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

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-phenyl

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.

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 mgÆmL)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

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 (kapp) 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).

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-syna-syn-uclein

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 l M (•, 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).

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 C18column 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

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 l M 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).

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 l M neurotoxin; lane 4, with 100 l M

neurotoxin and 50 l M dopamine; lane 5, with 200 l M 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 l M neu-rotoxin; lane 4, with 100 l M neurotoxin and 50 l M dopamine; lane 5, with 200 l M neurotoxin and 50 l M dopamine (D) Lane M, prestained molecular mass markers; lane 1, a-synuclein with

100 l M neurotoxin; lane 2, a-synuclein with 200 l M neurotoxin; lane 3, a-synuclein with 100 l M neurotoxin and 50 l M dopamine; lane 4, a-synuclein with 200 l M neurotoxin and 50 l M dopamine.

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 (formnucle-ation 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

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 l M MPTP (C), in the presence of 100 l M MPP + (D), in the presence of 200 l M MPP +

(E), in the presence of 100 l M MPTP and 50 l M dopamine (F), in the presence of 200 l M MPTP and 50 l M dopamine (G), in the presence

of 100 l M MPP+and 50 l M dopamine (H) and in the presence of 200 l M MPP+and 50 l M dopamine (I).

intermediate state (UNM 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 vitroresults 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

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

a-synuclein plasmid construct using a standard calcium chloride method [37] Transformed cells were grown at

was induced with 1 mm IPTG and the cells were further

of the induction period, the cells were centrifuged at 7000 g

were lysed in lysis buffer (10 mm sodium phosphate mono-basic, 40 mm sodium phosphate dimono-basic, 1 mm EDTA, pH

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

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

in miniVE electrophoresis unit (GE Healthcare, Hong Kong) [39] The resolved proteins were detected by silver staining

[40] For western blotting, after completion of the

(0.45 lm) with transfer buffer (25 mm Tris, 20 mm glycine

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

(1 : 50) for 1.5 h The blot was finally scanned on variable

mode image scanner (Typhoon Trio; GE Healthcare)

Aggregation of a-synuclein

(100 000 g) for 1 h to remove preformed aggregate The

were withdrawn at predefined time intervals The

western blotting and various biophysical techniques

a-Syn-uclein was also incubated in the presence of different

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

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]:

y¼ yiþ mxiþyfþ mxf

1¼ exx0s

where yi+ mxiis the initial line, yf+ mxf is the final line

constant (kapp) is 1⁄ s and lag time is calculated to be x0) 2s

Chromatographic analysis

RP-HPLC analysis of the samples was carried out to

aggregated samples After completion of aggregation, the

samples were centrifuged The supernatants (20 lL) were

a HPLC system (Shimadzu, Japan) Elution was carried out

phase [28] The column eluates were monitored online at

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

the samples were calculated using calibration curves plotted

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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