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

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

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

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

)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 (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-

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

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

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

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

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

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

+
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

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)

(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

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-

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1696 FEBS Journal 278 (2011) 1688–1698 ª 2011 The Authors Journal compilation ª 2011 FEBS
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