion has not been fully elucidated. As our results show, at lower concentrations of MPP + , the rate of nucle- ation (formation of toxic protofibrils) is delayed but once the nucleus is formed, the rate of fibrillation is accelerated. At a higher concentration of the metabo- lite, the lag time is similar to that observed with pesti- cides (32.5 h with rotenone) [25]. It has been hypothesized that pesticides may interact directly with the hydrophobic residues to bring about a conformational change and stabilize the partially folded intermediate conformation, thus shifting the equilibrium from the natively unfolded state to the ABC D EF GH I Fig. 6. Scanning electron micrographs of a-synuclein following aggregation for 240 h. Samples are of a-synuclein incubated alone (A), in the presence of 100 l M MPTP (B), in the presence of 200 lM MPTP (C), in the presence of 100 lM MPP + (D), in the presence of 200 lM MPP + (E), in the presence of 100 lM MPTP and 50 lM dopamine (F), in the presence of 200 lM MPTP and 50 lM dopamine (G), in the presence of 100 l M MPP + and 50 lM dopamine (H) and in the presence of 200 lM MPP + and 50 lM dopamine (I). Modulation of a-synuclein aggregation P. N. Jethva et al. 1694 FEBS Journal 278 (2011) 1688–1698 ª 2011 The Authors Journal compilation ª 2011 FEBS intermediate state (U N M I fi fibrils) [21]. The importance of hydrophobic interactions in the aggrega- tion of a-synuclein has recently been reinforced by agi- tation studies which have clearly shown the formation of amyloid-type of aggregates only at the hydrophobic air-water interface [29]. It is possible that either the species that interacts directly with MPTP remains insoluble in the presence of urea, or a metabolite of MPTP, different from MPP + , is responsible for the change in the aggregation kinetics of a-synuclein. This will require further experimental proof. The absence of MPTP in the aggregated protein points to an indirect, rather than a direct, role of MPTP in the fibrillation process. The most probable reason why direct role of MPTP in animal models has not been observed so far could be because in living systems, MPTP is metabo- lized to MPP + by MAO-B and aggregation of a-syn- uclein is then a result of the presence of mainly MPP + , and not MPTP. It has recently been shown that dopaminergic neu- rons from Ndufs4-deleted mice (Ndufs4 is required for the complete assembly of mitochondrial complex I) survive normally and do not exhibit any Parkinson’s disease-like symptoms [13]. Because the basis of action of MPP + had been hypothesized to be inhibition of mitochondrial complex I [32], the mode of action of MPP + needs to be re-evaluated. Even more impor- tantly, Ndufs4-deleted mice exhibited the same level of sensitivity to MPP + as wild-type mice. Alternative rea- sons for the damage caused by MPP + have been pro- posed; these include oxidative stress, microtubule destabilization and inhibition of glycolysis [13]. Our in vitro results provide direct evidence that MPTP and MPP + can facilitate aggregation of a-synuclein in the absence of any cellular machinery. It has been proposed that the auto-oxidation product of dopamine interacts with protofibrillar a-synuclein and converts it into a stable adduct, which cannot form fibrils [14,17]. According to this model, dopamine has a cytotoxic role and enhances the rate of neurodegenera- tion in the initial stages. In the presence of MPTP, dopamine presumably cannot undergo auto-oxidation. The rate of fibrillation of a-synuclein cannot be inhib- ited and is, in fact, accelerated. Thus the effect of dopa- mine is reversed and the presence of MPTP actually has a ‘beneficial’ effect in that it probably facilitates faster elimination of the toxic oligomers. The levels of antioxi- dant enzymes like glucose-6-phosphate dehydrogenase have been shown to be upregulated during protection against MPTP-induced neuronal damage [33,34]. The co-administration of antioxidants like coenzyme Q and creatine has also been shown to be beneficial against a-synuclein aggregation in the substantia nigra pars compacta of an MPTP-induced mouse model of Parkinson’s disease [35]. It has recently been shown that the protective action of rasagiline, a MAO-B inhib- itor, on the aggregation of a-synuclein, is because of its action as a free radical scavenger [36]. Thus, it may be speculated that dopamine exhibits a beneficial effect on the fibrillation kinetics of a -synuclein in the presence of MPTP by altering its redox potential. Materials and methods Plasmid pRSETB (a-synuclein) was a gift from Dr Roberto Cappai (Department of Pathology, University of Mel- bourne, Australia). Luria–Bertani broth, ampicillin, phen- ylmethanesulfonyl fluoride, isopropyl thio-b-d-galactoside, mouse monoclonal anti-(a-synuclein) IgG1, anti-(mouse fluorescein isothiocyanate-conjugated) secondary IgG, MPTP, dopamine, MPP + and DEAE-Sepharose were pur- chased from Sigma–Aldrich Chemicals Pvt. Ltd (Bangalore, India). Lysozyme was obtained from Bangalore Genei Ltd. (Bangalore, India). Expression and purification of human a-synuclein Escherichia coli BL21 cells were transformed with pRSETB– a-synuclein plasmid construct using a standard calcium chloride method [37]. Transformed cells were grown at 37 °C, 200 rpm in Luria–Bertani media containing ampicil- lin (0.6% w ⁄ v) until D 600 = 0.6. Expression of a-synuclein was induced with 1 mm IPTG and the cells were further incubated for 3.5 h at 37 °C, 200 rpm. After the completion of the induction period, the cells were centrifuged at 7000 g for 30 min at 4 °C and stored overnight at ) 80 °C. The cells were lysed in lysis buffer (10 mm sodium phosphate mono- basic, 40 mm sodium phosphate dibasic, 1 mm EDTA, pH 7.4) containing 0.5 mgÆmL )1 lysozyme and 1 mm phen- ylmethanesulfonyl fluoride. Purification of a-synuclein was carried out as described previously [18]. The supernatant was treated with 1 m HCl to reduce the pH to 3.5. After 30 min, the pH was raised immediately to 7.5 and centrifu- gation was carried out at 15 000 g for 1 h. The cleared supernatant was purified by DEAE-Sepharose anion exchange chromatography [18]. The eluates were pooled and the amount of protein was determined by the bicinchoninic acid assay [38] using bovine serum albumin as a standard protein. The pooled eluate fractions were dialysed against water and then lyophilized. Gel electrophoresis and immunoblotting The expression and purification of a-synuclein protein was confirmed by 15% SDS ⁄ PAGE at constant current (25 mA) in miniVE electrophoresis unit (GE Healthcare, Hong Kong) [39]. The resolved proteins were detected by silver staining P. N. Jethva et al. Modulation of a-synuclein aggregation FEBS Journal 278 (2011) 1688–1698 ª 2011 The Authors Journal compilation ª 2011 FEBS 1695 [40]. For western blotting, after completion of the electro- phoretic run, the proteins on the SDS ⁄ PAGE gel were trans- ferred electrophoretically to nitrocellulose membrane (0.45 lm) with transfer buffer (25 mm Tris, 20 mm glycine and 10% v ⁄ v methanol, pH 8.3) using a semi-dry blotting assembly (TE70 PWR; GE Healthcare). The nitrocellulose membrane was incubated with mouse anti-(a-synuclein) monoclonal IgG1 (1 : 5000 dilution) for 6 h. After washing, the membrane was transferred to a solution of anti-(mouse fluorescein isothiocyanate-conjugated) monoclonal IgG (1 : 50) for 1.5 h. The blot was finally scanned on variable mode image scanner (Typhoon Trio; GE Healthcare). Aggregation of a-synuclein The lyophilized protein was dissolved in 0.02 m Tris ⁄ HCl buffer, pH 7.8 and subjected to ultracentrifugation (100 000 g) for 1 h to remove preformed aggregate. The final concentration of the protein was adjusted to 7mgÆmL )1 (483 lm) and incubated at 37 °C [19]. Aliquots were withdrawn at predefined time intervals. The aggrega- tion pattern was analysed by performing 15% SDS ⁄ PAGE, western blotting and various biophysical techniques. a-Syn- uclein was also incubated in the presence of different con- centrations of MPTP and MPP + (100 and 200 lm each), in the absence and presence (50 lm) of dopamine and analy- sed as above. ThT fluorescence measurement A stock solution of ThT (5 mm) was prepared in 0.02 m Tris ⁄ HCl buffer, pH 7.8. Aliquots (20 lL) of a-synuclein were withdrawn at different time intervals and added to ThT so that the final concentrations of protein and ThT were 2 and 10 lm, respectively. The fluorescence intensity of the resultant sample was measured in the wavelength range of 470–560 nm after excitation at 450 nm. Slit widths were kept at 5 nm each for excitation and emission. The aggregation kinetics was followed by fitting the data using the formula [21]: y ¼ y i þ mx i þ y f þ mx f 1 ¼ e xÀx 0 s where y i + mx i is the initial line, y f + mx f is the final line and x 0 is the midpoint of maximum signal. The apparent rate constant (k app )is1⁄ s and lag time is calculated to be x 0 ) 2s. Chromatographic analysis RP-HPLC analysis of the samples was carried out to deter- mine the residual amounts of MPTP and MPP + in the aggregated samples. After completion of aggregation, the samples were centrifuged. The supernatants (20 lL) were injected into a C 18 column (Zorbax 300SB-C18) attached to a HPLC system (Shimadzu, Japan). Elution was carried out with 0.1 m H 2 SO 4 , 0.075 m triethylamine and 10 % acetoni- trile, pH 2.3 (at a flow rate of 1 mLÆmin )1 ) as the mobile phase [28]. The column eluates were monitored online at 245 nm (for MPTP) or 295 nm (for MPP + ) using a photo- diode array detector (SPD-M20A). All absorbance signals were quantified by integrating the peak of interest using the software LC solution version 1.22 SP1 supplied by the manufacturer. The concentrations of MPTP and MPP + in the samples were calculated using calibration curves plotted for known concentrations of MPTP and MPP + . Scanning electron microscopy After completion of aggregation, the samples were centri- fuged. The precipitated aggregate was washed twice with water and resuspended in a minimum volume of water. Two microlitres of each sample was deposited over broken cover slip and dried under air. The dried samples were gold coated and viewed under scanning electron microscope (S-3400N, Hitachi High-Technologies Corporation, Japan). Conclusion MPTP-induced parkinsonism bears important similari- ties with idiopathic Parkinson’s disease, as confirmed by similar response to levodopa therapy in both cases. However, there are differences as well, the most signifi- cant being the absence of Lewy bodies. In this study, we show that MPTP, and not its conversion to MPP + , is sufficient for a-synuclein to aggregate. It has been proposed that Lewy bodies are not seen in case of MPTP because their formation is an age-related phe- nomenon and administration of MPTP leads to ‘accel- erated’ parkinsonism. The results presented here support this hypothesis. They also indicate that in addition to the pathological consequence of MPP + acting as a mitochondrial toxin, both MPTP and MPP + speed up the aggregation of a-synuclein, thus hastening the disease onset. 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