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:
(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.
Acknowledgements
The authors are grateful to Department of Biotechnol-
ogy (Govt. of India) for partial financial support. The
authors thank Dinesh Kumar for recording the scan-
ning electron micrographs and Shivcharan Prasad and
Pinakin Makwana for technical assistance.
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