Neurochem Res (2009) 34:1838–1846

DOI 10.1007/s11064-009-9986-8

REVIEW ARTICLE

Modulation of a-Synuclein Aggregation by Dopamine: A Review

Su Ling Leong Æ Roberto Cappai Æ
Kevin Jeffrey Barnham Æ Chi Le Lan Pham

Accepted: 23 April 2009 / Published online: 15 May 2009

Ó Springer Science+Business Media, LLC 2009

Abstract Parkinson’s disease (PD) is a progressive neu- may influence the onset of the disease. Several genes
rodegenerative disorder that is characterized by (1) the including, a-synuclein (a-syn), parkin, DJ-1, PINK-1 and
selective loss of dopaminergic neurons in the substantia LRRK2, have been identified and linked to PD (reviewed
nigra and (2) the deposition of misfolded a-synuclein in [2]). Mutations within these genes lead to the familial
(a-syn) as amyloid fibrils in the intracellular Lewy bodies forms of PD, accounting for less than 10% of PD cases.
in various region of the brain. Current thinking suggests Both environmental risk factors and genetic mutations
that an interaction between a-syn and dopamine (DA) leads in PD-linked genes have been associated with oxidative
to the selective death of neuronal cells and the accumula- stress and mitochondria dysfunction and this could
tion of misfolded a-syn. However, the exact mechanism by cause impairment of the proteasomal pathways leading to
which this occurs is not fully defined. DA oxidation could abnormal protein aggregation and ultimately PD [2].
play a key role is the pathogenesis of PD by causing oxi- One of the major pathological hallmarks of PD is the
dative stress, mitochondria dysfunction and impairment of selective loss of dopaminergic neurons in the substantia
protein metabolism. Here, we review the literature on the nigra region of the brain, with a reduction of approximately
role of DA and its oxidative intermediates in modulating 70% of these neurons occurring in PD patients [3]. Another
the aggregation pathways of a-syn. key pathological characteristic of PD is the formation of
intracytoplasmic inclusions, known as Lewy bodies, in
Keywords a-synuclein
Dopamine
various regions of the brain including the surviving neurons
Parkinson’s disease
Oligomers
Amyloid fibrils of the substantia nigra [4, 5]. The Lewy body is found in
the cell body of the neuron rather than the neurites, despite
both DA and a-syn being present in the synapses of
Parkinson’s disease (PD) is a progressive neurodegenera- dopaminergic neurons. This may reflect the requirement for
tive disorder clinically characterized by severe motor certain buffer conditions or co-factors being present or
impairment including bradykinesia, muscular rigidity, and absent to either inhibit or promote Lewy body formation.
resting tremor (reviewed in [1]). The disease is largely The Lewy bodies are deposited in different parts of the
sporadic where environmental risk factors (such as expo- brain as the disease progresses, as described by Braak
sure to pesticides, environmental toxins and metal ions) staging for PD [4, 5]. The lesions start, Stage 1, in the
dorsal IX/X motor nucleus and/or intermediate reticular
zone. Stage 2 includes stage 1 pathology in addition to
S. L. Leong
R. Cappai
K. J. Barnham
C. L. L. Pham (&)
lesions in the caudal raphe nuclei, gigantocellular reticular
Department of Pathology, Bio21 Molecular Science and
Biotechnology Institute, The University of Melbourne, Parkville, nucleus, and coeruleus–subcoeruleus complex. Stage 3 has
VIC 3010, Australia stage 2 pathology and midbrain lesions, especially in the
e-mail: clpham@[Link] pars compacta of the substantia nigra. Stage 4 is composed
of stage 3 pathology plus prosencephalic lesions and the
S. L. Leong
K. J. Barnham
C. L. L. Pham
The Mental Health Research Institute, Parkville, VIC 3052, temporal mesocortex (transentorhinal region) and allocor-
Australia tex (CA2-plexus). The neocortex is unaffected. At stage 5

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there is stage 4 pathology and lesions in the neocortex and reactive intermediates [20]. a-Syn knock-out mice release
prefrontal neocortex which are associated with high order more DA at the synaptic terminal when stimulated with
sensory functions. At stage 6 there is stage 5 pathology and an electrical pulse, suggesting that a-syn is an activity-
lesions in the neocortex and premotor areas associated with dependent negative regulator of DA neurotransmission
first order sensory functions. Some mild changes in pri- [21]. The a-syn knock-out mice have a significant decrease
mary sensory areas and the primary motor field may be in the number of vesicles in the distal pool of the presyn-
present. aptic terminal [21], resulting in an increase in cytoplasmic
The Lewy bodies are composed of a large number of DA. The over-expression of a-syn in PC12 or chromaffin
molecules, of which a-syn amyloid fibrils are the most cells interferes with the release of DA from vesicles by
prominent component [5–7]. Missense mutations (A30P, affecting a late exocytoic step, further suggesting a-syn has
E46K and A53T) and genomic duplication and triplication a role in binding to lipids and regulating vesicle function
of the a-syn gene have strongly linked a-syn with both [22].
early onset familial PD and sporadic PD [8–12].

Oxidation of DA and Neuromelanin Biosynthesis

Structure and Function of a-Synuclein
It has been proposed by a number of groups that an
Human a-syn is a small natively unfolded protein com- interaction between a-syn and dopamine (DA) leads to the
posed of 140 amino acids with a predicted molecular selective death of neuronal cells and the accumulation of
weight of 14,459 Da. It has been observed that a-syn misfolded a-syn in Lewy bodies [23]. Cytosolic DA readily
migrates anomalously at approximately 17–18 kDa on undergoes redox reactions [24], generating toxic DA
SDS–PAGE. The amino acid sequence can be divided into reactive quinones along with superoxides and hydrogen
three regions, with each region having distinct structural peroxide [25]. The increase in reactive oxygen species
characteristics. The highly conserved N-terminal region (ROS) promotes oxidative stress and mitochondria dys-
encode for a series of six imperfect 11 amino acid repeats function lead to neuronal cell death. The oxidation of
with a consensus motif of KTKEGV, predicted to resemble excess cytosolic DA leads to the formation of neuromela-
the lipid-binding domain of apolipoprotein, containing nin within dopaminergic neurons [24]. Neuromelanin is
several amphipathic helices [13]. The N-terminal domain present at high levels in the substantia nigra region of the
binds to phospholipids and adopts an a-helical conforma- human brain [26] and accounts for the darken appearance
tion [13–15] suggesting that the biological function of of these neurons [27]. In PD, it is the neuromelanin con-
a-syn may involve binding to lipid membranes [13]. The taining dopaminergic neurons that are selectivity lost
central region, referred to as the non amyloid-like com- [26, 27]. In this proposed oxidative pathway [28] (Fig. 1)
ponent (NAC region) was originally isolated is association DA is initially oxidized to form dopamine-o-quinone (DQ)
with the Ab peptide in Alzheimer’s disease amyloid pla- which subsequently undergoes a cyclization step followed
ques [16, 17]. This region is hydrophobic and contains the by another oxidation step to form dopaminochrome (DC).
amino acid sequences essential for b-sheet amyloid fibril DC itself is an unstable molecule and can undergo rear-
formation [18]. The acidic C-terminal region of a-syn rangement to form 5,6-dihydroxyindole (DHI) which can
contains mostly negatively charged residues and is largely then polymerize into melanin. Alternatively, DHI can be
unfolded. further oxidized to form indole-5,6-quione (IQ) that will
The a-syn protein is ubiquitously expressed throughout subsequently form melanin.
the brain and is mainly localized at the presynaptic ter- In addition to the non-enzymatic oxidation pathway,
minal. The biological role of a-syn is not fully understood. DA can also undergo a two step enzymatic oxidation
There is strong evidence to suggest a-syn is involved in reaction. The first step involves the catalyzed oxidation of
regulating and maintaining DA homeostasis within the DA by monoamine oxidase (MAO) to form 3,4-dihy-
cytoplasm. a-Syn act as a negative regulator of DA bio- droxyphenylacetaldehyde (DOPAL) along with hydrogen
synthesis by interacts with tyrosine hydroxylase and peroxide [23]. DOPAL is a reactive aldehyde, also known
reduces the activity of this enzyme [19]. Tyrosine as the MAO metabolite, and in the presence of hydrogen
hydroxylase coverts tyrosine into L-dopa, the first step in peroxide it can generate toxic hydroxyl radicals [23, 29].
the biosynthetic pathway of DA. Upon synthesis, DA is In the second step of this enzymatic oxidation of DA,
translocated from the cytoplasm into small synaptic vesi- DOPAL can be converted to a non-toxic 3,4-dihydroxyl-
cles via the vesicular monoamine transporter 2 protein for phenylacetic acid (DOPAC) by aldehydyde dehydrogenase
storage [20]. The low pH environment within these vesicles or converted to 3,4-dihydroxyphenylethanol by aldehyde
stabilizes DA, preventing oxidation and the formation of reductase [23].

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Fig. 1 Proposed oxidation pathway of DA. The initial oxidation of 5,6-dihydroxyindole (DHI) and this can then polymerize into melanin.
dopamine (DA) results in the formation of dopamine-o-quinone (DQ) Alternatively, DHI can be further oxidized to form indole-5,6-quinone
which subsequently undergoes a cyclization step, followed by another (IQ) that will subsequently form melanin. Figure taken from Pham
oxidation step, to form a second quinone; dopaminochrome (DC). DC et al. [30] with permission from publisher (Elsevier)
itself is unstable and can readily undergo rearrangement to form

a-Synuclein Aggregation oxidized methionines or nitrated tyrosines on a-syn can

inhibit fibril formation of un-modified a-syn [39, 41, 42].
In vitro, a-syn aggregates into amyloid fibrils with similar The identity of ‘‘toxic a-syn species’’ within the fibril
morphology and properties to the amyloid fibrils extracted formation pathway remains controversial. The deposition
from Lewy bodies in vivo [31]. Amyloid fibril formation of a-syn amyloid fibrils into the Lewy bodies indicated that
by a-syn involves the initial aggregation of a-syn into small the insoluble amyloid fibrils may be the causative agent in
oligomers and protofibrils that elongate into mature fibrils. the pathology of PD. However, in recent years it have been
The kinetics of fibril formation by a-syn is believed to hypothesized that soluble oligomers, on-pathway to amy-
occur via a nucleation dependent pathway [32]. This loid fibril formation, may be the common toxic species
involves an initial conformational change of a-syn mono- observed in different amyloid-related diseases including
mers into a partially folded conformation that undergoes a the Ab peptide associated with Alzheimer’s disease
series of unfavorable self-associations to form a highly [43–46]. Thus, the formation of insoluble amyloid fibrils
unstable nucleus during a lag phase [33]. The formation of may serve as a protective pathway sequestrating toxic
the nucleus is followed by a more favorable growth phase species away from the cellular environment [43–46]. This
(elongation) where the nucleus rapidly grows into fibrils by would be consistent with the presence of Lewy bodies in
the addition of monomers. Various environmental and surviving neurons. The E46K and A53T familial mutants
solution factors induce conformational changes within the of a-syn promote the formation of oligomers and fibrils
a-syn monomer and modulates the aggregation state of [31, 47–50], although the effect of A30P on fibrillization is
a-syn [34]. Increases in solution pH and incubation tem- less defined [51–53]. The A30P oligomers are inefficient in
perature can induce a partially folded conformation [35, converting into fibrils. It has been suggested that the
36]. High protein concentration, the presence of metal ions, oligomeric species of a-syn are the neurotoxic species in
increase in the ionic strength of the solution and a decrease PD and that the mature fibrils within the Lewy bodies are
in solution pH can accelerate fibril formation [32, 37, 38]. neuroprotective [31, 47, 53–55]. Electron and atomic force
Oxidative modifications of a-syn can inhibit fibril forma- microscopy studies showed that the oligomers formed by
tion. The oxidation of the four methionine residues within either WT or mutant a-syn have a number of different
a-syn inhibits oligomerization and fibril formation [39, 40]. morphologies including spherical, chain-like, ring-like and
Oxidative modification of the tyrosine residues via nitra- annular structures [53, 56, 57]. The annular oligomeric
tion induces a partially folded conformation of a-syn structures have been proposed to have pore-like activity
leading to the formation of stable soluble oligomers that do that binds and permeabilizes lipid membranes [58, 59]. The
not elongate into fibrils [41, 42]. The presence of either lipid binding properties of a-syn appears to correlate with

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the formation of a high affinity intermediate formed during its oxidative intermediates have the ability to induce the
the early aggregation phase [50]. In agreement with its conversion of oligomers into fibrils or whether these
lipid binding properties, certain oligomeric species induce a-syn:DA oligomers have the ability to slowly form fibrils.
calcium influx in neuronal SH-SY5Y cells leading to cel- To fully address these issues there is a need to purify these
lular toxicity [60]. Furthermore, over-expression of WT oligomeric species in order to study their structural,
and mutant a-syn in PC12 cells showed that specific a-syn aggregation, cytotoxic and aggregation properties.
oligomers inhibit the activity of the ubiquitin-proteasome The screening of drug-like compounds that inhibit a-syn
system [61]. This would be consistent with a defect in the amyloid fibril formation identified 15 catecholamine
turnover of mis-folded protein in PD affected neurons. compounds that are related to dopamine [71]. The inhibi-
tory effect of the compounds is due to the formation, of a
small percentage (\10%), of covalently modified a-syn
DA can Modulate a-Synuclein Aggregation that has formed adducts with DA-quinone and these
modified species prevented the elongation of protofibrils
A number of in vitro studies have shown that DA can into amyloid fibrils. DA-quinones can modify proteins
modulate the aggregation of a-syn along distinct pathways through cysteine residues, but since a-syn lacks cysteine
[58, 62–67]. This occurs at a 1:1 (a-syn:DA) stoichiometry, residues then the formation of a-syn-DA covalent adducts
which is consistent with the relative physiological stoi- has been proposed to occur via radical coupling to form
chiometries found between a-syn and DA in cells and dityrosine linkages and/or a nucleophilic attack via lysine
dopaminergic neurons [62]. DA induces the aggregation of side chains [63, 71]. Incubation of a-syn with several DA
both WT a-syn and the A53T mutant in a temperature analogs, namely polyphenols, also showed inhibition of
dependent manner, with the A53T mutant having an fibril formation [72]. The polyphenols can be readily oxi-
increased propensity to aggregated in the presence of DA dized into quinones that react with the amino group of
with both recombinant and protein expressing in human a-syn to form a-syn-quinone covalent adduct [72]. Alter-
neuroblastma M17 cells [68]. Expressing different forms of ations in the homeostasis of DA can lead to DA oxidation
tyrosine hydroxylase-containing letiviruses can alter the and the formation and accumulation of DA-quinones
steady-state levels of intracellular DA. Co-expressing within the cytoplasm. While DA-quinone can interact with
a-syn and tyrosine hydroxylase in SH-SY5Y cells caused a-syn and inhibit the conversion of oligomers into mature
an increase in cytosolic DA levels and this induced the fibrils [71], DA-quinone also causes DA neuron-specific
formation of intracellular soluble oligomeric intermediates oxidation stress by interacting and altering the normal
[69]. However, these oligomeric intermediates do not lead functional activity of a number of other proteins including
to toxicity [69]. Brain tissues from transgenic mouse over- tyrosine hydroxylase [65, 66], DA transporter [67] and
expressing the A53T a-syn mutant showed the presence of parkin [73].
soluble oligomers and the absence of a-syn inclusions in Fe3? promotes the kinetics of a-syn aggregation into
dopaminergic neuron. These neurons maintained the ability amyloid fibrils [38, 62]. Iron is the most abundant transi-
to produce DA [69]. tion metal in the body and increased levels of Fe3? occurs
The observations made from the various in vitro studies in the substantia nigra due to the presence of neuromelanin
on the interaction between DA with a-syn is summarized in [74]. Fe3? can act as catalyst in the Fenton’s reaction that
Fig. 2. Under physiological conditions, a-syn self associ- converts hydrogen peroxide, via the oxidation of DA, into
ates into small oligomers and protofibrils that elongate into toxic hydroxyl free radical. We showed that DA com-
mature amyloid fibril (Fig. 2, Route 1). The addition of DA pletely inhibited the ability of Fe3? to promote fibril for-
to pre-formed fibrils can disaggregate fibrils into small mation [38, 62]. This established DA as a dominant
non-fibrillar soluble oligomers [63] (Fig. 2, Route 2). modulator of a-syn oligomerization into non-amyloid
DA can promote a-syn to form soluble SDS-resistant species.
and Thioflavin T negative oligomers [62], which are off- A number of studies suggest that the interaction of a-syn
pathway intermediates to amyloid fibril formation [62, 64] with DA leads to the formation of a-syn-DA quinone
(Fig. 2, Route 3a). However, prolonged incubation (after adducts [63, 71, 72]. This indicates that reactive interme-
18 days) of a-syn in the presence of equimolar concen- diates of DA oxidation, and not DA itself, are responsible
trations of DA can result in a-syn aggregating into fibrils as for modulating the aggregation properties of a-syn. Dopa-
observed by electron microscopy [70] (Fig. 2, Route 3b). minochrome, an oxidative product of DA, inhibits fibril
These fibrils, formed in the presence of DA, are unstable formation and promotes the oligomerization of a-syn
and susceptible to breakage [70]. Prolonged incubation of [63, 64]. In our recent studies, we attempted to purify
DA under physiological conditions can lead to the forma- dopaminochrome but isolated DHI instead [30]. This is
tion of melanin. This raises the question of whether DA or perhaps due to the instability of dopaminochrome and the

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Route 1

α-syn
monomer
α-syn DA α-syn fibrils
oligomers
Route 2

DA
very slow
Route 3b

α-syn:DA
adducts
SDS- resistant soluble
α-syn oligomers
Route 3a
Fig. 2 Model of a-synuclein aggregation pathways. Amyloid fibril off-pathway intermediates to amyloid fibril formation [62, 64] (Route
formation by a-syn occurs via a nucleation dependent pathway [32] 3a). One possible mechanism of how DA modulates the formation of
that involves an initial self-association step into small soluble SDS-resistant soluble a-syn oligomers involve the interaction of a-syn
oligomers that then elongate into fibrils as shown in the electron and DA to form covalent adducts that inhibit the conversion of
micrograph (Route 1). DA can disaggregate these insoluble fibrils into oligomers into amyloid fibrils [63, 71, 72]. a-syn incubated with DA
small soluble oligomeric species [63] (Route 2). In the presence of can slowly form fibrils after prolonged incubation [70] (Route 3b)
DA, a-syn monomer form SDS-resistant soluble oligomers, that are

tendency of this compound to undergo rearrangement and oxidation of DA), can inhibits fibril formation by a-syn by
form DHI (Fig. 1). In the presence of DHI a-syn formed stabilizing oligomeric species via non-covalent interaction
SDS-resistant soluble oligomers that do not aggregate into between DOPAC and a-syn [76].
amyloid fibril. SDS–PAGE/silver stain analysis showed
that the oligomers formed in the presence of DHI were
indistinguishable to those formed in the presence of DA Proposed Mechanism for the Modulation of
[30]. DHI is itself an unstable compound that can undergo a–Synuclein Aggregation by DA
further oxidation to form melanin. This indicates that redox
reactions involving DHI are most likely to mediate the As yet the molecular mechanism of how DA interacts with
formation of a-syn SDS-stable oligomers. Our findings are a-syn to modulate the aggregation pathway remains
in agreement with the studies by Bisaglia et al. [28] on the unclear. One possibility, as suggested by Conway et al.
kinetic and structural effects of DA oxidative products on [71], was that the formation of DA-a-syn adducts pre-
a-syn. They showed that indole-5,6-quione and not dopa- vented the conversion of oligomers into fibrils and resulted
mine-o-quinone or dopaminochrome, is the reactive qui- in the accumulation of soluble oligomers. In contrast,
none acting on a-syn [28]. Norris et al. [64] failed to detect any significant levels of
The MAO metabolite from the enzymatic oxidation of DA-a-syn adducts. Sequence activity studies identified the
125
DA, DOPAL, can also trigger the aggregation of a-syn into YEMPS129 motif in the acidic C-terminal domain of
oligomers in cell free assay [75]. Furthermore, the addition a-syn to be important for DA-mediated inhibition of a-syn
of DOPAL to a-syn over-expressing SH-SY5Y cells, fibril formation [64]. The removal of this motif in recom-
accelerates a-syn oligomerization and aggregation into binant a-syn, terminating at residue 125, or mutations
species that may be toxic to cells [75]. In addition, a very within the YEMPS motif of a-syn, restored the ability of
recent studies show that substoichiometric concentration of a-syn to form amyloid fibrils in the presence of DA or
DOPAC (a product of the second step in the enzymatic dopaminochrome [64]. Based on these findings, Norris

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et al. proposed that the oxidation of DA and the subsequent Conclusion Remarks
non-covalent interaction between the DA oxidative inter-
mediate and the C-terminus region of a-syn inhibits fibril The loss of dopaminergic neurons and the accumulation of
formation. The interaction of the ‘‘YEMPS’’ motif with DA a-syn amyloid fibrils in Lewy bodies is strongly associated
was also studied by Mazzulli et al. [77] where they created with PD. What is less clear is the direct relationship
stably transfected SH-SY5Y cells over-expressing the between a-syn and the other proteins (parkin, DJ-1 and
a-syn A53T mutant with the ‘‘YEMPS’’ residues mutated LRRK2) in the pathogenic pathways that lead to PD. This
to alanine (termed A53T-125m). They showed that A53T- review summarizes the observations made from various
125m was able to form large amyloidogenic inclusions groups on the relationship between DA, a-syn and PD. It is
[77]. Furthermore, co-expression of the A53T-125m with clear that the oxidation of DA plays a key role in the
tyrosine hydroxylase show that intracellular DA levels had pathology of PD. a-Syn expression can alter the homeo-
no effect on the ability of A53T-125m to form amyloid stasis of DA and this leads to an increase in cytosolic DA
inclusions [77]. These results suggested that DA modulated that readily undergoes oxidation. The oxidation of DA
a-syn oligomerization by interacting with the C-terminal generates reactive intermediates and ROS that causes oxi-
‘‘YEMPS’’ sequence. Furthermore, molecular dynamic dative stress, mitochondria dysfunction, impairments of
simulation, along with in vitro biophysical assays, sug- protein function. A significant body of data indicates that
gested that a-syn forms complexes with DA by non-spe- the DA and its oxidative intermediates modulating a-syn
cific hydrophobic interactions of the ‘‘YEMPS’’ sequence aggregation pathway. The pathogenic species for many
with the aromatic ring of DA. These a-syn:DA complexes amyloid-related diseases, including AD and PD, appear to
are stabilized by the electrostatic interaction with E83 be the small soluble oligomers on-pathway to amyloid
residue with the NAC region of a-syn [78]. fibril formation [43–46, 53]. In contrast, insoluble amyloid
Our recent data demonstrate that the formation of the fibrils are thought to be relatively inert. DA induces the
DA-mediates SDS-resistant a-syn soluble oligomers does formation of soluble oligomers that may not convert into
not require the YEMPS sequence [40]. Truncated a-syn mature amyloid fibrils. As yet, little is known about
mutant terminating at residue 124 could still formed the physical, structural and cytotoxic properties of the
soluble oligomers in the presence of DA [40]. Based on a DA-mediated SDS-resistant a-syn oligomers. Therefore, to
number of observations, we proposed that methionine better understand the relationship between a-syn and DA,
oxidation of a-syn is the key mechanism by which DA and their association with PD, there is a further need to
mediates the formation of SDS-stable soluble a-syn isolate, purify and study the action and structure of these
oligomers [40]. Firstly, mass spectrometry and NMR oligomers.
studies identified that all four methionine residues in
a-syn were oxidized following incubation with DA. Sec-
ondly, the substitution of all four methionine residues to
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